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ANTI-ADIPOGENIC: Adipocyte Differentiation Inhibitor 200:1 – New!

Name: ANTI-ADIPOGENIC: Adipocyte Differentiation Inhibitor 200:1 - New!
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Adipogenesis refers to the process of formation of adipose tissue. It is a multistep process starting with clonal expansion of mesenchymal cells and the Differentiation of these mesenchymal cells into Pre-Adipocytes and finally into mature Adipocytes.

Herbal Remedies against Adipogenesis

Many phytochemicals and herbal extracts show Inhibitory effects on Adipogenesis in cell models and mouse models. However, lack of clinical evidence and epidemiological data to support their roles in Inhibiting Adipogenesis hindered their development to become an Anti-Obesity therapeutic agent. Furthermore, many of these phytochemicals have low bioavailability. Chemical structural modification or targeted delivery system may help to increase their Anti-Adipogenesis or Anti-Obesity efficacies. Nevertheless, many of these phytochemicals are abundantly found in our daily food, or they are the herbs for cooking or seasoning. For example, resveratrol is rich in red wine and grapes; genistein is rich in soy; quercetin in apples and onions, and curcumin is a well-known component of the cook seasoning. Therefore, a healthy diet can definitely help us to increase the uptake of these phytochemicals in our daily life. Moreover, combination of phytochemicals or herbal extracts may act synergistically to Inhibit Adipogenesis.

Indeed, Obesity is not simply an excess accumulation of white Adipose Tissue but is usually associated with insulin resistance and an increased production of metabolic hormones coupled with chronic low-grade state of Inflammation . To effectively Reduce Obesity , a holistic strategy with the consumption of phytochemicals or herbal extracts can be designed not only to Reduce Adipose Tissue Mass, but also increase thermogenic energy expenditure, improve insulin sensitivity, Reduce plasma lipids which can help to Reduce the Obesity -associated dyslipidemia and other comorbid conditions.

Featuring:

Açai (Euterpe Oleracea Martius) Anthocyanins • Acer Okamotoanum Nakai Leaf • Achyranthes Bidentata Blum • Acorn (Quercus Acutissima Carr.) Shell • Adenanthin, A Natural Ent-Kaurane Diterpenoid Isolated From The Herb Isodon Adenantha • Aicar • Ajoene Sulfur-Containing Compound • Allium Hookeri Root • Andrographolide • Anthocyanins • Antofine Alkaloid • Antrodia Cinnamomea • Apigetrin (Apigenin-7-O-Glucoside) • Arctigenin • Arctiin • Aristolochia Manshuriensis Kom • Aristotelia Chilensis • Artemisia Capillaris • Aster Yomena (Kitam.) Honda Leaves • Astilbe Chinensis Franch. Et Savet • Astragalin, (3-O-Glucoside Of Kaempferol) • Averrhoa Carambola L. Peel • Avicularin • Bacaba (Oenocarpus Bacaba Mart.) Phenolic (Bpe) • Baicalin • Bamboo (Phyllostachys Bambusoides) Leaf • Benincasa Hispida • Berberine • Bergamottin • Bestatin (Ubenimex) • Betanin (Betanin-5-O-Β-Glucoside) • Bisdemethoxycurcumin • Blumea Balsamifera • Boldine • Boussingaulti Gracilis Miers Var. Pseudobaselloides Bailey • Butein Chalconoid • Capsaicin • Carduus Crispus • Carnosic Acid • Carnosine (L-Carnosine) • Chromolaena Odorata Leaves • Chrysanthemum Zawadskii Extact • Chrysin • Cinnamomum Verum • Cirsium Brevicaule A. Gray • Cirsium Setidens Nakai • Citrus Aurantium Peel • Clitoria Ternatea Flower Petal • Cocoa Tea (Camellia Ptilophylla) • Colocynth (Citrullus Colocynthis) Flesh • Conjugated Linoleic Acid (Cla) • Coptis Chinensis (Coptidis Rhizoma) • Cordycepin • Coumestrol • Cranberries (Oxycoccus Quadripetalus) • Crocin • Cryptotanshinone • Curcumin • Curcumin-3,4-Dichloro Phenyl Pyrazole (Cdpp) • Cyanidine-3-O-Galactoside Enriched Aronia Melanocarpa • Cyclopia Subternata • Delphinidin • Delphinidin-3-O-Β-Glucoside • Dhea • Diallyl Trisulphide • Dioscin (Ds) A Steroidal Saponin • Djulis (Chenopodium Formosanum) • Dolichos Lablab L. Seeds • Ecklonia Cava • Ecliptal, Isolated From Eclipta Alba • Egcg • Ellagic Acid • Epiberberine • Erigeron Annuus (L.) Pers. • Esculetin • Euphorbia Lunulata • Evodiamine • Ferulic Acid • Ficus Deltoidea Var. Deltoidea • Fisetin • Foenumoside B From Lysimachia Foenum-Graecum • Formononetin • Fucoxanthinol • Gallotannin Derivatives From Mango (Mangifera Indica L.) • Ganoderma Applanatum • Ganoderma Lucidum • Garcinia Cambogia • Garcinol • Gelidium Elegans • Genistein • Gentiopicroside • Gentiopicroside Isolated From Gentiana Scabra Bge. • Ginkgetin, A Biflavone From Ginkgo Biloba Leaves • Ginkgo Biloba Seed Coat • Ginkgolide C, Isolated From Ginkgo Biloba Leaves • Ginsenoside Rg1 • Ginsenoside Rg2 • Ginsenoside Rg3 • Glehnia Littoralis Root • Guarana (Paullinia Cupana) • Guggulsterone • Heshouwu (Polygonum Multiflorum Thunb) • Hesperidin • Hibiscus Rosa Sinensis Flower • Hibiscus Sabdariffa L • Hippophae Rhamnoides L. Leaves • Hwangryunhaedok-Tang • Hyperoside • Icaritin Hydrolytic Product Of Icarin From Epimedium Genus • Irvingia Gabonensis Seed • Ivy Gourd (Coccinia Grandis L. Voigt) Root • Kaempferol • Komulkosiraegi [Gracilaria Vermiculophylla (Ohmi) Papenfuss] • Lindera Obtusiloba • Lupenone • Lupeol • Lysimachia Foenum-Graecum • Magnolia Officinalis Magnolol • Mai Tong Fang (Radix Astragali • Mangifera Indica L. Peel • Mangiferin From Iris Rossii Baker • Maté (Ilex Paraguariensis) • Melissa Officinalis • Metadina Trichotoma • Methyl Cinnamate • Mollugin (From Rubia Cordifolia L Roots) • Momordica Charantia • Moringa Oleifera Leaves (Drumstick Tree) • Morus Alba • Murta (Myrceugenia Euosma) Dried Branches And Leaves • Myrica Gale • Nam Doc Mai • Naringenin • Nobiletin • Nonivamide • Oleanolic Acid • Oligonol • Orientin • Oroxylin A Flavonoid • Panicum Miliaceum L. • Pelargonidin • Petalonia Binghamiae • Peucedanum Japonicum Thunberg L. • Phyllanthus Emblica Fruit • Piperine • Platycodin D • Platyphylloside • Pleurotus Eryngii Var. Ferulae ‘Beesan No. 2’ (Cebt) • Pluchea Indica (L.) Less. Tea • Polygonum Cuspidatum • Porphyra Yezoensis • Prieurianin • Procyanidin B2 • Pterostilbene • Punicalagin • Quercetin • Raspberry Ketone • Rehmannia Glutinosa • Resveratrol • Retinoic Acid • Rhamnetin • Rhein Glycoside • Rhizoma Polygonati Falcatum • Rosmarinic Acid • S-Petasin • Salicortin • Sargassum Micracanthum • Sargassum Serratifolium • Saururus Chinensis • Securigera Securidaca (Seeds) • Sesamol • Shikonin • Sibiraea Angustata • Silibinin • Siphonaxanthin • Soyasaponin Ab • Stearidonic Acid (Sda) • Stinging Nettles (Utrica Dioica L.) • Styrax Japonica Fruit • Sulforaphene • Sulfuretin Is A Natural Flavonoid Found In The Plant Rhus Verniciflua Stokes. • Tannic Acid • Taraxacum Officinale (Dandelion) • Tocotrienol • Tricin • Trigonella Foenum-Graecum (Seeds) • Triticum Aestivum • Tyrosol (From Rhodiola Crenulata ) • Ulmus Pumila • Ursolic Acid • Vaccinium Floribundum • Vanillic Acid • Veratrum Nigrum • Viburnum Opulus L. • Vitexilactone, A Constituent From Vitex Trifolia L • Vitexin From Mung Bean • Vitisin A From Iris Lactea Pall. Var. Chinensis (Fisch.) Koidz • Wasabi (Wasabia Japonica Matsum.) Leaves • Wedelolactone Furanocoumarin • Widdrol • Xanthohumol • Zeaxanthin • Zizyphus Jujube • Α-Mangostin • Β-Asarone • Β-Cryptoxanthin • 13-Methylberberine • 17Β-Glycyrrhetinic Acid • 2,6-Dimethoxy-1,4-Benzoquinone • 25 Hydroxycholesterol • 3-Chloro-4,5-Dihydroxybenzaldehyde • 3,5-Dicaffeoyl-Epi-Quinic Acid Isolated From Edible Halophyte Atriplex Gmelinii • 3,5,6,7,7,3′,4′-Heptamethoxyflavone (Hmf), A Naturally Occurring Polymethoxyflavone Found In Citrus Peel • 6-Gingerol • 6,7,4′-Trihydroxyisoflavone

Potential of natural products in the Inhibition of Adipogenesis through regulation of PPARγ expression and/or its transcriptional activity

Obesity is a global health problem characterized as an increase in the Mass of Adipose Tissue . Adipogenesis is one of the key pathways that increases the Mass of Adipose Tissue , by which preAdipocyte s mature into Adipocytes through cell Differentiation . Peroxisome proliferator-activated receptor γ (PPARγ ), the chief regulator of Adipogenesis , has been acutely investigated as a molecular target for natural products in the development of anti-Obesity treatments. In this review, the regulation of PPARγ expression by natural products through Inhibition of CCAAT/enhancer-binding protein β (C/EBPβ) and the farnesoid X receptor (FXR), increased expression of GATA-2 and GATA-3 and activation of the Wnt/β-catenin pathway were analyzed.

Furthermore, the regulation of PPARγ transcriptional activity associated with natural products through the antagonism of PPARγ and activation of Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) were discussed. Lastly, regulation of mitogen-activated protein kinase (MAPK) by natural products, which might regulate both PPARγ expression and PPARγ transcriptional activity, was summarized. Understanding the role natural products play, as well as the mechanisms behind their regulation of PPARγ activity is critical for future research into their therapeutic potential for fighting Obesity .

Effects of Flavonoids and Phenolic Acids on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

Obesity has become a global epidemic in both developed and developing countries, and it is a significant risk factor for various diseases such as Diabetes , cancer, heart disease, and hypertension. In the present study, the effect of naturally occurring Antioxidants (Flavonoids and Phenolic Acids ) on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes was investigated. The results showed that o-coumaric acid and rutin had the highest Inhibition on intracellular triglyceride (61.3 and 83.0%, respectively) among 15 Phenolic Acids and 6 Flavonoids tested. However, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 Adipocytes was not influenced by those compounds.

For glycerol-3-phosphate dehydrogenase (GPDH) activity, the data indicated that o-coumaric acid and rutin had the highest Inhibition on GPDH activity (54.2 and 66.8%, respectively) among the compounds tested. o-Coumaric acid and rutin also Inhibited the expression of PPARγ , C/EBPα and leptin and then up-regulated expression of adiponectin at the protein level. Some naturally occurring Antioxidants efficiently suppressed Adipogenesis in 3T3-L1 Adipocytes. These results suggest that o-coumaric acid and rutin targeted for Adipocyte functions could be effective in improving the symptoms of Metabolic Syndrome.

INGREDIENTS:

açai (Euterpe oleracea Martius)

Anti-lipidaemic and anti-inflammatory effect of açai (Euterpe oleracea Martius) Polyphenols on 3T3-L1 Adipocytes

The anti-lipidaemic and anti-inflammatory effects of açai Polyphenols in 3T3-L1 mouse Adipocytes were investigated. Pre-Adipocytes were differentiated with and without açai-Polyphenols at concentrations of 2.5, 5 and 10 µg gallic acid equivalents (GAE)/mL. Results showed that açai Polyphenols Reduce the accumulation of intracellular lipids in differentiated Adipocytes in a dose-dependent manner and downregulated PPARγ 2. The gene-expression of Adipogenic transcription factors C/EBPα, C/ebpβ, Klf5 and Srebp1c was decreased.

This was accompanied by a reduction of Adipogenic genes, including aP2, LPL, FATP1 and FAS, leptin and total PAI and an increase of adiponectin. Additionally, açai Polyphenols protected cells against the production of ROS and decreased the expression of mRNA and protein levels of pro-inflammatory cytokines when 3T3-L1 cells were challenged with TNF-α. Thus, these results indicate that açai Polyphenols may be useful in the prevention of Adipogenesis, oxidative stress and Inflammation.

AntiAdipogenic effects of açai seed extract on high fat diet-fed mice and 3T3-L1 Adipocytes : A potential mechanism of action

Açai seed extract (ASE) exerts antiobesogenic effects.•ASE regulates lipogenic signaling pathways.•ASE is promising functional food ingredient for the management of Obesity.

Body Adiposity is an important risk factor for the development of chronic non-transmissible diseases. Studies on the process of Adipogenesis from ScienceDirect’s AI-generated Topic PagesAdipogenesis have been extensively performed in vivo and in vitro models to describe the molecular and cellular bases of Adipose Tissue development and the effect of natural products in this process. The açai seed extract (ASE) has been evidenced as a potential regulator of body Mass. In our work high-fat diet–fed mice treated with ASE (300 mg/Kg/d) (HFD-ASE) showed a lower adipose index (−32.63%, p < 0.001) than the high-fat diet–fed mice group (HFD) and the Adipocytes from the HFD group were considerably enlarged (p < 0.001) compared to those in the control group (CG) and HFD-ASE group (+175% and +123%, respectively).

We also evaluated the effects of ASE on the modulation of Adipogenesis in 3T3-L1 cells. ASE exposure (25 and 100 μg/mL) led to a decrease of 26.6 (p < 0.05) in proliferation and also Inhibited pre-Adipocyte Differentiation through the decreasing expression (p < 0.05) of transcription factors and Adipogenic proteins such as PPARɣ, SREBP-1, and FAS. These results show that the ASE Reduce Adipogenesis and suppress lipid accumulation in the in vivo model and in 3T3-L1 Adipocytes and reinforce ASE as a potential strategy to modulate Adipogenesis.

Acer okamotoanum Nakai Leaf

Acer okamotoanum Nakai Leaf Extract Inhibits Adipogenesis Via Suppressing Expression of PPAR γ and C/EBP α in 3T3-L1 cells

The genus Acer contains several species with various bioactivities including antioxidant, antitumor and anti-inflammatory properties. However, Acer okamotoanum Nakai, one species within this genus has not been fully studied yet. Therefore, in this study, we investigated the Anti-Adipogenic activities of leaf extract from A. okamotoanum Nakai (LEAO) on 3T3-L1 preadipocytes. Adipogenesis is one of the cell Differentiation processes, which converts preadipocytes into mature Adipocytes . Nowadays, Inhibition of Adipogenesis is considered as an effective strategy in the field of Anti-Obesity research. In this study, we observed that LEAO decreased the accumulation of lipid droplets during Adipogenesis and down-regulated the expression of key Adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/enhancer binding protein α (C/EBP α). In addition, LEAO inactivated PI3K/Akt signaling and its downstream factors that promote Adipogenesis by inducing the expression of PPAR γ. LEAO also activated β-catenin signaling, which prevents the Adipogenic program by suppressing the expression of PPAR γ. Therefore, we found that treatment with LEAO is effective for attenuating Adipogenesis in 3T3-L1 cells . Consequently, these findings suggest that LEAO has the potential to be used as a therapeutic agent for preventing Obesity.

Keywords: Acer okamotoanum, Adipogenesis, Anti-Obesity, 3T3-L1 preadipocytes

Achyranthes bidentata Blum

Effect of Achyranthes bidentata Blume on 3T3-L1 Adipogenesis and Rats Fed with a High-Fat Diet

The present study investigated the AntiObesity effect of Achyranthes bidentata Blume root water extract in a 3T3-L1 Adipocyte Differentiation model and rats fed with a high-fat diet. To investigate the effect of Achyranthes bidentata Blume on Adipogenesis in vitro, differentiating 3T3-L1 cells in Adipocyte -induction media were treated every two days with Achyranthes bidentata Blume at various concentrations (1 to 25 μg/mL) for eight days. We found that Achyranthes bidentata Blume root Inhibited 3T3-L1 Adipocyte Differentiation without affecting cell viability and Western blot analysis revealed that phospho-Akt expression was markedly decreased, whereas there was no significant change in perilipin expression.

Furthermore, administration of Achyranthes bidentataBlume root (0.5 g/kg body weight for six weeks) to rats fed with a high-fat diet significantly Reduced body Weight Gain without affecting food intake, and the level of triglyceride was significantly decreased when compared to those in rats fed with only a high-fat diet. These results suggest that Achyranthes bidentata Blume root water extract could have a beneficial effect on Inhibition of Adipogenesis and controlling body weight in rats fed with a high-fat diet.

Acorn (Quercus acutissima CARR.) shell

Anti-oxidant and Anti-Adipogenic effects of acorn (Quercus acutissima CARR.) shell extracts via regulation of wnt signaling in 3T3-L1 cells

Acorn (Quercus acutissima CARR.) is a nut from the Fagaceae family that has been used in traditional medicine for many years. However, shells from acorns are regarded as a by-product and are mostly discarded. Anti-Adipogenic activities of acorn shells were investigated using 3T3-L1 cells and methanol shell extracts (AE-M). AE-M demonstrated Cu²⁺-chelation activities and anti-oxidant activities via reduction of oxidative stress levels induced using AAPH. Six days after Adipocyte Differentiation, 50 and 100 μg/mL AE-M completely suppressed 3T3-L1 Adipogenesis and the Anti-Adipogenic effect was stronger than for the positive control 50 μM quercetin.

Treatment with AE-M in 3T3-L1 cells Reduced mRNA expression levels of Adipogenic genes. AE-M-Inhibition was found in Pre-Adipogenic, early, and intermediate stages of Adipogenesis in 3T3-L1 cells. The Wnt/β-catenin signaling pathway is required for AE-M-Inhibition of 3T3-L1 Adipogenesis.

Adenanthin

(Isodon adenantha)

Adenanthin, a Natural ent-Kaurane Diterpenoid Isolated from the Herb Isodon adenantha Inhibits Adipogenesis and the Development of Obesity by Regulation of ROS

Adenanthin, a natural ent-kaurane diterpenoid extracted from the herb Isodon adenantha, has been reported to increase intracellular reactive oxygen species in leukemic and hepatocellular carcinoma cells. However, the function and mechanism of the compound in Adipogenesis and the development of Obesity is still unknown. In this study, we demonstrated that adenanthin Inhibited Adipogenesis of 3T3-L1 and mouse embryonic fibroblasts, and the underlying mechanism included two processes: a delayed mitotic clonal expansion via G0/G1 cell cycle arrest by Inhibiting the RB-E2F1 signaling pathway and a Reduced C/EBPβ signaling by Inhibiting the expression and activity of C/EBPβ during mitotic clonal expansion. Furthermore, adenanthin significantly Reduced the growing body weight and Adipose Tissue Mass during high-fat diet-inducing Obesity of mice, indicating the beneficial effects of adenanthin as a potential agent for prevention of Obesity .

In this work, we showed that adenanthin functioned its Anti-Adipogenic effect by increasing the intracellular ROS level during MCE and then affected two processes: (1) Inhibited the RB-E2F1 signaling pathway and results in a G0/G1 cell cycle arrest during MCE at the early stage of Adipocyte Differentiation ; (2) Reduced C/EBPβ transcriptional activity by Inhibiting the expression and activity of C/EBPβ during MCE. These results were in line with the Inhibitory effect of adenanthin on HFD-induced development of Obesity in mice.

ROS, oxygen-derived small molecules, react readily with various chemical structures including proteins, lipids, sugars, and nucleic acids [36]. In recent years, there has been an accumulating understanding of ROS as signaling molecules [37]. Most research groups now believe that a regulated basal level of ROS is necessary and advantageous for maintenance of cell functions, such as proliferation, Differentiation, and survival [38,39]. Increasing evidence has indicated that ROS plays a key role for Adipogenesis, but whether the increased ROS is positive or negative for Adipogenesis is still controversial. Some studies demonstrate that ROS promotes Adipocytes to differentiate [22,23], however, other studies show that ROS is indicated to Inhibit Adipocyte Differentiation by reducing the DNA-binding activity of C/EBPβ [24] and suppressing MCE [25]. Consistent with these findings [24,25], in our case, increased ROS Inhibited Adipogenesis, which was rescued by treatment of cells with adenanthin supplemented with H₂O₂ cleaner NAC (Figure 4). Furthermore, we exogenously treated cells with various doses of H₂O₂ (50 to 1200 μM) and found that lower doses (100, 200, and 400 μM) of H₂O₂ promoted 3T3-L1 Differentiation , whereas higher doses (800, 1000, and 1200 μM) of H₂O₂ Inhibited 3T3-L1 Differentiation (Supplementary Figure S1a–c), implying that “positive” or “negative” roles of H₂O₂ relies on its level as previous report [26].

It is well known that Adipogenesis occurs in several stages and involves a cascade of transcription factors [40]. Our results showed that adenanthin functions in MCE (Figure 3 and Figure 4). When it was induced to Differentiation, growth-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle and undergo MCE followed by expression of genes that produce the Adipocyte phenotype. MCE is a prerequisite for Differentiation of 3T3-L1 preadipocytes into Adipocytes [16]. Therefore, cell cycle regulation at the early stage of Adipogenesis has been considered as a strategy for modulating Adipogenesis [41]. Recently, many natural products have been reported to be Anti-Obesity agents that can Inhibit MCE of 3T3-L1 Adipocytes [41,42,43]. In our work, we found that adenanthin could attenuate cell cycle progression through a G0/G1 arrest during MCE by decreasing protein levels of the phosphorylation of RB, E2F1, Cyclin B1, and Cdk1 and modestly increased protein levels of P21 and P18 (Figure 3 and Figure 4d–f). As the cells cross the G1/S checkpoint, C/EBPβ acquires DNA-binding activity. Coincident with the acquisition of DNA-binding activity, C/EBPβ binds to centromeres through consensus C/EBP-binding sites in centromeric satellite DNA [15,20]. Consistent with a previous study [43], our results showed that adenanthin Inhibits the expression and activity of C/EBPβ during MCE (Figure 4b,c).

Finally, our results demonstrated that adenanthin Reduced the growing body weight and Adipose Tissue Mass during the early stage of induction of Obesity (Figure 5a–d). Moreover, to evaluate the safety of adenanthin, the serum of HFD fed mice after a 30-day exposure to adenanthin was collected and serum aspartate amino transferase (AST), alamine amino transferase (ALT), and creatinine levels between vehicle and adenanthin-treated mice were evaluated, and the results were not significantly changed (Supplementary Table S1). Whether adenanthin is active during the late stage of Obesity or has an Anti-Obesity effect is still unknown. In the future, we can monitor the activity of adenanthin during different stages of HFD-induced Obesity.

In summary, adenanthin, a natural ent-kaurane diterpenoid isolated from the herb Isodon adenantha in 1987 [27], was able to Inhibit Adipogenesis of 3T3-L1 and mouse embryonic fibroblasts (MEFs) through regulation of ROS and significantly Reduced the growing body weight and Adipose Tissue Mass during HFD-induced development of Obesity in mice, revealing adenanthin may be a potential agent for prevention of Obesity.

AICAR (5-aminoimidazole-4-carboxamide ribonucleoside)

The AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has been found to Inhibit the Differentiation of 3T3-L1 Adipocytes, if added at an early phase of Differentiation. AICAR blocks the expression of the late Adipogenic markers, fatty acid synthase and acetyl-CoA carboxylase, and of the transcription factors, C/EBPα and PPARγ. It also Inhibits early clonal expansion of Pre-Adipocytes, prevents the fall in C/EBPβ expression during the intermediate stage of Differentiation and Inhibits the late phase expression of CHOP-10, an antagonist of C/EBPβ. These data suggest a possible Inhibitory role for AMPK in the process of adipose Differentiation and suggest that AMPK might be a target to block Adipogenesis.

AMP-activated protein kinase

AICAR (5-aminoimidazole-4-carboxamide ribonucleoside)

3T3-L1 Adipocytes

Acetyl-CoA carboxylase

C/EBP (CAAT/enhancer binding protein)

PPAR (peroxisome proliferator-activated receptor)

AICAR Inhibits Adipocyte Differentiation in 3T3L1 and restores metabolic alterations in diet-induced Obesity mice model

Background: Obesity is one of the principal causative factors involved in the development of Metabolic Syndrome. AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. The role of AMP-activated protein kinase in Adipocyte Differentiation is not completely understood, therefore, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK) on Adipocyte Differentiation in 3T3L1 cells and in a mouse D iet induced o besity (DIO) model.

Methods: To examine the effect of AICAR on Adipocyte Differentiation in 3T3L1 cells and in a mouse Diet i nduced o besity (DIO) model, 3T3L1 cells were differentiatied in the presence or absence of different concentration of AICAR and neutral lipid content and expression of various Adipocyte-specific transcription factors were examined. In vivo study, treated and untreated mice with AICAR (0.1–0.5 mg/g body weight) were fed high-fat diet (60% kcal% fat) to induce DIO and several parameters were studied.

Results: AICAR blocked Adipogenic conversion in 3T3L1 cells along with significant decrease in the neutral lipid content by downregulating several Adipocyte -specific transcription factors including peroxisome proliferators-activated receptor γ (PPARγ), C/EBPα and ADD1/SREBP1, which are critical for Adipogenesis in vitro. Moreover, intraperitoneal administration of AICAR (0.5 mg g/body weight) to mice fed with high-fat diet (60% kcal% fat) to induce DIO, significantly blocked the body Weight Gain and total content of epididymal fat in these mice over a period of 6 weeks. AICAR treatment also restored normal adipokine levels and resulted in significant improvement in glucose tolerance and insulin sensitivity. The reduction in Adipose Tissue content in AICAR treated DIO mice was due to reduction in lipid accumulation in the pre-existing Adipocytes. However, no change was observed in the expression of PPARγ, C/EBPα and ADD1/SREBP1 transcription factors in vivo though PGC1α expression was significantly induced.

Conclusion: Our study demonstrates that AICAR treatment significantly attenuates adipoctye Differentiation in vitro. However, its administration restricted the body weight, epididymal fat content and normalized metabolic alteration mediated by diet induced Obesity in mice. Increase in phosphorylation of ACC and AMPKα during Adipocyte Differentiation and AMPK activity in epididymal Adipose Tissue in DIO mice raises doubts about the involvement of AMPK in this process.

Ajoene Sulfur

Ajoene Exerts Potent Effects in 3T3-L1 Adipocytes by Inhibiting Adipogenesis and Inducing Apoptosis

This paper describes effects of several sulfur-containing compounds from garlic on the cell viability, apoptosis and Adipogenesis in 3T3-L1 Adipocytes. In both preadipocytes and mature Adipocytes, 100 and 200 microM ajoene significantly decreased cell viability and increased apoptosis. The effect on apoptosis was further confirmed with Hoechst staining. In contrast, diallyl sulfide, diallyl disulfide, diallyl trisulfide, deoxyalliin, and allyl methyl sulfide had no significant effect on cell viability or apoptosis in either preadipocytes or mature Adipocytes. In maturing preadipocytes ajoene significantly decreased lipid accumulation in a dose-dependent manner and these results were further confirmed by a decrease in lipid droplet number and lipid content through Oil Red O staining. There was no significant change in lipid accumulation in maturing preadipocytes treated with other garlic derivatives.

Thus, despite the same source of origin, garlic, ajoene was the only one with potent effects on cell viability, apoptosis and Adipogenesis in 3T3-L1 Adipocytes.

Allium hookeri root

Effects of Allium hookeri root water extracts on Inhibition of Adipogenesis and GLUT-4 expression in 3T3-L1 Adipocytes

Anti-Adipogenic and AntiDiabetic activities of Allium hookeri root water extracts (ARW) were assessed. Oil Red O staining showed that treatment with ARW caused a dose-dependent reduction in lipid accumulation. ARW was also involved in Adipocyte Lipolysis via LPL activity, and in the concentration of glycerol in a culture medium. On the basis of the concentration of adipokines following ARW treatment, ARW appeared to Inhibit expression of PPAR-γ, to Reduce concentrations of leptin and resistin, to increase the concentration of adiponectin, and to Inhibit lipid accumulation. ARW modulated adipokine expression associated with insulin resistance and sensitivity. 3T3-L1 Adipocytes treated with ARW showed increased GLUT-4 expression with increased glucose uptake into Adipocytes. ARW showed effectiveness for improvement of Diabetic conditions.

Allium hookeri Root Extract Inhibits Adipogenesis by Promoting Lipolysis in High Fat Diet-Induced Obese Mice

Allium hookeri (AH) is widely consumed as a herbal medicine. It possesses biological activity against metabolic diseases. The objective of this study was to investigate effects of AH root water extract (AHR) on Adipogenesis in 3T3-L1 cells and in high-fat diet (HFD)-induced obese mice. AHR Inhibited lipid accumulation during Adipocyte Differentiation by downregulation of gene expression, such as hormone sensitive lipase (HSL), lipoprotein lipase (LPL) and an Adipogenic gene, CCAAT/enhancer binding protein-α in 3T3-L1 preadipocytes.

Oral administration of AHR significantly suppressed body Weight Gain, Adipose Tissue weight, serum leptin levels, and Adipocyte cell size in HFD-induced obese mice. Moreover, AHR significantly decreased hepatic mRNA expression levels of cholesterol synthesis genes, such as 3-hydroxy-3-methylglutaryl CoA reductase, sterol regulatory element-binding transcription factor (SREBP)-2, and low-density lipoprotein receptor, as well as fatty acid synthesis genes, such as SREBP-1c and fatty acid synthase. Serum triglyceride levels were also lowered by AHR, likely as a result of the upregulating gene involved in fatty acid β-oxidation, carnitine palmitoyltransferase 1a, in the liver. AHR treatment activated gene expression of peroxisome proliferator-activated receptor-γ, which might have promoted HSL and LPL-medicated Lipolysis, thereby reducing white Adipose Tissue weight. In conclusion, AHR treatment can improve metabolic alterations induced by HFD in mice by modifying expression levels of genes involved in Adipogenesis, lipogenesis, and Lipolysis in the white Adipose Tissue and liver. View Full-Text

Keywords: Allium hookeri root; Anti-Obesity; Adipogenesis; Lipolysis ; high fat diet

Anti-Obesity Effect of Allium hookeri Leaf Extract in High-Fat Diet-Fed Mice

Allium hookeri has been widely cultivated and used as a vegetable and medicine in Asia, but its Anti-Obesity effects have not been previously reported. In this study, the effects of a leaf extract of A. hookeri on Obesity were investigated by administering a high-fat diet (HFD) to mice. Male Institute of Cancer Research mice (n = 32; 5 weeks old) were randomly divided into four groups: normal-diet group, HFD group, HFD containing 200 mg/kg/day A. hookeri leaf extract (HFD-A1), and HFD containing 400 mg/kg/day A. hookeri leaf extract (HFD-A2). A. hookeri leaf extract was orally administered daily for 4 weeks. We found that the body Weight Gain and organ tissue weights of mice in the HFD-A1 and HFD-A2 groups were significantly lower compared with those of mice in the HFD group.

Administration of A. hookeri leaf extract also significantly decreased the size of the epididymal Adipose Tissue (AT). Serum levels of triglyceride (TG), total cholesterol, low-density lipoprotein cholesterol, and the atherogenic index were significantly lower in the HFD-A1 and HFD-A2 groups than in the HFD group. The TG and total cholesterol levels in the hepatic, epididymal, and mesenteric ATs of the HFD-A2 group were significantly lower than the levels in the HFD group. In addition, mRNA levels of liver fatty acid synthase and lipoprotein lipase were decreased in the A. hookeri leaf extract groups compared with those of the HFD group. These results demonstrate that intake of A. hookeri leaf may have beneficial effects for suppressing Obesity -related disease.

Andrographolide (Andrographis paniculata)

•Andrographolide is a diterpenoid phytochemical.

•Andrographolide Inhibits Adipogenesis of 3 T3-L1 Adipocytes.

•Andrographolide Suppresses Differentiation cocktail-induced C/EBPβ expression.

•Andrographolide attenuates ERK and GSK3β-dependent C/EBPβ activation.

•Andrographolide arrests 3 T3-L1 Adipocytes at G0/G1 phase.

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-Inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays Anti-Obesity property by Inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 Preadipocyte Differentiation into Adipocyte were determined. Andrographolide dose-dependently (0–15 μM) Inhibited CCAAT/enhancer-binding protein α (C/EBPα ) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the Adipogenesis of 3T3-L1 cells.

Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of Differentiation was critical for andrographolide Inhibition of Adipocyte formation. Andrographolide Inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a Differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide.

Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent Anti-Obesity action by Inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression.

Anthocyanins (Vitis coignetiae Pulliat)

Regulation of Adipocyte Function by Anthocyanins ; Possibility of Preventing the Metabolic Syndrome

Obesity is defined as the accumulation of excess Adipose Tissue resulting from various metabolic disorders. Adipocyte dysfunction is strongly associated with the development of Obesity and insulin resistance. Metabolic Syndrome is characterized by a group of metabolic risk factors in one person. Abdominal Obesity and Adipocyte dysfunction play an important role in the development of this syndrome.

Anthocyanins are used as a food coloring, and they are widely distributed in human diets including berries, suggesting that large amounts of Anthocyanins are ingested from plant-based foods. This study shows that Anthocyanins have a significant potency of AntiObesity and ameliorate Adipocyte function in in vitro and in vivo systems and also that they have important implications for preventing Metabolic Syndrome.

KEYWORDS:

•Anthocyanins

•cyanidin 3-glucoside

Anthocyanins Inhibit Lipogenesis During Adipocyte Differentiation of 3T3-L1 preadipocytes

Anthocyanins have been shown to suppress body weight and fat Mass in animal studies. However, the effect of Anthocyanins on the process of lipid accumulation during Adipocyte Differentiation is not fully understood and the lipogenic transcription factors regulated by Anthocyanins have not been identified. We investigated the effects of Anthocyanins on lipogenesis pathways during Adipocyte Differentiation in 3T3-L1 cells. Anthocyanins Reduced triglyceride (TG) accumulation in a dose-dependent manner during Adipocyte Differentiation. Accumulation of TG was rapidly reversed by anthocyanin withdrawal.

Anthocyanins markedly Reduced gene and protein expression levels of lipogenic transcription factors such as liver X receptor α, sterol regulatory element-binding protein-1c, peroxisome proliferators-activated receptor-γ, and CCAAT enhancer-binding protein-α. In addition, the target gene and protein expression of these lipogenic transcription factors such as fatty acid synthase, stearoyl-CoA desaturase-1, and acetyl-CoA carboxylase α were markedly suppressed by Anthocyanins. Thus, Anthocyanins suppress lipid accumulation in Adipocytes due to broad Inhibition of the transcription factors regulating lipogenesis. This may partially explain the mechanism by which Anthocyanins exert their Anti-Obesity effect.

Antofine Alkaloid

Anti-Adipogenic Activity of the Naturally Occurring Phenanthroindolizidine Alkaloid Antofine via Direct Suppression of PPARγ Expression

Antofine (ANTF) is a phenanthroindolizidine alkaloid isolated from the root of Cynanchum paniculatum Kitagawa (Asclepiadaceae), which is used as an herbal remedy for pain and Inflammation. ANTF also possesses antiviral and antitumorigenic activities. In this study, we investigated the role of ANTF in Adipogenesis. Chronic ABTF administration suppressed Adipocyte Differentiation and marker expression in a dose-dependent manner. Furthermore, acute administration of ANTF at early stages of Differentiation process Inhibited lipid droplet formation and Adipogenic gene expression.

ANTF Treatment decreased expression of PPARγ protein, a master transcription factor in the regulation of Adipocyte Differentiation, leading to a suppression of aP2 promoter activity. These results suggest that ANTF exerts potent Anti-Adipogenic effects via direct suppression of PPARγ protein expression, with consequent downregulation of Adipogenic gene expression.

Antrodia cinnamomea

Aqueous extract of Antrodia cinnamomea Reduced high-fat diet-induced Obesity in mice and suppressed Adipogenesis in 3T3-L1 cells

•Antrodia cinnamomea (AC) has been used as a natural dietary supplement for health promotion.

•AC prevents high fat diet-induced Obesity in vivo.

•AC Inhibits Adipogenesis and mitotic clonal expansion in vitro.

•AC has potential applications to treat Obesity , insulin resistance and hepatic steatosis.

This study examined the Anti-Obesity and Anti-Adipogenic effects of Antrodia cinnamomea. Oral administration of aqueous extract of A. cinnamomea (ACW) significantly Reduced high-fat diet-induced body Weight Gain and relative perirenal and mesenteric fat weight in C57BL/6J mice. Administration of ACW significantly Reduced serum insulin, leptin, aspartate aminotransferase levels, HOMA-IR index, and hepatic cholesterol and triacylglycerol levels. Next, investigation of whether ACW and its polysaccharides (PS) and non-polysaccharides (NPS) subfractions possess Anti-Adipogenic action was conducted. Evidence showed that ACW, PS and NPS significantly Inhibited lipid deposits in 3T3-L1 Adipocytes.

Both ACW and NPS significantly Inhibited mitotic clonal expansion process of Adipocyte Differentiation. ACW and NPS also significantly decreased the expressions of PPARγ, C/EBPα, aP2 and FAS genes, while PS markedly Inhibited PPARγ and aP2 gene expression. Our data indicated that ACW Inhibits Preadipocyte Differentiation and Adipogenesis. These results suggested that ACW may have therapeutic potential for Obesity and related metabolic disorders.

Apigetrin (apigenin-7-O-glucoside) (Cosmos bipinnata Cav.)

Apigetrin Inhibits Adipogenesis in 3T3-L1 cells by downregulating PPARγ and CEBP-αAbstract

Background: Apigetrin, a flavonoid found in many plant leaves and seeds, has been known to possess antimutagenic, anti-cancer, antioxidant and anti-inflammatory properties. Here, we are investigating the effect of the apigetrin on Adipocytes Differentiation in 3T3-L1 Adipocytes, and elucidating the mechanism of its action.

Methods: Lipids accumulation was measured by Oil Red O staining and cell cycle was analyzed by flow cytometry. The antioxidant effect of apigetrin was evaluated against hydrogen peroxide. The expression of various genes, involved in Adipogenesis and Inflammation, was studied by real-time PCR.

Results: Our results showed that apigterin treatment Inhibited significantly lipid accumulation without effect on cell viability at 100 μM, and it exerted the Anti-Adipogenic effect during the early stages of Differentiation. Flow cytometry analysis showed that apigenin-7-O-glucoside (Ap7G) Inhibited cell proliferation during mitotic clonal expansion and caused cell cycle delay. Quantitative PCR analysis revealed that the mRNA levels of C/EBP-α, PPAR-γ, SREBP-1c and FAS were suppressed after apigetrin treatment at 100 μM. Moreover, the mRNA level of pro-inflammatory genes (TNF-α and IL-6) were suppressed after apigterin treatment, at high concentration Preadipocyte cells.

Conclusion: Taken together, these results indicated that apigenin-7-O-glucoside Inhibits Adipogenesis of 3T3-L1 preadipocytes at early stage of Adipogenesis.

Arctigenin

Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduce s Weight Gain in High‐Fat Diet‐Induced Obese Mice

Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti‐Inflammation, anti‐cancer, and antioxidant, there have been no reports on the anti‐Obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti‐Obesity effect and mediates the AMP‐activated protein kinase (AMPK) pathway. We investigated the anti‐Adipogenic effect of ARC using 3T3‐L1 pre‐Adipocytes and human Adipose Tissue‐derived mesenchymal stem cells (hAMSCs). In high‐fat diet (HFD)‐induced obese mice, whether ARC can Inhibit Weight Gain was investigated. We found that ARC Reduced Weight Gain, fat pad weight, and triglycerides in HFD‐induced obese mice.

ARC also Inhibited the expression of peroxisome proliferator‐activated receptor gamma (PPARγ ) and CCAAT/enhancer‐binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down‐modulation of Adipogenesis‐related factors including PPARγ, C/EBPα, fatty acid synthase, Adipocyte fatty acid‐binding protein, and lipoprotein lipase. This study demonstrates that ARC can Reduce key Adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for Obesity treatment.

Arctiin (Arctium lappa L.seed)

Arctiin Inhibits Adipogenesis in 3T3-L1 cells and decreases Adiposity and body weight in mice fed a high-fat diet

Background: The purpose of this study was to examine the effects and associated mechanisms of arctiin, a lignan compound found in burdock, on Adipogenesis in 3T3-L1 cells . Also, the effects of arctiin supplementation in obese mice fed a high-fat diet on Adiposity were examined.

Materials and Methods: 3T3-L1 cells were treated with arctiin (12.5 to 100 µM) during Differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining and intracellular triglyceride contents. The expressions of genes related to Adipogenesis were measured by real-time RT-PCR and Western blot analyses. For in vivo study, C57BL/6J mice were first fed either a control diet (CON) or high-fat diet (HF) to induce Obesity, and then fed CON, HF, or HF with 500 mg/kg BW arctiin (HF + AC) for four weeks.

Results: Arctiin treatment to 3T3-L1 Pre-Adipocytes markedly decreased Adipogenesis in a dose-dependent manner. The arctiin treatment significantly decreased the protein levels of the key Adipogenic regulators PPARγ and C/EBPα, and also significantly Inhibited the expression of SREBP-1c, fatty acid synthase, fatty acid-binding protein and lipoprotein lipase. Also, arctiin greatly increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase. Furthermore, administration of arctiin significantly decreased the body weight in obese mice fed with the high-fat diet. The epididymal, perirenal or total visceral Adipose Tissue weights of mice were all significantly lower in the HF + AC than in the HF. Arctiin administration also decreased the sizes of lipid droplets in the epididymal Adipose Tissue.

Conclusions: Arctiin Inhibited Adipogenesis in 3T3-L1 Adipocytes through the Inhibition of PPARγ and C/EBPα and the activation of AMPK signaling pathways. These findings suggest that arctiin has a potential benefit in preventing Obesity.

Keywords: Arctiin, Adipogenesis, AMP kinase, 3T3-L1 cells, high-fat diet.

Aristolochia Manshuriensis Kom

Aristolochia Manshuriensis Kom Inhibits Adipocyte Differentiation by Regulation of ERK1/2 and Akt Pathway

Aristolochia manshuriensis Kom (AMK) is a traditional medicinal herb used for the treatment of arthritis, rheumatism, hepatitis, and Anti-Obesity. Because of nephrotoxicity and carcinogenicity of AMK, there are no pharmacological reports on Anti-Obesity potential of AMK. Here, we showed AMK has an Inhibitory effect on Adipocyte Differentiation of 3T3-L1 cells along with significantly decrease in the lipid accumulation by downregulating several Adipocyte-specific transcription factors including peroxisome proliferation-activity receptor γ (PPAR-γ), CCAAT/enhancer binding protein α (C/EBP-α) and C/EBP-β, which are critical for Adipogenesis in vitro.

AMK also markedly activated the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway including Ras, Raf1, and mitogen-activated protein kinase kinase 1 (MEK1), and significantly suppressed Akt pathway by Inhibition of phosphoinositide-dependent kinase 1 (PDK1). Aristolochic acid (AA) and ethyl acetate (EtOAc) fraction of AMK with AA were significantly Inhibited TG accumulation, and regulated two pathway (ERK1/2 and Akt) during Adipocyte Differentiation, and was not due to its cytotoxicity. These two pathways were upstream of PPAR-γ and C/EBPα in the Adipogenesis. In addition, gene expressions of secreting factors such as fatty acid synthase (FAS), adiponectin, lipopreotein lipase (LPL), and aP2 were significantly Inhibited by treatment of AMK during Adipogenesis. We used the high-fat diet (HFD)-induced Obesity mouse model to determine the Inhibitory effects of AMK on Obesity.

Oral administration of AMK (62.5 mg/kg/day) significantly decreased the fat tissue weight, total cholesterol (TC), and low density lipoprotein-cholesterol (LDL-C) concentration in the blood. The results of this study suggested that AMK Inhibited lipid accumulation by the down-regulation of the major transcription factors of the adipogensis pathway including PPAR-γ and C/EBP-α through regulation of Akt pathway and ERK 1/2 pathway in 3T3-L1 Adipocytes and HFD-induced Obesity mice, and AA may be main act in Inhibitory effects of AMK during Adipocyte Differentiation.

Herbal Remedies against Adipogenesis

Aristolochia manshuriensis Kom is a traditional medicinal herb used for treatment of arthritis, rheumatism, hepatitis. Its extract Inhibited Adipocyte Differentiation by regulating ERK1/2 and Akt pathway. Besides, expressions of FAS, LPL and aP2 were significantly Reduced by the extract treatment during Adipogenesis.

Aristotelia chilensis

Antioxidant Capacity and in Vitro Inhibition of Adipogenesis and Inflammation by Phenolic Extracts of Vaccinium floribundum and Aristotelia chilensis

Interest in berries from South America has increased due to their potential health benefits. The objective of this study was to characterize the Anthocyanins and proanthocyanidins of Vaccinium floribundum and Aristotelia chilensis, total phenolics, and antioxidant capacity and to evaluate, in vitro, the ability of their phenolic extracts to Reduce Adipogenesis and lipid accumulation in 3T3-L1 Adipocytes. The anti-inflammatory property of these extracts on RAW 264.7 macrophages was also investigated. Antioxidant capacity, measured as oxygen radical scavenging capacity and expressed as Trolox equivalents, was higher in the berries of A. chilensis. Phenolic extracts Inhibited lipid accumulation by 4.0−10.8% when Adipocytes were treated at maturity and by 5.9−37.9% when treated throughout Differentiation.

Furthermore, a proanthocyanidin-enriched fraction from V. floribundum significantly increased Pref-1 expression in preadipocytes. Phenolic extracts decreased the production of nitric oxide (3.7−25.5%) and prostaglandin E₂ (9.1−89.1%) and the expression of inducible nitric oxide synthase (9.8−61.8%) and cycloxygenase-2 (16.6−62.0%) in lipopolysaccharide-stimulated RAW 264.7 macrophages. V. floribundum and A. chilensisphytochemicals limit Adipogenesis and inflammatory pathways in vitro, warranting further in vivo studies.

KEYWORDS (keywords):

•Aristotelia chilensis

•Vaccinium floribundum

Artemisia capillaris

Anti-Adipogenic Effect of Artemisia Annua in Diet-Induced-Obesity Mice Model

Obesity has increased continuously in western countries during the last several decades and recently become a problem in developing countries. Currently, Anti-Obesity drugs originating from natural products are being investigated for their potential to overcome adverse effects associated with chemical drugs. Artemisinic acid, which was isolated from the well-known anti-malaria herb Artemisia annua (AA) L., was recently shown to possess Anti-Adipogenic effects in vitro. However, the Anti-Adipogenic effects of AA in animal models have not yet been investigated.

Therefore, we conducted daily oral administration with AA water extract in a diet-induced Obesity animal model and treated 3T3-L1 cells with AA to confirm the Anti-Adipogenic effects in the related protein expressions. We then evaluated the physiology, Adipose Tissue histology and mRNA expressions of many related genes. Inhibition of Adipogenesis by the AA water extract was observed in vitro. In the animal model, Weight Gain was significantly lower in the AA treated group, but there were no changes in food intake volume or calories. Reductions in lipid droplet size and mRNA expression associated with Adipogenesis were also observed in animal epididymal fat. This study is the first to report that AA has an anti-obese effects in vivo.

Aster yomena (Kitam.) Honda leaves

Ethanol extracts of Aster yomena (Kitam.) Honda Inhibit Adipogenesis through the activation of the AMPK signaling pathway in 3T3-L1 preadipocytes

The leaves of Aster yomena (Kitam.) Honda have long been used as a traditional herb for treating disorders including coughs, asthma, and insect bites. According to recent studies, A. yomena leaf extracts have several pharmacological properties, including anti-inflammatory, antioxidant, and anti-asthmatic activities. However, little information is available regarding their Anti-Obesity effect. In this study, we investigated the Inhibitory effect of the ethanol extracts of A. yomena leaves (EEAY) on Adipocyte Differentiation and Adipogenesis using 3T3-L1 preadipocytes. When 3T3-L1 preadipocytes were treated with various concentrations of EEAY (ranging from non-toxic), the number of lipid droplets, lipid content, and triglyceride production, the typical characteristics of Adipocytes, were suppressed in a concentration-dependent manner.

During this process, EEAY significantly Reduced the expression of Adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein α and β, and sterol regulatory element-binding protein-1c. In addition, EEAY was also found to potently Inhibit the expression of Adipocyte-specific genes, including Adipocyte fatty acid-binding protein and leptin. In particular, EEAY treatment effectively enhanced the activation of the AMP-activated protein kinase (AMPK) signaling pathway; however, the co-treatment with compound C, an Inhibit or of AMPK, significantly restored the EEAY-induced Inhibition of Pro-Adipogenic transcription factors and Adipocyte-specific genes. These results indicate that EEAY may exert an Anti-Obesity effect by controlling the AMPK signaling pathway, suggesting that the leaf extract of A. yomena may be a potential Anti-Obesity agent.

Astilbe chinensis Franch. et Savet

AntiObesity Effect of Astilbe chinensis Franch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice

Astilbe chinensis Franch. et Savat. (AC) has been used in traditional medicine for the treatment of chronic bronchitis, arthralgia, and gastralgia. In this study, we investigated the AntiObesity effect of AC extract on 3T3-L1 preadipocytes and high-fat-diet-fed C57BL/6N obese mice. We found that AC extracts dramatically decreased the lipid content of 3T3-L1 cells in a concentration-dependent manner without cytotoxicity. The action mechanism of AC extract was demonstrated to be the Inhibition of lipid accumulation and dose-dependent decrease in the expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPAR-γ), and sterol regulatory element-binding protein 1 (SREBP1).

Furthermore, AC extract increased the mitochondrial phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), mitochondrial biogenesis, and Lipolysis -related factors. In amice model of high-fat-diet-induced Obesity, the mice administered AC extract experienced significant decrease of 64% in Weight Gain, 55% in insulin resistance index, 22% in plasma triglycerides (TG), 56% in total cholesterol (TC), and 21% in nonesterified fatty acid (NEFA) levels compared with those in the high-fat diet-fed control mice. Collectively, these results indicated that AC extract exerted antiobesogenic activity through the modulation of the AMPK signaling pathway, Inhibition of Adipogenesis, decreased lipid content, and Reduced Adipocyte size.

Astragalin, (3-O-glucoside of kaempferol) (persimmon leaves)

AntiObesity Effect of Astilbe chinensisFranch. et Savet. Extract through Regulation of Adipogenesis and AMP-Activated Protein Kinase Pathways in 3T3-L1 Adipocyte and High-Fat Diet-Induced C57BL/6N Obese Mice

Furthermore, AC extract increased the mitochondrial phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), mitochondrial biogenesis, and Lipolysis-related factors. In amice model of high-fat-diet-induced Obesity, the mice administered AC extract experienced significant decrease of 64% in Weight Gain, 55% in insulin resistance index, 22% in plasma triglycerides (TG), 56% in total cholesterol (TC), and 21% in nonesterified fatty acid (NEFA) levels compared with those in the high-fat diet-fed control mice.

Collectively, these results indicated that AC extract exerted antiobesogenic activity through the modulation of the AMPK signaling pathway, Inhibition of Adipogenesis, decreased lipid content, and Reduced Adipocyte size.

Averrhoa carambola L. peel

Averrhoa carambola L. peel extract Suppresses Adipocyte Differentiation in 3T3-L1 cells

Obesity is associated with an increased risk of many chronic diseases. Recently, a growing body of evidence has shown that phytochemicals may Inhibit Adipogenesis and Obesity. In this study, we report for the first time, the ability of Averrhoa carambola L. peel extract commonly known as star fruit (SFP) to effectively suppress Adipocyte Differentiation in 3T3-L1 preadipocytes and therefore, address it as a potential candidate to treat Obesity and its related diseases. (−)-Epicatechin was identified as a bioactive compound likely responsible for this suppression.

As the genetic expression studies revealed that the Adipogenic activity of SFP extract was due to the simultaneous downregulation of the C/EBPα and PPARγ as well as the upregulation of PPARα receptor genes, a detailed computational docking study was also elucidated to reveal the likely binding mode of (−)-epicatechin to the receptor of interest, accounting for the likely mechanism that results in the overall suppression of Adipocyte Differentiation.

Avicularin (Guava leaves)

Avicularin, a Plant Flavonoid, Suppresses Lipid Accumulation through Repression of C/EBPα -Activated GLUT4-Mediated Glucose Uptake in 3T3-L1 cells

Avicularin (quercetin-3-O-α-l-arabinofuranoside) is a plant flavonoid and a quercetin glycoside. In this study, we found that avicularin suppressed the accumulation of intracellular lipids through repression of glucose transporter 4 (GLUT4)-mediated glucose uptake in mouse adipocytic 3T3-L1 cells. Avicularin was highly purified (purity of more than at least 99%) from Taxillus kaempferi (DC.) Danser (Loranthaceae) by high-performance liquid chromatography, and its structure was determined by nuclear magnetic resonance and Mass spectrometry. Avicularin decreased the intracellular triglyceride level along with a reduction in the expression of Adipogenic genes such as peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and aP2 (fatty acid-binding protein 4).

In contrast, avicularin did not affect the expression of lipogenic and lipolytic genes. Interestingly, the expression of the GLUT4 gene was significantly suppressed in an avicularin-concentration-dependent manner. Moreover, the binding of C/EBPα to the promoter region of the GLUT4 gene was repressed by adding avicularin to the medium in 3T3-L1 cells, as demonstrated by the results of a chromatin immunoprecipitation assay. These results indicate that avicularin Inhibited the accumulation of the intracellular lipids by decreasing C/EBPα-activated GLUT4-mediated glucose uptake in Adipocytes.

Bacaba (Oenocarpus bacaba Mart.) phenolic (BPE)

•Bacaba (Oenocarpus bacaba Mart.) phenolic extract (BPE) Inhibits Differentiation in 3T3-L1 preadipocytes.

•BPE down-regulates protein expression of PPARγ and C/EBPα in a dose-dependent manner.

•During mitotic clonal expansion (early stage of Differentiation) BPE Inhibits lipid accumulation.

•Longer BPE incubation period is needed to effect protein expression.

Bacaba (Oenocarpus bacaba Mart.) is a native Brazilian palm fruit with a high amount of Polyphenol ics, reported to have an apoptotic effect on cancer cells [1]. Here, we examined the effect of Bacaba phenolic extract (BPE) on Adipogenesis using 3T3-L1 preadipocytes . Proliferating and differentiating Adipocytes were incubated with BPE at 6, 12, and 24 μg of gallic acid equivalents (GAE)/ml. BPE Reduced accumulation of intracellular lipids and protein expression of Adipogenic markers including PPARγ, C/EBPα, FABP4, IR-β, and adiponectin in a dose-dependent manner during Differentiation of 3T3-L1 cells into Adipocytes.

Furthermore, lipid accumulation decreased with BPE (24 μg of GAE/ml) during the early stage of mitotic clonal expansion (Days 0–2). In contrast, the Inhibition of protein expression of Adipogenic markers needed a longer duration (Days 0–4, 0–7, 2–7) of BPE incubation. These results suggest that BPE Inhibits adipogenisis in vitro via targeting transcriptional factors during the early and middle stages of Differentiation.

Bacaba (Oenocarpus bacaba Mart.) phenolic extract

3T3-L1 cells

Adipogenesis

PPARγ

C/EBPα

Baicalin (Scutellaria baicalensis)

AntiObesity effect of baicalin involves the modulations of proAdipogenic and AntiAdipogenic regulators of the Adipogenesis pathway

In this study, the AntiObesity effects of baicalin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, were characterized using an in vitro system of Adipogenesis, i.e. fat cell formation. Baicalin‐treatment of 3T3‐L1 preadipocytes was shown to Inhibit triglyceride accumulation and lipid droplet formation during induced Adipogenesis. Microarray analyses showed that baicalin modulated the expression of genes located in pathways such as Adipogenesis, cholesterol biosynthesis, focal adhesion and others. In the Adipogenesis pathway, treatment with baicalin significantly down‐regulated terminal Differentiation markers of Adipocytes including fatty acid binding protein 4. The effects of baicalin on the core part of the Adipogenesis pathway, however, were paradoxical; the expression levels of CCAAT/enhancer binding protein (C/EBP)β and C/EBPδ were up‐regulated, while the expression levels of the peroxisome proliferator‐activated receptor (PPAR)γ and C/EBPα were down-regulated.

The antiAdipogenic mechanisms of baicalin can be explained by its effects on the upstream part of Adipogenesis pathway; baicalin not only up‐regulates the anti Adipogenic regulators, C/EBPγ, C/EBP homologous protein and Kruppel‐like factor (KLF)2, but also down‐regulates the pro Adipogenic regulator, KLF15. The overall effects of baicalin on these upstream regulators of Adipogenesis were antiAdipogenic, resulting in the down‐regulation of downstream genes and the Inhibition of cellular Fat Accumulation.

Bamboo (Phyllostachys bambusoides) leaf

Bamboo (Phyllostachys bambusoides) leaf extracts Inhibit Adipogenesis by regulating Adipogenic transcription factors and enzymes in 3T3-L1 Adipocytes

In this study, the Inhibitory effects of bamboo leaf extracts on Adipogenesis were investigated by evaluating their activity against Adipogenic transcription factors and enzymes in 3T3-L1 Adipocytes. Bamboo leaf extracts significantly decreased triglyceride levels, and increased glycerol release in Adipocytes. Cells treated with the water extract showed significantly higher glycerol release as well as lower triglyceride contents than those treated with the ethanol extract. Both bamboo leaf extracts significantly Inhibited the expression of Adipogenic transcription factors and enzymes, such as CCAAT/enhancer-binding protein α, sterol regulatory element binding protein 1c, peroxisome proliferator-activated receptor γ, acetyl-coenzyme A carboxylase, and fatty acid synthase, and increased the expression of phospho-adenosine monophosphate-activated protein kinase.

These results show that bamboo leaf extracts Inhibited Adipogenesis in 3T3-L1 Adipocytes and that the water extract was more efficacious than the ethanol extract.

Banaba (Lagerstroemia speciosa L.)

An Extract of Lagerstroemia speciosa L. Has Insulin-Like Glucose Uptake–Stimulatory and Adipocyte Differentiation–Inhibitory Activities in 3T3-L1 Cells

The effects of extracts isolated from Lagerstroemia speciosa L. (banaba) on glucose transport and adipocyte differentiation in 3T3-L1 cells were studied. Glucose uptake–inducing activity of banaba extract (BE) was investigated in differentiated adipocytes using a radioactive assay, and the ability of BE to induce differentiation in preadipocytes was examined by Northern and Western blot analyses. The hot water BE and the banaba methanol eluent (BME) stimulated glucose uptake in 3T3-L1 adipocytes with an induction time and a dose-dependent response similar to those of insulin. Furthermore, there were no additive or synergistic effects found between BE and insulin on glucose uptake, and the glucose uptake activity of insulin could be reduced to basal levels by adding increasing amounts of BE.

Unlike insulin, BE did not induce adipocyte differentiation in the presence of 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (DEX). BE inhibited the adipocyte differentiation induced by insulin plus IBMX and DEX (IS-IBMX-DEX) of 3T3-L1 preadipocytes in a dose-dependent manner. The differences in the glucose uptake and differentiation inhibitory activities between untreated cells and those treated with BE were significant (P < 0.01). The inhibitory activity was further demonstrated by drastic reductions of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and glucose transporter-4 (GLUT4) protein in cells induced from preadipocytes with IS-IBMX-DEX in the presence of BE. The unique combination of a glucose uptake stimulatory activity, the absence of adipocyte differentiation activity and effective inhibition of adipocyte differentiation induced by IS-IBMX-DEX in 3T3-L1 cells suggest that BE may be useful for prevention and treatment of hyperglycemia and obesity in type II diabetics.

Benincasa hispida

Effects of Fractions from Benincasa hispida on Inhibition of Adipogenesis in 3T3-L1 preadipocytes

The effects of three fractions, hexane (BHHH), chloroform (BHHC), and ethyl acetate (BHHE), from water extract of Benincasa hispida on the underlying mechanisms of Adipogenesis were investigated in 3T3-L1 cells. Intracellular lipid droplets were stained with Oil Red O dye and quantified. Compared to control, lipid accumulation significantly decreased by 11% and 13% upon treatment with BHHC and BHHE, respectively at a concentration of 50 $μ g / m L$ . Intracellular triglyceride (TG) levels were also Reduced by 21% and 16%, respectively, at the same concentration. To determine the mechanism behind the reductions in TG content and lipid accumulation, glycerol release and expression levels of Adipogenic marker genes were measured. The levels of free glycerol released into culture medium increased by 13% and 17% upon treatment with BHHC and BHHE, respectively.

In subsequent measurements using real-time polymerization chain reaction, the mRNA levels of $P P A R γ$ , C/ $E B P α$ , and leptin significantly decreased upon treatment with BHHE (45%, 67%, and 35%) in comparison with non-treated control. These results suggest that BHHE Inhibits Adipocyte Differentiation by blocking $P P A R γ$ , C/ $E B P α$ , and leptin gene expression in 3T3-L1 cells , resulting in Reduced lipid accumulation, increased glycerol release, and intracellular triglycerides.

Berberine

Berberine Inhibits Adipocyte Differentiation , proliferation and Adiposity through down-regulating galectin-3

This study is designed to investigate the effects of berberine (BBR) on galectin-3 (Gal-3) and the relationships to its suppressive activities on Adipocyte Differentiation , proliferation and Adiposity . Our results showed that BBR greatly suppressed the Differentiation and proliferation of mouse primary preadipocytes isolated from epididymal white Adipose Tissue (eWAT), during which the expression level of Gal-3 was down-regulated significantly. Overexpression of Gal-3 totally abolished the suppressive activities of BBR on Gal-3 expression, Preadipocyte Differentiation and proliferation. BBR Reduced Gal-3 promoter activity, destabilized its mRNA and Inhibited firefly luciferase activity of a recombinant plasmid containing the Gal-3 3′ untranslated region (UTR). Furthermore, BBR up-regulated microRNA (miRNA) let-7d expression and the suppressive activity on Gal-3 3′UTR was abolished by point mutation on the let-7d binding site. In mice fed a high-fat diet (HFD), BBR up-regulated let-7d and down-regulated Gal-3 expression in eWAT; it also suppressed Adipocyte Differentiation and proliferation and Reduced Adiposity greatly.

In summary, our study proves that BBR Inhibits the Differentiation and proliferation of Adipocytes through down-regulating Gal-3, which is closely associated with its Anti-Obesity effect. Our results may support the future clinical application of BBR for the treatment of Obesity or related diseases.

Bergamottin (Bergamot)

Bergamottin Inhibits Adipogenesis in 3T3-L1 cells and Weight Regulation in Diet-Induced Obese Mice

Obesity is a serious and increasing health problem worldwide, and the Inhibition of Adipogenesis is considered to be a potential therapeutic target for it. Bergamottin (BGM), a component of grapefruit juice, has been reported to regulate Lipolysis. However, the physiological role of BGM in Obesity has not been evaluated so far. In the present study, we investigated the effects of BGM on Obesity in 3T3-L1 cells and in mice fed a high-fat diet (HFD). BGM Inhibited Adipogenic Differentiation of 3T3-L1 cells along with a significant decrease in the lipid content by downregulating the expression of two critical Adipogenic factors, CCAAT enhancer-binding protein-alpha (C/EBP $α$ ) and peroxisome proliferator activated receptor-gamma (PPAR $γ$ ).

The expressions of target proteins such as Adipocyte fatty acid-binding protein (aP2), adiponectin, and resistin were also decreased by BGM. It activated AMP-activated protein kinase (AMPK) by increasing phosphorylation of AMPK and the downstream target acetyl-CoA carboxylase (ACC), indicating that BGM exerted its antiAdipogenic effect through AMPK activation. In the HFD-induced obese mouse model, BGM administration significantly Reduced the weight and sizes of white Adipose Tissue as well as the Weight Gain of mice fed HFD. Moreover, UCP1 and PGC1 $α$ expressions, well-known as brown Adipocyte marker genes, were higher in the BGM-treated HFD mice than that in the HFD-induced obese mice. This study suggests that BGM suppress Adipogenesis by AMPK activation in vitro and Reduce s body weight in vivo.

Betanin (betanin-5-O-β-glucoside) 97% (Beetroot)

The Inhibitory Effect of Betanin on Adipogenesis in 3T3-L1 Adipocytes

Betanin, a natural pigment that presents ubiquitously in plants, has been reported to show biological effects. However, not much is known on the effectiveness of betanin in regulating Fat Accumulation. Therefore, the aim of this study is to explore the Inhibitory effect of betanin on Adipogenesis in 3T3-L1 Adipocytes and its mechanism action. The results show betanin significantly Inhibited oil red O-stained material (OROSM) and triglyceride levels in 3T3-L1 Adipocytes, indicating betanin Inhibited lipid accumulation in 3T3-L1 Adipocytes. In addition, the peroxisome proliferator–activated receptor γ (PPARγ) expression was significantly Inhibited in the betanin-treated Adipocytes, implying that betanin suppressed the cellular PPARγ expression in 3T3-L1 Adipocytes.

Moreover, the suppression of lipid accumulation by betanin occurred by decreasing the gene expression of PPARγ, CCAAT-enhancer-binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c). Taken together, these findings suggest betanin may be a mediator of Adipocyte accumulation, leading to the Inhibition of lipogenesis in 3T3-L1 Adipocytes and betanin is therefore potentially useful for designing new antiAdipogenic agent.

Keywords: Adipogenesis, betanin, gene expression, lipogenesis, lipid accumulation.

Bisdemethoxycurcumin (Curcuma longa L. root)

Bisdemethoxycurcumin Inhibits Adipogenesis in 3T3-L1 preadipocytes and Suppresses Obesity in High-Fat Diet-Fed C57BL/6 Mice

Obesity is caused by excessive accumulation of body fat and is closely related to complex metabolic diseases. Adipogenesis is a key process that is required in Adipocyte hypertrophy in the development of Obesity. Curcumin (Cur) has been reported to Inhibit Adipocyte Differentiation, but the Inhibitory effects of other curcuminoids present in turmeric, such as demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), on Adipogenesis have not been investigated. Here, we investigated the effects of curcuminoids on Adipogenesis and the molecular mechanisms of Adipocyte Differentiation. Among three curcuminoids, BDMC was the most effective suppressor of lipid accumulation in Adipocytes. BDMC suppressed Adipogenesis in the early stage primarily through attenuation of mitotic clonal expansion (MCE). In BDMC-treated preadipocytes, cell cycle arrest at the G₀/G₁phase was found after initiation of Adipogenesis and was accompanied by downregulation of cyclin A, cyclin B, p21, and mitogen-activated protein kinase (MAPK) signaling.

The protein levels of the Adipogenic transcription factors peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding proteins (C/EBP)α were also Reduced by BDMC treatment. Furthermore, 0.5% dietary BDMC (w/w) significantly lowered body Weight Gain and Adipose Tissue Mass in high-fat diet (HFD)-fed mice. The results of H&E staining showed that dietary BDMC Reduced hypertrophy in Adipocytes. These results demonstrate for the first time that BDMC suppressed Adipogenesis in 3T3-L1 Adipocytes and prevented HFD-induced Obesity. Our results suggest that BDMC has the potential to Prevent Obesity.

Blumea balsamifera

Anti-Obesity Effect of Blumea balsamifera Extract in 3T3-L1 preadipocytes and Adipocytes

Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. We investigated the Anti-Obesity effect of Blumea balsamifera extract on Adipocyte Differentiation of 3T3-L1 preadipocytes and Anti-Obesity effect of 3T3-L1 Adipocytes. We found that treatment with an extract of Blumea balsamifera suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability in 3T3-L1 preadipocytes and Adipocytes.

Furthermore, Blumea balsamifera extract brought significant attenuation of expressions of key Adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT element binding protein (C/EBPs) and leptin, however, induced up-regulation of adiponectin at the protein level in 3T3-L1 preadipocytes and Adipocytes. These results suggest that Blumea balsamifera extract may block Adipogenesis, at least in part, by decreasing key Adipogenic transcription factors in 3T3-L1 preadipocytes and may have antiatherogenic, anti-inflammatory, and AntiDiabetic effects through up-regulation of adiponectin in 3T3-L1 Adipocytes.

Boldine (Peumus boldus Molina leaf)

The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 cells

Adiponectin is an adipokine secreted by differentiated Adipocytes . Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with Metabolic Syndrome or cardiovascular disease. Natural compounds that can Prevent oxidative stress mediated Inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells.

Differentiated 3T3-L1 Adipocytes were exposed to either hydrogen peroxide (H₂O₂) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the Inhibitory effect of H₂O₂ or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H₂O₂ or TNFα was also able to induce adiponectin at the inductive phase of Adipogenesis.

Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in Obesity-related cardiovascular disease.

Boussingaulti gracilis Miers var. pseudobaselloides Bailey

Anti-Obesity Effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey via Activation of AMP-Activated Protein Kinase in 3T3-L1 cells

In a previous study, we demonstrated the Anti-Obesity and hypolipidemic effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey in high-fat diet-induced obese rats. The present study investigated the molecular mechanisms by which B. gracilis Miers var. pseudobaselloides Bailey ethanol extract (BGE) conferred antiDifferentiation and Anti-Adipogenic effects in the 3T3-L1 Preadipocyte Differentiation model. BGE treatment significantly and dose-dependently suppressed lipid accumulation and down-regulated the expression of major transcription factors involved in Adipogenesis, such as peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein α, sterol regulatory element-binding proteins, and their target genes.

It is important that treatment with BGE increased phosphorylation of AMP-activated protein kinase (AMPK), which is one of the rate-limiting enzymes in the fatty acid synthesis pathway, and its direct downstream protein, acetyl-coenzyme A carboxylase. These results suggest that BGE may exert Anti-Adipogenic effects through regulation of AMPK activity and expression of genes involved in lipogenesis.

Bromophenol (5‐bromo‐3,4‐dihydroxybenzaldehyde) isolated from red alga Polysiphonia morrowii

Bromophenol (5‐bromo‐3,4‐dihydroxybenzaldehyde) isolated from red alga Polysiphonia morrowii Inhibits Adipogenesis by regulating expression of Adipogenic transcription factors and AMP‐activated protein kinase activation in 3T3‐L1 Adipocytes

The aim of the present study was to investigate the effect of 5‐bromo‐3,4‐dihydroxybenzaldehyde (BD) isolated from Polysiphonia morrowii on Adipogenesis and Differentiation of 3T3‐L1 preadipocytes into mature Adipocytes and its possible mechanism of action. Levels of lipid accumulation and triglyceride were significantly lower in BD treated cells than those in untreated cells. In addition, BD treatment Reduced protein expression levels of peroxisome proliferator‐activated receptor‐γ, CCAAT/enhancer‐binding proteins α, and sterol regulatory element‐binding protein 1 compared with control (no treatment).

It also Reduced expression levels of adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. AMP‐activated protein kinase activation was found to be one specific mechanism involved in the effect of BD. These results demonstrate that BD possesses Inhibitory effect on Adipogenesis through activating AMP‐activated protein kinase signal pathway.

Butein Chalconoid

Butein is a novel Anti-Adipogenic compound

Rhus verniciflua Stokes (RVS) has been used as a traditional herbal medicine for its various biological activities including Anti-Adipogenic effects. Activity-guided separation led to the identification of the Anti-Adipogenic functions of butein. Butein, a novel Anti-Adipogenic compound, robustly suppressed lipid accumulation and Inhibited expression of Adipogenic markers. Molecular studies showed that activated transforming growth factor-β (TGF-β) and suppressed signal transducer and activator of transcription 3 (STAT3) signaling pathways were mediated by butein. Analysis of the temporal expression profiles suggests that TGF-β signaling precedes the STAT3 in the butein-mediated Anti-Adipogenic cascade.

Small interfering RNA-mediated silencing of STAT3 or SMAD2/3 blunted the Inhibitory effects of butein on Adipogenesis indicating that an interaction between two signaling pathways is required for the action of butein. Upon butein treatments, stimulation of TGF-β signaling was still preserved in STAT3 silenced cells, whereas regulation of STAT3 signaling by butein was significantly impaired in SMAD2/3 silenced cells, further showing that TGF-β acts upstream of STAT3 in the butein-mediated anti-Adipogenesis.

Taken together, the present study shows that butein, a novel Anti-Adipogenic compound from RVS, Inhibits Adipocyte Differentiation through the TGF-β pathway followed by STAT3 and peroxisome proliferator-activated receptor γ signaling, further implicating potential roles of butein in TGF-β- and STAT3-dysregulated diseases.

Capsaicin

Effects of Capsaicin on Induction of Apoptosis and Inhibition of Adipogenesis in 3T3-L1 cells

Currently, at the beginning of the 21st century, Obesity has become the leading metabolic disease in the world. It is a serious health problem in industrialized countries. Previous research has suggested that decreased Preadipocyte Differentiation and proliferation and decreased lipogenesis are mechanisms to Reduce Obesity. In the present study, the effects of capsaicin on the induction of apoptosis and Inhibition of lipid accumulation in 3T3-L1 preadipocytes and Adipocytes were investigated. The results demonstrated that capsaicin decreased cell population growth of 3T3-L1 preadipocytes, assessed with the MTT assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to capsaicin showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with capsaicin decreased the number of normal cells and increased the number of early apoptotic and late apoptotic cells in a dose-dependent manner.

The treatment of cells with capsaicin caused the loss of mitochondria membrane potential (ΔΨm). The induction of apoptosis in 3T3-L1 preadipocytes by capsaicin was mediated through the activation of caspase-3, Bax, and Bak, and then through the cleavage of PARP and the down-regulation of Bcl-2. Moreover, capsaicin significantly decreased the amount of intracellular triglycerides and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 Adipocytes. Capsaicin also Inhibited the expression of PPARγ, C/EBPα, and leptin, but induced up-regulation of adiponectin at the protein level. These results demonstrate that capsaicin efficiently induces apoptosis and Inhibits Adipogenesis in 3T3-L1 preadipocytes and Adipocytes.

Keywords: Capsaicin; Adipogenesis; 3T3-L1 cells; apoptosis; protein expression.

Carduus crispus

Inhibition of Adipocyte Differentiation by MeOH Extract from Carduus crispus through ERK and p38 MAPK Pathways

In this study, the effects of a methanol (MeOH) extract of Carduus crispus L. (Asteraceae) on Adipogenesis was investigated in 3T3-L1 cells. To differentiate preadipocytes to Adipocytes, confluent 3T3-L1 preadipocytes were treated with a hormone mixture, which included isobutylmethylxanthine, dexamethasone, and insulin (MDI). The methanol extract of C. crispus significantly decreased Fat Accumulation by Inhibiting Adipogenic signal transcriptional factors in MDI-induced 3T3-L1 cells in a dose-dependent manner.

In MTT assays and on PI-staining, methanol extract of C. crispus Inhibited the proliferation of 3T3-L1 cells during mitotic clonal expansion (MCE). The Anti-Adipogenic effect of the Carduus extract seemed to be associated with the up-regulation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways within the first 2 days after MDI treatment. These results suggest that methanol extract of C. crispus might be beneficial for the treatment of Obesity.

Carnosic acid (Salvia japonica Thunb.)

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic”quinone-type of CA or CS. Here, we found that CA and CS Inhibited the in vitro Differentiation of mouse preadipocytes, 3T3-L1 cells, into Adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element(ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes.

Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the Inhibition of Adipocyte Differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against Obesity-related diseases.

•The antiAdipogenic mechanism of carnosic acid is multifactorial.

•Carnosic acid Inhibits mitotic clonal expansion of 3T3-L1 cells.

•The expression of C/EBPα , PPARγ and FABP2 are Inhibited by carnosic acid.

•Carnosic acid alters the ratio of C/EBPβ forms and its subnuclear distribution.

•Carnosic acid is a potential agent for the treatment/prevention of Obesity.

Background: Obesity is a serious health problem all over the world, and Inhibition of Adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, Inhibits 3T3-L1 preadipocytes Differentiation. However, very little is known about the molecular mechanism responsible for its antiAdipogenic effect.

Methods: We evaluated the effect of CA on the Differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of Adipogenic markers, and the subcellular distribution of C/EBPβ.

Results: CA treatment only during the first day of 3T3-L1 Differentiation process was enough to Inhibit Adipogenesis. This Inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPβ and C/EBPδ mRNA levels but blocked PPARγ , and FABP4 expression. C/EBPβ has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24 h of Differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPβ.

Conclusions: CA exerts its Anti-Adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPβ forms, inducing the loss of C/EBPβ proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ.

General significance: Understanding the molecular mechanism by which CA blocks Adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of Obesity.

Carnosine (L-Carnosine) (β – alanine and L-histidine)

Synergistic effect of carnosine on browning of Adipose Tissue in exercised obese rats; a focus on circulating irisin levels

The recent appreciation of the energy burning capacity of brown Adipose Tissue turns it to an attractive target for anti‐Obesity therapy. We sought to evaluate the effect of L‐carnosine on browning of white Adipose Tissue in exercised obese rats. Sixty adult male Wistar albino rats, 7–8 week‐old weighing 130–150 g, were allocated into six groups; with 10 rats in each, for an experimentation period of 12 weeks: (i) normal control rats fed a standard fat diet (SFD/control), (ii) normal control rats fed a standard diet and injected with L‐carnosine (250 mg/kg, i.p,) for 6 weeks (SFD/CAR), (iii) high‐fat diet (HFD)‐induced obese rats for 12 weeks, (iv) HFD rats subjected to exercise training (HFD/EXE) for 6 weeks, (v) HFD rats injected with L‐carnosine (250 mg/kg,i.p.) for 6 weeks (HFD/CAR) and, (vi) HFD rats subjected to exercise training and L‐carnosine (HFD/EXE/CAR). At the end of the 12‐week‐experiment, the body weights and the serum levels of lipid profile, oxidative stress, and inflammatory markers as well as circulating myokines were investigated. Gastrocnemius muscles and inguinal Adipose Tissues were excised for the measurement of gene expression of muscle irisin, Adipose Tissue uncoupling protein1 (UCP1), CD137 and the protein level of p38MAPK.

In addition, histopathological examination for the studied groups was performed. Both exercise training for 6 weeks and carnosine treatment significantly decreased body Weight Gain, ameliorated Obesity‐induced dyslipidemia, Reduced the thiobarbituric acid reactive species (TBARS) and TNF‐α, while increased total antioxidant capacity and IL‐10. Furthermore, increases in serum irisin levels and the expression of adipose uncoupling protein‐1 (UCP‐1), adipose CD137, p38 MAPK, and muscular fibronectin type III domain‐containing protein 5(FNDC5), the precursor of irisin gene expression, were correlated with these carnosine‐ and exercise‐induced physiological improvements. The highest improvement was evident in the combined exercise and carnosine group which indicates that their beneficial effects in obese animals were synergistic. These findings suggest that L‐carnosine may induce browning of Adipose Tissue through irisin stimulation, a phenomenon that could be related to its antioxidant, Anti‐Inflammatory, and anti‐Obesity effects.

Celastrol (Tripterygium wilfordii)

Objective: Celastrol, a triterpene from the root bark of the Chinese medicinal plant Tripterygium wilfordii, has been shown to exhibit anti-oxidant, anti-inflammatory, anti-cancer and insecticidal activities. Also, it has been demonstrated that celastrol has obesity-controlling effects in diet-induced obesity mice. However, direct evidence that celastrol contributes to the development of adipocyte differentiation and lipolysis has not been fully elucidated. Moreover, no previous studies have evaluated whether celastrol may regulate adipogenic transcriptional markers in adipocytes.

Materials/Methods: In order to address the questions above, we extended previous observations and investigated in vitro celastrol signaling study whether celastrol may regulate differentiation, lipolysis and key adipogenic transcriptional pathways in 3T3-L1 adipocytes.

Results: Treatment of celastrol not only inhibited adipocyte differentiation (lipid accumulation, glyceraldehyde-3-phosphate dehydrogenase activity and triglyceride content) but also increased lipolysis (glycerol release and free fatty acid release) in 3T3-L1 adipocytes. In addition, all celastrol-regulated functional activities were controlled by PPARγ² and C/EBPα signaling pathways in duration of celastrol’s treatment in 3T3-L1 adipocytes.

Conclusion: Our initial data from in vitro celastrol signaling studies suggest novel insights into the role of PPARγ² and C/EBPα as probable mediators of the action of celastrol in regulating adipocyte differentiation and lipolysis in 3T3-L1 adipocytes.

Chromolaena odorata leaves

Objective: The leaves of Chromolaena odorata, a highly invasive shrub found growing wild worldwide, are traditionally used for wound healing. Due to its high flavonoidcontents, we aimed to find a new application for this plant. Preliminary tests using its ethanolic leaf extract showed that it could suppress the accumulation of lipids in Adipocytes. We therefore studied the Anti-Adipogenic effect of several C. odorata leaf extracts and the relationship between molecular structure and bio-activity of its isolated flavonoid constituents using 3T3-L1 preadipocytes /Adipocytes as a model.

Methods: Three leaf extracts and thirteen Flavonoids isolated from C. odorata were tested for their effect on lipid accumulation in 3T3-L1 Adipocytes using AdipoRed reagent, with quercetin as the positive control. The effects of active Flavonoids on the Adipocytes were confirmed by oil red O staining and visualized under a light microscope.

Results: n-Hexane and ethyl acetate extracts of C. odorata leaves displayed Anti-Adipogenic activity. The latter extract was the more potent one, especially at 40 µg/mL. Four Flavonoids, pectolinarigenin, kaempferide, 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone and dillenetin, exhibited significant, concentration-dependent Inhibitory effects on lipid accumulation in 3T3-L1 Adipocytes . The most potent flavonoid obtained in this study was 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone, which caused 75% and 90% Inhibition of cellular lipid accumulation at 30 and 50 µmol/L, respectively. Both kaempferide and 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone were major constituents in the ethyl acetate extract of this plant.

Conclusion: C. odorata leaves contained several Flavonoids with Anti-Adipogenic effects against lipid accumulation in 3T3-L1 Adipocytes. The plant, normally considered a useless weed, may actually provide an abundant source of biologically active Flavonoids.

Chrysanthemum zawadskii

Anti-Adipogenic Effects of Ethanol Extracts Prepared from Selected Medicinal Herbs in 3T3-L1 cells

Obesity is a major risk factor for various metabolic diseases such as cardiovascular disease, hypertension, and type 2 Diabetes mellitus. In this study, we prepared ethanol extracts from Agastache rugosa (ARE), Chrysanthemum zawadskii (CZE), Mentha arvensis (MAE), Perilla frutescens (PFE), Leonurus sibiricus (LSE), Gardenia jasminoides (GJE), and Lycopus coreanus (LCE). The anti-oxidant and Anti-Adipogenic effects were evaluated. The IC₅₀ values for ascorbic acid and LCE against 2,2-diphenyl-1-picrylhydrazyl radicals were 246.2 μg/mL and 166.2 μg/mL, respectively, followed by ARE (186.6 μg/mL), CZE (198.6 μg/mL), MAE (337.1 μg/mL), PFE (415.3 μg/mL), LSE (548.2 μg/mL), and GJE (626.3 μg/mL). In non-toxic concentration ranges, CZE had a strong Inhibitory effect against 3T3-L1 adipogenes (84.5%) than those of the other extracts.

Furthermore, the Anti-Adipogenic effect of CZE is largely limited in the early stage of Adipogenesis, and we revealed that the Inhibitory role of CZE in Adipogenesis is required for the activation of Wnt signaling. Our results provide scientific evidence that the Anti-Adipogenic effect of CZE can be applied as an ingredient for the development of functional foods and nutri-cosmetics for Obesity prevention.

Chrysin (Oroxylum indicum(L.)Vent.seed)

Objectives: Many studies have to do with promising therapeutic phytochemicals such as Flavonoids to treat Obesity and related complications, and a number of dietary compounds have been proposed as tools for increasing energy expenditure and decreasing Fat Accumulation in mammals. Here, we show that the flavonoid chrysin induces browning of 3T3-L1 Adipocytes via enhanced expression of brown fat–specific genes and proteins as well as enhances lipid metabolism.

Methods: Chrysin-induced fat browning was investigated by determining expression levels of brown fat–specific genes and proteins by real-time polymerase chain reaction and immunoblot analysis, respectively.

Results: Chrysin enhanced expression of brown fat–specific markers and increased protein levels of peroxisome proliferator-activated receptor (PPAR)α, PPARγ, PPARδ, phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated acetyl-CoA carboxylase, hormone sensitive lipase, perilipin, carnitine palmitoyltransferase 1, acyl-coenzyme A oxidase 1, peroxisome proliferator-activated receptor-1 alpha (PGC-1α), and uncoupling protein 1 (UCP-1), suggesting its possible role in augmentation of Lipolysis, fat oxidation, and thermogenesis as well as reduction of lipogenesis. Increased expression of UCP-1 and other brown fat–specific markers was possibly mediated by chrysin-induced activation of AMPK based on the fact that Inhibition of AMPK by dorsomorphin abolished expression of PR domain-containing 16, UCP-1, and PGC-1α while the activator 5-aminoimidazole-4-carboxamide ribonucleotide elevated expression of these brown marker proteins.

Conclusion: Our findings suggest that chrysin plays a dual modulatory role in the form of inducing the brown-like phenotype as well as enhancing lipid metabolism and thus may be explored as a potentially promising food additive for prevention of Obesity.

Cinnamomum verum

Inhibition of Adipocyte Differentiation and Adipogenesis by supercritical fluid extracts and marc from Cinnamomum verum

This study was performed to evaluate the AntiObesity effect of supercritical fluid extracts (SFC) and marc methanol extracts (SFM) from Cinnamomum verum in 3T3-L1 preadipocytes. In inducing the Differentiation of 3T3-L1 preadipocytes in the presence of an Adipogenic cocktail, iso-butylmethylanthine (IBMX), dexamathasone, and insulin, treatment with fraction residue SFC and SFM. SFC significantly Reduced the mRNA expression of the transcription factor peroxisome proliferator-activatedreceptor-γ (PPARγ), the sterol regulatory-element-binding protein-1c (SREBP1c), and the CCAAT enhancer-binding-protein α (C/EBPα) in a concentration-dependent manner.

Moreover, SFC markedly down-regulated acyl-CoA synthetase-1 (ASC1), fatty acid synthesis (FAS), fatty acid transport-1 (FATP1), fatty acid binding protein 4 (FABP4), and perilipin. These findings suggest that SFC may be a potential therapeutic adjunct for Obesity by targeting the Differentiation of preadipocytes, as well as their functions.

Cirsium brevicaule A. GRAY

Cirsium brevicaule A. GRAY leaf Inhibits Adipogenesis in 3T3-L1 cells and C57BL/6 mice

Background: Various Flavonoids obtained from the genus Cirsium have been reported to exhibit beneficial effects on health. The present study evaluated the AntiObesity effects of Cirsium brevicaule A. GRAY leaf (CL) by using 3T3-L1 cells and C57BL/6 mice that were fed a high-fat diet (HFD).

Methods: Dried CL powder was serially extracted with solvents of various polarities, and these extracts were tested for antiAdipogenic activity using 3T3-L1 Adipocytes . Mice were fed experimental HFD supplemented with dried CL powder for 4 wk. Lipid levels and mRNA levels of genes related to lipid metabolism were determined in 3T3-L1 Adipocytes and the white Adipose Tissue (WAT) and liver of mice fed on a HFD.

Results: Treatment of 3T3-L1 Adipocytes with a hexane extract of CL significantly Reduced cellular lipid accumulation and expression of the fatty acid synthase (FASN) gene. Dietary CL Reduced the serum levels of non-esterified fatty acids in HFD-fed mice. Significant decreases in subcutaneous WAT weight and associated FASN gene expression were observed in the mice fed the experimental CL diet. Dietary CL also Reduced the hepatic lipid and serum levels of a hepatopathic indicator in the HFD-fed mice. A significant reduction in mRNA levels of FASN and HMG-CoA reductase were observed in the livers of the CL-diet group. Dietary CL, on the other hand, increased in the hepatic mRNA levels of genes related to β-oxidation, namely peroxisome proliferator-activated receptor α, calnitine palmitoyltrasferase 1A, and uncoupling protein 2. Expression of the insulin receptor gene was also significantly increased in the livers of mice-fed the CL diet.

Conclusions: The present study therefore demonstrated that CL Suppresses lipid accumulation in the WAT and liver partly through Inhibiting mRNA levels of FASN gene and enhancing the Lipolysis -related gene expression.

Cirsium setidens Nakai

Standardized Cirsium setidens Nakai Ethanolic Extract Suppresses Adipogenesis and Regulates Lipid Metabolisms in 3T3-L1 Adipocytes and C57BL/6J Mice Fed High-Fat Diets

Cirsium setidens Nakai, a wild perennial herb, grows mainly in Gangwon province, Korea, and has been reported to contain bioactive ingredients with various medicinal activities, including the treatment of edema, bleeding, and hemoptysis. However, the potential AntiObesity effects of C. setidens Nakai have not been fully investigated. This study evaluated the AntiObesity effect of standardized C. setidens Nakai ethanolic extract (CNE) in 3T3-L1 Adipocytes and in obese C57BL/6J mice fed a high-fat diet. CNE suppressed the expression of lipogenic genes and increased the expression of lipolytic genes. The AntiAdipogenic and antilipogenic effects of CNE appear to be mediated by the Inhibition of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) expressions.

Moreover, CNE stimulated fatty acid oxidation in an AMPK-dependent manner. CNE-treated groups of C57BL/6J mice showed Reduced body weights and Adipose Tissue weight and improved serum lipid profiles through the downregulation of PPARγ, C/EBPα, fatty acid binding protein 4 (FABP4), sterol regulatory element binding protein-1c (SREBP-1c), and fatty acid synthase (FAS) and the upregulation of adiponectin and carnitine palmitoyltransferase-1 (CPT-1) in obese C57BL/6J mice fed a high-fat diet. These results suggest that CNE may have an AntiObesity effect on Adipogenesis and lipid metabolism in vitro and in vivo and present the possibility of developing a treatment for Obesity with nontoxic natural resources.

Assessment of the pectolinarin content and the radical scavenging-linked AntiObesity activity of Cirsium setidens Nakai extracts

Cirsium setidens Nakai contains bioactive compounds that exert biological activities. Method validation for analysis of the pectolinarin content in Cirsium setidens extracts (CSE) and radical scavenging-linked AntiObesity activities using 3T3-L1 cells and C57BL/6 mice were performed. The pectolinarin content of CSE was 2.81±0.01 mg/g with a high degree of linearity in calibration curves (R²=0.9999).

CSE exhibited free radical-scavenging activities and a reducing power. CSE and pectolinarin Inhibited lipid accumulation during Adipogenesis of 3T3-L1 cells via down-regulation of Adipogenic transcription factors. CSE supplementation suppressed body weight in C57BL/6 mice fed a high fat diet and Reduced plasma total cholesterol, triglyceride, insulin, and glucose levels. Pectolinarin-enriched CSE can be considered as a good source of natural Antioxidants and AntiObesity ingredients.

Citrus aurantium peel

Citrus aurantium Flavonoids Inhibit Adipogenesis through the Akt signaling pathway in 3T3-L1 cells

Background: Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 Diabetes , hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium Flavonoids (CAF) on the Inhibition of Adipogenesis and Adipocyte Differentiation in 3T3-L1 cells.

Methods: During Adipocyte Differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of Adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during Adipocyte Differentiation.

Results: The insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by Adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9), which may Reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only Inhibited triglyceride accumulation during Adipogenesis but also contributed to the Lipolysis of Adipocytes.

Conclusions: In the present study, we demonstrate that CAF suppressed Adipogenesis in 3T3-L1 Adipocytes . Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently Inhibits the activation of PPARγ and C/EBPα. The Anti-Adipogenic activity of CAF was mediated by the Inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately Inhibiting Adipocyte Differentiation.

Clitoria ternatea Flower Petal

Clitoria ternatea Flower Petal Extract Inhibits Adipogenesis and Lipid Accumulation in 3T3-L1 preadipocytes by Downregulating Adipogenic Gene Expression

Clitoria ternatea (commonly known as blue pea) flower petal extract (CTE) is used as a natural colorant in a variety of foods and beverages. The objective of study was to determine the Inhibitory effect of CTE on Adipogenesis in 3T3-L1 preadipocytes. The phytochemical profiles of CTE were analyzed by liquid chromatography and tandem Mass spectrometry (LC-MS/MS). Anti-Adipogenesis effect of CTE was measured by using Oil Red O staining, intracellular triglyceride assay, quantitative real-time PCR and western blot analysis in 3T3-L1 Adipocytes. Cell cycle studies were performed by flow cytometry. Lipolysis experiments were performed using a colorimetric assay kit. In early stages, CTE demonstrated Anti-Adipogenic effects through Inhibition of proliferation and cell cycle retardation by suppressing expression of phospho-Akt and phospho-ERK1/2 signaling pathway.

The results also showed that CTE Inhibited the late stage of Differentiation through diminishing expression of Adipogenic transcription factors including PPARγ and C/EBPα. The Inhibitory action was subsequently attenuated in downregulation of fatty acid synthase and acetyl-CoA carboxylase, causing the reduction of TG accumulation. In addition, CTE also enhanced catecholamine-induced Lipolysis in Adipocytes. These results suggest that CTE effectively attenuates Adipogenesis by controlling cell cycle progression and downregulating Adipogenic gene expression.

Cocoa tea (Camellia ptilophylla)

Cocoa tea (Camellia ptilophylla) water extract Inhibits Adipocyte Differentiation in mouse 3T3-L1 preadipocytes

Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced Obesity , hepatic steatosis and hyperlipidemia in mice. The present study aimed to investigate the Anti-Adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic Differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly Inhibited triglyceride accumulation in mature Adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key Adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α).

The tea extract was subsequently found to Reduce the expressions of Adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were Inhibited during cocoa tea Inhibition of 3T3-L1 Adipogenic Differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress Adipogenesis in pre-Adipocyte 3T3-L1 similar to traditional green tea

Colocynth (Citrullus colocynthis) Flesh

Adipogenesis is the overall process of Adipocyte Differentiation. With a positive energy balance, intracellular accumulation of triglyceride increases through the generation of functional Adipocytes. This occurs with Adipogenesis of undifferentiated preadipocytes. Citrullus colocynthis (CC), a member of the Cucurbitaceae family, is a desert viny plant native to North Africa. The effect of ethanolic seed extracts of colocynth (SCEE) on Adipogenesis was investigated using 3T3-L1 preadipocytes. Cell viability was measured using the MTT assay; triglycerides were stained with Oil Red O and Adipogenesis-related gene expressions were quantified using qRT-PCR. Results showed that SCEE helps to Inhibit intracellular triglyceride accumulation during Adipogenesis without affecting cell viability. Likewise, SCEE not only showed Anti-Adipogenic activities, essentially during the early stage, but their effects during the middle and late stages were very low.

These effects have been confirmed by the down-regulation of C/EBPβ and C/EBPδ at 24, 48 and 72 h. In addition, SCEE treatment in 3T3-L1 cells induced down-regulation of the transcription factors CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator activated receptor γ (PPARγ), and sterol regulatory element-binding protein 1c (SREBP-1c) and their target genes. UPLC-ESI-MS/MS profiling showed 10 bioactive compounds including quinic acid, isovitexin, scoparin, vitexin-2’’-O-rhamnoside, reserpine, digitoxin, triprolidine, naringenin, linoleic acid and oleic acid. Thus, these results suggested that the seeds of colocynth may have beneficial health effects.

Conjugated linoleic acid (CLA) (Ruminant fat)

Impact of conjugated linoleic acid on bone physiology: proposed mechanism involving Inhibition of Adipogenesis

Conjugated linoleic acid (CLA) supplementation decreases adipose Mass and increases bone Mass in mice. Recent clinical studies demonstrate a beneficial effect of CLA on reducing weight and adipose Mass in humans. This article reviews possible biological mechanisms of action of CLA on bone metabolism, focusing on modulation of nuclear receptor peroxisome proliferator‐activated receptor gamma activity to steer mesenchymal stem cell Differentiation toward an adipose and away from an osteoblast lineage. Clinical studies of the effects of CLA on bone Mass and clinical implications of the effects of CLA on bone health in humans are summarized and discussed.

Coptis chinensis (Coptidis Rhizoma)

Effect of Atractylodes Rhizoma Alba, Houttuyniae Herba, Lonicerae Flos, Scutellariae Radix and Coptidis Rhizoma Extracts Combined with Metformin on the Antioxidant and Adipocyte Differentiate Inhibition

Objectives: This study was to investigate the antioxiative capacity, AntiObesity effects of Atractylodes Rhizoma Alba, Houttuyniae Herba, Lonicerae Flos, Scutellariae Radix, and Coptidis Rhizoma on Raw 264.7 and 3T3-L1 cell lines.

Methods: Three different types of herb extracts (A. Rhizoma Alba, H. Herba, L. Flos, S. Radix, and C. Rhizoma; water 100%, ethanol 30%, ethanol 100%) were used in this study. Total Polyphenol compound, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, reactive oxygen species (ROS) activity, NO production and cell proliferation were measured.

Results: Total Polyphenol compound measurement of L. Flos, A. Rhizogenes, and C. Rhizoma extracts were higher than A. Rhizoma Alba, H. Herba. DPPH radical scavenging activity, ROS activity and NO production of S. Radix, C. Rhizoma extracts were lower than L. Flos, A. Rhizoma, and H. Herba.

Conclusions: Metformin and S. Radix, C. Rhizoma, A. Rhizoma Alba, and L. Flos extracts combination groups showed synergistic effect on Adipocyte Differentiation Inhibition and antioxidative activity.

Cordycepin (Cordyceps sinensis)

The Effects of Cordyceps sinensis (Berk.) Sacc. and Gymnema inodorum (Lour.) Decne. Extracts on Adipogenesis and Lipase Activity In Vitro

This study aimed to investigate the effects of Cordyceps sinensis extract (CSE) and Gymnema inodorum extract (GIE), used alone and combined, on AntiAdipogenesis in 3T3-L1 cells . Oil Red O staining was used to examine the effects of these extracts on Inhibition of intracellular lipid accumulation in 3T3-L1 Adipocytes and on lipid droplet morphology. Fourier transform-infrared (FTIR) microspectroscopy was used to examine biomolecular changes in 3T3-L1 Adipocytes . The pancreatic lipase assay was used to evaluate the Inhibitory effects of CSE and GIE on pancreatic lipase activity.

Taken together, the results indicated that CSE, GIE, and their combination suppressed lipid accumulation. The FTIR microspectroscopy results indicated that CSE, GIE, and their combination had Inhibitory effects on lipid accumulation in the Adipocytes. Compared with the untreated Adipocytes, the signal intensity and integrated areas of glycogen and other carbohydrates, the acyl chain of phospholipids, and the lipid/protein ratios of the CSE, GIE, alone, and combined treated Adipocytes were significantly lower (p < 0.05). Combination treatment resulted in a synergistic effect on lipid accumulation reduction in the Adipocytes. Principal component analysis of the biomolecular changes revealed six distinct clusters in the FTIR spectra of the sample cells. The pancreatic lipase assay results indicated that CSE and GIE Inhibited the pancreatic lipase activity in a dose-dependent manner (mean ± standard error of the mean IC₅₀ values, 2312.44 ± 176.55 μg mL⁻¹ and 982.24 ± 44.40 μg mL⁻¹, resp.). Our findings indicated that FTIR microspectroscopy has potential application for evaluation of the effectiveness of medicinal plants and for the development of infrared biochemical Obesity markers useful for treating patients with Obesity.

These results suggested that use of CSE and GIE alone and in combination may be efficacious as a complementary therapy for hyperlipidemia and Obesity management. However, clinical trials in animals and humans must first be completed.

Coumestrol (Tonka bean)

Coumestrol modulates Akt and Wnt/β-catenin signaling during the attenuation of Adipogenesis

Coumestrol is a natural phytochemical present in plants such as red clover and soy, and has been reported to stimulate the estrogen receptor as a major phytoestrogen. While the molecular mechanisms responsible for the Anti-Adipogenic effects of phytoestrogens such as genistein and daidzein have been previously investigated, the effects of coumestrol on Adipogenesis remain to be elucidated. We observed that coumestrol dose-dependently attenuates MDI (mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin)-induced lipid accumulation, consistent with an earlier study, while significantly Inhibiting MDI-induced Adipogenesis in the first 48 hours of Differentiation, a critical time window for Anti-Adipogenic effects. Coumestrol treatment suppressed MDI-induced protein expression of PPARγ and C/EBPα in Adipocytes, leading to the subsequent downregulation of FAS and aP2 expression. Akt and GSK3β were phosphorylated shortly after MDI stimulation, and these responses were Inhibited by coumestrol treatment.

Coumestrol also increased LRP6 protein expression, resulting in the recovery of β-catenin downregulation by MDI, while attenuating MDI-induced downregulation of Wnt10b. In addition, mRNA and protein expression of c-Myc and cyclin D1, target genes of β-catenin, were both recovered by coumestrol treatment. These results suggest that coumestrol Inhibits Adipocyte Differentiation via regulation of Akt and Wnt/β-catenin signaling and may have potential for development as an agent to Prevent Adipogenesis.

Cranberries (Oxycoccus quadripetalus)

Cranberries (Oxycoccus quadripetalus) Inhibit Adipogenesis and lipogenesis in 3T3-L1 cells

Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the Inhibiting effects of Adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries Reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner.

Treatment with cranberries decreased the number of Adipocytes and Reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an Inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced Lipolysis in Adipocytes and down-regulated the expression of major transcription factors of the Adipogenesis pathway, such as PPARγ, C/EBPα and SREBP1. These findings indicate that cranberries are capable of suppressing Adipogenesis and therefore they seem to be natural bioactive factors effective in Adipose Tissue Mass modulation.

Crocin (saffron extract)

Crocin Inhibits Obesity via AMPK-dependent Inhibition of Adipocyte Differentiation and promotion of Lipolysis

Obesity has become a severe public health problem worldwide. Crocin, a natural product, has been reported to have a number of pharmacological activities, including anti-inflammatory, anti-cancer, neuroprotective, antihypertensive, and cardioprotective action. The aims of the current study were to identify the beneficial effects of crocin on Obesity, Adipocyte Differentiation, and Lipolysis and to evaluate the possible role of AMPK. Results indicated that crocin significantly increased AMPK phosphorylation in differentiated Adipocytes in vitro and in Adipose Tissue in db/db mice. Crocin Reduced lipid accumulation in differentiated Adipocytes. In addition, crocin Inhibited the expression of mRNA of important Adipogenesis -related regulators, including CEBPα, CEBPβ, PPARγ, aP2, FAS, and CD36, in both differentiated Adipocytes and Adipose Tissue in db/db mice. Crocin increased the expression of mRNA of key Lipolysis -associated factors, including PPARα, LPL, and HSL, in both differentiated Adipocytes and Adipose Tissue in db/db mice.

In Adipocytes, knockdown of AMPK significantly suppressed the crocin-induced Inhibition of Adipocyte Differentiation and increase in Lipolysis. BML-275 is an Inhibit or of AMPK. In Adipose Tissue in db/db mice, BML-275 suppressed crocin-induced Inhibition of fat formation and alleviation of a metabolic disorder. The current results suggest that crocin alleviates Obesity in db/db mice and that it Inhibits Adipocyte Differentiation in preadipocytes. Crocin Inhibits Adipogenesis and promotes Lipolysis via activation of AMPK. Therefore, crocin may have promise as an option for the clinical treatment for Obesity and associated metabolic diseases.

Cryptotanshinone, a compound of Salvia miltiorrhiza

Background: Cryptotanshinone (CT), a major tanshinonefound in Salvia miltiorrhiza Bunge (Lamiaceae), has various pharmacological effects such as antitumor, anti-inflammatory, and antioxidant properties. Despite its well-documented benefits in a wide range of diseases, the effect of CT on Adipocyte Differentiation has not been well characterized.

Purpose: The present study was designed to determine the in vitro Anti-Adipogenic effect and underlying molecular mechanisms of CT using 3T3-L1 murine Pre-Adipocytes .

Methods: We measured the levels of intracellular triglyceride accumulation and mRNA and protein expression of key Adipogenic transcription factors and their target genes.

Results: Treatment with CT drastically Reduced lipid accumulation in a dose- and time-dependent manner. Molecular assays showed that CT effectively suppressed the expression of C/EBPβ, C/EBPα , and PPARγ and of their target Adipocyte -specific genes aP2, adiponectin, and GLUT4 but activated the expression of Anti-Adipogenic genes such as GATA2, CHOP10, and TNF-α. CT treatment also Inhibited the phosphorylation of STAT3 in the early phase of Adipogenesis . A small-interfering-RNA-mediated knock-down of STAT3 potentiated the Anti-Adipogenic effect of CT.

Conclusion: Taken together, the results suggest that CT may be a good Anti-Adipogenic candidate because it regulates STAT3 during early Adipogenesis .

Curcumin

Modification of Curcumin with Polyethylene Glycol Enhances the Delivery of Curcumin in preadipocytes and Its AntiAdipogenic Property

Conjugation of curcumin (CCM) by polyethylene glycol (PEG) has been previously developed to improve water solubility of the natural form of CCM and its antiproliferative role in some human cancer cell lines. This study examined the cellular uptake kinetics of the natural form of CCM and CCM−PEG. Their cytotoxic effect in proliferating preadipocytes and AntiAdipogenic property in differentiating preadipocytes had also been investigated. CCM and CCM−PEG were found to be differently absorbed in 3T3-L1 preadipocytes and Adipocytes with a limited amount of CCM−PEG absorption in the cell. The improved water solubility of CCM−PEG was correlated with increased cellular retention of CCM in 3T3-L1 cells, particularly in preadipocytes.

Consequently, CCM−PEG treatment sensitized proliferating preadipocytes to CCM-induced cell toxicity. Furthermore, incubation of differentiating 3T3-L1 cells with CCM−PEG resulted in improvement of the Inhibitory role of CCM in Adipocyte Differentiation with no toxic effect. These results suggest that pegylation-improved water solubility and cellular retention of CCM may be uniquely useful for improving the delivery of CCM in preadipocytes and its AntiAdipogenic ability.

Curcumin Inhibits Adipocyte Differentiation through modulation of mitotic clonal expansion

Adipocyte Differentiation is a key process in determining the number of mature Adipocytes in the development of Obesity. Here, we examined the function of curcumin, a dietary Polyphenol found in turmeric, and its underlying mechanisms in Adipocyte Differentiation. Our study reveals that curcumin exerts an Anti-Adipogenic function both in 3T3-L1 murine cells and in human primary preadipocytes as determined by intracellular lipid accumulation assay, quantitative analysis of Adipocyte marker gene expression and a noninvasive multimodal Coherent Anti-Stokes Raman Scattering (CARS) microscopic analysis of intracellular curcumin. The Inhibitory action of curcumin was largely limited to the early stage of Adipocyte Differentiation, where curcumin was found to Inhibit mitotic clonal expansion (MCE) process as evidenced by impaired proliferation, cell-cycle entry into S phase and the S to G2/M phase transition of confluent cells, and levels of cell cycle-regulating proteins with no significant effect on cell viability and cytotoxicity.

This, in turn, resulted in Inhibition of mRNA levels of early Adipogenic transcription factors, particularly Krüppel-like factor 5 (KLF5), CCAAT/enhancer binding proteinα (C/EBPα ) and peroxisome proliferator-activated receptorγ (PPARγ), in the early stage of Adipocyte Differentiation. Supplementation with rosiglitazone, a PPARγ ligand, during the early stage of Adipocyte Differentiation partially rescued curcumin-Inhibited Adipocyte Differentiation. Collectively, our results show that curcumin is an Anti-Adipogenic dietary bioactive component largely involved in the modulation of the MCE process during the early stage of Adipocyte Differentiation.

Curcumin-induced suppression of Adipogenic Differentiation is accompanied by activation of Wnt/β-catenin signaling

In summary, curcumin, the major Polyphenol found in turmeric, effectively Inhibits Preadipocyte Differentiation. Dephosphorylation of MAPKs may participate in the Anti-Adipogenic effects of curcumin. Curcumin also Inhibited the induction of Adipogenic transcription factors such as PPARγ and C/EBPα. This study is the first to demonstrate that the Wnt/β-catenin signaling pathway plays a crucial role in curcumin-induced suppression of Adipogenesis.

Curcumin Inhibits Adipogenesis in 3T3-L1 Adipocytes and angiogenesis and Obesity in C57/BL mice

Angiogenesis is necessary for the growth of Adipose Tissue. Dietary Polyphenols may suppress growth of Adipose Tissue through their antiangiogenic activity and by modulating Adipocyte metabolism. We investigated the effect of curcumin, the major Polyphenol in turmeric spice, on angiogenesis, Adipogenesis, Differentiation, apoptosis, and gene expression involved in lipid and energy metabolism in 3T3-L1 Adipocyte in cell culture systems and on body Weight Gain and Adiposity in mice fed a high-fat diet (22%) supplemented with 500 mg curcumin/kg diet for 12 wk. Curcumin (5–20 μmol/L) suppressed 3T3-L1 Differentiation, caused apoptosis, and Inhibited adipokine-induced angiogenesis of human umbilical vein endothelial cells.

Supplementing the high-fat diet of mice with curcumin did not affect food intake but Reduced body Weight Gain, Adiposity, and microvessel density in Adipose Tissue, which coincided with Reduced expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2. Curcumin increased 5′AMP-activated protein kinase phosphorylation, Reduced glycerol-3-phosphate acyl transferase-1, and increased carnitine palmitoyltransferase-1 expression, which led to increased oxidation and decreased fatty acid esterification. The in vivo effect of curcumin on the expression of these enzymes was also confirmed by real-time RT-PCR in subcutaneous Adipose Tissue. In addition, curcumin significantly lowered serum cholesterol and expression of PPARγ and CCAAT/enhancer binding protein α, 2 key transcription factors in Adipogenesis and lipogenesis. The curcumin suppression of angiogenesis in Adipose Tissue together with its effect on lipid metabolism in Adipocytes may contribute to lower body fat and body Weight Gain. Our findings suggest that dietary curcumin may have a potential benefit in preventing Obesity.

Curcumin and Obesity

Turmeric has been long recognized for its Anti‐Inflammatory and health‐promoting properties. Curcumin is one of the principal Anti‐Inflammatory and healthful components of turmeric comprising 2–8% of most turmeric preparations. Experimental evidence supports the activity of curcumin in promoting weight loss and reducing the incidence of Obesity‐related diseases. With the discovery that Obesity is characterized by chronic low‐grade metabolic Inflammation, phytochemicals like curcumin which have Anti‐Inflammatory activity are being intensely investigated. Recent scientific research reveals that curcumin directly interacts with white Adipose Tissue to suppress chronic Inflammation. In Adipose Tissue, curcumin Inhibits macrophage infiltration and nuclear factor κB (NF‐κB) activation induced by inflammatory agents. Curcumin Reduces the expression of the potent pro inflammatory adipokines tumor necrosis factor‐α (TNFα), monocyte chemoattractant protein‐1 (MCP‐1), and plasminogen activator Inhibit or type‐1 (PAI‐1), and it induces the expression of adiponectin, the principal Anti‐Inflammatory agent secreted by Adipocytes . Curcumin also has effects to Inhibit Adipocyte Differentiation and to promote antioxidant activities. Through these diverse mechanisms curcumin Reduces Obesity and curtails the adverse health effects of Obesity.

The direct role of Wnt/β‐catenin signaling in the anti‐Adipogenic activity of curcumin was examined using mouse 3T3‐L1 Preadipocyte cells [72] … All of these activities driven by curcumin were associated with suppressed Adipogenesis in 3T3‐L1 cells.

Curcumin Inhibits 3T3-L1 Preadipocyte proliferation by mechanisms involving post-transcriptional p27 regulation

Inhibition of Differentiation resulted from suppression of PPARγ and C/EBPα, the dominant transcriptional regulators of Adipogenesis. Further, data presented here elucidate an early anti–Adipogenic role for curcumin via post-transcriptional regulation of p27.

•Curcumin Inhibited early stages of Adipogenesis during cell cycle progression.

•Curcumin increased p27 protein through post-translational mechanisms.

•Curcumin suppressed Skp2 protein which directs p27 ubiquitylation.

•p27 accumulation may account for late G1 arrest and suppression of Adipogenesis.

•Potential mechanism regarding Anti-Obesity effects of curcumin.

Previous reports from our lab have shown that Skp2 is necessary for p27 degradation and cell cycle progression during Adipocyte Differentiation. Data presented here demonstrate that the anti-inflammatory, Anti-Obesity phytochemical curcumin blocked Skp2 protein accumulation during early Adipocyte hyperplasia. In addition, curcumin dose-dependently induced p27 protein accumulation andG1 arrest of synchronously replicating 3T3-L1 preadipocytes. Of note, p27 protein accumulation occurred in the presence of decreased p27 mRNA suggesting a role for post-transcriptional regulation. In support of this hypothesis, curcumin markedly increased p27 protein half-life as well as attenuated ubiquitin proteasome activity suggesting that Inhibition of targeted p27 proteolysis occurred through curcumin-mediated attenuation of Skp2 and 26S proteasome activity. While we observed no cytotoxic effects for curcumin at doses less than 20 µM, it is important to note an increase in apoptotic signaling at concentrations greater than 30 µM.

Finally, data presented here demonstrate that the anti-proliferative effect of curcumin was critical for the suppression of Adipocyte Differentiation and the development of the mature Adipocyte. Collectively, our data demonstrate that curcumin-mediated post-transcriptional accumulation of p27 accounts in part for the anti-proliferative effect observed in 3T3-L1 preadipocytes.

Curcumin-3,4-Dichloro Phenyl Pyrazole (CDPP)

Curcumin-3, 4-Dichloro Phenyl Pyrazole (CDPP) overcomes curcumin’s low bioavailability and Inhibits Adipogenesis

Adipocyte dysfunction, Obesity and associated metabolic disorders are of prime healthcare concern worldwide. Among available medications, natural products and inspired molecules hold 40% space in clinically prescribed medicines. In queue, this study overcomes the drawback of curcumin’s low bioavailability with potent Anti-Adipogenic and anti-dyslipidemic activity.

Methods: To evaluate the role of CDPP on Adipocyte Differentiation, 3T3-L1 Adipocytes were used as an in-vitro model. Flow cytometry was performed for cell cycle analysis. Syrian golden hamsters were used to study pharmacokinetic profile and dyslipidemic activity exhibited by CDPP.

Result: CDPP was found to be a potent Inhibit or of Adipogenesis in-vitro. It blocked mitotic clonal expansion by causing cell cycle arrest. CDPP showed marked improvement in gastrointestinal stability and bioavailability in-vivo as compared to curcumin. Administration of CDPP (100 mg/kg) significantly improved HFD induced dyslipidemic profile in hamsters and activated reverse cholesterol transportmachinery.

Conclusion: CDPP could be used as a potential drug candidate against Adipogenesis and dyslipidemia with enhanced gastrointestinal stability and bioavailability.

Cyanidine-3-O-Galactoside Enriched Aronia melanocarpa

Cyanidin-3-O-galactoside-enriched Aronia melanocarpa extract attenuates Weight Gain and Adipogenic pathways in high-fat diet-induced obese C57BL/6 mice

Aronia melanocarpa are a rich source of Anthocyanins that have received considerable interest for their relations to human health. In this study, the Anti-Adipogenic effect of cyanidin-3-O-galactoside-enriched Aronia melanocarpa extract (AM-Ex) and its underlying mechanisms were investigated in an in vivo system. Five-week-old male C57BL/6N mice were randomly divided into five groups for 8-week feeding with a control diet (CD), a high-fat diet (HFD), or a HFD with 50 (AM-Ex 50), 100 (AM-Ex 100), or 200 AM-Ex (AM-Ex 200) mg/kg body weight/day. HFD-fed mice showed a significant increase in body weight compared to the CD group, and AM-Ex dose-dependently Inhibited this Weight Gain. AM-Ex significantly Reduced the food intake and the weight of white fat tissue, including epididymal fat, retroperitoneal fat, mesenteric fat, and inguinal fat. Treatment with AM-Ex (50 to 200 mg/kg) Reduced serum levels of leptin, insulin, triglyceride, total cholesterol, and low density lipoprotein (LDL)-cholesterol.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AM-Ex suppressed Adipogenesis by decreasing CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma coactivator-1α, acetyl-CoA carboxylase 1, ATP-citrate lyase, fatty acid synthase, and Adipocyte protein 2 messenger RNA (mRNA) expressions. These results suggest that AM-Ex is potentially beneficial for the suppression of HFD-induced Obesity by modulating multiple pathways associated with Adipogenesis and food intake.

Cyanidine-3-O-Galactoside Enriched Aronia melanocarpa Extract Inhibits Adipogenesis and Lipogenesis via Down-Regulation of Adipogenic Transcription Factors and Their Target Genes in 3T3-L1 cells

Our results showed that the Anti-Obesity effect of AM-Ex occured through down-regulation of the transcription factors PPARγ, C/EBPα, SREBP-1c, and Adipogenesis and lipogenesis-related genes, aP2, LPL, ACC1, ACL, and FAS. These results demonstrate that AM-Ex can be used as a preventive or therapeutic agent for Obesity. Future, animal and human studies are needed to further investigate the mechanism and proper concentration of AM to be used as Anti-Obesity agents.

Cyclopia subternata

Cyclopia maculata and Cyclopia subternata (honeybush tea) Inhibits Adipogenesis in 3T3-L1 Pre-Adipocytes

The stems, leaves and flowers of Cyclopia have been consumed as aherbal tea ‘honeybush tea’ to treat various medical ailments since the 19th century. Plant Polyphenols are reported to Inhibit Adipogenesis in cell and animal models of Obesity. The aim of this study was to assess the effect of hot water extracts of two Cyclopia species, C. maculata and C. subternata on Obesity in an in vitro model. The total Polyphenol content of unfermented C. subternata, unfermented C. maculata and fermented C. maculata extracts was 25.6, 22.4 and 10.8 g GAE/100 g, respectively.

The major compounds present in the extracts were: the flavonoid, phloretin-3′,5′-di-C-glucoside in C. subternata, the xanthone, mangiferin in unfermented C. maculata and the flavanone, hesperidin in fermented C. maculata. All of the plant extracts Inhibited intracellular triglyceride and Fat Accumulation, and decreased PPARγ 2 expression. The higher concentrations of unfermented C. maculata (800 and 1600 μg/ml) and C. subternata(1600 μg/ml) were cytotoxic in terms of decreased mitochondrialdehydrogenase activity. Both fermented and unfermented C. maculata, at concentrations greater than 100 μg/ml, decreased cellular ATPcontent. Cyclopia maculata and C. subternata Inhibit Adipogenesis in vitro, suggesting their potential as Anti-Obesity agents.

Cyclopia maculata (honeybush tea) stimulates Lipolysis in 3T3-L1 Adipocytes

We have previously, for the first time, demonstrated that hot water extracts of Cyclopia maculata and Cyclopia subternata, endemic South African plants that are consumed as herbal teas, Inhibit Adipogenesis in 3T3-L1 Adipocytes. The aim of this study was to extend the Anti-Obesity investigations of these plants by quantifying Lipolysis in mature 3T3-L1 Adipocytes. Glycerol concentration in culturesupernatants was used as a marker of Adipocyte Lipolysis. Isoproterenol, a β-adrenergic agonist and a known lipolytic agent, was used as a positive control in our assays. Lipolysis was stimulated by all extracts, although statistical significance was noted for fermented (oxidised) C. maculata only. A concentration of 80 μg/ml ofC. maculata extract induced maximal Lipolysis (1.8-fold, p < 0.001).

The increased Lipolysis was accompanied by an increase in the expression of hormone sensitive lipase (1.6-fold, p < 0.05) and perilipin (1.6-fold,p < 0.05). The plant extracts, at the concentration range assayed (0–100 μg/ml), were not cytotoxic in terms of mitochondrialdehydrogenase and adenosine-5′-triphosphate activity. These results showed that C. maculata stimulates Lipolysis in mature 3T3-L1 Adipocytes, providing further support for the Anti-Obesity effects ofCyclopia spp.

A Polyphenol -enriched fraction of Cyclopia intermedia decreases lipid content in 3T3-L1 Adipocytes and Reduce s body Weight Gain of obese db/db mice

Extracts of Cyclopia species, indigenous South African fynbos plants used for the production of honeybush tea, have potential as Anti-Obesity nutraceuticalingredients. Previously, we demonstrated that aqueous extracts of C. maculata and C. subternata exhibited Anti-Obesity effects in 3T3-L1 Adipocytes. In this study, we further explored these Anti-Obesity effects of C. maculata and C. subternata as well as C. intermedia for the first time. Extracts were prepared using a 40% methanol–water mixture (40% MeOH) in order to enhance the Polyphenol ic content of the extracts.

Moreover, these extracts were separated into aqueous and organic fractions using liquid–liquid partitioning with n-butanol and water to further enrich the Polyphenol content of the organic fractions. Extracts of all three Cyclopia species decreased the lipid content in 3T3-L1 Adipocytes, although differences in bioactivity of their aqueous and organic fractions were observed. The organic fraction of C. intermedia was further investigated. This fraction dose-dependently decreased the lipid content in 3T3-L1 Adipocytes without affecting cell viability, while increasing mRNA expression of HSL (1.57-fold, P < 0.05), SIRT1 (1.5-fold, P = 0.07), UCP3 (1.5-fold, P < 0.05) and PPARγ (1.29-fold, P < 0.05). Daily treatment of obese db/db mice with 351.5 mg/kg bodyweight of the organic C. intermedia fraction for 28 days decreased body Weight Gain by 21% (P < 0.05) without any effect on food or water consumption.

The organic fraction was enriched in phenolic content relative to the extract with neoponcirin, a flavanone not previously identified in Cyclopia species, mangiferin, isomangiferin and hesperidin comprising 17.37% of the organic fraction of C. intermedia compared to 4.96% of its “large scale” prepared 40% MeOH extract. Their specific roles as Anti-Obesity agents in these models needs to be studied to guide product development.

An investigation into the Anti–Obesity properties of Cyclopia

Polyphenols have a range of health promoting effects against chronic diseases such as Obesity and type 2 Diabetes mellitus. Previous research in our group showed that Cyclopia species (honeybush), endemic South African plants, display Anti-Obesity effects. The aim of this study was to investigate the Anti-Obesity potential of Polyphenol-enriched extracts of three Cyclopia spp. (C. subternata, C. intermedia and C. maculata) using bioactivity guided fractionation to facilitate the identification of Anti-Obesity Polyphenols. Aqueous methanol extracts of C. subternata, C. intermedia and C. maculata were prepared and separated into their aqueous and organic fractions, which were tested for their Anti-Obesity effects in 3T3-L1 Adipocytes using the Oil Red O, glycerol release (marker of Lipolysis), and triglyceride assays. Cytotoxicity was evaluated using the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays. The Anti-Obesity properties of the organic fraction of the aqueous methanol extract of C. intermedia were evaluated in obese Leprdb/db mice. Thereafter, the organic fraction was separated into four fractions (CCC F1 to CCC F4) using high performance counter-current chromatography (HPCCC), which were tested for their Anti-Obesity and cytotoxic effects in 3T3-L1 Pre-Adipocytes and in mature Adipocytes. Liquid chromatography tandem Mass spectrometry (LC-MS/MS) and quantitative high performance liquid chromatography diode array detection (HPLC-DAD) were used for determination of phenolic composition.

The molecular mechanisms of action of the organic fraction and its CCC fractions were assessed with quantitative real-time PCR and western blot analysis. The Polyphenol-enriched extracts of C. subternata, C. intermedia and C. maculata, and their aqueous and organic fractions exhibited differences in phenolic composition and bioactivity. The aqueous fractions of C. maculata and C. subternata, containing lower phenolic content than the organic fractions, decreased lipid content in 3T3-L1 Adipocytes compared to their organic counterparts. However, the organic fraction of C. intermedia, with higher phenolic content than its aqueous fraction, decreased lipid content compared to its aqueous fraction in 3T3-L1 Adipocytes. In Leprdb/db mice, the organic fraction of C. intermedia decreased body Weight Gain, without affecting food or water consumption.

Further fractionation of the organic fraction of C. intermedia by HPCCC provided four fractions with differences in phenolic composition and Anti-Obesity effects. None of the extracts or fractions, except for the aqueous C. intermedia and organic C. maculata fractions which decreased ATP content, affected cell viability as measured with the MTT and ATP assays during acute treatment. The highest concentrations of CCC F1 and CCC F3 decreased ATP content during chronic treatment in Pre-Adipocytes . In 3T3-L1 Adipocytes, the organic fraction of C. intermedia and CCC fractions modulated the expression of genes implicated in lipid and energy metabolism, whereas the organic fraction of C. intermedia had no effect on the expression of these genes in Leprdb/db mice. In conclusion, the organic fraction of C. intermedia exhibited Anti-Obesity properties in vitro and in vivo. A flavanone with Anti-Obesity potential, neoponcirin, was identified in C. intermedia, the first discovery of this compound in Cyclopia spp. Bioactivity guided fractionation of the organic fraction of C. intermedia resulted in CCC fractions retaining different Polyphenols with varying Anti-Obesity effects.

These results demonstrate that the Anti-Obesity potential of C. intermedia is due to the presence of more than one compound, with different mechanisms of action, or the synergistic effects of more than one compound, may contribute to the bioactivity of C. intermedia.

Delphinidin (Consolida ajacis (L.) Schur)

Anti-Adipogenic Effects of Delphinidin-3-O–β-Glucoside in 3T3-L1 preadipocytes and Primary White Adipocytes

Delphinidin-3-O–β-glucoside (D3G) is a health-promoting anthocyanin whose Anti-Obesity activity has not yet been thoroughly investigated. We examined the effects of D3G on Adipogenesis and lipogenesis in 3T3-L1 Adipocytes and primary white Adipocytes using real-time RT-PCR and immunoblot analysis. D3G significantly Inhibited the accumulation of lipids in a dose-dependent manner without displaying cytotoxicity. In the 3T3-L1 Adipocytes, D3G downregulated the expression of key Adipogenic and lipogenic markers, which are known as peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBP1), CCAAT/enhancer-binding protein alpha (C/EBPα ), and fatty acid synthase (FAS).

Moreover, the relative protein expression of silent mating type information regulation 2 homolog 1 (SIRT1) and carnitine palmitoyltransferase-1 (CPT-1) were increased, alongside Reduced lipid levels and the presence of several small lipid droplets. Furthermore, D3G increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), which suggests that D3G may play a role in AMPK and ACC activation in Adipocytes. Our data indicate that D3G attenuates Adipogenesis and promotes lipid metabolism by activating AMPK-mediated signaling, and, hence, could have a therapeutic role in the management and treatment of Obesity.

Delphinidin-3-O-β-Glucoside

Anti-Adipogenic effects of delphinidin–3–O–β–glucoside in 3T3-L1 preadipocytes and primary white Adipocytes

Abstract Delphinidin–3–O–β–glucoside (D3G) is a health-promoting anthocyanin whose anti–Obesity activity has not yet been thoroughly investigated. We examined the effects of D3G on Adipogenesis and lipogenesis in 3T3-L1 Adipocytes and primary white Adipocytes.

DHEA

Dehydroepiandrosterone (DHEA), a precursor sex steroid, circulates in sulphated form (DHEAS). Serum DHEAS concentrations are inversely correlated with Metabolic Syndrome components and in vivo/in vitro studies suggest a role in modulating adipose Mass. To investigate further, we assessed the in vitro biological effect of DHEA in white (3T3-L1 ) and brown (PAZ6) Preadipocyte cell lines and human primary preadipocytes.

DHEA (from 10⁻⁸ M) caused concentration-dependent proliferation Inhibition of 3T3-L1 and PAZ6 preadipocytes. Cell cycle analysis demonstrated unaltered apoptosis but indicated blockade at G1/S or G2/M in 3T3-L1 and PAZ6, respectively. Preadipocyte cell-line Adipogenesis was not affected.

In human primary subcutaneous and omental preadipocytes, DHEA significantly Inhibited proliferation from 10⁻⁸ M. DHEA 10⁻⁷ M had opposing effects on Adipogenesis in the two fat depots. Subcutaneous Preadipocyte Differentiation was unaffected or increased whereas omental preadipocytes showed significantly Reduced Adipogenesis.

We conclude that DHEA exerts fat depot-specific differences which modulate body composition by limiting omental fat production.

Diallyl trisulphide (onion)

The aim of the present study was to examine the effect of quercetin-rich onion peel extract (OPE) on anti-Differentiation in 3T3-L1 preadipocytes and the AntiObesity in high-fat fed rats. We found that lipid accumulations and TG contents in 3T3-L1 cells were markedly suppressed by OPE. The mRNA levels of activating protein (AP2) were down-regulated and those of carnitine palmitoyl transferase-1 α (CPT-1α) and fatty acid binding protein 4 (FABP4) were up-regulated by 75 and 100 μg/ml OPE. Body weight, retroperitoneal and mesenteric fat weights of SD rats were significantly lower in the 8 week high fat (HF) diet + 0.72% OPE group than in the HF group.

Peroxisome proliferator-activated receptor (PPAR)γ mRNA levels were down-regulated in the epididymal fat of OPE than those of control and HF, and significant down-regulation of CCAAT/enhancer binding protein (C/EBP)α mRNA levels in OPE was also observed than the control. The mRNA levels of CPT-1α and uncoupling protein-1 (UCP-1) were up-regulated by the OPE, while those of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were down-regulated in HF and OPE groups compared to control group. These results suggest that quercentin-enriched OPE may have AntiObesity effects by suppressing Preadipocyte Differentiation and Inhibiting Adipogenesis.

Dioscin (DS) a steroidal saponin (Dioscorea japonica Thunb.)

Dioscin Inhibits Adipogenesis through the AMPK/MAPK pathway in 3T3-L1 cells and modulates Fat Accumulation in obese mice

Dioscin (DS) is a steroidal saponin present in a number of medicinal plants and has been shown to exert anticancer, antifungal and antiviral effects. The present study aimed to deternube the effects DS on the regulation of Adipogenesis and to elucidate the underlying mechanisms. In vitro experiments were performed using differentiating 3T3-L1 cells treated with various concentrations (0-4 µM) of DS for 6 days. A cell viability assay was performed on differentiating cells following exposure to DS. Oil Red O staining and triglyceride content assay were performed to evaluate the lipid accumulation in the cells. We also carried out the following experiments: i) flow cytometry for cell cycle analysis, ii) quantitative reverse transcription polymerase chain reaction for measuring Adipogenesis-related gene expression, and iii) western blot analysis to measure the expression of Adipogenesis transcription factors and AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and mitogen-activated protein kinase (MAPK) phosphorylation.

In vivo experiements were performed using mice with Obesity induced by a high-fat diet (HFD) that were treated with or without DS for 7 weeks. DS suppressed lipid accumulation in the 3T3-L1 cells without affecting viability at a dose of up to 4 µM. It also delayed cell cycle progression 48 h after the initiation of Adipogenesis. DS Inhibited Adipocyte Differentiation by the downregulation of Adipogenic transcription factors and attenuated the expression of Adipogenesis-associated genes. In addition, it enhanced the phosphorylation of AMPK and its target molecule, ACC, during the Differentiation of the cells. Moreover, the Inhibition of Adipogenesis by DS was mediated through the suppression of the phosphorylation of MAPKs, such as extracellular-regulated kinase 1/2 (ERK1/2) and p38, but not c-Jun-N-terminal kinase (JNK).

DS significantly Reduced Weight Gain in the mice with HFD-induced Obesity; this was evident by the suppression of Fat Accumulation in the abdomen. the present study reveals an Anti-Adipogenic effect of DS in vitro and in vivo and highlights AMPK/MAPK signaling as targets for DS during Adipogenesis.

Djulis (Chenopodium formosanum)

The Inhibitory Effects of Djulis (Chenopodium formosanum) and Its Bioactive Compounds on Adipogenesis in 3T3-L1 Adipocytes

The aim of this study was to provide new insights into the role of the ethanolic extracts of Djulis (Chenopodium formosanum, EECF) and its bioactive compounds in preventing Adipogenesis in 3T3-L1 Adipocytes. The results demonstrated EECF significantly Inhibited oil red O-stained material (OROSM), triglyceride levels and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 Adipocytes. The expression of the critical molecules involved in lipid synthesis such as PPARγ, C/EBPα and SREBP-1c was attenuated in EECF-treated cells. According to HPLC-DAD and HPLC-MS/MS analysis, rutin, kaempferol, betanin and another nine compounds were present in EECF. The suppression of lipid accumulation by rutin, kaempferol and betanin occurred by decreasing the gene expression of PPARγ, C/EBPα and SREBP-1c. Taken together, these findings suggest the presence of bioactive compounds in EECF may partly account for the anti-Adipogenesis of EECF and EECF is therefore a potentially lipid lowering functional food.

Dolichos lablab L. Seeds

Anti–Obesity activities of chikusetsusaponin IVa and Dolichos lablab L. seeds

Obesity, a condition where excess body fat accumulates to the extent, causes a negative effect on health. Previously, we reported the extract of Dolichos lablab L. (DLL-Ex) Inhibited high-fat diet (HFD)-induced increases in body weight and body fat Mass and ameliorated increases in body weight. In the present work, we studyed the molecular mechanism for the Inhibitory effect of DLL-Ex or Chikusetsusaponin IVa (CS-IVa), as isolated from Dolichos lablab L. (DLL) seeds extract, on Adipocyte Differentiation . We evaluated the effect of DLL-Ex, an Anti-Obesity agent, and CS-IVa, an active component of DLL-Ex, on 3T3-L1 cell Differentiation via Oil red O assay and Q-PCR, along with their effects on CCAAT element binding protein alpha (C/EBPα ), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) mRNA transcriptions. FAS and FABP4 protein expression levels after exposure to CS-IVa were also tested. The results showed that DLL-Ex and CS-IVa have potent Inhibitory activity on Adipocyte Differentiation. Therefore, DLL and CS-IVa may be developed as a functional food material to treat Obesity.

Ecklonia Cava

Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, Inhibits Adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1

•The Ecklonia cava extract tested herein evidenced profound Adipogenesis Inhibition.

•The three Polyphenol compounds of phlorotannins were isolated from E. cava.

•Dieckol exhibited greatest potential Adipogenesis Inhibition.

•Dieckol significantly Reduced the expression of Adipogenic-specific genes and proteins.

•Dieckol Inhibits Adipogenesis by activating the AMPK pathway.

In this study, we assessed the potential Inhibitory effect of 5 species of brown seaweeds on Adipogenesis the Differentiation of 3T3-L1 preadipocytes into mature Adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound Adipogenesis Inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three Polyphenol compounds of phlorotannins were obtained and their Inhibitory effect on Adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential Adipogenesis Inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPα), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner.

The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate Inhibitory effect of dieckol compound on Adipogenesis through the activation of the AMPK signal pathway.

Anti-Adipogenic effect of dioxinodehydroeckol via AMPK activation in 3T3-L1 Adipocytes

Dioxinodehydroeckol (DHE) isolated from Ecklonia cava, has previously been investigated for its Inhibition of the Differentiation of 3T3-L1 preadipocytes into Adipocytes. Levels of lipid accumulation were measured, along with changes in the expression of genes and proteins associated with Adipogenesis and Lipolysis. Confluent 3T3-L1 preadipocytes in medium with or without different concentrations of DHE for 7 days were differentiated into Adipocytes. Lipid accumulation was quantified by measuring direct triglyceride contents and Oil-Red O staining. The expression of genes and proteins associated with Adipogenesis and Lipolysis was measured using RT-PCR, quantitative real-time RT-PCR and Western blotting analysis. It was found that the presence of DHE significantly Reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1) and CCAAT/enhancer-binding proteins (C/EBPα) in a dose-dependent manner.

Moreover, DHE suppressed regulation of the Adipocyte-specific gene promoters such as fatty acid binding protein (FABP4), fatty acid transport protein (FATP1), fatty acid synthase (FAS), lipoprotein lipase (LPL), acyl-CoA synthetase 1 (ACS1), leptin, perilipin and HSL compared to control Adipocytes. The specific mechanism mediating the effects of DHE was confirmed by activation of phosphorylated AMP-activated protein kinase (pAMPK). Therefore, these results suggest that DHE exerts Anti-Adipogenic effect on Adipocyte Differentiation through the activation and modulation of the AMPK signaling pathway.

Anti-hyperlipidemic effect of Polyphenol extract (Seapolynol™) and dieckol isolated from Ecklonia cava in in vivo and in vitro models

The Inhibitory effect of Polyphenol extracts (Seapolynol^™, SPN) of the marine brown algae Ecklonia cavaand dieckol, a major component of SPN, on hyperlipidemia was investigated in ICR mice fed a high-fat diet (HFD) for five weeks. For analysis of the anti-hyperlipidemic effects of SPN and dieckol, these two agents were given orally on a daily basis to HFD-fed mice for four weeks, starting one week after the beginning of HFD feeding. Groups administered with SPN as well as dieckol showed lower body Weight Gains than the HFD only group. Administration of SPN and dieckol also resulted in a significant reduction of the level of total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein (LDL) cholesterol in the serum of HFD-fed mice. In Oil Red O staining using 3T3-L1 preadipocytes, it was shown that both SPN and dieckol markedly Inhibited lipid accumulation of 3T3-L1 cells. Furthermore, SPN and dieckol (50 μg/mL) significantly Inhibited 3-hydroxyl-methyl glutaryl coenzyme A (HMGCoA) reductase activity in vitro.

Taken together, these results suggest that Polyphenols of Ecklonia cava (SPN) and dieckol Reduce body Weight Gain and Fat Accumulation in HFD-induced obese mice, and that their hypolipidemic effect is related to the Inhibition of Adipogenesis of Adipocytes and HMGCoA reductase activity.

Enzyme-treated Ecklonia cava extract Inhibits Adipogenesis through the downregulation of C/EBPα in 3T3-L1 Adipocytes

In this study, we examined the Inhibitory effects of enzyme- treated Ecklonia cava (EEc) extract on the Adipogenesis of 3T3-L1 Adipocytes. The components of Ecklonia cava (E. cava) were first separated and purified using the digestive enzymes pectinase (Rapidase® X‑Press L) and cellulase (Rohament® CL). We found that the EEc extract contained three distinct phlorotannins: eckol, dieckol and phlorofucofuroeckol-A. Among the phlorotannins, dieckol was the most abundant in the EEc extract at 16 mg/g. Then we examined the Inhibitory effects of EEc extract treatment on Differentiation‑related transcription factors and on Adipogenesis‑related gene expression in vitro using 3T3-L1 Adipocytes. 3T3‑L1 pre‑Adipocytes were used to determine the concentrations of the EEc extract and Garcinia cambogia (Gar) extract that did not result in cytotoxicity. Glucose utilization and triglyceride (TG) accumulation in the EEc‑treated Adipocytes were similarly Inhibited by 50 µg/ml EEc and 200 µg/ml Gar, and these results were confirmed by Oil Red O staining.

Protein expression of Adipogenesis Differentiation‑related transcription factors following treatment with the EEc extract was also examined. Only the expression of CCAAT/enhancer‑binding protein (C/EBP)α was decreased, while there was no effect on the expression of C/EBPβ, C/EBPδ, and peroxisome proliferator‑activated receptor γ (PPARγ). Treatment with the EEc extract decreased the expression levels of Adipogenesis‑related genes, in particular sterol regulatory element binding protein‑1c (SREBP‑1c), Adipocyte fatty acid binding protein (A‑FABP), fatty acid synthase (FAS) and adiponectin. These results suggest that EEc extract treatment has an Inhibitory effect on Adipogenesis, specifically by affecting the activation of the C/EBPα signaling pathway and the resulting Adipogenesis-related gene expression.

Edible Brown Alga Ecklonia cava Derived Phlorotannin‐Induced Anti‐Adipogenic Activity in Vitro

The present study performed the bioactivity‐monitored isolation of anti‐Adipogenic phlorotannins from E cklonia cava as a part of the ongoing research to develop AntiObesity nutraceuticals from natural origin. Extracts were partitioned into n‐hexane, 85% aqueous MeOH , n‐BuOH and water fractions. Their Adipogenic activities were examined by measuring glycerol release level and Adipogenic‐related gene expression in differentiating 3T 3‐L 1 preadipocytes. Among them, n‐BuOH fractions significantly increased glycerol secretion and Reduced the regulation of Adipogenic transcription factors, peroxisome proliferator‐activated receptor‐γ (PPAR γ) and CCAAT /enhancer‐binding protein α (C/EBP α), as well as tumor necrosis factor α (TNF α). Further separation from n‐BuOH fraction led to the isolation of phlorotannins, triphlorethol‐A, eckol and dieckol.

Phlorotannins increased the glycerol secretion and Reduced the glucose consumption levels of 3T 3‐L 1 Adipocytes. Moreover, the phlorotannins Reduced the expression levels of PPAR γ, C /EBP α and Differentiation‐dependent factor 1/sterol regulatory element‐binding protein 1c, as well as downstream genes such as fatty acid binding protein‐4, fatty acid transport protein‐1, fatty acid synthase, L eptin and acyl‐CoA synthetase 1. In addition, phlorotannins increased the mRNA expression of hormone‐sensitive lipase while suppressing perilipin and TNF α expressions.

Evaluation of Inhibitory effect of phlorotannins from Ecklonia cava on triglyceride accumulation in Adipocyte

In the present study, a methanolic extract of Ecklonia cava and its solvent-partitioned fractions were evaluated for their AntiAdipogenic effect in 3T3-L1 Adipocytes. One of them, the n-BuOH fraction, effectively Reduced lipid accumulation and glucose consumption. In addition, the presence of the n-BuOH fraction in Adipocytes suppressed the regulations of Adipogenic transcription factors, PPARγ and SREBP1c, and Adipogenic specific genes, FABP4, FABP1, FAS, LPL, HSL, and ACS1. Further purification of n-BuOH fractions led to the isolation of six phlorotannins. The six phlorotannins effectively suppressed triglyceride accumulation. Comparative analysis showed that lipid accumulation in Adipocytes was dramatically attenuated in the presence of eckstolonol.

Phlorotannins isolated from the edible brown alga Ecklonia stolonifera exert Anti-Adipogenic activity on 3T3-L1 Adipocytes by downregulating C/EBPα and PPARγ

The dramatic increase in Obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. Inhibition of Adipocyte Differentiation has been suggested to be an important strategy for preventing or treating Obesity. In our previous study, we characterized an Ecklonia stolonifera extract and non-polar fractions thereof, including dichloromethane and ethyl acetate fractions. We showed that these fractions Inhibited Adipocyte Differentiation and lipid formation/accumulation in 3T3-L1 preadipocytes, as assessed by Oil Red O staining. As part of our ongoing search for Anti-Obesity agents derived from E. stolonifera, in this work, we characterized five known phlorotannins, including phloroglucinol, eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol A, all of which were isolated from the active ethyl acetate fraction of E. stolonifera.

We determined the chemical structures of these phlorotannins through comparisons of published nuclear magnetic resonance (NMR) spectral data. Furthermore, we screened these phlorotannins for their abilities to Inhibit Adipogenesis over a range of concentrations (12.5–100 μM). Of these five phlorotannins, phloroglucinol, eckol, and phlorofucofuroeckol A significantly concentration-dependently Inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability. In addition, the five isolated phlorotannins also significantly Reduced the expression levels of several Adipocyte marker genes, including proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), although they did so to different extents.

These results suggest that the molecular weight of a phlorotannin is an important factor affecting its ability to Inhibit Adipocyte Differentiation and modulate the expression levels of Adipocyte marker genes.

Seapolynol extracted from Ecklonia cava Inhibits Adipocyte Differentiation in vitro and decreases Fat Accumulation in vivo

Our study showed that SN Inhibited lipid accumulation in 3T3-L1 cells and high-fat-diet-fed zebrafish and mice. Furthermore, we examined the underlying mechanisms of the SN-induced AntiAdipogenic effect including Inhibition of MCE via cell cycle arrest, and activation of AMPKα signaling. Ecklonia cava has been shown to Inhibit Adipogenesis in cell lines. Several Polyphenols (dieckol, phloroeckol, dioxinodehydroeckol) derived from Ecklonia cava were shown to have AntiAdipogenic effects in Adipocytes. A clinical study on Ecklonia cava Polyphenols was also performed in overweight subjects. In particular, the most abundant Ecklonia cava Polyphenol, dieckol, has been identified to have AntiAdipogenic effects in cells and animal models. SN, a standardized extract of Ecklonia cava, includes active compounds including the Polyphenols mentioned above, and is commercially available; SN contains dieckol, eckol, and bieckol derivatives. Recent studies have shown the biological effects of this extract. Ecklonia cava extract or Ecklonia cava-derived Polyphenols have been reported to have antioxidant effects. Kang et al., showed the potential of Ecklonia cava Polyphenols as chemopreventive agents [25], and the antioxidant and anti-inflammatory effects of Ecklonia cava in osteoarthritis were evaluated. Ecklonia cava extract was shown to have antibacterial effects on antibiotic-resistant food-borne pathogens. Additionally, Heo et al., showed protective effects of Ecklonia cavaphlorotannins against photo-oxidative stress from UV-B radiation.

Shibata et al., reported the Inhibitory effect of Ecklonia cava phlorotannins on pro-inflammatory responses. In particular, an AntiAdipogenic effect of SN has been reported recently. However, the mechanisms underlying the AntiAdipogenic effects of SN should be investigated. The current study is the first to show the AntiAdipogenic effect of SN in two animal models with early Adipogenic and signaling mechanisms, in which zebrafish was used to show the effects of SN in early Adipogenesis. Lipid accumulation in zebrafish is known to be detectable from 15 dpf. Zebrafish were grown for 17–20 dpf to show early Fat Accumulation by Nile-red staining; SN Inhibited Fat Accumulation in zebrafish from an early stage.

In addition, the diet induced obese mice has been used here were 5 weeks old, fed on HFD for 10 weeks. The HFD mice caused a marked increase the lipid accumulation in the liver or TG and total cholesterol in the blood compared to control diet mice, while SN dramatically repressed the lipid accumulation in the liver or TG and total cholesterol in the blood. In addition, SN significantly decreased HFD-induced abnormal expression of Adipogenic and fatty liver associated genes. On the other hand, the high levels of glucose and insulin concentration are generally occurring in HFD-induced obese mice [30]. However, we did not observed the sign of hyperglycemia and hyperinsulinemia in HFD-induced obese mice compared to the control diet group in this study.

The afore-mentioned evidence prompt us to speculate that some of the factors such as mouse strain, ages, diet composition or duration of HFD may affect to develop the intermediate stage Obesity between Obesity and type 2 Diabetes. This model may occur the some of genetic changes in the tissues due to the HFD consumption in mice, but it has not yet appeared the hyperglycemia and hyperinsulinemia. However, additional animal study should be analyzed in further studies.

Our data showed that SN induced Inhibition in early Adipogenesis via regulating early Adipogenic factors, such as KLF2, KLF4, and C/EBPβ. SN-mediated Inhibition of early Adipogenesis was associated with the Inhibition of MCE via cell cycle arrest, and the Inhibition of ERK and Akt signaling was underlined as a reason for the SN-induced Inhibition of early Adipogenesis. Recent studies have shown that several phytochemicals induce Inhibition of MCE via cell cycle arrest as one of the major mechanisms of an AntiAdipogenic effect in 3T3-L1 cells. In particular, our data showed that SN activates AMPKα, a sensor in energy metabolism, and ACC, a major enzyme in fatty acid synthesis, in both Adipocytes and a mouse model. Such AMPKα activation was also shown previously in a study of dieckol. Accordingly, the SN-induced activation of AMPKα is likely due to dieckol, a major component of SN. However, the other compounds in SN should also be investigated. The SN-mediated repression of ACC could contribute to the suppression of TG accumulation in Adipocytes and mice models. Our Inhibit or study showed SN-induced repression of ACC was mediated via activation of AMPKα signaling.

Our study showed that SN supplementation effectively improved the plasma lipid status of mice fed HFD, as was seen with dieckol. However, SN was superior to dieckol in terms of the suppression of lipid accumulation and cholesterol levels. SN decreased triglyceride levels by 40%—while dieckol Reduced them by only ~10%—in mice fed, an HFD. LDL cholesterol levels were Reduced by 66% in SN-supplemented HFD-fed mice, while dieckol supplementation showed a 54% reduction in LDL cholesterol [32]. This result was supported by the study of Yeo et al., which showed SN is more effective than dieckol on Inhibition of lipid accumulation and weight increase. Given that dieckol comprises 8% of SN, other SN-derived compounds likely contribute to the difference in effects on lipid accumulation and cholesterol levels between SN and dieckol. SN showed significant effects in decreasing triglyceride and cholesterol levels in HFD-fed mice.

The reductions in triglycerides and lipid accumulation by SN are supported by its suppression of Adipogenic factors. However, the SN-induced reduction of (LDL) cholesterol level was not fully addressed in this study. In particular, downstream targets of AMPKα include various factors—including HMGCoA and SREBP1, which are responsible for cholesterol metabolism. Although SREBF-1 was shown to be regulated by SN in zebrafish, other cholesterol-related factors should be examined. AMPKα also regulates carbohydrate metabolism via factors such as ChREBP, but glucose and insulin levels were unaffected by SN, suggesting that SN does not greatly affect carbohydrate metabolism.

AMPKα is known to be associated with factors related to the energy expenditure, such as the NAD-dependent protein deacetylase sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). These signaling processes are known to activate thermogenic responses, such as heat production with upregulation of uncoupling proteins. A recent study showed that a phytochemical, indole-3-cabinol, directly activated SIRT1 via binding in 3T3-L1 cells [34]. Thus, the roles of SN and SN-derived compounds should be investigated in thermogenic signaling in future studies.

We also used the zebrafish model, and showed SN-mediated Inhibition in early Adipogenesis and lipid accumulation. Zebrafish provides various advantages for in vivoresearch but also has several limitations. Full sets of antibodies for lipid metabolism studies have not yet been established. Further studies should include data for the AntiAdipogenic effects of SN in protein levels. Finally, our data demonstrated that SN effectively suppressed HFD-induced lipid accumulation in cell line, zebrafish, and mouse models.

Ecklonia cava contains various Polyphenols, known as phlorotannins. Phloroeckol, dioxinodehydroeckol, and dieckol have been shown to have AntiAdipogenic activities in Adipocytes and animals, and dieckol, a major compound of SN, could give an important contribution to AntiAdipogenic effects of SN. However, the effect may be derived from the synergistic or combinational effects of constituents in SN. Since other Ecklonia cava-derived Polyphenols, which may also have AntiAdipogenic effects, have yet to be fully investigated, further study should be performed on SN-derived compounds, additionally, comparative and combinational studies on SN-derived compounds would be executed in the future.

Ecliptal, isolated from Eclipta alba

Ecliptal, a promising natural lead isolated from Eclipta alba modulates Adipocyte function and Ameliorates Metabolic Syndrome

A swift increase has been observed in the number of individuals with metabolic syndrome worldwide. A number of natural compounds have been identified towards combating metabolic syndrome. Adding to this premise, here we report the pleiotropic activities of Ecliptal (EC); a natural compound isolated from the herb Eclipta alba. Administration of EC was shown to have prominent anti-adipogenic effects in 3T3-L1 and hMSC derived adipocytes. It was shown to activate Wnt-pathway and alter AKT signaling. Additionally, it caused cell cycle arrest and inhibited mitotic clonal expansion. EC treatment augmented mitochondrial biogenesis as well as function as estimated by expression of PGC1α, UCP-1, mitochondrial complexes and estimation of oxygen consumption rate. EC also reduced LPS-induced inflammation and tunicamycin induced ER stress.

Further, EC enhanced insulin sensitivity by increasing AKT phosphorylation, inhibiting PKCα/βII phosphorylation and reducing leptin/adiponectin ratio. Finally, EC administration in Syrian golden hamsters was shown to have potent anti-dyslipidemic effects. Cumulatively, encompassing pleiotropic activities of EC, it could prove to be a potential drug candidate against obesity, insulin resistance and related metabolic syndrome.

Ethyl acetate fraction of Eclipta alba: a potential phytopharmaceutical targeting Adipocyte Differentiation

Natural products have always fascinated mankind for their miraculous properties. Eclipta alba (E. alba), a medicinal herb has long been used in traditional medicine for curing several pathologies. It has been shown to have anti-diabetic effect as well as hepato-protective activity. Here, in order to address metabolic derangements, the study was designed to evaluate the efficacy of E. alba and its fractions in adipogenesis inhibition and dys-lipidemia. Of the crude extract and fractions screened, ethyl acetate fraction of E. alba inhibited adipocyte differentiation in 3T3-L1 pre-adipocytes and hMSC derived adipocytes.

It inhibited mitotic clonal expansion and caused cell cycle arrest in G1 and S phase as suggested by western blot analysis and flow cytometry. It was also shown to have lipolytic effects. Oral administration of ethyl acetate fraction of E. alba to hamsters unveiled its anti-adipogenic as well as anti-dyslipidemic activity in-vivo. Mass spectrometry analysis of ethyl acetate fraction confirmed the presence of several bioactive components, projecting it as an effective phytopharmaceutical agent. In conclusion, ethyl acetate fraction of E. alba possesses potent anti-adipogenic as well as anti-dyslipidemic activity and could be projected as an herbal formulation towards obesity.

Ethnopharmacological significance of Eclipta alba (L.) hassk.(Asteraceae)

Eclipta alba can be found growing wild in fallow lands of Bangladesh where it is considered as a weed by farmers. Traditional medicinal systems of the Indian subcontinent countries as well as tribal practitioners consider the plant to have diverse medicinal values and use it commonly for treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss and graying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, and cuts and wounds. The plant has several phytoconstituents like wedelolactone, eclalbasaponins, ursolic acid, oleanolic acid, luteolin, and apigenin.

Pharmacological activities of plant extracts and individual phytoconstituents have revealed anticancer, hepatoprotective, snake venom neutralizing, anti-inflammatory, and antimicrobial properties. Phytoconstituents like wedelolactone and ursolic and oleanolic acids as well as luteolin and apigenin can form the basis of new drugs against cancer, arthritis, gastrointestinal disorders, skin diseases, and liver disorders.

Egcg

The Anti-Adipogenic effects of (-) epigallocatechin gallate are dependent on the WNT/β-catenin pathway

(-)Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and reportedly has anti-obesity and anti-adipogenic effects. In this study, we determined that the up-regulation of the WNT/β-catenin pathway is the anti-adipogenic mechanisms of EGCG in 3T3-L1 cells. EGCG treatment down-regulates the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP)α, fatty acid binding protein (FABP)4 and fatty acid synthase (FASN), while up-regulating the nuclear level of β-catenin. Knockdown of β-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. β-catenin siRNA transfection also recovered terminal adipocyte markers such as FABP4, FASN, lipoprotein lipase and adiponectin, which were down-regulated by EGCG. The DNA binding activities as well as the expression levels of PPARγ and C/EBPα, which were down-regulated by EGCG, were significantly restored by β-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the expressions of low density lipoprotein receptor-related protein (LRP)5, LRP6, disheveled (DVL)2 and DVL3 were significantly up-regulated, while AXIN expression was down-regulated by EGCG, and the phosphorylation of glycogen synthase kinase 3β was increased. These results suggest that EGCG activates the WNT/β-catenin pathway, resulting in the up-regulation of β-catenin, which down-regulates the major genes of the adipogenesis pathway. Taken together, our findings clearly show that the anti-adipogenic effects of EGCG are, at least partially, dependent on the WNT/β-catenin pathway.

Genistein, EGCG, and capsaicin Inhibit Adipocyte Differentiation process via activating AMP-activated protein kinase

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure.

We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20-200muM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.

Comparison of cytotoxicity and the Anti-Adipogenic effect of green tea Polyphenols with epigallocatechin-3-gallate in 3T3-L1 preadipocytes

Recent studies have demonstrated the effects of green tea polyphenols (GTP) and epigallocatechin-3-gallate (EGCG) on obesity. However, high doses of EGCG have also exhibited cytotoxicity. The aim of this study was to compare total GTP with purified EGCG on cytotoxicity, and to investigate the effects and the molecular mechanism of total GTP and EGCG on adipogenesis. Cytotoxicity was determined by cell viability assay. For the adipogenesis study, 3T3-L1 preadipocytes were incubated with three doses of GTP (1, 10, and 100 μg/ml) and the effect of EGCG (6.8 μg/ml) was compared with 10 μg/ml GTP containing 68% EGCG. Oil Red O staining and triglyceride content assay were carried out 10 days after differentiation and treatment. Adipogenic regulators CCAAT element binding protein α (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein-1c (SREBP-1c) were determined by qRT-PCR and immunoblotting. GTP at 1000 μg/ml and EGCG (68 and 680 μg/ml) significantly affected cell viability.

Purified EGCG had greater cytotoxicity than corresponding doses of GTP. About 10 μg/ml of GTP showed stronger reduction in triglyceride accumulation than EGCG treatment. Transcriptional factors of C/EBPα, SREBP-1c and PPARγ were markedly decreased in both GTP and EGCG-treated cells and GTP exhibited stronger inhibitory effects on C/EBPα and PPARγ protein expression than EGCG (p < 0.05). In conclusion, total GTP exerted greater inhibitory effects than purified EGCG on adipogenesis through down-regulating the adipogenic factor C/EBPα, SREBP-1c and PPARγ expression. These findings support that a polyphenol mixture is safer and more effective than EGCG alone for preventing obesity and obesity-related chronic diseases.

Epigallocatechin-3-gallate Inhibits Adipogenesis through down-regulation of PPARγ and FAS expression mediated by PI3K-AKT signaling in 3T3-L1 cells

Epigallocatechin-3-gallate (EGCG), a major component in green tea, functions as extensive bioactivities including anti-inflammation, anti-oxidation, and anti-cancer. However, little is known about its anti-adipogenesis and underlying mechanisms. The purport of this study sought to investigate effects of EGCG on 3T3-L1 preadipocyte differentiation and to explore its possible mechanisms. The 3T3-L1 cells were induced to differentiate under the condition of pro-adipogenic cocktail with or without indicated EGCG concentrations (10, 50, 100, 200µM) for 2, 4, 6 and 8 days, respectively. Also, another batch of 3T3-L1 cells was induced under the optimal EGCG concentration (100µM) with or without SC3036 (PI3K activator, 10µM) or SC79 (AKT activator, 0.5µM) for 8 days.

Subsequently, the cell viability was examined by MTT assay and the cell morphology was visualized by Oil red O staining. Finally, the mRNA levels including peroxisome proliferator activated receptor γ (PPARγ) and fatty acid synthase (FAS) were detected by quantitative real time PCR, while the protein levels of PPARγ, FAS, phosphatidylinositol 3 kinase (PI3K), insulin receptor substrate1(IRS1), AKT, and p-AKT were measured by immunoblotting analysis. Our results showed that EGCG inhibited adipogenesis of 3T3-L1 preadipocyte in a concentration-dependent manner. Moreover, the inhibitory effects were reversed by SC3036 or SC79, suggesting that the inhibitory effects of EGCG are mediated by PI3K-AKT signaling to down-regulate PPARγ and FAS expression levels. The findings shed light on EGCG anti-adipogenic effects and its underlying mechanism and provide a novel preventive-therapeutic potential for obesity subjects as a compound from Chinese green tea.

Keywords: Adipocyte; Adipogenesis; Epigallocatechin-3-gallate; Peroxisome proliferator-activated receptor gamma; Phosphatidylinositol 3-kinase.

Anti–Obesity effect of EGCG and glucosamine-6-phosphate through decreased expression of genes related to Adipogenesis and cell cycle arrest in 3T3-L1

Purpose: Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes.

Methods: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expression level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells.

Results: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG 60 μM and PGlc 200 μg/m compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of PPARγ, C/EBPα, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2.

Conclusion: Combination treatment of EGCG and PGlc inhibited adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.

Ellagic Acid (Rhus chinensis Mill.)

Fatty acid synthase (FAS) has been recognized as a potential therapeutic target for Obesity. In this study, for the first time, the Inhibitory effect of pomegranate husk extract, punicalagin and ellagic acid on FAS was investigated. We found them potently Inhibiting the activity of FAS with half-Inhibitory concentration values (IC₅₀) of 4.1 μg/ml (pomegranate husk extract), 4.2 μg/ml (4.50 μM, punicalagin) and 1.31 μg/ml (4.34 μM, ellagic acid), respectively. Moreover, they all exhibited time-dependent inactivation of FAS.

Punicalagin and ellagic acid Inhibited FAS with different mechanisms compared to previously reported Inhibitors, through inactivating acetyl/malonyl transferase and β-ketoacyl synthase domains, respectively. Additionally, 100 μg/ml pomegranate husk extract, 5.24 μg/ml (5 μM) punicalagin and 4.5 μg/ml (15 μM) ellagic acid effectively Reduced lipid accumulation inside FAS over-expressed 3T3-L1 Adipocytes. Since FAS plays a key role in the biosynthesis pathway of fatty acid, these findings suggest that pomegranate husk extract, punicalagin and ellagic acid have potential in the prevention and treatment of Obesity.

Epiberberine (Berberis thunbergii DC)

Anti-Adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways

It has been reported that alkaloids derived from Coptis chinensis exert Anti-Adipogenic activity on 3T3-L1 Adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the Inhibitory role of epiberberine in the early stages of 3T3-L1 Adipocyte Differentiation is uncharacterized. Here, we show that epiberberine had Inhibitory effects on Adipocyte Differentiation and significantly decreased lipid accumulation by downregulating an Adipocyte -specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1).

Furthermore, we observed that epiberberine markedly suppressed the Differentiation -mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly Inhibited by treatment with epiberberine during Adipogenesis . These results indicate that the Anti-Adipogenic mechanism of epiberberine is associated with Inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of Adipogenesis , such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the Anti-Adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 Adipocyte Differentiation . Moreover, the Anti-Adipogenic effects of epiberberine were not accompanied by modulation of β-catenin.

Erigeron annuus (L.) Pers.

Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 cells by Activating an AMP-Dependent Kinase Signaling Pathway

Obesity is one of the major public health problems in the world because it is implicated in Metabolic Syndrome s, such as type 2 Diabetes , hypertension, and cardiovascular diseases. The objective of this study was to investigate whether Erigeron annuus (L.) Pers. (EAP) extract Suppresses reactive oxygen species (ROS) production and Fat Accumulation in 3T3-L1 cells by activating an AMP-dependent kinase (AMPK) signaling pathway. Our results showed that EAP water extract significantly Inhibits ROS production, Adipogenesis , and lipogenesis during Differentiation of 3T3-L1 preadipocytes . In addition, EAP decreased mRNA and protein levels of proliferator-activated receptor γ (PPARγ ) and CCAAT/enhancer-binding protein alpha (C/EBPα ). Moreover, EAP suppressed mRNA expressions of fatty acid synthase (FAS), lipoprotein lipase (LPL), Adipocyte protein 2 (aP2) in a dose-dependent manner. Whereas, EAP upregulated adiponectin expression, phosphorylation levels of AMPK and carnitine palmitoyltransferase 1 (CPT-1) protein level during Differentiation of 3T3-L1 preadipocytes.

These results suggest that EAP water extract can exert ROS-linked Anti-Obesity effect through the mechanism that might involve Inhibition of ROS production, Adipogenesis and lipogenesis via an activating AMPK signaling pathway.

Esculetin

Esculetin Induces Apoptosis and Inhibits Adipogenesis in 3T3‐L1 Cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 Adipocyte apoptosis and Adipogenesis .

Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled Adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation.

Results: In mature Adipocytes , esculetin caused a time‐ and dose‐related increase in Adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes , apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in Preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also Inhibited Adipogenesis of 3T3‐L1 preadipocytes . Esculetin‐mediated Inhibition of Adipocyte Differentiation occurred during the early, intermediate, and late stages of the Differentiation process. In addition, esculetin induced apoptosis during the late stage of Differentiation .

Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, Adipogenesis, and apoptosis in 3T3‐L1 cells.

Euphorbia lunulata

The insulin-like and/or insulin-sensitising effects of Syzygium aqueum leaf extract and its six bioactive compounds; 4-hydroxybenzaldehyde, myricetin-3-O-rhamnoside, europetin-3-O-rhamnoside, phloretin, myrigalone-G and myrigalone-B were investigated in 3T3-L1 Adipocytes. We observed that, S. aqueum leaf extract (0.04–5 μg/ml) and its six bioactive compounds (0.08–10 μM) at non-cytotoxic concentrations were effectively enhance Adipogenesis, stimulate glucose uptake and increase adiponectin secretion in 3T3-L1 Adipocytes. Clearly, the compounds myricetin-3-O-rhamnoside and europetin-3-O-rhamnoside showed insulin-like and insulin-sensitising effects on Adipocytes from a concentration of 0.08 μM.

These compounds were far better than rosiglitazone and the other isolated compounds in enhancing Adipogenesis, stimulating 2-NBDG uptake and increasing adiponectin secretion at all the concentrations tested. These suggest the AntiDiabetic potential of S. aqueum leaf extract and its six bioactive compounds. However, further molecular interaction studies to explain the mechanisms of action are highly warranted.

Evodiamine (Evodia rutaecarpa)

The molecular mechanism of the Anti-Adipogenic effect of evodiamine (which has several capsaicin-like pharmacological actions) was investigated. The evodiamine effect was not blocked by the specific TRPV1 antagonist capsazepine in 3T3-L1 preadipocytes , whereas its effect was greatly curtailed by Inhibitors of protein kinase C (PKC) and epidermal growth factor receptor (EGFR). Signal analyses showed that evodiamine stimulated the phosphorylation of EGFR, PKCα, and ERK, all of which were Reduced by an Inhibitors EGFR Inhibitor.

Silencing experiments of EGFR mRNA supported the involvement of these signaling molecules in the Inhibitory effect of evodiamine. An unidentified mechanism whereby evodiamine Inhibits Adipogenesis via the EGFR–PKCα–ERK signaling pathway was revealed.

Ferulic Acid (Ferula assafoetida L)

World epidemic Obesity is a major contributing factor to metabolic diseases including insulin resistance and cardiovascular diseases. In this study, aqueous and ethanolic extracts of hulled barley with roasting temperatures of up to 250 °C were investigated for their Anti-Adipogenic effects in vitro and in vivo. An aqueous extract of hulled barley roasted at 210 °C (AHB210) effectively Inhibited Adipocyte Differentiation. Intraperitoneal injections of 15 or 50 mg/kg AHB210 dose dependently prevented body Weight Gain, fat Mass increase, and dysregulated lipid profiles in high fat diet-induced obese male mice. In addition, oral administration of AHB210 to ovariectomized rats also prevented body Weight Gain.

A high performance liquid chromatographic analysis identified coumaric acid and ferulic acid as primary Anti-Obesity mediators. The presence of beta glucan in AHB210 was less likely to be responsible for the lipid accumulating actions. Taken together, AHB210 may be useful to Prevent Obesity and its related metabolic diseases.

Ficus deltoidea var. deltoidea

Anti-Adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 Adipocytes

Objective: This study examined the Anti-Adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 Adipocytes.

Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible Anti-Adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (½MNTD), after which the preadipocytces were induced to form mature Adipocytes. Visualisation and quantification of lipid content in mature Adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively.

Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) µg/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) µg/ml for methanol extracts and (8.0±1.0) µg/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ½MNTD significantly Inhibited the maturation of preadipocytes.

Conclusions: The Inhibition of the formation of mature Adipocytes indicated that leaf extracts of F. deltoidea could have potential Anti-Obesity effects.

Fisetin (Rhus succedanea L)

Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of Adipocyte Differentiation in 3T3-L1 cells

Adipocytes are the key player in Adipose Tissue Inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and Differentiation of preadipocytes. Fistein, a Polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-Diabetic effects. In this study, we aimed to investigate the effect of fisetin on Adipocyte proliferation and Differentiation in 3T3-L1 Preadipocyte cell line and its mechanism of action. We found that fisetin Inhibits Adipocyte Differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature Adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ.

Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the Differentiation medium. We also found that fisetin Inhibition of Adipocyte Differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of Preadipocyte proliferation at early stage of Differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the Inhibition of Adipocyte Differentiation by fisetin may be at least in part mediated by cell cycle arrest during Adipogenesis.

Foenumoside B from Lysimachia foenum-graecum

Foenumoside B from Lysimachia foenum–graecum Inhibits Adipocyte Differentiation and Obesity induced by high-fat diet

We have previously reported anti-obesity effects of Lysimachia foenum-graecum in high-fat diet (HFD)-induced obesity model. Here we isolated a triterpene saponin foenumoside B as an active component of L. foenum-graecum. Foenumoside B blocked the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner with an IC50 of 0.2 μg/ml in adipogenesis assay and suppressed the induction of PPARγ, the master regulator of adipogenesis. Foenumoside B induced the activation of AMP-activated protein kinase (AMPK), and modulated the expression of genes involved in lipid metabolism towards lipid breakdown in differentiated adipocytes. In mouse model, oral administration of foenumoside B (10mg/kg/day for 6 weeks) reduced HFD-induced body weight gain significantly without affecting food intake. Treatment of foenumoside B suppressed lipid accumulation in white adipose tissues and the liver, and lowered blood levels of glucose, triglycerides, ALT, and AST in HFD-induced obese mice.

Consistent with the in vitro results, foenumoside B activated AMPK signaling, suppressed the expression of lipogenic genes, and enhanced the expression of lipolytic genes in vivo. Foenumoside B also blocked HFD-induced proinflammatory cytokine production in adipose tissue, suggesting its protective role against insulin resistance. Taken together, these findings demonstrate that foenumoside B represents the anti-obesity effects of L. foenum-graecum, and suggest therapeutic potential of foenumoside B in obesity and obesity-related metabolic diseases.

Suppression of Adipocyte Differentiation by Foenumoside B from Lysimachia foenum–graecum Is Mediated by PPARγ Antagonism

Lysimachia foenum-graecum extract (LFE) and its active component foenumoside B (FSB) have been shown to inhibit adipocyte differentiation, but their mechanisms were poorly defined. Here, we investigated the molecular mechanisms responsible for their anti-adipogenic effects. Both LFE and FSB inhibited the differentiation of 3T3-L1 preadipocytes induced by peroxisome proliferator-activated receptor-γ (PPARγ) agonists, accompanied by reductions in the expressions of the lipogenic genes aP2, CD36, and FAS. Moreover, LFE and FSB inhibited PPARγ transactivation activity with IC₅₀s of 22.5 μg/ml and 7.63 μg/ml, respectively, and showed selectivity against PPARα and PPARδ. Rosiglitazone-induced interaction between PPARγ ligand binding domain (LBD) and coactivator SRC-1 was blocked by LFE or FSB, whereas reduced NCoR-1 binding to PPARγ by rosiglitazone was reversed in the presence of LFE or FSB.

In vivo administration of LFE into either ob/ob mice or KKAy mice reduced body weights, and levels of PPARγ and C/EBPα in fat tissues. Furthermore, insulin resistance was ameliorated by LFE treatment, with reduced adipose tissue inflammation and hepatic steatosis. Thus, LFE and FSB were found to act as PPARγ antagonists that improve insulin sensitivity and metabolic profiles. We propose that LFE and its active component FSB offer a new therapeutic strategy for metabolic disorders including obesity and insulin resistance.

Foenumoside B isolated from Lysimachia foenum–graecum extract Suppresses LPS-induced inflammatory response via NF-κB/AP-1 inactivation in murine

Foenumoside B (FSB), a bioactive component isolated from the Lysimachia foenum-graecum extract (LFE), has been shown to possess anti-inflammatory effects, but the underlying molecular mechanisms involved have not been elucidated. Accordingly, the authors investigated the mechanisms responsible for the anti-inflammatory effects of FSB in murine macrophages activated by LPS. FSB suppressed the LPS-induced expressions of iNOS and COX-2 at protein and mRNA levels and consequently decreased NO and PGE2 production in RAW264.7 and primary macrophages. FSB also reduced the LPS-induced inductions of TNF-α, IL-6 and IL-1β at protein and mRNA levels.

Studies of the molecular mechanisms involved in the anti-inflammatory effects of FSB showed that it inhibited the transcriptional activities of NF-κB and AP-1, and the nuclear translocation of NF-κB via inhibition of the phosphorylations of AKT, p38 and STAT3. In a sepsis model, pretreatment with FSB inhibited the LPS-stimulated mRNA and protein levels of proinflammatory mediators, such as, iNOS, COX-2, TNF-α, IL-6 and IL-1β in plasma and liver. Importantly, FSB increased the survival rate of mice in the LPS-induced sepsis model. Taken together, these results show that the anti-inflammatory effects of FSB against LPS-induced inflammatory conditions are associated with inhibitions of the phosphorylations of AKT, p38 and STAT3 followed by the transcriptional suppressions of NF-κB and AP-1, and thus, reduced expressions of pro-inflammatory genes.

Formononetin (Astragalus membranaceus)

Formononetin, an isoflavone, activates AMP-activated protein kinase/β-catenin signalling to Inhibit Adipogenesis and rescues C57BL/6 mice from high-fat diet-induced Obesity and bone loss

Balance between Adipocyte and osteoblast Differentiation is the key link of disease progression in Obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and Adipocytes prompted us to analyse the effect of FNT on Adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced Obesity. The Anti-Obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT Suppresses the Adipogenic Differentiation of 3T3-L1 fibroblasts, through down-regulation of key Adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and Inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of β-catenin were attributed to the Anti-Adipogenic action of FNT. In vivo, 12 weeks of FNT treatment Inhibited the development of Obesity in mice by attenuating HFD-induced body Weight Gain and visceral Fat Accumulation. The Anti-Obesity effect of FNT results from increased energy expenditure.

FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT’s rescuing action against Obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased Adipogenic potentials. Our findings suggest that FNT Inhibits Adipogenesis through AMPK/β-catenin signal transduction pathways and protects against HFD-induced Obesity and bone loss.

fucoxanthinol

Fucoxanthin and its metabolite, fucoxanthinol, suppress Adipocyte Differentiation in 3T3-L1 cells

Fucoxanthin is a major carotenoid found in edible seaweed such as Undaria pinnatifida and Hijikia fusiformis. We investigated the suppressive effects of fucoxanthin and its metabolite, fucoxanthinol, on the differentiation of 3T3-L1 preadipocytes to adipocytes. Fucoxanthin inhibited intercellular lipid accumulation during adipocyte differentiation of 3T3-L1 cells. Furthermore, fucoxanthin was converted to fucoxanthinol in 3T3-L1 cells. Fucoxanthinol also exhibited suppressive effects on lipid accumulation and decreased glycerol-3-phosphate dehydrogenase activity, an indicator of adipocyte differentiation.

The suppressive effect of fucoxanthinol was stronger than that of fucoxanthin. In addition, in 3T3-L1 cells treated with fucoxanthin and fucoxanthinol, peroxisome proliferator-activated receptor gamma (PPARgamma), which regulates adipogenic gene expression, was down-regulated in a dose-dependent manner. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through down-regulation of PPARgamma. Fucoxanthinol had stronger suppressive effects than fucoxanthin on adipocyte differentiation in 3T3-L1 cells.

Simultaneous determination of fucoxanthin and its deacetylated metabolite fucoxanthinol in rat plasma by liquid chromatography-tandem Mass spectrometry

Fucoxanthin and its deacetylated metabolite fucoxanthinol are two major carotenoids that have been confirmed to possess various pharmacological properties. In the present study, fucoxanthinol was identified as the deacetylated metabolite of fucoxanthin, after intravenous (i.v.) and intragastric gavage (i.g.) administration to rats at doses of 2 and 65 mg/kg, respectively, by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Next, an accurate and precise LC-MS/MS method was developed to quantitatively determine fucoxanthin and fucoxanthinol in rat plasma.

Plasma samples were resolved by LC-MS/MS on a reverse-phase SB-C18 column that was equilibrated and eluted with acetonitrile (A)/aqueous 0.1% formic acid (B; 92/8, v/v) at a flow rate of 0.5 mL/min. Analytes were monitored by multiple-reaction monitoring (MRM) under positive electrospray ionization mode. The precursor/product transitions (m/z) were 659.3→109.0 for fucoxanthin, 617.2→109.0 for fucoxanthinol, and 429.4→313.2 for the internal standard (IS). Calibration curves for fucoxanthin and fucoxanthinol were linear over concentrations ranging from 1.53 to 720 and 1.17 to 600 ng/mL, respectively. The inter- and intraday accuracy and precision were within ±15%. The method was applied successfully in a pharmacokinetic study and the resulting oral fucoxanthin bioavailability calculated.

Overview on the antihypertensive and anti-Obesity effects of secondary metabolites from seaweeds

Hypertension and obesity are two significant factors that contribute to the onset and exacerbation of a cascade of mechanisms including activation of the sympathetic and renin-angiotensin systems, oxidative stress, release of inflammatory mediators, increase of adipogenesis and thus promotion of systemic dysfunction that leads to clinical manifestations of cardiovascular diseases. Seaweeds, in addition to their use as food, are now unanimously acknowledged as an invaluable source of new natural products that may hold noteworthy leads for future drug discovery and development, including in the prevention and/or treatment of the cardiovascular risk factors. Several compounds including peptides, phlorotannins, polysaccharides, carotenoids, and sterols, isolated from brown, red and green macroalgae exhibit significant anti-hypertensive and anti-obesity properties.

This review will provide a comprehensive overview of the recent advances on bioactive pure compounds isolated from different seaweed sources focusing on their potential use as drugs to treat or prevent hypertension and obesity. On the other hand, although it is obvious that macroalgae represent promising sources of antihypertensive and anti-obesity compounds, it is also clear that further efforts are required to fully understand their cellular mechanisms of action, to establish structure-inhibition relationships and mainly to evaluate them in pre-clinical and clinical trials.

Keywords: anti-hypertension; anti-obesity; fucoxanthin; peptides; phlorotannins; seaweeds.

Potential of natural products in the Inhibition of Adipogenesis through regulation of PPARγ expression and/or its transcriptional activity

Obesity is a global health problem characterized as an increase in the mass of adipose tissue. Adipogenesis is one of the key pathways that increases the mass of adipose tissue, by which preadipocytes mature into adipocytes through cell differentiation. Peroxisome proliferator-activated receptor γ (PPARγ), the chief regulator of adipogenesis, has been acutely investigated as a molecular target for natural products in the development of anti-obesity treatments. In this review, the regulation of PPARγ expression by natural products through inhibition of CCAAT/enhancer-binding protein β (C/EBPβ) and the farnesoid X receptor (FXR), increased expression of GATA-2 and GATA-3 and activation of the Wnt/β-catenin pathway were analyzed.

Keywords: obesity, adipogenesis, PPARγ, natural products, PPARγ expression, PPARγ transcriptional activity.

Gallotannin derivatives from mango (Mangifera indica L.)

Gallotannin derivatives from mango (Mangifera indica L.) suppress Adipogenesis and increase thermogenesis in 3T3-L1 Adipocytes in part through the AMPK

Expansion of adipose tissue in obesity is associated with dysregulation of adipokines, which can lead to long-term metabolic disorders. Gallotannin derivatives from mango possess anti-inflammatory activities, but their potential role in lipid metabolism is not well investigated. In this study, 3T3-L1 preadipocytes were differentiated into adipocytes and treated with mango polyphenols (MG), or pyrogallol (PG) for 6 days. The anti-adipogenic activity of PG was demonstrated by reduced number of lipid droplets and expressions of adipogenic markers, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα).

In mature adipocytes, PG promoted adipocyte browning and increased the expressions of uncoupling protein 1 (UCP1) and sirtuin 1 (Sirt1). Knockdown of AMP-activated protein kinase (AMPK) α1 with siRNA partially abolished the effect of PG on adipogenesis. Results indicate that gallotannin derivatives modulate lipid metabolism, at least in part, though the AMPK pathway, and possess potential to prevent obesity-related conditions.

Role of the AMPK-C/EBPα / PPARγ and AMPK-UCP1/Sirt1 Axes in the Anti-obesogenic Activities of Gallotannin Derivatives from Mango (Mangifera indica L.)

Obesity is an escalating global epidemic associated with increased risk of developing type 2 diabetes, cardiovascular diseases and cancer. Dietary modifications, particularly increasing consumption of polyphenol-rich foods, are considered some of the most effective strategies in the prevention of obesity-related chronic diseases. Mango (Mangifera indica L.) contains high content of phenolic compounds (e.g., gallic acid (GA), gallotannin (GT), and galloyl glycosides), showing anti-inflammatory and antiobesogenic potential in chronic diseases. Lactobacillus plantarum (L.plantarum) possesses enzymatic activities to degrade GT into GA and PG, allowing for absorption and excretion. Thus, the interaction between gut microbiota and GT derivatives may affect the subsequent biological activities exerted by the microbial metabolites.

Anti-obesogenic potential of GT derivatives from mango in modulating lipid metabolism was investigated in 3T3-L1 adipocytes. GT derivatives suppressed adipogenesis and increased thermogenesis in adipocytes in part through the interactions with the AMPK-C/EBPα/PPARγ and AMPK-UCP1/Sirt1 axes. In gnotobiotic mice fed a high-fat diet (HFD), GT alone decreased lipid accumulation in white adipose tissue and increased thermogenesis in brown adipose tissue. Intestinal colonization with L.plantarum enhanced these effects and additionally lowered levels of inflammation and insulin resistance. GT and L.plantarum reduced HFD-induced inflammation and insulin resistance and promoted thermogenesis in adipose tissue potentially through the activity of GT-metabolizing bacterial enzymes yielding absorbable bioactive of GT-metabolizing bacterial enzymes yielding absorbable bioactive GT metabolites, which implies the potential role of prebiotic-probiotic interactions in the prevention of diet-induced metabolic disorders.

These findings were expected to translate into a human clinical trial, which examined the influence of 6 weeks of daily mango supplementation on inflammation and metabolic functions. Mango supplementation improved the plasma levels of proinflammatory cytokines and metabolic hormones in obese participants partly due to increased systemic exposure to polyphenolic metabolites. In summary, health benefits of mango-derived polyphenols in obesity and insulin resistance are mainly attributed to the production of microbial metabolites of GT, which is in part through the interactions with the AMPK-C/EBPα/PPARγ and AMPKUCP1/Sirt1 axes in adipose tissue. Improving the abundance of probiotics in gut microbiota may improve the bioavailability of mango-derived polyphenols, resulting in enhanced efficacy of the microbial metabolites in the prevention of lipid accumulation and metabolic dysfunction in obesity.

Ganoderma applanatum Polysaccharide

Effect of Ganoderma applanatum Mycelium Extract on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

Ganoderma applanatum (GA) and related fungal species have been used for over 2000 years in China to Prevent and treat various human diseases. However, there is no critical research evaluating the functionality of GA grown using submerged culture technology. This study aimed to evaluate the effects of submerged culture GA mycelium (GAM) and its active components (protocatechualdehyde [PCA]) on Preadipocyte Differentiation of 3T3-L1 cells. Mouse-derived Preadipocyte 3T3-L1 cells were treated with Differentiation inducers in the presence or absence of GAM extracts. We determined triglyceride accumulations, glycerol-3-phosphate dehydrogenase (GPDH) activities, and Differentiation makers. PCA, the active component of GAM extract, was also used to treat 3T3-L1 cells. The MTT assay showed that the GAM extract (0.01–1 mg/mL) was not toxic to 3T3-L1 preAdipocyte. Treatment of cells with GAM extracts and its active components significantly decreased the GPDH activity and lipid accumulation, a marker of Adipogenesis, in a dose-dependent manner.

Western blot analysis results showed that the protein expression levels of peroxisome proliferator-activated receptor γ (PPARγ ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) were Inhibited by the GAM extract. In addition, Adipogenic-specific genes such as perilipin, fatty acid synthase (FAS), fatty acid transport protein 1 (FATP1), and fatty acid-binding protein 4 (FABP4) decreased in a dose-dependent manner. Quantitative high-performance liquid chromatography analysis showed that the GAM extract contained 1.14 mg/g PCA. GAM extracts suppressed Differentiation of 3T3-L1 preadipocytes, in part, through altered regulation of PPARγ, C/EBPα, and SREBP1. These results suggest that GAM extracts and PCA may suppress Adipogenesis by Inhibiting Differentiation of preadipocytes.

Ganoderma lucidum Polysaccharide

Lanostane Triterpenes from the Fruiting Bodies of Ganoderma lucidum and Their Inhibitory Effects on Adipocyte Differentiation in 3T3-L1 cells

Four new lanostane triterpenes, butyl ganoderate A, butyl ganoderate B, butyl lucidenate N, and butyl lucidenate A, were isolated from the fruiting bodies of Ganoderma lucidum together with 14 known compounds. The structures of the new triterpenes were established by extensive spectroscopic studies and chemical evidence. In addition, the Inhibitory effect of isolated compounds on Adipocyte Differentiation in 3T3-L1 cells was examined.

Garcinia cambogia (HCA(Hydroxy Citric acid)

Gut microbiota Modulated by Probiotics and Garcinia cambogia Extract Correlate with Weight Gain and Adipocyte Sizes in High Fat-Fed Mice

Results of recent studies on gut microbiota have suggested that obesogenic bacteria exacerbate Obesity and metabolic dysfunction in the host when fed a high fat diet (HFD). In order to explore Obesity-associated bacterial candidates and their response to diet, the composition of faecal bacterial communities was investigated by analyzing 16S rRNA gene sequences in mice. Dietary intervention with probiotics and Garcinia cambogia extract attenuated Weight Gain and Adipocyte size in HFD-fed mice.

To identify Obesity-causative microbiota, two statistical analyses were performed. Forty-eight bacterial species were found to overlap between the two analyses, indicating the commonly identified species as diet-driven and Obesity-associated, which would make them strong candidates for host-microbiome interaction on Obesity.

Finally, correlation based network analysis between diet, microbe, and host revealed that Clostridium aminophilum, a hyper-ammonia-producing bacterium, was highly correlated with Obesity phenotypes and other associated bacteria, and shown to be suppressed by the combination of probiotics and Garcinia cambogia extract. Results of the present study suggest that probiotics and Garcinia cambogia extract alleviate Weight Gain and Adiposity, in part via differentially modulating the composition of gut microbiota in HFD fed mice.

Garcinol (Mangosteen shell)

Inhibitory effects of garcinol and pterostilbene on cell proliferation and Adipogenesis in 3T3-L1 cells

The aim of this work was to study the effects of garcinol and pterostilbene on cell proliferation and Adipogenesis in 3T3-L1 cells. The results showed that garcinol and pterostilbene decreased the cell population growth and caused cell cycle arrest at the G2/M phase in 3T3-L1 preadipocytes. During Adipocyte Differentiation, both garcinol and pterostilbene had Inhibitory effects on fat droplet formation and triacylglycerol accumulation. The data indicated that garcinol and pterostilbene could Inhibit the glycerol-3-phosphate dehydrogenase (GPDH) activity by 97.8 and 61.5%, respectively, as compared to the control. Both garcinol and pterostilbene significantly attenuated the protein expressions of PPARγ and C/EBPα during 3T3-L1 Adipocyte Differentiation.

Moreover, garcinol and pterostilbene caused an Inhibition of lipid accumulation in the 3T3-L1 Adipocyte Differentiation phase. Garcinol and pterostilbene also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin, resistin, and fatty acid synthase (FAS) in 3T3-L1 Adipocyte Differentiation . In 3T3-L1 Adipocytes, garcinol significantly down-regulated the protein expressions of PPARγ and FAS as well as up-regulated the protein expressions of adipose triglyceride lipase (ATGL) and adiponectin. Garcinol also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin and FAS. These results suggest that garcinol and pterostilbene have Anti-Adipogenic effects on preadipocytes and Adipocytes.

Gelidium elegans

Altered Gelidium elegans Extract-stimulated Beige-like Phenotype Attenuates Adipogenesis in 3T3-L1 cells

Previously, we showed that Gelidium elegans extract (GE) Suppresses oxidative stress and lipid accumulation. However, the molecular mechanism underlying the Anti-Adipogenic ability of GE is still unclear. The levels of Adipogenesis markers and triglyceride synthesis enzymes were measured by western blot. To evaluate the lipid accumulation in 3T3-L1 cells, oil red o staining was performed. We investigated whether GE induces Lipolysis by measuring Adipocyte triglyceride lipase (ATGL) during Adipocyte Differentiation. We also examined the expression of beige cell-associated genes and the production of carbon dioxide in 3T3-L1 cells. We showed that GE increased the protein expression of CAAT/enhancer binding protein (C/EBP) homologous protein 10 and Inhibited the expression of C/EBPβ. GE discouraged triglyceride synthesis via deregulation of lysophosphatidic acid acyltransferase-θ (LPAATθ) and diacylglycerolacyltransferase 1 (DGAT1) during late-stage Adipogenesis in 3T3-L1 cells. GE also dramatically increased ATGL in 3T3-L1 cells.

Finally, in 3T3-L1 cells treated with GE, markers of beige Adipocytes such as PRDM16 and UCP1 were upregulated, and large amounts of carbon dioxide were produced. These data indicate that GE Suppresses Adipogenesis by stimulating a beige-like phenotype in 3T3-L1 cells.

Genistein

Genistein Mediates the Anti-Adipogenic Actions of Sophora japonica L. Extracts

Previous studies showed that feeding diets containing the mature fruits of Sophora japonica L. prevented body Weight Gain and Reduced fat Mass in high-fat diet-induced obese mice.

Enhanced pro-apoptotic and Anti-Adipogenic effects of genistein plus guggulsterone in 3T3-L1 Adipocytes

Genistein (G), an isoflavone, and guggulsterone (GS), the active substance in guggulipid, have been reported to possess therapeutic effects for Obesity . In the present study, we investigated the effects of combinations of G plus GS on apoptosis.

Genistein Inhibits CCAAT/enhancer-binding protein β (C/EBPβ) activity and 3T3-L1 Adipogenesis by increasing C/EBP homologous protein expression

genistein’s Anti-Adipogenic effect coincides with the expression of C\EBPβ, C\EBPα and PPARγ , we hypothesized that genistein Inhibits 3T3-L1 cell …

Genistein and daidzein repress Adipogenic Differentiation of human Adipose Tissue ‐derived mesenchymal stem cells via Wnt/β‐catenin signalling or Lipolysis

Microarrays showed that anti‐Adipogenic effects of genistein were principally attributable to activation of Wnt signalling via ERs‐dependent pathway, such as Erk/JNK signalling and LEF/TCF4 co … Unlike genistein, daidzein Inhibited Adipogenesis through stimulation of …

Differential effects of Flavonoids on 3T3-L1 Adipogenesis and Lipolysis

The AntiAdipogenic effect of genistein was not due to Inhibition of insulin receptor subtrate-1 …Genistein (4′,5,7-trihydroxyisoflavone) and all tissue culture materials were from GIBCO … Rabbit polyclonal anti-signal transducers and activators of transcription (STAT) 3 antibody was …

Genistein Inhibits Differentiation of primary human Adipocytes

Genistein has been shown to have potential anti–Obesity effects, decreasing food intake, bodyweight … to ERα [28], but in a cell-based gene transcription assay, genistein was only … Therefore,

the AntiAdipogenic effect of genistein in our study might be partially mediated through …

Genistein affects Adipose Tissue deposition in a dose-dependent and gender-specific manner

Genistein Affects Adipose Tissue Deposition in a Dose-Dependent and Gender-Specific Manner.

Combined effects of genistein, quercetin, and resveratrol in human and 3T3-L1 Adipocytes

compounds genistein (G), quercetin (Q), and resveratrol (R) have been reported to each exhibit Anti-Adipogenic activities in Adipocytes and antiproliferative and pro-apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on Adipogenesis and …

The phytoestrogen genistein enhances osteogenesis and represses Adipogenic Differentiation of human primary bone marrow stromal cells

At d 3 of Adipogenesis , TGFβ1 was strongly up-regulated by genistein in an ER-dependent manner …Adipogenic Differentiation and maturation, on the other hand, were Reduced by genistein (and 17β-estradiol) via an ER-dependent mechanism involving autocrine or …

Genistein, EGCG, and capsaicin Inhibit Adipocyte Differentiation process via activating AMP-activated protein kinase

and apoptotic potential [3]. Also, it has been reported that genistein exhibits Anti-Adipogenic effects in several Adipocytes, although its precise mechanism of action is not known [4]. We have investigated the molecular events leading to the Inhibition of Adipogenesis by genistein …

Gentiopicroside isolated from Gentiana scabra Bge.

Gentiopicroside isolated from Gentiana scabra Bge. Inhibits Adipogenesis in 3T3-L1 cells and Reduce s body weight in diet-induced obese mice

Gentiopicroside is a major active component of the Gentiana scabra Bge., which is commonly used as herbal medicine for the treatment of Inflammation in Asia. Gentiopicroside significantly down-regulated expression of key Adipogenic transcription factors (PPARγ, C/EBPα, SREBP-1c) and dose-dependently Inhibited the lipid uptake-related gene (LPL), fatty acid transport-related gene (FABP4) and triglyceride (TG) synthesis-related gene (DGAT2), as well as fatty acid synthesis-related genes (FAS, SCD1), which resulted in Reduced intracellular lipid droplet accumulation and TG content in 3T3-L1 cells.

Gentiopicroside also down-regulated expression of inflammatory cytokine genes (NFκB1, TNFα, IL6) compared with vehicle. Oral administration of gentiopicroside (50 mg/kg) in mice fed with high-fat diet for 12 weeks resulted in Reduced body weight and visceral fat Mass compared with the control group. Overall, the results of this study showed that gentiopicroside had positive Anti-Obesity effects by regulating the expression of Adipogenesis /lipogenesis-related genes and inflammatory genes in 3T3-L1, and that it effectively Reduced body weight and visceral fat Mass in vivo.

AntiObesity Effects of Gentiana lutea Extract on 3T3-L1 preadipocytes and a High-Fat Diet-Induced Mouse Model

Obesity is one of the most common metabolic diseases resulting in Metabolic Syndrome. In this study, we investigated the AntiObesity effect of Gentiana lutea L. (GL) extract on 3T3-L1 preadipocytes and a high-fat-diet (HFD)-induced mouse model. For the induction of preadipocytes into Adipocytes , 3T3-L1 cells were induced by treatment with 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 μg/mL insulin. Adipogenesis was assessed based on the messenger ribonucleic acid expression of Adipogenic -inducing genes (adiponectin (Adipoq), CCAAT/enhancer-binding protein alpha (Cebpa), and glucose transporter type 4 (Slc2a4)) and lipid accumulation in the differentiated Adipocytes was visualized by Oil Red O staining. In vivo, obese mice were induced with HFD and coadministered with 100 or 200 mg/kg/day of GL extract for 12 weeks. GL extract treatment Inhibited Adipocyte Differentiation by downregulating the expression of Adipogenic -related genes in 3T3-L1 cells.

In the obese mouse model, GL extract prevented HFD-induced Weight Gain, fatty hepatocyte deposition, and Adipocyte size by decreasing the secretion of leptin and insulin. In conclusion, GL extract shows AntiObesity effects in vitro and in vivo, suggesting that this extract can be beneficial in the prevention of Obesity.

Ginkgetin, a biflavone from Ginkgo biloba leaves

Ginkgetin, a biflavone from Ginkgo biloba leaves, prevents Adipogenesis through STAT5-mediated PPARγ and C/EBPα regulation

Adipogenesis involved in hypertrophy and hyperplasia of Adipocytes is responsible for expanding the Mass of Adipose Tissue s in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ ) and CCAAT/enhancer-binding protein α(C/EBPα ) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in Adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 Inhibit or that blocks the Differentiation of preadipocytes into Adipocytes. During the Differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly Inhibited the formation of lipid-bearing Adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during Adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPβ or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of Adipogenesis.

In addition, ginkgetin-mediated the Inhibition of Adipogenesis was recapitulated in the Differentiation of primary preadipocytes. Lastly, we confirmed the Inhibitory effects of ginkgetin on the hypertrophy of white Adipose Tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-Adipogenesis and Anti-Obesity drug.

Ginkgetin: A natural biflavone with versatile pharmacological activities

Natural products, being richly endowed with curative powers, have become spotlight for biomedical and pharmaceutical research to develop novel therapeutics during recent years. Ginkgetin, a natural non-toxic biflavone, has been shown to exhibit anti-cancer, anti-inflammatory, anti-microbial, Anti-Adipogenic , and neuroprotective activities. Ginkgetin combats cancer progression by arresting cell cycle, inducing apoptosis, stimulating autophagy, and targeting many deregulated signaling pathways such as JAK/STAT and MAPKs. Ginkgetin halts Inflammation mediators like interleukins, iNOS, COX-2, PGE2, NF-κB, and acts as an Inhibit or of PLA2. GK shows strong neuroprotection against oxidative stress-promoted cell death, Inhibits cerebral micro-hemorrhage, decreases neurologic deficits, and halts apoptosis of neurons.

Ginkgetin also acts as anti-fungal, anti-viral, anti-bacterial, leishmanicidal and anti-plasmodial agent. Ginkgetin shows substantial preventive or therapeutic effects in in vivo models of many diseases including atherosclerosis, cancer, neurodegenerative, hepatic, influenza, and inflammatory diseases. Based on various computational, in vitro and in vivo evidences, this article demonstrates the potential of ginkgetin for development of therapeutics against various diseases. Although GK has been systematically studied from pharmacological point of view, a vast field of pharmacokinetics, pre-clinical and clinical studies is still open for the researchers to fully validate its potential for the treatment of various diseases.

Isoginkgetin enhances adiponectin secretion from differentiated adiposarcoma cells via a novel pathway involving AMP-activated protein kinase

The recent discovery of adiponectin and its role in Metabolic Syndrome has pointed to a new direction of therapeutics. The fact that adiponectin levels are inversely correlated with the degree of Obesity and insulin resistance suggests the benefit of lifting plasma adiponectin levels. The feasibility of this approach has been demonstrated in various animal models (Shklyaev et al. 2003, Yamauchi et al. 2003a). We are interested in exploring approaches based on up-regulation of adiponectin using small molecules. At present, the only available small molecules with this property are the synthetic PPARγ agonists (Maeda et al. 2001, Combs et al. 2002). The PPARγ agonists, such as the drug Avandia (rosiglitazone), not only ameliorate insulin resistance but also cause Weight Gain which further increases the risk of type II Diabetes and limits its application for Diabetes. PPARα agonists are also being investigated for this application due to their weight loss effect in rodents (Liu et al. 2005), but the potency of these agonists are generally lower than PPARγ agonists.

In this study, we report a natural compound, isoginkgetin, up-regulating adiponectin production with potency comparable to that of rosiglitazone, but devoid of the potent Adipogenesis-promoting effect of rosiglitazone. Isoginkgetin is one of the major active constituents in G. biloba extract, which has been used as an ancient Chinese remedy for its multiple biological activities, including anti-oxidant, anti-inflammatory, and anti-tumor activities (Yoshikawa et al. 1999, DeFeudis et al. 2003). The cellular signaling induced by this compound is largely unknown. This is the first study to reveal the potential insulin-sensitizing effects of isoginkgetin based on its regulation of adiponectin. Our observation of the effect of isoginkgetin on Lipolysis is different from the results reported by Dell’Agli & Bosisio (2002). This could be due to the different experimental settings, such as incubation time. In addition, Dell’Agli observed a bi-phase effect of isoginkgetin on Lipolysis, an enhancement of Lipolysis was only observed at 0.03–0.3 μM isoginkgetin, and the abolishment of this effect is not explainable by cytotoxicity. Therefore, more detailed studies under different experimental conditions need to be carried out to address this distinction.

The mechanism of isoginkgetin action is apparently distinct from that of rosiglitazone for a number of reasons. First, Inhibition of PPARγ activity did not affect the effect of isoginkgetin, whereas the activity of rosiglitazone was significantly diminished. Second, isoginkgetin and rosiglitazone exhibited differential stimulation kinetics. Third, results on the adiponectin mRNA and protein levels suggest the differential regulation of adiponectin synthesis and secretion by these two compounds. Fourth, although both compounds have comparable effects on adiponectin production, they have differential effects on Adipogenesis and PPARγ activation.

Regulation of adiponectin production seems complex and has not been fully understood. Hormones and cytokines, including insulin, TNF-α, and interleukin-6, exhibited different effects on adiponectin production in vitro, while the signaling pathways mediating these regulations are largely unknown. The results that isoginkgetin potentiates adiponectin secretion in a likely PPARγ -independent manner suggest new types of targets and agents that can be explored for new therapy.

AMPK is a key regulator for energy metabolism in vivo. Activation of AMPK is involved in fat oxidation via the Inhibition of downstream acetyl CoA carboxylase (ACC; Tomas et al. 2002). PPARγ agonists activate AMPK and Inhibit ACC both in vitro and in vivo in skeletal muscle (Saha et al. 2004). Our data suggest that AMPK or genes upstream of the AMPK pathway maybe effectors of isoginkgetin action. Further experiments, for example introducing the kinase-dead AMPK into Adipocytes to investigate its effect on isoginkgetin-regulated adiponectin production, would be helpful to confirm the involvement of AMPK. Whether isoginkgetin activates AMPK by increasing the ratio of AMP:ATP, as rosiglitazone does (Fryer et al. 2002a), needs to be further investigated. An interesting finding that Inhibition of AMPK-Reduced basal adiponectin production in differentiated Adipocytes suggests the general role of AMPK in the regulation of adiponectin. Whatever the mechanism, the activation of AMPK and Inhibition of phosphor-ACC by isoginkgetin will be beneficial for lipid regulation. Moreover, AMPK pathway can be further explored for adiponectin up-regulation.

In conclusion, isoginkgetin is a promising candidate compound for the treatment of insulin resistance based on our in vitro studies. Further in vivo studies are warranted. Analogs of isoginkgetin possessing higher potency in adiponectin production and lower PPARγ agonist activity can also be explored to identify more active compounds in vivo. Currently, insulin-sensitizing drugs mainly include PPARγ agonists and compounds that target the insulin signaling pathway. This study presents for the first time a potential new strategy for the discovery of insulin sensitizers by screening adiponectin production. Moreover, the critical genes involved in isoginkgetin action may provide novel targets for anti-Diabetic therapy.

Potential anti-obesogenic effects of ginkgo biloba observed in epididymal white Adipose Tissue of obese rats

The definition of white Adipose Tissue (WAT) as an inert Mass for energy storage is long gone; over the last two decades the Adipose Tissue has been recognized as a dynamic tissue and key player in the modulation of energy metabolism (1, 2). Adipokines such as leptin, adiponectin, and tumour necrosis factor-α (TNF-α) have a direct effect on energy homeostasis and modulation of low-grade Inflammation (3). The intake of high fat diets has the potential to not only disturb normal adipokine secretion but also to remodel Adipose Tissue by increasing Adipocyte size and/or number, contributing to the development of a pro-inflammatory microenvironment (4, 5). These perturbations have been positively associated with metabolic disorders such as Obesity, type 2 Diabetes , non-alcoholic fatty liver disease (NAFLD), insulin resistance, and cardiovascular diseases (6, 7).

In Obesity , particularly visceral Obesity , enlarged WAT visceral Adipocytes show dysregulated Lipolysis , inducing high levels of circulating non-esterified fatty acids (NEFAs) (8, 9). NEFAs in normal circumstances are utilized as energy by tissues such as liver and muscle; however, when in excess they contribute to the development of insulin resistance (4, 9–11). Furthermore, in response to overnutrition, hypertrophic Adipocytes contribute to increased circulating triacylglycerol (TAG) levels mainly from de novo lipogenesis, in which fatty acids (FA) are synthetized from non-lipid substrates, particularly carbohydrates, or from FA obtained from ex-situ lipid sources such as chylomicrons and very-low-density lipoproteins (VLDL) (12, 13). Visceral Obesity seems to play a central role in the development of metabolic disorders, being associated with low-grade chronic Inflammation and the production of pro-inflammatory cytokines which have the potential to trigger insulin resistance and endothelial dysfunction (14–16).

In this context, several pharmacological approaches have been tried for the treatment of Obesity. However, more often than not such approaches were followed by undesired side effects, including psychiatric manifestations, increased risk of cardiovascular events, and others (17). Considering the dramatic increase in the prevalence of Obesity over the last decades globally, a range of anti-obesogenic alternative supplementation therapies based on plant extracts (18) have been investigated.

More recently, Ginkgo biloba Extract (GbE) has been investigated as an alternative therapy for metabolic disorders associated with Obesity. GbE, a herbal extract containing Flavonoids, terpenoids, and terpene lactones (19), is a well-known phytotherapic compound often employed as coadjuvant supplement in neurodegenerative diseases (20, 21), NAFLD (22, 23), type 1 and 2 Diabetes (24, 25). Previous findings from our research group showed that diet-induced obese (DIO) rats supplemented with GbE showed Reduced food and energy intake, Reduced body Adiposity, improved insulin signalling and sensitivity, enhanced insulin receptor and AKT phosphorylation, and Reduced NFκB-p65 phosphorylation in retroperitoneal Adipose Tissue (26, 27).

GbE may have a potentially therapeutic use for menopause-associated Obesity; supplementation with 500 mg/kg of GbE stimulated hypothalamic serotonergic activity in ovariectomized rats (28). GbE isolated bioactive compounds have been demonstrated to stimulate Lipolysis in 3T3-L1 Adipocytes (29), and to Inhibit Adipogenesis through activation of the AMPK pathway (30). However, the effects of GbE supplementation on metabolic processes of visceral Adipose Tissue in DIO rats remain largely unknown. In view of the considerations highlighted above, the aim of the present study was to investigate the effects of GbE supplementation as a potentially anti-obesogenic effector for improvement in lipid metabolism of epididymal Adipose Tissue of DIO rats.

Ginkgo Biloba

During the Differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly Inhibited the formation of lipid-bearing Adipocytes . PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during Adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPβ or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of Adipogenesis. In addition, ginkgetin-mediated the Inhibition of Adipogenesis was recapitulated in the Differentiation of primary preadipocytes. Lastly, we confirmed the Inhibitory effects of ginkgetin on the hypertrophy of white Adipose Tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-Adipogenesis and Anti-Obesity drug.

Ginkgolide C, isolated from Ginkgo biloba leaves

Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK signaling pathway

Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the AntiAdipogenic effect of ginkgolide C in 3T3-L1 Adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to Adipogenesis and Lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated Adipocytes.

It also decreased Adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for Lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK Inhibit or (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing Lipolysis and Inhibiting Adipogenesis in Adipocytes through the activated AMPK pathway.

Ginkgolide C Reduced oleic acid-induced lipid accumulation in HepG2 cells

Ginkgolide C, isolated from Ginkgo biloba, is a diterpene lactone that has multiple biological functions and can improve Alzheimer disease and platelet aggregation. Ginkgolide C also Inhibits Adipogenesis in 3T3-L1 Adipocytes . The present study evaluated whether ginkgolide C Reduced lipid accumulation and regulated the molecular mechanism of lipogenesis in oleic acid-induced HepG2 hepatocytes. HepG2 cells were treated with 0.5 mM oleic acid for 48 h to induce a fatty liver cell model. Then, the cells were exposed to various concentrations of ginkgolide C for 24 h. Staining with Oil Red O and the fluorescent dye BODIPY 493/503 revealed that ginkgolide C significantly Reduced excessive lipid accumulation in HepG2 cells. Ginkgolide C decreased peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein 1c to block the expression of fatty acid synthase.

Ginkgolide C treatment also promoted the expression of adipose triglyceride lipaseand the phosphorylation level of hormone-sensitive lipase to enhance the decomposition of triglycerides. In addition, ginkgolide C stimulated CPT-1 to activate fatty acid β-oxidation, significantly increased sirt1 and phosphorylation of AMP-activated protein kinase (AMPK), and decreased expression of acetyl-CoA carboxylase for suppressed fatty acid synthesis in hepatocytes. Taken together, our results suggest that ginkgolide C Reduced lipid accumulation and increased Lipolysis through the sirt1/AMPK pathway in oleic acid-induced fatty liver cells.

Ginsenoside Rg1

AntiObesity effects of ginsenoside Rg1 on 3T3-L1 preadipocytes and high fat diet-induced obese mice mediated by AMPK

Ginsenosides Rg1 is one of the major pharmacologically active saponins in ginseng, which as an antioxidant Reduces oxidative damage in the liver and can also be used to Prevent cardiovascular diseases and Diabetes. However, there is no research targeting the effect of lipid metabolism in high-fat diet (HFD)-induced mice. In this study, we evaluated the Anti-Obesity effects of Rg1 in 3T3-L1 Adipocyte cells and HFD-induced obese C57BL/6J mice. Administration of Rg1 to HFD-induced obese mice significantly decreased body weight, total cholesterol, and total triglyceride levels. In addition to effects in 3T3-L1 cells, Rg1 Reduced the accumulation of lipid droplets in a dose-dependent manner.

Furthermore, Rg1 exhibits an Anti-Adipogenic effect via regulation of the expression of the transcriptional factors and lipid metabolism-related genes in vivo and in vitro. We observed that Rg1 administration significantly increased the phosphorylation level of AMP-activated protein kinase (AMPK) in both epididymal white Adipose Tissue and 3T3-L1 cells. These results indicated that Rg1 works both in an Anti-Adipogenic and Anti-Obesity manner through inducing AMPK activation, Inhibiting lipogenesis, and decreasing intracellular lipid content, Adipocyte size, and adipose weight.

Ginsenoside Rg2

Ginsenoside Rg2 Inhibits Adipogenesis in 3T3-L1 preadipocytes and Suppresses Obesity in high-fat-diet-induced obese mice through the AMPK pathway

Ginsenoside Rg2 is one of the specific ginsenosides in red ginseng, and has been reported to exhibit protective effects against neurotoxicity and memory impairment, and also Inhibition of hepatic glucose production. However, the effect of Rg2 on the prevention of Obesity has not been investigated. In this study, we evaluated the Anti-Obesity and Anti-Adipogenic effects of Rg2 in high-fat diet-induced obese mice (HFD mice) and 3T3-L1 preadipocytes . Oral administration of Rg2 (10 mg kg⁻¹) to HFD mice significantly decreased body Weight Gain, total triglycerides, and free fatty acid levels.

In 3T3-L1 preadipocytes, Rg2 (80 μM) Inhibited Adipocyte Differentiation and Reduced the accumulation of intracellular lipids. Quantitative PCR and western blot analysis revealed that Rg2 decreased the expression levels of Adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1-c), and then regulated target genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Rg2 significantly promoted AMP-activated protein kinase (AMPK) both in vivo and in vitro, which is known to suppress Adipogenesis. It was also found that pretreating with compound C, a typical Inhibit or of AMPK, attenuated the Inhibitory effect of Rg2 on AMPK phosphorylation. These findings suggested that Rg2-induced activation of AMPK leads to a decrease in the expression of Adipogenic transcription factors, and suppression of Adipogenesis in vivo and in vitro. Hence, Rg2 has the potential for the development of healthy foods and the prevention of Obesity.

Ginsenoside Rg3

Anti–Adipogenic effects and mechanisms of ginsenoside Rg3 in Pre-Adipocytes and obese mice

Red or black ginseng has been reported more powerful than white/fresh ginseng in dealing with various diseases/conditions including Obesity. The major reason is that heating/steaming, the process of making red or black ginseng, produces large amount of bioactive compounds including ginsenoside Rg3 (Rg3), which are trace in fresh or white ginseng. In the present study, Rg3 was applied both in Pre-Adipocytes and obese mice to investigate the Anti-Adipogenic effects and relevant mechanisms. Our results show that Rg3 dose-dependently Inhibited cell Differentiation both in 3T3-L1 cells (30, 50, and 100 μM) and human primary Pre-Adipocytes (10, 20, and 30 μM).

This Inhibitory effect is accompanied by the attenuation of the expressions of Adipogenic markers including peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer binding protein alpha (C/EBP-α), fatty acid synthase (FAS), fatty acid binding protein 4 (FABP4) and perilipin. Although dietary intake of Rg3 (0.1 mg Rg3/kg diet, 8 weeks) did not significantly affect body Weight Gain , fat pads and food intake as well as of PPAR-γ expression in fat tissues, we found that hepatic PPAR-γ and C/EBP-α protein expressions and hepatic glutathione reductase and glutathione S-transferase, two major Antioxidants molecules were significantly Reduced by Rg3. These results suggest that ginsenoside Rg3 may be a potential agent in reducing/preventing Obesity.

AntiObesity effect of ginsenoside Rg3 involves the AMPK and PPAR‐γ signal pathways

Ginsenosides, the active component of ginseng, exerts AntiDiabetic and anticancer effects. This study investigated the molecular basis of ginsenoside Rg3, a red ginseng rich constituent, focusing on its ability to Inhibit Adipocyte Differentiation in 3T3‐L1 cells. The data show that ginsenoside Rg3 was effective in the Inhibition of Adipocyte Differentiation.

This Inhibitory effect of ginsenoside Rg3 on Adipocyte Differentiation was accompanied by PPAR‐γ Inhibition in rosiglitazone‐treated cells. The study also tested whether AMP‐activated protein kinase (AMPK) activation was involved in the Inhibitory effects of ginsenoside Rg3. AMPK plays a role in maintaining health in the context of diseases such as type 2 Diabetes, Obesity and cancer. AMPK was reported to control nutritional and hormonal signal modulating. Rg3 significantly and time‐dependently activated AMPK. Taken together, these results suggest that the AntiObesity effect of red ginseng rich constituent, ginsenoside Rg3, involves the AMPK signaling pathway and PPAR‐γ Inhibition.

Ginsenoside Rg3 ameliorated HFD-induced hepatic steatosis through downregulation of STAT5-PPARγ

Ginsenoside Rg3 Reduced lipogenesis in 3T3-L1 cells

The chemical structure of the natural compound ginsenoside Rg3 is shown in Fig. 1A. It has been reported to regulate Adipocyte Differentiation (Hwang et al. 2009), but it is not clear how Rg3 regulates lipogenesis. Healthy Adipose Tissue expansion, which involves the accumulation of lipid inside adipose cells, functions in stark contrast with Obesity-induced pathologies such as Inflammation and insulin resistance (Sun et al. 2011, Kusminski et al. 2016). We first assessed whether Rg3 influences lipogenesis in 3T3-L1 cells. Rg3 has been reported to induce apoptosis and cause cytotoxicity (Kim et al. 2014, Shan et al. 2015, Tian et al. 2016); therefore, we analyzed cell viability to rule out such side effects. The high concentration of 100-μM Rg3 induced cytotoxicity and Reduced the viability of 3T3-L1 cells.

The LC₅₀ value of Rg3 in 3T3-L1 cell lipogenesis is 200 μM, and our working concentrations (5, 25 and 50 μM) of Rg3 Reduced lipogenesis without cell toxicity (Fig. 1B and C). To investigate the Inhibiting effect on lipid accumulation by Rg3, we treated 3T3-L1 cells for 3 or 9 days. Oil Red O staining confirmed lipid accumulation in the cell. At 3 days, the lipogenic cocktail MDI (insulin, dexamethasone and isobutylmethylxanthine) induced in Pre-Adipocytes a very low increase in their accumulated fat deposits. When Rg3 was combined with MDI-induced cells, their lipid accumulation was suppressed (Fig. 1D), in an Rg3 dose-dependent manner, as assessed at 9 days after treatment using Oil Red O staining microscopy data and Oil Red O absorbance at 520 nm (Fig. 1E). In Adipocytes, lipid accumulation is related to the amount of TGs in the cell. Similar to the Oil Red O staining results, TG storage was Reduced by Rg3 treatment in 3T3-L1 cells in a dose-dependent manner (Fig. 1F). These data suggest that Rg3 can Prevent lipogenesis and lipid accumulation in the 3T3-L1 cell line.

Ginsenoside Rg3 induces browning of 3T3-L1 Adipocytes by activating AMPK signaling

Ginsenoside Rg3, one of the major components in Panax ginseng, has been reported to possess several therapeutic effects including Anti-Obesity properties. However, its effect on the browning of mature white Adipocytes as well as the underlying mechanism remains poorly understood. In this study, we suggested a novel role of Rg3 in the browning of mature 3T3-L1 Adipocytes by upregulating browning-related gene expression. The browning effects of Rg3 on differentiated 3T3-L1 Adipocytes were evaluated by analyzing browning-related markers using quantitative PCR, immunoblotting, and immunostaining. In addition, the size and sum area of lipid droplets in differentiated 3T3-L1 Adipocytes were measured using Oil-Red-O staining. In mature 3T3-L1 Adipocytes, Rg3 dose-dependently induced the expression of browning-related genes such as Ucp1, Prdm16, Pgc1α, Cidea, and Dio2.

Moreover, Rg3 induced the expression of beige fat-specific genes (CD137 and TMEM26) and lipid metabolism-associated genes (FASN, SREBP1, and MCAD), which indicated the activation of lipid metabolism by Rg3. We also demonstrated that activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) is required for Rg3-mediated up-regulation of browning gene expression. Moreover, Rg3 Inhibited the accumulation of lipid droplets and Reduced the droplet size in mature 3T3-L1 Adipocytes.

Taken together, this study identifies a novel role of Rg3 in browning of white Adipocytes, as well as suggesting a potential mechanism of an Anti-Obesity effect of Panax ginseng.

Glehnia littoralis Root

Glehnia littoralis root extract Inhibits Fat Accumulation in 3T3-L1 cells and high-fat diet-induced obese mice by downregulating Adipogenic gene expression

Glehnia littoralis has been reported to have several pharmacological properties but no reports describing the AntiAdipogenic effect of this plant have been published. This study was conducted to investigate the effects of Glehnia littoralis root hot water extract (GLE) and its underlying mechanism on 3T3-L1 cell Adipogenesis and in high-fat diet- (HFD-) induced obese mice. We measured intracellular lipid accumulation using oil red O staining in vitro. For in vivo study, twenty-eight C57BL/6J male mice were randomly divided into four groups, Control, HFD, HFD + 1% GLE, and HFD + 5% GLE, which was performed for eight weeks.

We determined the expression levels of the Adipogenesis -related proteins by RT-PCR and western blotting in HFD-induced obese mice. The GLE dose-dependently Inhibited 3T3-L1 Adipocyte Differentiation and intracellular lipid accumulation in differentiated Adipocytes. Further, body Weight Gain and Fat Accumulation were significantly lower in the GLE-treated HFD mice than in the untreated HFD mice. GLE treatment suppressed the expression of Adipogenic genes such as peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding protein (C/EBP) α, fatty acid synthase (aP2), and fatty acid synthase (FAS).

These results suggest that the GLE Inhibits Adipocyte Differentiation and intracellular lipid accumulation by downregulating the Adipogenic gene expression both in vitro and in vivo.

Guarana (Paullinia cupana)

Modulatory Effects of Guarana (Paullinia cupana) on Adipogenesis

Guarana (Paullinia cupana) is a plant originated in Brazil that presents a beneficial effect on body weight control and metabolic alterations. The aim of this study was to evaluate the effects of guarana on genes and miRNAs related to Adipogenesis in 3T3L1 cells. The Anti-Adipogenic effect of guarana was evaluated by Oil Red-O staining. Gene and miRNA expression levels were determined by real time PCR. The Cebpα and β-catenin nuclear translocation were evaluated using immunocytochemistry. Our data indicated that the triglyceride-reducing effect of guarana was dose-dependent from 100 to 300 µg/mL (−12%, −20%, −24% and −40%, respectively, p < 0.0001). An up-regulation of the Anti-Adipogenic genes Wnt10b, Wnt3a, Wnt1, Gata3 and Dlk1 and a down-regulation of Pro-Adipogenic genes Cebpα, PPARγ and Creb1 were also observed. Furthermore, guarana repressed mmu-miR-27b-3p, mmu-miR-34b-5p and mmu-miR-760-5p, that contributed for up-regulation of their molecular targets Wnt3a, Wnt1 and Wnt10b.

Additionally, cells treated with guarana presented an increase on β-catenin nuclear translocation (p < 0.0018). In summary, our data indicate that guarana has an Anti-Adipogenic potential due to its ability to modulate miRNAs and genes related to this process. Together our data demonstrate the important role of guarana as a putative therapeutic agent.

Guggulsterone (Commiphora mukul tree)

Guggulsterone Inhibits Adipocyte Differentiation and Induces Apoptosis in 3T3‐L1 Cells

Objective: To determine the effects of guggulsterone (GS), the active substance in guggulipid, on apoptosis, Adipogenesis , and Lipolysis using 3T3‐L1 cells.

Methods and Procedures: For apoptosis and Lipolysis experiments, mature Adipocytes were treated with GS isomers. Viability, apoptosis, and caspase 3/7 activation were quantified using MTS, enzyme‐linked immunosorbent assay (ELISA), caspase‐Glo 3/7 activity assay, respectively. The expression of cytochrome c was demonstrated by western blot. Lipolysis was quantified by measuring the release of glycerol. For Adipogenesis experiments, postconfluent preadipocytes were incubated with GS isomers for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye. Western blot was also used to demonstrate the Adipocyte ‐specific transcription factors peroxisome proliferator–activated receptor γ2 (PPARγ 2), CCAAT/enhancer binding protein α (C/EBPα ), and C/EBPβ.

Results: In mature Adipocytes cis‐GS decreased viability, whereas the trans‐GS isomer had little effect. Both isomers caused dose‐dependent increases in apoptosis and cis‐GS was more effective than trans‐GS in inducing apoptosis. cis‐ and trans‐GS also increased caspase‐3 activity and release of cytochrome c from mitochondria. In maturing preadipocytes , both isomers were equally effective in reducing lipid content. The Adipocyte ‐specific transcription factors PPARγ 2, C/EBPα , and C/EBPβ were downregulated after treatment with cis‐GS during the maturation period. Furthermore, cis‐GS increased basal Lipolysis of mature Adipocytes , but trans‐GS had no effect.

Discussion: These results indicate that GS isomers may exert AntiObesity effects by Inhibiting Differentiation of preadipocytes , and by inducing apoptosis and promoting Lipolysis of mature Adipocytes . The cis‐GS isomer was more potent than the trans‐GS isomer in inducing apoptosis and Lipolysis in mature Adipocytes .

Heshouwu (Polygonum multiflorum Thunb)

This study investigated whether Heshouwu (Polygonum multiflorum Thunb.) root ethanol extract (PME) has Anti-Obesity activity using 3T3-L1 cells and high-fat diet (HFD)-induced obese mice. Treatment with PME (5 and 10 μg/mL) dose-dependently suppressed 3T3-L1 pre-Adipocyte Differentiation to Adipocytes and cellular triglyceride contents. In addition, PME Inhibited mRNA and protein expression of Adipogenic transcription factors such as CCAAT/enhancer-binding protein α (C/EBPα ) and peroxisome proliferator-activated receptor γ (PPARγ ), which led to down-regulation of fatty acid synthase gene expression. After feeding mice PME (0.05%) with HFD for 12 weeks, their visceral fat Mass , size and body weight were significantly Reduced compared with the HFD group.

Furthermore, PME supplementation significantly up-regulated the PPARα, CPT1, CPT2, UCP1 and HSL mRNA levels compared with the HFD group, whereas it down-regulated expression of the PPARγ and DGAT2 genes. Finally, HFD increased serum leptin, insulin, glucose and insulin and glucose levels; however, PME reversed these changes. These results demonstrated that PME might relieve Obesity that occurs via Inhibition of Adipogenesis and lipogenesis as well as through Lipolysis and fatty acid oxidation in 3T3-L1 cells and HFD-induced obese mice.

Hesperidin (Citrus aurantium L)

Combination of Capsaicin and Hesperidin Reduce s the Effectiveness of Each Compound To Decrease the Adipocyte Size and To Induce Browning Features in Adipose Tissue of Western Diet Fed Rats

We explored the potential of hesperidin and capsaicin, separately and in combination, to induce white Adipose Tissue (WAT) browning and to help body weight management in Western diet-fed rats. Adult male Wistar rats were fed for 8 weeks with Western diet and treated daily with hesperidin (100 mg/kg/day), capsaicin (4 mg/kg/day), hesperidin (100 mg/kg/day) + capsaicin (4 mg/kg/day), or the vehicle. Hesperidin and capsaicin separately, but not (or to a lesser extent) the combination, resulted in a decreased size of Adipocytes and induced emergence of multilocular brown-like Adipocytes positive for UCP1 and CIDEA in retroperitoneal WAT.

Expression levels of browning markers, such as Prdm16, in inguinal WAT also increased with capsaicin treatment compared with the vehicle (145% ± 17% vs 92% ± 21%, P < 0.05), but no significant effects were found with the combination (106% ± 12%). Thus, the combination of both bioactives Reduces the effectiveness of each compound to decrease the Adipocyte size and induce WAT browning.

Hibiscus rosa sinensis flower

AMPK activating and anti Adipogenic potential of Hibiscus rosa sinensis flower in 3T3-L1 cells

Ethnopharmacological relevance: The flowers of Hibiscus rosa sinensis has array of pharmacological actions. They are used in preparation of herbal decoction and teas, which have been used traditionally to reduce body weight and for its effect on metabolic syndrome.

Aim of the study: To investigate the anti adipogenic efficacy of major fraction from ethyl acetate extract of the Hibiscus rosa sinensis flower at 25 and 50 µg/mL (HRF 25 and 50 µg/mL) in 3T3-L1 cells and delineate its possible mechanism of action.

Materials and methods: Pre adipocyte 3T3-L1 cells were differentiated in the presence and absence of HRF 25 and 50 µg/mL, their lipid accumulation was measured qualitatively by Oil red O staining and quantitatively by triglyceride estimation. Effect on adipolysis was determined, adipogenic and its regulatory gene and protein expression were studied and effect of HRF 25 and 50 µg/mL on AMPK was confirmed in the presence of dorsomorphin.

Results: Treatment with HRF 25 and 50 µg/mL activated AMP-activated protein kinase (AMPK) and was found to alleviate triglyceride accumulation significantly (p < 0.001) by 1.6 and 2.3 times respectively in pre adipocytes during differentiation. HRF 25 and 50 µg/mL also nonsignificantly reduced lipolysis which releases free fatty acids, a major contributing factor for insulin resistance. Activation of AMPK by phosphorylation has led to reduced gene and protein expression of adipogenic factors Peroxisome proliferator- activated receptor gamma (PPAR-γ), CCAT/enhancer binding protein alpha (C/EBPα), Sterol regulatory element- binding protein-1c (SREBP-1c) and their targets Fatty acid binding protein 4 (FABP4), Fatty acid synthase (FAS), Perilipin and enhanced Adiponectin expression. Treatment with HRF 25 and 50 µg/mL also resulted in inactivation of Acetyl-CoA carboxylase (ACC) by enhancing ACC phosphorylation, which reduced the levels of malonyl-CoA an allosteric inhibitor of carnitine palmitoyl transferase 1 (CPT1). Enhanced CPT1 levels causes induction of fatty acid β- oxidation. Effects of HRF were nullified in the presence of AMPK antagonist dorsomorphin.

Conclusion: In summary, HRF treatments reduced adipogenesis, enhanced factors regulating fatty acid oxidation and this is mediated by AMPK activation. The results conclusively showed anti-obesity potential of HRF and it might be helpful in treatment of associated complications.

Keywords: 3T3-L; AMPK; Adipogenesis; Hibiscus rosa sinensis; Lipolysis.

Hibiscus extract Inhibits the lipid droplet accumulation and Adipogenic transcription factors expression of 3T3-L1 preadipocytes

Objectives: This study was designed to investigate the effect of hibiscus (Hibiscus sabdariffa) on adipogenic differentiation of 3T3-L1 cells at the cellular and molecular levels.

Design: Various concentrations of hibiscus extract were added to confluent 3T3-L1 preadipocytes at the outset of the differentiation program and further incubated for 36 hours. Cells were maintained in postdifferentiation medium containing insulin with hibiscus extract in complete culture medium.

Results: Hibiscus extract inhibited the adipocyte differentiation of 3T3-L1 preadipocytes induced by insulin, dexamethasone, and isobutylmethylxanthine (IBMX) in a dose-dependent manner. Hibiscus blocked the cytoplasmic lipid accumulation when administered at the onset of differentiation and 4 days after induction of differentiation. The inhibitory effect of hibiscus on adipogenic lipid accumulation of preadipocytes was significant (p < 0.01) between control cells and cells treated with hibiscus. Hibiscus extract significantly attenuated the expression of key adipogenic transcription factors, including CCAAT element binding protein (C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR)gamma at protein levels.

Conclusion: These results suggest that hibiscus extract blocks adipogenesis, in part, by its suppression on the expression of adipogenic transcription factors, including C/EBPalpha and PPARgamma.

Hibiscus sabdariffa L

Beneficial effects of natural bioactive compounds from Hibiscus sabdariffa L. on Obesity

Obesity is a condition associated with the accumulation of excess fat in the body, energy imbalance, lipogenesis, etc., which increases adipose tissue mass through adipogenesis and poses a health risk. Its prevalence has become an economic burden to the health care system and the world at large. One of the alternatives to tackling obesity involves the use of bioactive compounds. We critically examined the effects of Hibiscus sabdariffa extract (HSE) on various parameters associated with the development of obesity such as; the effect of HSE on body weight, the effect of HSE on lipid accumulation, cholesterol metabolism and plasma parameters, the inhibitory effect of HSE on pancreatic lipase, and the effect of HSE on adipocyte differentiation/adipogenesis.

This review has gathered reports on the various anti-obesity effects of H. sabdariffa bioactive compounds in cell and animal models, as well as in humans. Available toxicology information on the consumption of H. sabdariffa revealed that its toxicity is dose-dependent and may cause an adverse effect when administered over a long period of time. Reports have shown that H. sabdariffa derived bioactive compounds are potent in the treatment of obesity with an evident reduction in body weight, inhibition of lipid accumulation and suppression of adipogenesis through the PPARγ pathway and other transcriptional factors.

Hibiscus sabdariffa L. water extract Inhibits the Adipocyte Differentiation through the PI3-K and MAPK pathway

Hibiscus sabdariffa L., a tropical beverage material and medical herb, is used commonly as in folk medicines against hypertension, pyrexia, inflammation, liver disorders, and obesity. This report was designed to investigate the inhibitory mechanisms of hibiscus extract on adipocyte differentiation in 3T3-L1 preadipocytes. The possible inhibitory pathways that regulate the adipocyte differentiation contain the adipogenic transcription factors, C/EBPalpha and PPARgamma, PI3-kinase, and MAPK pathway. In this study, we examined whether hibiscus extract affected the adipogenesis via these three pathways.

To differentiate preadipocyte in adipocyte, confluent 3T3-L1 preadipocytes were treated with the hormone mixture including isobutylmethylxanthine, dexamethasone, and insulin (MDI). Hibiscus extract inhibited significantly the lipid droplet accumulation by MDI in a dose-dependent manner and attenuated dramatically the protein and mRNA expressions of adipogenic transcriptional factors, C/EBPalpha and PPARgamma, during adipogenesis.

The increase of phosphorylation and expression of PI3-K/Akt during adipocytic differentiation was markedly inhibited by treatment with hibiscus extract or PI3-K inhibitors. Furthermore, the phosphorylation and expression of MEK-1/ERK known to regulate the early phase of adipogenesis were clearly decreased with the addition of hibiscus extract. Taken together, this report suggests that hibiscus extract inhibits the adipocyte differentiation through the modulation of PI3-K/Akt and ERK pathway that play pivotal roles during adipogenesis.

Hibiscus extract Inhibits the lipid droplet accumulation and Adipogenic transcription factors expression of 3T3-L1 preadipocytes

Objectives: This study was designed to investigate the effect of hibiscus (Hibiscus sabdariffa) on adipogenic differentiation of 3T3-L1 cells at the cellular and molecular levels.

Conclusion: These results suggest that hibiscus extract blocks adipogenesis, in part, by its suppression on the expression of adipogenic transcription factors, including C/EBPalpha and PPARgamma.

Polyphenol ic extract from Hibiscus sabdariffa Reduce s body fat by Inhibiting hepatic lipogenesis and Preadipocyte Adipogenesis

Diets high in fat lead to excess lipid accumulation in adipose tissue, which is a crucial factor in the development of obesity, hepatitis, and hyperlipidemia. In this study, we investigated the anti-obesity effect of Hibiscus sabdariffa extract (HSE) in vivo. Hamsters fed a high-fat diet (HFD) develop symptoms of obesity, which were determined based on body weight changes and changes in plasma and serum triglycerides, free fatty acid concentrations, total cholesterol levels, LDL-C levels, HDL-C levels, and adipocyte tissue weight. HFD-fed hamsters were used to investigate the effects of HSE on symptoms of obesity such as adipogenesis and fatty liver, loss of blood glucose regulation, and serum ion imbalance. Interestingly, HSE treatment effectively reduced the effects of the HFD in hamsters in a dose-dependent manner.

Further, after inducing maturation of preadipocytes, Hibiscus sabdariffa polyphenolic extract (HPE) was shown to suppress the adipogenesis of adipocytes. However, HPE does not affect the viability of preadipocytes. Therefore, both HSE and HPE are effective and viable treatment strategies for preventing the development and treating the symptoms of obesity.

Synergism of plant-derived Polyphenols in Adipogenesis : perspectives and implications

Dietary polyphenols may exert their pharmacological effect via synergistic interactions with multiple targets. Putative effects of polyphenols in the management of obesity should be primarily evaluated in adipose tissue and consequently in well-documented cell model. We used Hibiscus sabdariffa (HS), a widely recognised medicinal plant, as a source of polyphenols with a number of salutary effects previously reported. We present here the full characterisation of bioactive components of HS aqueous extracts and document their effects in a model of adipogenesis from 3T3-L1 cells and in hypertrophic and insulin-resistant adipocytes. Aqueous extracts were up to 100 times more efficient in inhibiting triglyceride accumulation when devoid of fibre and polysaccharides. Significant differences were also observed in reactive oxygen species generation and adipokine secretion.

We also found that, when polyphenols were fractionated and isolated, the benefits of the whole extract were greater than the sum of its parts, which indicated a previously unnoticed synergism. In conclusion, polyphenols have interactive and complementary effects, which suggest a possible application in the management of complex diseases and efforts to isolate individual components might be irrelevant for clinical medicine and/or human nutrition.

Hibiscus sabdariffa L.: Phytochemical Composition and Nutraceutical Properties

Hibiscus sabdariffa (HS) is a plant cultivated around the world for food uses, its medicinal properties and health benefits. Infusions of the calices of this plant are consumed for their unique taste and pleasant color either hot as tea or cold as a refreshing beverage. There are reports in the scientific literature about the effects of H. sabdariffa on decreasing the risk of hypertension, diabetes mellitus, inflammation, cancer and obesity, among other health conditions, due to the presence of biologically active compounds.

The purpose of this chapter is to review the scientific evidence in support of the potential health effects of HS, using studies performed in vitro, and in preclinical and clinical models. The results suggest that biologically active compounds in HS extracts are responsible for the potential prevention of the various disease conditions associated with the consumption of HS extracts.

Effect of Hibiscus sabdariffa extract on high fat diet–induced Obesity and liver damage in hamsters

Background: Obesity is a chronic metabolic disorder associated with an increase in adipogenesis and often accompanied with fatty liver disease.

Objective: In this study, we investigated the anti-obesity effects of Hibiscus sabdariffa water extract (HSE) in vivo.

Method: Eight-weeks-old male mice were divided into six groups (n=8 per group) and were fed either normal feed, a high fat diet (HFD), HFD supplemented with different concentrations of HSE, or HFD supplemented with anthocyanin. After 10 weeks of feeding, all the blood and livers were collected for further analysis.

Results: Mesocricetus auratus hamster fed with a high-fat diet developed symptoms of obesity, as determined from their body weight change and from their plasma lipid levels. Meanwhile, HSE treatment reduced fat accumulation in the livers of hamsters fed with HFD in a concentration-dependent manner. Administration of HSE reduced the levels of liver cholesterol and triglycerides, which were elevated by HFD. Analysis of the effect of HSE on paraoxonase 1, an antioxidant liver enzyme, revealed that HSE potentially regulates lipid peroxides and protects organs from oxidation-associated damage. The markers of liver damage such as serum alanine aminotransferase and aspartate aminotransferase levels that were elevated by HFD were also reduced on HSE treatment. The effects of HSE were as effective as treatment with anthocyanin; therefore the anthocyanins present in the HSE may play a crucial role in the protection established against HFD-induced obesity.

Conclusions: In conclusion HSE administration constitutes an effective and viable treatment strategy against the development and consequences of obesity.

Keywords: Hibiscus sabdariffa; adipogenesis; obesity.

Hibiscus sabdariffa extract Inhibits Obesity and Fat Accumulation , and improves liver steatosis in humans

Obesity is associated with a great diversity of diseases including non-alcoholic fatty liver disease. Our previous report suggested that Hibiscus sabdariffa extracts (HSE) had a metabolic-regulating and liver-protecting potential. In this study, we performed a clinical trial to further confirm the effect of HSE. Subjects with a BMI ≧ 27 and aged 18-65, were randomly divided into control (n = 17) and HSE-treated (n = 19) groups, respectively, for 12 weeks. Our data showed that consumption of HSE reduced body weight, BMI, body fat and the waist-to-hip ratio.

Serum free fatty acid (FFA) was lowered by HSE. Anatomic changes revealed that HSE improved the illness of liver steatosis. Ingestion of HSE was well tolerated and there was no adverse effect during the trial. No alteration was found for serum α-amylase and lipase. The clinical effect should mainly be attributed to the polyphenols of HSE, since composition analysis showed that branched chain-amino acids, which is associated with obesity, is not obviously high.

In conclusion, consumption of HSE reduced obesity, abdominal fat, serum FFA and improved liver steatosis. HSE could act as an adjuvant for preventing obesity and non-alcoholic fatty liver.

Therapeutic potential of Hibiscus sabdariffa: a review of the scientific evidence

Background and objective: Infusion of Hibiscus sabdariffa (H. sabdariffa) is a very popular drink in many parts of the world. Its phytochemical composition is associated to antioxidant, hypotensive, and antiatherosclerotic effects. However, the molecular mechanisms involved in these processes are not well known. The aim of this review was to report the scientific evidence supporting that regular use of H. sabdariffa decreases oxidative stress, atherosclerosis, lipid profile, and blood pressure.

Material and methods: A search of recent publications was made in the following specialized electronic databases: Elsevier Journal, SciELO, FSTA, Science Direct, Springer Link, and NCBI. Results of research conducted in clinical trials in humans and in animal models and cell cultures were recorded. Keywords used included Hibiscus sabdariffa, oxidative stress, polyphenols, hypertension, atherosclerosis, and lipid profile.

Results: Results of the different articles suggested a possible therapeutic effect of H. sabdariffa extracts on oxidative stress, lipid profile, hypertension, and atherosclerosis thanks to its composition rich in phenolic compounds. Anthocyanins significantly decrease LDL oxidation, inhibit adipogenesis by regulating adipogenic signaling pathways and transcription factors, and modulate gene expression of certain microRNAs. No adverse events or side effects were reported.

Conclusions: Further more homogeneous, placebo-controlled studies in humans are needed to state that H. sabdariffa has therapeutic efficacy in humans.

Keywords: Aterosclerosis; Atherosclerosis; Estrés oxidativo; Hibiscus sabdariffa; Hipertensión; Hypertension; Lipid profile; Oxidative stress; Perfil lipídico; Polifenoles; Polyphenols.

Effect of Hibiscus sabdariffa on Obesity in MSG mice

The aim of the present investigation was determine whether a standardized Hibiscus sabdariffa calyces aqueous extract has an effect on body weight in an obese animal model induced by the administration of monosodium glutamate. Hibiscus sabdariffa aqueous extract, containing 33.64 mg of total anthocyanins per each 120 mg of extract, was orally administered (120 mg/kg/day) for 60 days to healthy and obese mice, and body weight gain, food and liquid intake, aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, and triglycerides levels were measured.

Hibiscus sabdariffa administration significantly reduced body weight gain in obese mice and increased liquid intake in healthy and obese mice. ALT levels were significantly increased on the 15th and 45th days in obese mice, but AST levels did not show significant changes. Mortality was not observed in the Hibiscus sabdariffa treated groups. Triglycerides and cholesterol levels showed non-significant reductions in animals treated with Hibiscus sabdariffa. Our data confirm the anti-obesity effect of Hibiscus sabdariffa reported by the Mexican population.

Hippophae Rhamnoides L. Leaves

Ethanolic extract of seabuckthorn (Hippophae rhamnoides L) prevents high-fat diet–induced Obesity in mice through down-regulation of Adipogenic and lipogenic gene expression

Phenolic compounds and Flavonoids ameliorate bodyweight, blood glucose, and serum lipid profile. Since seabuckthorn (Hippophae rhamnoides L.) is known as a rich source of isoflavones and Flavonoids , we hypothesized that ethanolic extract of seabuckthorn leaves (SL) may have Anti-Obesity and hypoglycemic effects. To investigate the effect of ethanolic extract of SL, 32 C57BL/6J mice were randomly divided into 4 dietary groups, containing 8 mice in each group: normal diet group; high-fat diet (HD) control group; high-fat diet with SL extract, 500 mg/kg body weight (BW) (SL1) group; and high-fat diet with SL extract, 1000 mg/kg BW (SL2) group. After 13 weeks, it was observed that oral administration of SL extract significantly Reduced the energy intake; BW gain; epididymal fat pad weight; hepatic triglyceride, hepatic, and serum total cholesterol levels; and serum leptin levels in the SL groups compared to the HD group. However, differences in serum triglyceride and insulin levels in the SL groups were not significant in comparison to the HD group.

The hepatic mRNA expression of peroxisome proliferator-activated receptor (PPAR) α and carnitine palmitoyltransferase 1 along with PPAR-γ were significantly increased in SL groups, whereas the level of acetyl-CoA carboxylase was significantly Reduced in SL groups compared to HD group. Our results indicated that SL is effective in preventing BW gain and Fat Accumulation in the liver; it also Reduced Adipose Tissue Mass , hepatic lipid profile, and serum leptin level in the mouse. Together, these observations suggest that SL is a potential agent to study in the management of Obesity and related disorders.

Hwangryunhaedok-tang

Inhibitory Effects of Hwangryunhaedok-Tang in 3T3-L1 Adipogenesis by Regulation of Raf/MEK1/ERK1/2 Pathway and PDK1/Akt Phosphorylation

Hwangryunhaedok-tang (HRT) has been long used as traditional medicine in Asia. However, Inhibitory role of HRT is unclear in early stage of 3T3-L1 Adipocyte Differentiation related to signaling. In the present study, we investigated the Inhibitory effects of HRT on upstream signaling of peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-β (C/EBP-β) expression in Differentiation of 3T3-L1 preadipocytes. We found that HRT significantly Inhibited the Adipocyte Differentiation by downregulating several Adipocyte -specific transcription factors including PPAR-γ, C/EBP-α, and C/EBP-β in 3T3-L1 preadipocytes.

Furthermore, we observed that HRT markedly Inhibited the Differentiation media-mediated phosphorylation of Raf/extracellular mitogen-activated protein kinase 1 (MEK1)/signal-regulated protein kinase 1/2 (ERK1/2) and phosphorylation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. These results indicate that anti-Adipogenesis mechanism involves the downregulation of the major transcription factors of Adipogenesis including PPAR-γ and C/EBP-α through Inhibition of Raf/MEK1/ERK1/2 phosphorylation and PDK1/Akt phosphorylation by HRT.

Furthermore, high performance liquid chromatography (HPLC) analysis showed HRT contains active AntiObesity constituents such as palmatine, berberine, geniposide, baicalin, baicalein, and wogonin. Taken together, this study suggested that anti-Adipogenesis effects of HRT were accounted by downregulation of Raf/MEK1/ERK1/2 pathway and PDK1/Akt pathway during 3T3-L1 Adipocyte Differentiation .

Hyperoside (Hypericum perforatum L)

EFFECT OF HYPEROSIDE ON THE Inhibition OF Adipogenesis IN 3T3-L1 Adipocytes

Context: The Inhibition of Adipocyte Differentiation has a significant role on the prevention of Obesity and Obesity-associated complications.

Objective: In this study, we aimed to detect whether hyperoside is able to Inhibit the conversion of pre-adiposits into mature adiposits.

Design and Methods: 3T3-L1 Pre-Adipocytes were stimulated so as to differentiate into mature Adipocytes. Hyperoside in non-cytotoxic concentrations (1, 2, 5, and 10 µM) were separately applied to differentiated 3T3-L1 cells. Oil red O staining was performed and triacylglycerol contents were measured. Furthermore, gene and protein expressions of transcription factors, Adipogenic genes and adipokines were examined in order to investigate the effect of hyperoside on Adipocyte Differentiation.

Results: Hyperoside in high concentrations significantly suppressed the Adipogenic process by Inhibiting the expression of transcription factors and Adipogenic genes and reducing lipid accumulation in Adipocytes (p<0.05). Low doses of hyperoside are able to Inhibit Adipogenesis , but higher doses are needed to Reduce Fat Accumulation in mature Adipocytes. In the case of maturing preadipocytes, 5 μM of hyperoside exerts its AntiAdipogenic effect at the early stages of Adipogenesis, whereas 10 μM of hyperoside acts at the later stages (p<0.05).

Conclusion: These results suggest that hyperoside has a beneficial effect on the prevention and treatment of Obesity.

Hypericum perforatum

Hypolipidemic and AntiObesity -like activity of standardised extract of Hypericum perforatum L. in rats

In our study, 15 days of repeated oral administration of Hypericum perforatum extract and clofibrate have shown a significant hypolipidemic activity in normal rats. Elevated plasma lipoprotein level, especially hypercholesterolemia, results from increased absorption of cholesterol from the intestine or enhanced endogenous synthesis [25]. Therefore, there could be two possible underlying mechanisms of observed hypolipidemic activity of plant extracts, that is, the blockade of biosynthesis of cholesterol or decrease in dietary cholesterol absorption from the intestine by binding with bile acids within the intestine and increasing bile acids excretion.

In the present study, HpE not only showed hypolipidemic activity in normal rats but also normalised the lipid abnormalities induced by HFD or fructose feeding. Effects of HpE on lipid parameters are consonant with the earlier finding of Laggner et al. [26]. HpE also significantly Reduced the increased plasma glucose level in fructose or HFD-fed rats indicating improvement in insulin functions. Thus, reversal of fructose-induced insulin resistance appears to be the likely mechanism responsible for the observed effects of HpE on lipid parameters.

HpE showed significant Inhibition in Weight Gain induced by high-fat diet or fructose feeding. Serotonin is important neurotransmitter that controls increase in body Mass and is involved in the pathophysiology of Obesity as well as depression [27]. Serotonin release is increased upon intake of carbohydrates. Serotonin regulates the overconsumption of carbohydrate-rich foods. Serotonin has also been reported to decrease food intake in fructose-fed rats [28]. Hypericum perforatum increases the quantity of serotonin present within synaptosomes by Inhibiting synaptosomal uptake of serotonin [29]. This increased level of serotonin caused by HpE Reduces the food intake and Suppresses the appetite [30, 31]. Thus increased serotonergic transmission might be the connecting link between antidepressant- and AntiObesity -like activity of HpE.

Icaritin

Icaritin stimulates osteogenic Differentiation and Inhibits Adipogenesis of mesenchymal stem cells

This was one of the first studies demonstrating that the semisynthetic molecule Icaritin could promote osteogenic Differentiation and Inhibit their Adipogenesis of MSCs. These results demonstrate also that the pro-osteogenic effect of Icaritin is associated with regulation of GSK3β activity, and its Anti-Adipogenic effect is associated with blocking PPARγ signaling. The in vitro study indicates that Icaritin has the potential for further development into anti-osteoporosis drug.

Irvingia gabonensis seed

Inhibition of Irvingia gabonensis seed extract (OB131) on Adipogenesis as mediated via down regulation of the PPARgamma and Leptin genes and up-regulation of the adiponectin gene

Background: Endeavors to manage Obesity have been heavily reliant on controlling energy intake and expenditure equilibrium, but have failed to curtail the overweight and Obesity epidemic. This dynamic equilibrium is more complex than originally postulated and is influenced by lifestyle, calorie and nutrient intake, reward cravings and satiation, energy metabolism, stress response capabilities, immune metabolism and genetics. Fat metabolism is an important indicator of how efficiently and to what extent these factors are competently integrating. We investigated whether an Irvingia gabonensis seed extract (IGOB131) would provide a more beneficial comprehensive approach influencing multiple mechanisms and specifically PPAR gamma, leptin and adiponectin gene expressions, important in Anti-Obesity strategies.

Methods: Using murine 3T3-L1 Adipocytes as a model for adipose cell biology research, the effects of IGOB131 were investigated on PPAR gamma, adiponectin, and leptin. These Adipocytes were harvested 8 days after the initiation of Differentiation and treated with 0 to 250 microM of IGOB131 for 12 and 24 h at 37 degree C in a humidified 5 percent CO2 incubator. The relative expression of PPAR gamma, adiponectin, and leptin in 3T3-L1 Adipocytes was quantified densitometrically using the software LabWorks 4.5, and calculated according to the reference bands of beta-actin.

Results: The IGOB131 significantly Inhibited Adipogenesis in Adipocytes . The effect appears to be mediated through the down-regulated expression of Adipogenic transcription factors (PPAR gamma) [P less than 0.05] and Adipocyte -specific proteins (leptin) [P less than 0.05], and by up-regulated expression of adiponectin [P less than 0.05].

Conclusion: IGOB131 may play an important multifaceted role in the control of Adipogenesis and have further implications in in-vivo anti Obesity effects by targeting the PPAR gamma gene, a known contributory factor to Obesity in humans.

Ivy gourd (Coccinia grandis L. Voigt) root

Ivy gourd (Coccinia grandis L. Voigt) root Suppresses Adipocyte Differentiation in 3T3-L1 cells

Our results have shown for the first time, that ivy gourd root possessed an Anti-Obesity property. It acted directly on Pre-Adipocytes by Inhibiting their Differentiation through down-regulation of at least the key Adipogenic transcription factor-PPARγ. The presence of possible Anti-Adipogenic agent in this plant might be relevant to its use to improve metabolic diseases induced by Obesity, in addition to having a blood sugar lowering effect. We are now attempting to identify its active component(s). Further study is also necessary to evaluate the Anti-Obesity effect of ivy gourd root in experimental animals.

Kaempferol

TAZ is required for the osteogenic and Anti-Adipogenic activities of kaempferol

Kaempferol (KMP) exerts protective effects against both osteoporosis and obesity by regulating cellular activities, but the underlying molecular mechanisms have not been fully elucidated. TAZ (transcriptional coactivator with PDZ-binding motif) modulates both osteoblast and adipocyte differentiation from mesenchymal stem cells by stimulating the activities of RUNX2 (runt-related transcription factor 2) and suppressing the activities of PPARγ (peroxisome proliferator-activated receptor γ). In this study, we investigated the effects of KMP on TAZ regulated osteoblast and adipocyte differentiation. KMP increased the osteoblast differentiation of mesenchymal cells by facilitating the physical interaction between TAZ and RUNX2, thus the increasing transcriptional activities of RUNX2.

KMP also enhanced the association of TAZ with PPARγ, thereby suppressing the gene transcription of PPARγ targets and resulting in diminished adipocyte differentiation. Interestingly, the regulatory effects of kaempferol on RUNX2 and PPARγ-mediated transcriptional activity were impaired in TAZ-null mouse embryonic fibroblasts but recovered by restoration of TAZ expression. Our results demonstrate that KMP fortifies TAZ activity, which enhances RUNX2-mediated osteoblast differentiation and suppresses PPARγ-stimulated adipocyte differentiation, indicating the potential of KMP as an effective therapeutic reagent for controlling bone loss and adiposity through TAZ activation.

Anti-Adipogenic effect of kaempferol, a component of Polygonati rhizoma

Objective: It has been reported that Polygonati rhizoma (Pr) has anti-hyperglycemia, anti-triglycemia, anti-diabetic, and anti-tumor activity. Total extract of Pr was tested to identify anti-adipogenic activity in 3T3-L1 differentiation and molecular mechanism of Pr in 3T3-L1 differentiation.

Methods: Differentiation of 3T3-L1 pre-adipocyte was induced in the presence of Pr extract and kaempferol. The level of lipid accumulation was measured by Oil Red O staining. The expression of genes associated with adipocyte differentiation was measured by RT-PCR. Results: Extract of Pr and its component kaempferol reduced lipid accumulation in 3T3-L1 during adipogenesis and also reduced mRNA levels of genes associated with adipogenesis, such as adipsin, aP2, LPL, SERBP-1c and

P P A R γ

Conclusions: In this study, we showed that the molecular mechanism of Pr and kaempferol activity is related to regulation of

P P A R γ

expression and activation.

Anti–Obesity effects of kaempferol by Inhibiting Adipogenesis and increasing Lipolysis in 3T3-L1 cells

Kaempferol is a natural flavonoid widely found in fruits, vegetables, and tea. Kaempferol possesses beneficial biological properties such as anti-inflammatory and antioxidant activities. Positive energy balance during obesity correlates with a pro-inflammatory chronic state. In this context, we hypothesized that kaempferol might promote anti-obesity effects by modulating adipogenesis and lipolytic pathways. Adipocyte viability at 24, 48, and 72 h was measured by an ATP-based assay. Pre-adipocytes (day 0) or mature adipocytes (day 12) were treated with 60 μM kaempferol until day 21 to evaluate its potential anti-adipogenic and lipolytic effect, respectively.

Total lipid accumulation was assessed using Oil Red O staining assay. Gene expression was measured by RT-qPCR to evaluate the effect of kaempferol on adipogenesis and lipolysis gene expression. Our results showed a dose-dependent effect of kaempferol treatment on cell viability promoting cell death at higher than 60 μM concentration. Pre-adipocytes stimulation by 60 μM kaempferol resulted in 62% adipogenesis inhibition whereas in mature adipocytes, it reduced 39% intracellular lipid accumulation. Also, 60 μM kaempferol treatment decreased Cebpa mRNA expression when compared to control cells.

In contrast, Pnpla2 and Lipe gene expression were upregulated in 3T3-L1 cells incubated with 60 μM kaempferol. In summary, our results showed that kaempferol modulates adipogenic differentiation in 3T3-L1 cells by promoting downregulation of Cebpa gene expression and decreasing lipid accumulation in mature adipocytes by its positive effects on Pnpla2 and Lipe mRNA levels. Kaempferol might display an anti-obesity effect.

Kaempferol Suppresses lipid accumulation by Inhibiting early Adipogenesis in 3T3-L1 cells and zebrafish

Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression.

Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ).

These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism.

Negative regulation of Adipogenesis by kaempferol, a component of Rhizoma Polygonati falcatum in 3T3-L1 cells

Rhizoma Polygonati falcatum (RPF) has been used as a traditional herbal medicine in Asia, because of its anti-hyperglycemic, anti-triglycemic, and anti-tumor activity. In this study, we determined the anti-adipogenic potential of RPF extract and its component kaempferol in 3T3-L1 adipocytes, and the underlying molecular mechanism(s) using microarray analysis. Adipocyte differentiation of 3T3-L1 cells was significantly impaired by RPF extract and kaempferol as monitored by Oil Red O staining and quantitative measurement of lipid accumulation.

Additionally, the mRNA expression of adipogenesis genes decreased on treatment with kaempferol. The role of kaempferol at the genome-wide level was further assessed by a microarray approach. Our analysis indicated that kaempferol decreased the expression of adipogenic transcription factors (Pparγ, Cebpβ, Srebp1, Rxrβ, Lxrβ, Rorα) and genes involved in triglyceride biosynthesis (Gpd1, Agpat2, Dgat2), while increasing lipolysis-related genes, such as Tnfα, Lsr, and Cel. Finally, co-transfection assays using luciferase reporter gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis using peroxisome proliferator-activated receptor-γ (PPARγ) target genes indicated that kaempferol significantly repressed rosiglitazone-induced PPARγ transcriptional activity.

Overall, our data suggests that kaempferol, a major component of RPF, may be beneficial in obesity, by reducing adipogenesis and balancing lipid homeostasis partly through the down-regulation of PPARγ.

Screening of Korean natural products foranti–Adipogenesis properties and isolation of kaempferol-3-O-rutinoside as a potent anti-adipogenetic compound from …

In this study, the anti-adipogenetic activity of 300 plant extracts was investigated using an Oil Red O staining assay in a 3T3-L1 cell line. Our results indicate that three plants, including the stem and leaf of Physalis angulata, the whole grass of Solidago virgaurea, and the root of Dioscorea nipponica, produced over 90% inhibition of adipogenesis.

Kaempferol-3-O-rutinoside, which demonstrated a 48.2% inhibitory effect on adipogenesis without cytotoxicity, was isolated from the butanol layer of a water extract of S. virgaurea guided by the anti-adipogenesis assay in 3T3-L1. PPAR-γ and C/EBPα expression levels were determined using western blot, and our results indicate that kaempferol-3-O-rutinoside has a strong anti-adipogenic effect in 3T3-L1 cells through the suppression of increases in PPAR-γ and C/EBPα expression.

Screening of potential Anti-Adipogenic effects of phenolic compounds showing different chemical structure in 3T3-L1 preadipocytes

This study was designed to analyze the anti-adipogenic effect of fifteen phenolic compounds from various chemical groups in 3T3-L1 pre-adipocytes. Cells were treated with 25 μM, 10 μM or 1 μM of apigenin, luteolin, catechin, epicatechin, epigallocatechin, genistein, daizein, naringenin, hesperidin, quercetin, kaempferol, resveratrol, vanillic acid, piceatannol and pterostilbene for 8 days. At 25 μM lipid accumulation was reduced by all the compounds, with the exception of catechin, epicatechin and epigallocatechin.

At a dose of 10 μM apigenin, luteolin, naringenin, hesperidin, quercetin and kaempferol induced significant reductions, and at 1 μM only naringenin, hesperidin and quercetin were effective. The expression of c/ebpα was not. C/ebpβ was significantly reduced by genistein and kaempferol, pparγ by genistein and pterostilbene, srebp1c by luteolin, genistein, hesperidin, kaempferol, pterostilbene and vanillic acid, and lpl by kaempferol. In conclusion, the most effective phenolic compounds are naringenin, hesperidin and quercetin. Differences were found in terms of effects on the expression of genes involved in adipogenesis among the analyzed compounds.

Phenolic compounds apigenin, hesperidin and kaempferol Reduce in vitro lipid accumulation in human Adipocytes

Background: Adipocytes derived from human mesenchymal stem cells (MSCs) are widely used to investigate adipogenesis. Taking into account both the novelty of these MSCs and the scarcity of studies focused on the effects of phenolic compounds, the aim of the present study was to analyze the effect of apigenin, hesperidin and kaempferol on pre-adipocyte and mature adipocytes derived from this type of cells. In addition, the expression of genes involved in TG accumulation was also measured.

Methods: Pre-adipocytes were cultured from day 0 to day 8 and mature adipocytes for 48 h with the polyphenols at doses of 1, 10 and 25 µM.

Results: Apigenin did not show an anti-adipogenic action. Pre-adipocytes treated with hesperidin and kaempferol showed reduced TG content at the three experimental doses. Apigenin did not modify the expression of the main adipogenic genes (c/ebpβ, c/ebpα, pparγ and srebp1c), hesperidin inhibited genes involved in the three phases of adipogenesis (c/ebpβ, srebp1c and perilipin) and kaempferol reduced c/ebpβ. In mature adipocytes, the three polyphenols reduced TG accumulation at the dose of 25 µM, but not at lower doses. All compounds increased mRNA levels of atgl. Apigenin and hesperidin decreased fasn expression. The present study shows the anti-adipogenic effect and delipidating effects of apigenin, hesperidin and kaempferol in human adipocytes derived from hMSCs. While hesperidin blocks all the stages of adipogenesis, kaempferol only inhibits the early stage. Regarding mature adipocytes, the three compounds reduce TG accumulation by activating, at least in part, lipolysis, and in the case of hesperidin and apigenin, also by reducing lipogenesis.

Conclusions: The present study shows for the first time the anti-adipogenic effect and delipidating effect of apigenin, hesperidin and kaempferol in human adipocytes derived from MSCs for the first time.

Keywords: Adipocytes; Apigenin; Hesperidin; Human mesenchymal stem cells; Kaempferol; Obesity.

Kaempferol slows intervertebral disc degeneration by modifying LPS-induced osteogenesis/Adipogenesis imbalance and Inflammation response in BMSCs

Intervertebral disc (IVD) degeneration is a common disease that represents a significant cause of socio-economic problems. Bone marrow-derived mesenchymal stem cells (BMSCs) are a potential autologous stem cell source for the nucleus pulposus regeneration. Kaempferol has been reported to exert protective effects against both osteoporosis and obesity. This study explored the effect of kaempferol on BMSCs differentiation and inflammation. The results demonstrated that kaempferol did not show any cytotoxicity at concentrations of 20, 60 and 100μM. Kaempferol enhanced cell viability by counteracting the lipopolysaccharide (LPS)-induced cell apoptosis and increasing cell proliferation. Western blot analysis of mitosis-associated nuclear antigen (Ki67) and proliferation cell nuclear antigen (PCNA) further confirmed the increased effect of kaempferol on LPS-induced decreased viability of BMSCs.

Besides, kaempferol elevated LPS-induced reduced level of chondrogenic markers (SOX-9, Collagen II and Aggrecan), decreased the level of matrix-degrading enzymes, i.e., matrix metalloprotease (MMP)-3 and MMP-13, suggesting the osteogenesis of BMSC under kaempferol treatment. On the other hand, kaempferol enhanced LPS-induced decreased expression of lipid catabolism-related genes, i.e., carnitine palmitoyl transferase-1 (CPT-1). Kaempferol also suppressed the expression of lipid anabolism-related genes, i.e., peroxisome proliferators-activated receptor-γ (PPAR-γ). The Oil red O staining further convinced the inhibition effect of kaempferol on BMSCs adipogenesis.

In addition, kaempferol alleviated inflammatory by reducing the level of pro-inflammatory cytokines (i.e., interleukin (IL)-6) and increasing anti-inflammatory cytokine (IL-10) via inhibiting the nucleus translocation of nuclear transcription factor (NF)-κB p65. Taken together, our research indicated that kaempferol may serve as a novel target for treatment of IVD degeneration.

Keywords: Adipogenesis; BMSCs; IVD degeneration; Inflammation; Kaempferol; Osteogenesis.

Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without Adipogenesis activity

Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist.

When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

Komulkosiraegi [Gracilaria vermiculophylla (Ohmi) Papenfuss]

Anti-Obesity effect of komulkosiraegi [Gracilaria vermiculophylla (Ohmi) Papenfuss] extract in 3T3-L1 cells

The ingestion of edible seaweed has long been believed to be beneficial to human health due to its numerous biological actions. In the present study, the ethyl acetate fraction of a komulkosiraegi [Gracilaria vermiculophylla (Ohmi) Papenfuss] ethanol extract (GEFr) was found to potently Inhibit Adipogenesis of 3T3-L1 preadipocytes, decreasing triglycerol accumulation and the expression of peroxisome proliferator-activated receptor γ (PPARγ ), members of the CCAAT/enhancer-binding protein (C/EBP) family, and fatty acid binding protein 2 (aP2).

In mature Adipocytes, GEFr was found to significantly activate AMP-activated protein kinase (AMPK) by activating liver kinase B1 (LKB1) and stimulating intracellular reactive oxygen species generation. The mRNA levels of genes involved in lipid catabolism were up-regulated. Also, GEFr increased Lipolysis in a dose-dependent manner.

Taken together, these results suggest that GEFr has potential for use in therapies designed to improve Obesity.

Lindera obtusiloba extract 200:1

Obesity, the related Metabolic Syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea Polyphenols influencing Adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of Inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and Differentiation of (im)mature Adipocytes using 3T3-L1, an established cell line for studying Adipogenesis. L. obtusiloba extract Reduced the de‑novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC₅₀ of ∼135 μg/ml paralleled by induction of caspase‑3/7 mediated apoptosis.

Hormone-induced 3T3‑L1 Differentiation in the presence of L. obtusiloba extract resulted in a Reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the Adipogenesis -associated proteins glucose transporter-4 and vascular endothelial growth factor, in Reduced secretion of the proAdipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein β-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-γ. Treatment of mature Adipocytes with L. obtusiloba extract also significantly Reduced intracellular lipid droplets.

In addition to this strong interference of L. obtusiloba extract with Adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor α-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing Obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for Obesity associated with the Metabolic Syndrome.

Lupenone (Adenophora triphylla var. japonica)

Lupenone Isolated from Adenophora triphylla var. japonica Extract Inhibits Adipogenic Differentiation through the Downregulation of PPARγ in 3T3‐L1 Cells

Adenophora triphylla var. japonica (Campanulaceae) is known to have Anti‐Inflammatory and anti‐tussive effects. Dysfunction of Adipocytes and Adipose Tissue in Obesity is related to various inflammatory cytokines or adipokines. In this study, we investigated whether lupenone isolated from A. triphylla var. japonica extract Inhibits Adipocyte Differentiation and expression of Adipogenic marker genes in 3T3‐L1 preadipocytes . We demonstrated that lupenone resulted in a significant reduction in lipid accumulation and expression of Adipogenic marker genes in a dose‐dependent manner.

In addition, lupenone decreased the transcriptional activity of peroxisome proliferator‐activated receptor γ (PPARγ ) induced by troglitazone, and we also demonstrated that lupenone suppressed the PPARγ and CCAAT‐enhancer‐binding protein α (C/EBPα ) protein levels. These findings demonstrated that lupenone isolated from A. triphylla var. japonica extract effectively Inhibited Adipocyte Differentiation through downregulation of related transcription factor, particularly the PPARγ gene.

Lupeol (Lupinus micranthus Guss.)

In the Differentiation of a 3T3-L1 Preadipocyte to a mature Adipocyte , intensive triglyceride (TG) synthesis and intracellular accumulation of lipid droplets are induced. Lupeol, a lupane triterpene, markedly blocked TG synthesis and the accumulation of lipid droplets in 3T3-L1 cells stimulated with Differentiation inducers.

The analysis of gene expressions by quantitative real-time RT-PCR demonstrated that lupeol markedly Inhibited Adipogenic transcription factors, Adipogenic enzymes, and adipocytokines in mature 3T3-L1 cells. These findings suggested that lupeol strongly Inhibited 3T3-L1 cell Differentiation into mature Adipocytes in vitro.

Lysimachia foenum-graecum

Anti-Obesity effects of Lysimachia foenum-graecum characterized by decreased Adipogenesis and regulated lipid metabolism

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The Anti-Obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against Adipogenesis. To characterize its Anti-Obesity effects and to evaluate its potential as an Anti-Obesity drug, we performed various Obesity-related experiments in vitro and in vivo. In Adipogenesis assay, LFE blocked the Differentiation of 3T3-L1 Preadipocyte in a dose-dependent manner with an IC50 of 2.5 µg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 µg/ml and in vivo at 100 mg/kg/day. The Anti-Adipogenic and anti-lipogenic effect of LFE seems to be mediated by the Inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro.

Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body Weight Gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white Adipose Tissue s including abdominal subcutaneous, epididymal, and perirenal Adipose Tissue was Reduced markedly in LFE-treated HFD mice in a dose-dependent manner.

Treatment of LFE also greatly improved serum levels of Obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed Anti-Obesity effects of LFE on Adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as Anti-Obesity therapeutics.

Magnolol (Magnolia officinalis)

Aims: The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays an important role in Adipocyte Differentiation . Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 Diabetes . We investigate the effect of magnolol, an ingredient of Magnolia officinalis on Adipogenesis and glucose uptake using 3T3-L1 cells .

Main methods: The effect of magnolol on Adipocyte Differentiation was quantified by measuring Oil Rd O staining using 3T3-L1 cells and C3H10T1/2 cells. And real-time PCR and western blot were used to determine the expression of PPARγ or PPARγ target genes, respectively. The effect of magnolol on glucose uptake was performed using 3T3-L1 Adipocytes .

Key findings: Magnolol dose-dependently enhanced Adipocyte Differentiation in 3T3-L1 cells and C3H10T1/2 cells. In the early stage of Adipogenesis , magnolol induced gene expression of C/EBPδ, C/EBPα and PPARγ 2 and during Adipocyte Differentiation , it also induced the expression of PPARγ target genes such as aP2, LPL and adiponectin. In addition, magnolol it also increase expression of PAPRγ target gene such as C/EBPα and aP2 at mRNA and aP2 protein level in mature Adipocytes . In PPARγ ligand binding assays, magnolol exhibited binding affinity to PPARγ but its activity was weaker than rosiglitazone. At the same time, magnolol-induced Adipogenesis was Inhibited by co-treatment of GW9662 both 3T3-L1 cells and C3H10T1/2 cells. In mature 3T3-L1 Adipocytes , magnolol increased basal and insulin-stimulated glucose uptake accompanied by the up-regulation of mRNA and protein level of Glut4.

Significance: Our results suggest that magnolol could improve insulin sensitivity through the activation of PPARγ as a ligand.

Kudinoside-D, a triterpenoid saponin derived from Ilex kudingcha

Kudinoside–D, a triterpenoid saponin derived from Ilex kudingcha Suppresses Adipogenesis through modulation of the AMPK pathway in 3T3-L1 Adipocytes

The leaves of Ilex Kudingcha, locally named “Kudingcha” in China, has been traditionally applied for treating Obesity . Studies have demonstrated that the ethanol extract of Ilex kudingcha have Anti-Adipogenic effects. However, the constituent which was responsible for its Anti-Obesity and its underlying molecular mechanism has not yet been elucidated. This research explored the Anti-Obesity effect of kudinoside-D which was a main natural component of triterpenoid saponin from the ethanol extract of Ilex kudingcha, on lipid accumulation and the potential mechanism of action of Adipogenesis in 3T3-L1 Adipocytes.

The Adipocytes were treated with various concentrations of kudinoside D (0 to 40 μM) during Differentiation . The image-based Oil Red O staining analyses revealed that KD-D, dose dependently Reduced cytoplasmic lipid droplet in 3T3-L1 Adipocytes with the IC₅₀ is 59.49 μM. Meanwhile, major Adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα) and sterol regulatory element-binding protein 1c (SREBP-1c) were significantly repressed as well as their target genes. The phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase (ACC) expression were also increased.

In addition, the Inhibitory effects of KD-D on the expressions of PPARγ and C/EBPα were weakened when cells were cotreated with AMPK Inhibit or Compound C. These results indicated KD-D exerts Anti-Adipogenic effects through modulation of Adipogenic transcription factors via AMPK signaling pathway. And the current findings demonstrated that KD-D was a potential therapeutic candidate for alleviating Obesity and hyperlipidemia.

Mai Tong Fang

Pharmacological effects of the water fraction of key components in the traditional chinese prescription Mai Tong Fang on 3T3-L1 Adipocytes and ob/ob Diabetic Mice

Mai Tong Fang (MTF), a Chinese herbal combination, has been used for the treatment of Diabetic nephropathy in traditional medical clinics in China. However, the Anti-Adipogenic and anti-hyperglycemic effects of MTF have not been fully elucidated, so this study explored these pharmacological activities in 3T3-L1 Adipocytes and ob/ob mice, respectively, of the water fraction of milkvetch root, salviae miltiorrhizae and mulberry as key components of MTF. MTF was found to Inhibit Adipogenesis and triglyceride accumulation in 3T3-L1 Adipocytes.

Oral administration of MTF in ob/ob mice for 8 weeks, exhibited positive controls on blood glucose and body weight, and further improved glucose tolerance according to an oral glucose tolerance test. Importantly, MTF extract alleviated fat deposition and ballooning degeneration in liver tissue and blocked the increase of Adipocyte size in Adipose Tissue from treated ob/ob mice. These results indicated that the extract of key components in the traditional Chinese prescription MTF continue a potent Anti-Adipogenic and glucose-lowering agent.

Mangifera indica L. Peel

Expansion of Adipose Tissue in Obesity is associated with dysregulation of adipokines, which can lead to long-term metabolic disorders. Gallotannin derivatives from mango possess anti-inflammatory activities, but their potential role in lipid metabolism is not well investigated. In this study, 3T3-L1 preadipocytes were differentiated into Adipocytes and treated with mango Polyphenols (MG), or pyrogallol (PG) for 6 days. The Anti-Adipogenic activity of PG was demonstrated by Reduced number of lipid droplets and expressions of Adipogenic markers, such as peroxisome proliferator-activated receptor γ (PPARγ ) and CCAAT/enhancer binding protein α (C/EBPα).

In mature Adipocytes, PG promoted Adipocyte browning and increased the expressions of uncoupling protein 1 (UCP1) and sirtuin 1 (Sirt1). Knockdown of AMP-activated protein kinase (AMPK) α1 with siRNA partially abolished the effect of PG on Adipogenesis. Results indicate that gallotannin derivatives modulate lipid metabolism, at least in part, though the AMPK pathway, and possess potential to Prevent Obesity-related conditions.

mangiferin from Iris rossii Baker

Evaluation of AntiObesity Effect of Mangiferin in Adipogenesis ‐Induced Human Mesenchymal Stem Cells by Assessing Adipogenic Genes

We aimed to examine the anti‐adipogenic effects of mangiferin (xanthone glucoside) and its related mechanism. Human mesenchymal stem cells (hMSCs) were cultured in Dulbecco’s modified Eagle medium, after 60% confluence adipocyte differentiation was induced. Differentiation‐induced hMSCs were cultured in the presence and absence of 10, 20 and 40 μmol of mangiferin from day 0 to day 10. Adipocyte differentiation and lipid accumulation were significantly decreased in 40 μmol mangiferin‐treated groups when compared with the reference drugs (quercetin‐ and orlistat‐treated groups).

Using quantitative reverse transcription polymerase chain reaction, we studied the mRNA expression levels of resistin, adipocyte fatty acid‐binding protein 2 (aP2), lipoprotein lipase (LPL), peroxisome proliferator‐activated receptor (PPAR‐γ) and tumor necrosis factor‐α, in hMSCs undergoing adipocyte differentiation; treatment with mangiferin attenuated the expression of those adipogenic genes and decreased adipocyte differentiation. Mangiferin significantly inhibited hMSCs to preadipocyte differentiation within the first 2 days of treatment, indicating that the anti‐adipogenic effects of mangiferin are achieved through the inhibition of differentiation and maturation.

It is well known that various plant derived compounds presents antiobesity effect. However still there is lack of drugs for the arrest or inhibition of adipocyte differentiation from mesenchymal stem cell precursors. hMSCs treated with adipocyte differentiation medium along with mangiferin inhibits preadipocyte differentiation and maturation, via suppressing lipid accumulation. Mangiferin treatment resulted in a significant (p < 0.05) decrease in GPDH and triglyceride levels as well as an increase in LDH activity and attenuated lipid accumulation compared with untreated differentiating preadipocytes. This effect was demonstrated by the observed down‐regulation of adipogenesis related genes after hMSCs underwent induced adipocyte differentiation.

6′-O-acetyl mangiferin from Iris rossii Baker Inhibits lipid accumulation partly via AMPK activation in Adipogenesis

Effective control of white adipose tissue accumulation would provide a therapeutic strategy for obesity, which poses a growing global problem. The plant chemical mangiferin stimulates adenosine monophosphate-activated protein kinase (AMPK), which inhibits adipogenesis and has therefore been considered a therapeutic target for obesity and related diseases. We previously reported the anti-inflammatory properties of 6′-O-acetyl mangiferin (OAM). In this study, we evaluated the potential of OAM as an AMPK activator in vitro in 3T3-L1 preadipocytes.

OAM inhibited adipogenesis as indicated by lower intracellular lipid and triglyceride accumulation as well as reduced adipogenic gene and protein expression upon treatment. OAM-treated 3T3-L1 cells excreted more glycerol, indicating increased lipolysis, which was supported by increased expression of lipolysis-related genes, including adipose triglyceride lipase and hormone-sensitive lipase. We determined that OAM upregulates lipolysis via phosphorylation-dependent activation of AMPK. Further, OAM upregulated the β-oxidation pathway as indicated by enhanced expression of phosphorylated acetyl-CoA-carboxylase and long-chain acyl-CoA synthetase 1. In conclusion, OAM markedly decreased intracellular lipid accumulation by enhancing lipolysis via AMPK activation and by upregulating β-oxidation. Thus, OAM has potential as a drug for the prevention and/or improvement of obesity and related diseases and deserves further study.

Mangifera indica leaves extract and mangiferin modulate CB1 and PPARγ receptors and others markers associated with Obesity

Natural bioactive compounds may be used in obese patients because of their ability to impact on various key mechanisms involved in the complex pathophysiological mechanisms of such condition. The aim of this study was to investigate the effect of a Mangifera indica L. leaf extract (MLE) on adipogenic differentiation of murine preadipocyte cells. 3T3-L1 cells were treated during their differentiation with various concentrations of (Mangifera indica L.) leaves extract (MLE) (750, 380, 150, 75 and 35 μg) in order to assess their lipid content, adiponectin production, expression profile of genes involved in lipid metabolism, oxidative stress and inflammation.

Our results showed that MLE was particularly enriched in polyphenols (46.30 ± 0.083 mg/g) and that pharmacological treatment of cells resulted in a significant increase of adiponectin levels and reduction of intracellular lipid content. Consistently with these results, MLE resulted in a significant decrease of the expression of genes involved in lipid metabolism (FAS, PPARG, DGAT1, DGAT2, and SCD-1). In conclusion, our results suggest that MLE may represent a possible pharmacological tool for obese or metabolic syndrome patients.

Maté (Ilex paraguariensis)

Phenolic Compounds from Maté (Ilex paraguariensis) Inhibit Adipogenesis in 3T3-L1 preadipocytes

Leaves of Ilex paraguariensis are used to prepare a tea known as maté which is a common beverage in several South American countries. The ethanol extract was fractionated to identify the compounds responsible for the Anti-Adipogenic activity in 3T3-L1 cells . Extracts of both fresh and dried maté leaves were subjected to column chromatography using molecular permeation to obtain the saponin (20 % yields) and the Polyphenol extracts (40 % yields) from the fresh and dried leaves. The phenolic content was determined using high-performance liquid chromatography analysis and the Folin-Ciocalteau method. Also, maté extracts (50 μg/ml to 1,000 μg/ml) did not display citotoxicity using MTT.

The Polyphenol extract from the dried leaves was the most effective (50 μg/ml) in the Inhibition of triglyceride accumulation in 3T3-L1 Adipocytes, and rutin (100 μg/ml) likely accounted for a large portion of this activity. Additionally, maté extracts had a modulatory effect on the expression of genes related to the Adipogenesis as PPARγ 2, leptin, TNF-α and C/EBPα.

Melissa officinalis

The anti-angiogenic herbal extracts Ob-X from Morus alba, Melissa officinalis, and Artemisia capillaris Suppresses Adipogenesis in 3T3-L1 Adipocytes

Context: Growing adipose tissue is thought to require adipogenesis, angiogenesis, and extracellular matrix (ECM) remodeling. Close examination of developing adipose tissue microvasculature reveals that angiogenesis often precedes adipogenesis. Since our previous study demonstrated that Ob-X, the anti-angiogenic herbal composition composed of Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae), reduced adipose tissue mass in obese mice, we hypothesized that adipogenesis can be inhibited by Ob-X.

Objective: To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes.

Materials and methods: After differentiated 3T3-L1 adipocytes were treated with Ob-X, we studied the effects of Ob-X on triglyceride accumulation and expression of genes involved in adipogenesis, angiogenesis, and ECM remodeling.

Results: Treatment of cells with Ob-X inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or monocyte differentiation-inducing (MDI) mix. Ob-X reduced mRNA levels of angiogenic factors (vascular endothelial growth factor-A, -B, -C, -D, and fibroblast growth factor-2) and matrix metalloproteinases (MMPs; MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors [(thrombospondin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2)] in differentiated cells. MMP-2 and MMP-9 activities were also decreased in Ob-X-treated cells.

Discussion and conclusion: These results suggest that the anti-angiogenic herbal composition Ob-X inhibits differentiation of preadipocytes into adipocytes. These events may be mediated by changes in the expression of genes involved in lipogenesis, angiogenesis, and the MMP system. Thus, by reducing adipogenesis, anti-angiogenic Ob-X provides a possible therapeutic approach for the prevention and treatment of human obesity and its related disorders.

The anti-angiogenic herbal extract from Melissa officinalis Inhibits Adipogenesis in 3T3-L1 Adipocytes and Suppresses Adipocyte hypertrophy in high fat diet-induced …

Melissa officinalis L. (Labiatae; lemon balm) has been used traditionally and contemporarily as an anti-stress herb. Current hypotheses suggest that not only chronic stress promotes angiogenesis, but angiogenesis also modulates adipogenesis and obesity. Because the herbal extract ALS-L1023 from M. officinalis L. (Labiatae; lemon balm) has an anti-angiogenic activity, we hypothesized that ALS-L1023 could inhibit adipogenesis and adipocyte hypertrophy.

Materials and methods: ALS-L1023 was prepared by a two-step organic solvent fractionation from M. officinalis. The effects of ALS-L1023 on adipogenesis in 3T3-L1 adipocytes and adipocyte hypertrophy in high fat diet (HFD)-fed obese mice were measured using in vivo and in vitro approaches.

Results: ALS-L1023 inhibited angiogenesis in a dose-dependent manner in the HUVEC tube formation assay in vitro. Treatment of cells with ALS-L1023 inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or MDI differentiation mix. ALS-L1023 reduced mRNA expression of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9) in differentiated cells. In contrast, mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) increased. Protease activity, as measured by zymography, showed that activity of MMP-2 and MMP-9 decreased in ALS-L1023-treated cells. ALS-L1023 also inhibited MMP-2 and MMP-9 reporter gene expression in the presence of the MMP inducer phorbol 12-myristate 13-acetate. An in vivo study showed that ALS-L1023 not only decreased adipose tissue mass and adipocyte size, but also reduced mRNA levels of adipose tissue angiogenic factors and MMPs in HFD-fed obese mice.

Conclusions: These results suggest that the anti-angiogenic herbal extract ALS-L1023 suppresses adipogenesis and adipocyte hypertrophy, and this effect may be mediated by inhibiting angiogenesis and MMP activities. Thus, by curbing adipogenesis, anti-angiogenic ALS-L1023 yields a possible therapeutic choice for the prevention and treatment of human obesity and its associated conditions.

Metadina trichotoma

Pyrocincholic acid 3β–O–β–D-quinovopyranosyl-28-O–β–D-glucopyranoside Suppresses Adipogenesis and regulates lipid metabolism in 3T3-L1 Adipocytes

Obesity is crucially involved in many metabolic diseases, such as type 2 Diabetes, cardiovascular disease and cancer. Regulating the number or size of Adipocytes has been suggested to be a potential treatment for Obesity. In this study, we investigated the effect of pyrocincholic acid 3β–O–β–D-quinovopyranosyl-28-O–β–D-glucopyranoside (PAQG), a 27-nor-oleanolic acid saponin extracted from Metadina trichotoma, on Adipogenesis and lipid metabolism in 3T3-L1 Adipocytes. The 3T3-L1 Pre-Adipocytes were incubated with vehicle or PAQG for 6 days in Differentiation process. PAQG significantly Reduced the Adipogenesis, adiponectin secretion and the expression level of key transcription factors related to Adipogenesis, such as PPARγ, C/EBPβ, C/EBPα , and FABP4.

Moreover, PAQG increased the levels of FFA and glycerol in medium and Reduced TG level in mature Adipocytes. Interestingly, PAQG not only promoted the activation of AMPK and genes involved in fatty oxidation including PDK4 and CPT1a, but also Inhibited those genes involved in fatty acid biosynthesis, such as SREBP1c, FAS, ACCα and SCD1. In conclusion, PAQG Inhibits the Differentiation and regulates lipid metabolism of 3T3-L1 cells via AMPK pathway, suggesting that PAQG may be a novel and promising natural product for the treatment of Obesity and hyperlipidemia.

Methyl cinnamate (Zanthoxylum armatum)

Methyl Cinnamate Inhibits Adipocyte Differentiation via Activation of the CaMKK2–AMPK Pathway in 3T3-L1 preadipocytes

Methyl cinnamate, an active component of Zanthoxylum armatum, is a widely used natural flavor compound with antimicrobial and tyrosinase Inhibit or activities. However, the underlying bioactivity and molecular mechanisms of methyl cinnamate on Adipocyte function and metabolism remain unclear.

The aim of this study was to investigate the Inhibitory effect of methyl cinnamate on Adipogenesis in 3T3-L1 preadipocytes. Methyl cinnamate markedly suppressed triglyceride accumulation associated with down-regulation of Adipogenic transcription factor expression, including sterol regulatory element binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor γ (PPARγ ), and CCAAT/enhancer-binding protein α (C/EBPα).

Additionally, methyl cinnamate-Inhibited PPARγ activity and Adipocyte Differentiation were partially reversed by the PPARγ agonist troglitazone. Furthermore, methyl cinnamate stimulated Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and phospho-AMP-activated protein kinase (AMPK) expression during Adipogenesis. This study first revealed methyl cinnamate has AntiAdipogenic activity through mechanisms mediated, in part, by the CaMKK2–AMPK signaling pathway in 3T3-L1 cells.

Mollugin (from Rubia cordifolia L roots)

Effect of mollugin on apoptosis and Adipogenesis of 3T3‐L1 preadipocytes

The effect of mollugin, isolated from the roots of Rubia cordifolia L., on cell viability, apoptosis and Adipogenesis in 3T3‐L1 preadipocytes was investigated. The Inhibitory effect of mollugin (40–60 µm) on cell viability was more significant in differentiated Adipocytes than in 3T3‐L1 preadipocytes . In 3T3‐L1 cells, the cytotoxicity of mollugin was accompanied by apoptotic events including mitochondrial membrane potential (Δψm) loss and activation of caspase‐9, ‐3 and ‐7, leading to PARP degradation. Although the presence of 20 µm mollugin during induced adipocytic Differentiation of 3T3‐L1 cells for 6 days failed to affect the cell viability, it could almost completely abrogate the Differentiation‐associated morphology change and intracellular lipid accumulation.

A similar level of Inhibition was observed, when 20 µm mollugin was present during the early stage (D0–D2) of the Differentiation period. In addition, the expression of C/EBPα, PPARγ 1 and PPARγ 2 was significantly down‐regulated. The presence of 20 µm mollugin during either middle stage (D2–D4) or late stage (D4–D6) of the Differentiation period, however, caused the Inhibition to a lesser extent. These results indicated that mollugin at high concentrations (40–60 µm) exerted cytotoxicity via inducing apoptosis, whereas mollugin at a low concentration (20 µm) suppressed adipocytic Differentiation without exerting cytotoxicity in 3T3‐L1 preadipocytes.

Momordica charantia

Momordica charantia (bitter melon) Inhibits primary human Adipocyte Differentiation by modulating Adipogenic genes

Background: Escalating trends of Obesity and associated type 2 Diabetes (T2D) has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM) that is traditionally used to treat Diabetes and complications has been demonstrated to alleviate hyperglycemia as well as Reduce Adiposity in rodents. However, its effects on human Adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ) on lipid accumulation and Adipocyte Differentiation transcription factors in primary human differentiating preadipocytes and Adipocytes.

Methods: Commercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after Differentiation. Cytotoxicity, lipid accumulation, and Adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR).

Results: Preadipocytes treated with varying concentrations of BMJ during Differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of Adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ ) and sterol regulatory element-binding protein 1c (SREBP-1c) and adipocytokine, resistin. Similarly, Adipocytes treated with BMJ for 48 h demonstrated Reduced lipid content, perilipin mRNA expression, and increased Lipolysis as measured by the release of glycerol.

Conclusion: Our data suggests that BMJ is a potent Inhibit or of lipogenesis and stimulator of Lipolysis activity in human Adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to Reduce Adipogenesis in humans.

Moringa oleifera leaves (drumstick tree)

A dietary vegetable, Moringa oleifera leaves (drumstick tree) induced fat cell apoptosis by Inhibiting Adipogenesis in 3T3-L1 Adipocytes

M. oleifera belonging to the family Moringaceae is an effective remedy for malnutrition and it’s often referred as panacea because it cures more than 300 diseases. It’s widely been used by Indians and Africans. In our present study, Moringa oleifera leaf extract (MLE) has been explored for the ability to alleviate adipogenesis and promote apoptosis in 3T3-L1 cells. Iso-quercetin was a major component isolated from the leaves of M. oleifera. The anti-obesity and apoptotic effect of iso-quercetin was identified by performing molecular docking studies.

The iso-quercetin and MLE showed anti-proliferative activity at the concentration of 4.95 µg/mL and 280.5 µg/mL respectively. The expression of adipogenesis related genes were downregulated with increased concentration of MLE treatment and caused the decreased accumulation of triglyceride content. Furthermore, MLE induced apoptosis in adipocyte cells were confirmed by TUNEL, Hoechst and PI staining. The apoptosis pathway related pathway genes such as BAX, BCL2 were also studied. Upon MLE treatment, BAX, a pro-apoptotic protein was up-regulated and BCL2, an anti-apoptotic protein was down-regulated and showed increased activity of caspase 3 indicating that apoptosis of adipocyte cells.

Overall, we have demonstrated that the leaves of M. oleifera may reduce adipocyte formation by promoting apoptosis and thus can be considered as functional food for controlling obesity.

Bioactivity-Guided Identification of Anti-Adipogenic Isothiocyanates in the Moringa (Moringa oleifera) Seed and Investigation of the Structure-Activity …

Due to the side effects of obesity medications, many studies have focused on the natural products used in the daily diet to control weight. Moringa seed pods and leaves are widely used as vegetables or diet supplements due to the high nutrition value.

However, no bioactivity-guided anti-adipogenic study was previously conducted.

Therefore, a preadipocyte cell line was adopted as the bioactivity assay to identify the anti-adipogenic compounds in the peeled Moringa seed. Two known sulphur-containing compounds (1 and 2) were isolated and identified. Compound 2, 4-(α-l-rhamnosyloxy) benzyl isothiocyanate, showed a great anti-adipogneic effect with an IC50 value of 9.2 μg/mL. The isothiocyanate (ITC) group in compound 2 could be responsible for the inhibitory activity.

In addition, a series of compounds with the ITC group were used to further investigate the structure-activity relationship, indicating foods containing ITC derivatives have the potential of being used to control weight.

Astragalin, (3-O-glucoside of kaempferol), isolated from Moringa oleifera leaves modulates leptin, adiponectin secretion and Inhibits Adipogenesis in 3T3-L1

Inhibition of adipogenesis is crucial and is a key area of research to develop antiobesity drugs. In this study, 3-O-glucoside of kaempferol (astragalin) was isolated from Moringa oleifera leaves and evaluated for its lipolytic and antiadipogenic activity in 3T3-L1 adipocytes. Astragalin has substantially reduced the triglycerides content and lipid accumulation in 3T3-L1 adipocytes and enhanced the glycerol release in a dose dependent manner. The assay for secreted adipocytokines confirmed that, astragalin at a concentration of 20 µg/mL significantly (p < .01) increased the secretion of adiponectin, but decreased leptin secretion in 3T3-L1 adipocytes.

In molecular studies, both the mRNA expression and corresponding protein expression of PPAR-γ, C/EBP-α, FAS, and leptin genes were downregulated while that of adiponectin was upregulated in astragalin treated groups. Taken together, astragalin of M. oleifera promotes lipolysis, suppresses adipogenesis in 3T3-L1 adipocytes, and may be considered as an effective candidate to treat obesity aliments.

Moringa oleifera leaf petroleum ether extract Inhibits lipogenesis by activating the AMPK signaling pathway

In recent years, obesity has become a key factor affecting human health. Moringa oleifera Lam. is a perennial tropical deciduous tree, which is widely used in human medicine due to its nutritional and unique medicinal value. It has a cholesterol-lowering effect, but its mechanism of action is unclear. In this study, we elucidated the inhibitory effect of M. oleifera leaf petroleum ether extract (MOPEE) on lipid accumulation by in vitro and in vivo experiments, and we described its mechanism of action. MOPEE suppressed adipogenesis in 3T3-L1 adipocytes in a dose-dependent manner and had no effect on cell viability at doses up to 400 μg/ml. Furthermore, MOPEE (400 μg/ml) significantly downregulated the expression of adipogenesis-associated proteins [peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α and β (C/EBPα and C/EBPβ), and fatty acid synthase (FAS)] and upregulated the expression of a lipolysis-associated protein [hormone-sensitive lipase (HSL)] in 3T3-L1 adipocytes.

Additionally, MOPEE (400 μg/ml) significantly increased the degree of phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase (ACC). An AMPK inhibitor reversed the MOPEE-induced activation of AMPKα and ACC in 3T3-L1 adipocytes. Animal experiments showed that, in high-fat diet (HFD) mice, MOPEE [0.5 g/kg body weight (BW)] effectively decreased BW; relative epididymal, perirenal, and mesenteric fat weight and fat tissue size; and hepatic fat accumulation. Furthermore, MOPEE markedly reduced the serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and aspartate aminotransferase (AST). Moreover, MOPEE significantly downregulated the expression of adipogenesis-associated proteins (PPARγ and FAS) and upregulated the expression of a lipolysis-associated protein [adipose triglyceride lipase (ATGL)] in HFD mice hepatic and epididymal fat tissue.

Additionally, MOPEE markedly increased the degree of phosphorylation of AMPKα and ACC in HFD mice hepatic and epididymal fat tissue. Following ultrahigh-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) analysis, three phytocompounds (isoquercitrin, chrysin-7-glucoside, and quercitrin) were identified as compounds with relatively high levels in MOPEE. Among them, quercitrin showed excellent fat accumulation inhibitory activity, and the three compounds had synergistic effects in inhibiting adipogenesis.

Taken together, MOPEE inhibits fat accumulation by inhibiting the adipogenesis and promoting the lipolysis, and this process is related to AMPK activation.

Moringa oleifera and Obesity : A review

Moringaoleifera Lam [Moringaceae] is a highly valued plant, distributed in many countries of the tropics and subtropics. It has an impressive range of medicinal uses with high nutritional value. Different parts of this plant contain a profile of important minerals, and are a good source of protein, vitamins, β-carotene, amino acids and various phenolics. In addition to its compelling water purifying powers and high nutritional value, M. oleifera is very important for its medicinal value. Various parts of this plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumor, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities, and are being employed for the treatment of different ailments in the indigenous system of medicine.

Obesity arises from increase size of individual adipose cells due to lipid accumulation and from increased number of adipocytes arising from differentiation of adipose precursor cells to mature adipocytes under the appropriate nutritional and hormonal influence. An herbal formulation LI85008F also known as adipromin has potent antiadipogenic activity in mouse adipocytes invitro. LI85008F is comprised of three medicinal plants, Moringaoleifera, Murrayakoeingii and Curcuma longa.

Morus alba

Chemical constituents of Morus alba L. and their Inhibitory effect on 3T3-L1 Preadipocyte proliferation and Differentiation

Mulberry leaf, an important traditional Chinese medicine, possesses many biological activities, including effects of anti-obesity. However, which constituents of mulberry leaf are responsible for its anti-adipogenic action is unclear. This study primarily investigated the chemical constituents from mulberry leaf and their bioactivity on the proliferation and differentiation of 3T3-L1 preadipocytes.

A new flavane derivative, (2S)-4′-hydroxy-7-methoxy-8-prenylflavan (1), together with twelve known compounds including three flavanes (2-4), three chalcones (5-7), two flavones (8-9), two benzofurans (10-11) and two coumarin (12-13) was isolated from mulberry leaf. The structure of the new compound was elucidated by various spectroscopic methods including UV, HR-ESI-MS, (1)H and (13)C NMR and CD. The results of activity screening showed that compound 2, 6 and 7 inhibited the proliferation and differentiation of 3T3-L1 preadipocytes.

Keywords: (2S)-4′-hydroxy-7-methoxy-8-prenylflavan; (2S)-4′-hydroxy-7-methoxy-8-prenylflavan (PubChem CID: No CID); 2′,4′-Dihydroxy-7′-methoxy-8-prenylflavan (PubChem CID: No CID); 2′,7-Dihydroxy-4′-methoxy-8-prenylflavan (PubChem CID: No CID); 3T3-L1 preadipocyte; 7-Hydroxy-6-methoxycoumarin (PubChem CID: 5280460); 7-Hydroxycoumarin (PubChem CID: 5281426); Astragalin (PubChem CID: 5282102); Brosimine B (PubChem CID: No CID); Flavane derivative; Isobavachalcone (PubChem CID: 5281255); Isoquercitrin (PubChem CID: 5280804); Morachalcone A (PubChem CID: 9862769); Morachalcone B (PubChem CID: No CID); Moracin C (PubChem CID: 155248); Moracin M (PubChem CID: 185848); Mulberry leaf; Obesity.

The anti-angiogenic herbal extracts Ob-X from Morus alba, Melissa officinalis, and Artemisia capillaris Suppresses Adipogenesis in 3T3-L1 Adipocytes

Objective: To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes.

Anti–Obesity Effect of Extract from Nelumbo Nucifera L., Morus Alba L., and Raphanus Sativus Mixture in 3T3-L1 Adipocytes and C57BL/6J Obese Mice

The antioxidant and anti-adipogenic activities of a mixture of Nelumbo nucifera L., Morus alba L., and Raphanus sativus were investigated and their anti-obesity activities were established in vitro and in vivo. Among the 26 different mixtures of extraction solvent and mixture ratios, ethanol extract mixture no. 1 (EM01) showed the highest antioxidant (α,α-Diphenyl-β-picrylhydrazyl, total phenolic contents) and anti-adipogenic (Oil-Red O staining) activities. EM01 inhibited lipid accumulation in 3T3-L1 adipocytes compared to quercetin-3-O-glucuronide.

Furthermore, body, liver, and adipose tissue weights decreased in the high-fat diet (HFD)-EM01 group compared to in the high-fat diet control group (HFD-CTL). EM01 lowered blood glucose levels elevated by the HFD. Lipid profiles were improved following EM01 treatment. Serum adiponectin significantly increased, while leptin, insulin growth factor-1, non-esterified fatty acid, and glucose significantly decreased in the HFD-EM01 group. Adipogenesis and lipogenesis-related genes were suppressed, while fat oxidation-related genes increased following EM01 administration. Thus, EM01 may be a natural anti-obesity agent.

Keywords: anti-obesity, Nelumbo nucifera L., Morus alba L., Raphanus sativus, 3T3-L1 adipocyte, C57BL/6J mice

Anti-oxidant and anti-obese effects of mulberry (Morus alba L.) leaf extract in 3T3-L1 cells

Recently, numerous studies reported that excessive generation of ROS stimulated expression of adipogenesis transcription factors, such as C/EBPα, β, and PPARγ in 3T3-L1 cells. Therefore, effective scavenging of ROS could be regarded as strategy for anti-adipogenic effect through suppression of adipogenesis transcription factors. In this study, we examined the anti-oxidant and anti-adipogenic effects of mulberry (Morus alba L.) leaf extract (MLE). MLE was prepared by boiling water extraction method at 121°C for 15 min. DPPH radical scavenging activity of MLE was significantly increased to 3.48%, 8.28%, 16.63%, and 25.84% between 100 and 1,000 μg/mL.

The MLE treatment for 24 h did not affect to the cell viability of confluent 3T3-L1 preadipocytes at concnetrations of 10, 50, 100, 200, 400, and 800 μg/mL. Thus, nontoxic concentration rages of MLE were treated during adipogenesis period (Day -2 to 6) from preadipocytes into adipocytes in 3T3-L1 cells. Intracellular lipid accumulation in MLE-treated 3T3-L1 adipocytes (Day 6) were quantitatively judged by ORO staining and a number of lipid droplet and size were observed using microscopy. Inhibitory activity of lipid accumulation by MLE treatment in 3T3-L1 adipocytes was significantly increased 19.52%, 16.98%, and 32.48% at 200, 400, and 800 μg/mL, respectively.

The size and a number of lipid droplet in 3T3-L1 adipocytes were observed to dose-dependently decrease by MLE treatment. The mRNA expression levels of C/EBPα, β and PPARγ were decreased at 10 and 100 μg/mL in MLE-treated 3T3-L1 adipocytes compared with adipocytes. These results showed that antioxidant activity of MLE was contributed to anti-obesity effect by inhibition of intracellular lipid accumulation through suppression of adipogenesis transcription factors, such as C/EBPα, β, and PPARγ. Therefore, MLE is expected to utilize as anti-oxidant and anti-obesity cosmetic material.

Lipolytic effect of compounds isolated from leaves of mulberry (Morus alba L.) in 3T3-L1 Adipocytes

In this study, 19 known compounds were isolated from mulberry (Morus alba L.) leaves. The lipid accumulation inhibitory activity of the isolated compounds was investigated. Compounds 4 and 12 showed good anti-adipogenic activity based on 3T3-L1 adipocytes with values of 36.6 ± 9.0 and 34.7 ± 4.0%, respectively. In addition, compounds 3, 6 and 15 showed significant inhibitory activity with values from 15.4 to 21.2% and compounds 2, 8–9 and 17–18 exhibited weak activity with values ranging from 2.1 to 10.7% at a concentration of 40.0 μM.

These results show the potentiality that mulberry leaf is an excellent inhibitory phytochemical source against lipid accumulation.

Inhibitory Effects of Ethyl Acetate-Soluble Fraction from Morus alba on Lipid Accumulation in 3T3-L1 cells

Fruits of mulberry (Morus alba) have been widely used for therapeutic purposes in Asian countries for centuries. Treatment of 3T3-L1 cells with ethanolic extracts of M. alba decreased adipocyte differentiation at 100 microg/mL by 18.6%. Treatment suppressed mRNA levels of PPARgamma and C/EBPalpha expression in 3T3-L1 cells. However, the extract did not change free glycerol release from mature adipocytes. Thus, M. alba inhibited lipid accumulation by regulating transcription factors in 3T3-L1 adipocytes without a lipolytic effect. Among the soluble- fractions, the ethyl acetate-soluble fraction had the highest antiadipogenic effects on 3T3-L1 cells.

This fraction decreasing intracellular lipid accumulation by 38.5% in response to treatment with 100 microg/mL. In addition, HPLC analysis of the ethyl acetate-soluble fraction of M. alba contained 167.7 microM of protocatechulic acid in 1 mg/mL of fraction, which inhibited lipid accumulation by 44.8% in response to treatment with 100 microM. From these results, M. alba is a possible candidate for regulating lipid accumulation in obesity.

Anti-Adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 Adipocytes

Background/Objectives: Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes.

Materials/Methods: The 3T3-L1 adipocytes were treated with different doses of MLEE for 8 days starting 2 days post-confluence. Cell viability, fat accumulation, and adipogenesis-related factors including CCAAT-enhancer-binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator 1 alpha (PGC-1α), fatty acid synthase (FAS), and adiponectin were analyzed.

Results: Results showed that MLEE treatments at 10, 25, 50, and 100 µg/ml had no effect on cell morphology and viability. Without evident toxicity, all MLEE treated cells had lower fat accumulation compared with control as shown by lower absorbances of Oil Red O stain. MLEE at 50 and 100 µg/ml significantly reduced protein levels of PPARγ, PGC-1α, FAS, and adiponectin in differentiated adipocytes. Furthermore, protein level of C/EBPα was significantly decreased by the treatment of 100 µg/ml MLEE.

Conclusion: These results demonstrate that MLEE treatment has an anti-adipogenic effect in differentiated adipocytes without toxicity, suggesting its potential as an anti-obesity therapeutic.

Structural analysis and anti–Obesity effect of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.)

A water-soluble polysaccharide JS-MP-1 was isolated from Korean mulberry fruits Oddi (Morus alba L.). Sugar linkage analysis and NMR data confirmed that it is a rhamnogalacturonan type I (RG I) polymer carrying arabinan and arabinogalactan (AG II) side chains. JS-MP-1 reduced dose-dependently the viability of 3T3-L1 pre-adipocyte cells, significantly stimulated the cleavage of caspases 9 and 3 and poly (ADP-ribose) polymerase (PARP) and decreased the ratio of Bcl-2 to Bax expression level that led to mitochondrial dysfunction and apoptosis in pre-adipocyte cells. The apoptotic death was mediated by stimulation of MAPKs (ERK and p38) signalling pathway.

These results suggest that JS-MP-1 is able to reduce the number of fat cells and the mass of adipose tissue via inhibition of pre-adipocyte proliferation and thus JS-MP-1 itself or a crude aqueous Oddi extract containing this polysaccharide can be used as functional ingredient of health-beneficial food supplements for the treatment or prevention of obesity disorders.

Keywords: Apoptosis; Mulberry fruit; Pectic polysaccharide; Pre-adipocytes; Structure.

Murta (Myrceugenia euosma) dried branches and leaves

Inhibitory effects of compounds isolated from the dried branches and leaves of murta (Myrceugenia euosma) on lipid accumulation in 3T3-L1 cells

As Obesity is a global health concern the demand for Anti-Obesity drugs is high. In this study, we investigated the Anti-Obesity effect of the dried branches and leaves of murta (Myrceugenia euosma Legrand, Myrtaceae). A methanol extract of the dried branches and leaves of murta Inhibited Adipogenesis in 3T3-L1 cells. Three known flavanones—cryptostrobin (1), pinocembrin (4), and 5,7-dihydroxy-6,8-dimethylflavanone (6), and three chalcones—2′,6′-dihydroxy-3′-methyl-4′-methoxychalcone (2), pinostrobin chalcone (3), and 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethylchalcone (5) were isolated from the active fraction.

Structures of these compounds were identified using various spectral data. Each of these compounds also Inhibited Adipogenesis in 3T3-L1 cells. In particular, compound 3 was a more potent Inhibit or of triglyceride accumulation than the positive control berberine. Gene expression studies revealed that treatment of 3T3-L1 cells with 3 lowers the expression levels of CCAAT/enhancer-binding protein α and peroxisome proliferator activator γ2 during Adipogenesis without affecting cell viability.

Treatment of 3T3-L1 cells with 3 Reduced the expression levels of mRNAs encoding sterol regulatory element-binding protein 1c and several lipogenic enzymes, including fatty acid synthase and stearoyl CoA desaturase-1. These results indicate that the methanol extract and compounds isolated from the dried branches and leaves of murta exert their Anti-Obesity effects through the Inhibition of Adipogenesis.

Myrica gale

Evaluation of Antioxidant, AntiDiabetic and AntiObesity Potential of Selected Traditional Medicinal Plants

This study evaluated potential AntiDiabetic and AntiObesity properties in vitro of selected medicinal plants. The hot water (WE) and ethanol extracts (EE) of sweet gale (Myrica gale L.), roseroot (Rhodiola rosea L.), sheep sorrel (Rumex acetosa L.), stinging nettles (Utrica dioica L.) and dandelion (Taraxacum officinale L.) were tested for total antioxidant capacity using ferric reducing antioxidant power (FRAP) and DPPH^• scavenging capacity assays, followed by α-amylase, α-glucosidase and formation of advanced glycation end products (AGE) Inhibition assays in vitro.

Myrica gale EE had the highest total phenolic content (12.4 mmol GAE/L), FRAP value (17.4 mmol TE/L) and DPPH^• scavenging activity (IC₅₀ = 3.28 mg/L). Similarly, Myrica gale also exhibited significantly lower IC₅₀ values for the percentage Inhibition of α-amylase (IC₅₀ = 62.65 mg/L) and α-glucosidase (IC₅₀ = 27.20 mg/L) compared to acarbose (IC₅₀ = 91.71 mg/L; IC₅₀ = 89.50 mg/L, respectively) (p ≤ 0.05). The 3T3-L1 Preadipocyte study also revealed that Myrica gale EE (54.8%) and stinging nettles (62.2% EE; 63.2% WE) significantly Inhibited the Adipogenesis in Adipocytes in vitro (p ≤ 0.05). Polyphenols present in these medicinal plants have the potential to use in managing type 2 Diabetes and Obesity.

Type 2 Diabetes (T2D), characterized by hyperglycemia and abnormal carbohydrate metabolism, is a leading cause of morbidity and mortality worldwide and a major economic burden (1). According to the International Diabetes Federation, about 382 million people had Diabetes in the year 2013, and the numbers are expected to get double by 2035 (2). These increased numbers of Diabetes patients are mainly (>95%) T2D which is developed due to insulin resistance and pancreatic β-cell dysfunction, leading to hyperglycemia (3). The insulin resistance and pancreatic β-cell apoptosis can be traced back to Obesity and also provide the link between T2D and Obesity (4). The postprandial blood glucose levels have been found to play an important role in the onset and developing complications of T2D (5).

One of the therapeutic strategies for managing postprandial hyperglycemia involves the Inhibition of α-amylase and α-glucosidase (6). The synthetic drugs such as acarbose and miglitol have strong Inhibitory action against α-amylase and α-glucosidase; however, they may result in abdominal distention, flatulence, vomiting, and diarrhea (7). The hyperglycemia can also result in the excessive non-enzymatic glycation of proteins and formation of advanced glycation end products (AGE). The glycation modifications can further deteriorate the pathology of Diabetes by contributing to nephropathy, cataracts, vasculopathy, and atherosclerosis (8).

A number of studies and reviews have reported that phytochemicals such as phenolics offer potential therapeutic benefits in alleviating Diabetes and Obesity complications and Inhibitory effects against α-amylase and α-glucosidase (9–13). A safer alternative for management of Obesity and T2D are medicinal plants which help to maintain low blood glucose and Prevent high blood pressure, as well as boost body antioxidant system and insulin regulation (14). Like many other metabolic disorders, T2D is also linked to Obesity (15). Infusions and decoctions of traditional medicinal plants have been used for centuries to treat various health ailments including type 2 Diabetes and Obesity -related metabolic disorders (16). The water infusion prepared from wild raspberry (Rubus idaeus L.) stem exhibited the Inhibition of α-amylase, α-glucosidase activities and anti-AGE activity (17). Dandelion (Taraxacum officinale L.) has been used in traditional cancer medicines due to its hydroxycinnamic acid derivatives and flavonoid glycosides content (18). The plants containing antioxidant compounds can protect β-cells from reactive oxygen species (ROS) and therefore, can Prevent Diabetes induced by ROS (14). Therefore, it is pertinent to explore the traditional medicinal plants, which can be utilized for lowering blood glucose levels and also Obesity -related biomarkers.

This study aimed to assess the anti-Diabetic activity in vitro of plant resources used in traditional medicine in north Newfoundland region of Canada. The extracts prepared from sweet gale (Myrica gale L.), roseroot (Rhodiola rosea L.), sheep sorrel (Rumex acetosa L.), stinging nettles (Utrica dioica L.), and dandelion (Taraxacum officinale L.) were used to compare their antioxidant and AntiDiabetic properties in vitro. The study was extended further to explore and evaluate the AntiAdipogenic ability of selected plant extracts in vitro using 3T3-L1 preadipocytes.

Nam Doc Mai

Mango fruit peel and flesh extracts affect Adipogenesis in 3T3-L1 cells †

Obesity is associated with many chronic disease states, such as Diabetes mellitus, coronary disease and certain cancers, including those of the breast and colon. There is a growing body of evidence that links phytochemicals with the Inhibition of Adipogenesis and protection against Obesity. Mangoes (Mangifera indica L.) are tropical fruits that are rich in a diverse array of bioactive phytochemicals. In this study, methanol extracts of peel and flesh from three archetypal mango cultivars; Irwin, Nam Doc Mai and Kensington Pride, were assessed for their effects on a 3T3-L1 pre-Adipocyte cell line model of Adipogenesis.

High content imaging was used to assess: lipid droplets per cell, lipid droplet area per cell, lipid droplet integrated intensity, nuclei count and nuclear area per cell. Mango flesh extracts from the three cultivars did not Inhibit Adipogenesis; peel extracts from both Irwin and Nam Doc Mai, however, did so with the Nam Doc Mai extract most potent at Inhibiting Adipogenesis. Peel extract from Kensington Pride promoted Adipogenesis. The Inhibition of Adipogenesis by Irwin (100 μg mL⁻¹) and Nam Doc Mai peel extracts (50 and 100 μg mL⁻¹) was associated with an increase in the average nuclear area per cell; similar effects were seen with resveratrol, suggesting that these extracts may act through pathways similar to resveratrol.

These results suggest that differences in the phytochemical composition between mango cultivars may influence their effectiveness in Inhibiting Adipogenesis, and points to mango fruit peel as a potential source of nutraceuticals.

Naringenin (Amacardi-um occidentale L.)

Naringenin Inhibits Adipogenesis and Reduce s Insulin Sensitivity and Adiponectin Expression in Adipocytes

Adipose Tissue development and function are widely studied to examine the relationship between Obesity and the Metabolic Syndrome. It is well documented that the inability of Adipose Tissue to properly increase its lipid storage capacity during the obese state can lead to metabolic dysfunction. In a blind screen of 425 botanicals, we identified naringenin as an Inhibit or of Adipocyte Differentiation. Naringenin is one of the most abundant citrus Flavonoids , and recent studies have demonstrated antihyperlipidemic capabilities.

These studies have largely focused on the effects of naringenin on the liver. Our biochemical studies clearly demonstrate that naringenin Inhibits Adipogenesis and impairs mature fat cell function. Naringenin specifically Inhibited Adipogenesis in a dose-dependent fashion as judged by examining lipid accumulation and induction of Adipocyte marker protein expression. In mature 3T3-L1 Adipocytes , naringenin Reduced the ability of insulin to induce IRS-1 tyrosine phosphorylation and substantially Inhibited insulin-stimulated glucose uptake in a dose-dependent manner and over a time frame of 1.5 to 24 hours.

Exposure to naringenin also Inhibited adiponectin protein expression in mature murine and human Adipocytes. Our studies have revealed that naringenin may have a negative impact on Adipocyte-related diseases by limiting Differentiation of preadipocytes, by significantly inducing insulin resistance, and by decreasing adiponectin expression in mature fat cells.

Nobiletin (Citrus nobilis Lour)

Aims: The polymethoxyFlavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these Flavonoids on the hypertrophy of mature Adipocytes .

Main methods: All experiments were performed on the murine Preadipocyte cell line 3T3-L1 . We studied the formation of intracellular lipid droplets in Adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyFlavonoids on Adipocyte Differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA.

Key findings: We demonstrated that the combined treatment of Differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into Adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 Adipocytes . Both Flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 Adipocytes . Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 Adipocytes , nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation.

Significance: Our results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature Adipocytes .

Nonivamide

Nonivamide Enhances miRNA let‐7d Expression and Decreases Adipogenesis PPARγ Expression in 3T3‐L1 Cells

Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti‐Obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and Inhibiting Adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP‐analog, nonivamide, on Adipogenesis and underlying mechanisms in 3T3‐L1 cells were studied.

Nonivamide was found to Reduce mean lipid accumulation, a marker of Adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific Inhibit or trans‐tert‐butylcyclohexanol revealed that the anti‐Adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during Adipogenesis, protein levels of the pro‐Adipogenic transcription factor peroxisome‐proliferator activated receptor γ (PPARγ ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu‐let‐7d‐5p, which has been associated with decreased PPARγ levels.

nuciferine (Lotus Leaf)

Lotus Leaf Aqueous Extract Reduces Visceral Fat Mass and Ameliorates Insulin Resistance in HFD-Induced Obese Rats by Regulating PPARγ2 Expression

Objectives: Lotus leaf is a kind of traditional Chinese medicine. We aimed to explore the effects of lotus leaf aqueous extract (LLAE) on peroxisome proliferative activated receptor γ2 (PPARγ2) expression in preadipocytes and adipocytes and further investigate its effects on high fat diet (HFD)-induced obese rats.

Methods: pGL3-Enhancer-PPARγ2 (625 bp)-Luc plasmid, a luciferase reporter gene expression plasmid containing PPARγ2 promoter, was stably transfected into 3T3-L1 preadipocytes. PPARγ2 promoter activities were determined by assaying the luciferase activities. Then PPARγ2 promoter activities in preadipocytes and PPARγ2 mRNA levels in human subcutaneous adipocytes were measured after the administration with LLAE. Additionally, the effects of LLAE on body weight, fat mass, glucose and lipid metabolism and the expression of PPARγ2, insulin receptor substrate 1 and glucose transporter 4 (GLUT4) in visceral adipose tissue (VAT) were measured in HFD-induced obese rats treated with low or high dose [0.5 or 3.0 g crude drug/(kg.d)] LLAE for 6 weeks.

Results: Ten μg/ml LLAE significantly increased the luciferase activities in 3T3-L1 cells and the stimulatory action reached 2.51 folds of controls when LLAE was 1000 μg/ml (P < 0.01). After treating 3T3-L1 cells with 100 μg/ml LLAE, the stimulatory role peaked at 32 h where it was 2.58 folds of controls (P < 0.01). Besides, 100 μg/ml LLAE significantly increased PPARγ2 mRNA levels in human adipocytes to 1.91 folds of controls (P < 0.01). In HFD-induced obese rats, administration with both low and high dose LLAE notably reduced visceral fat mass by 45.5 and 58.4%, respectively, and significantly decreased fasting serum insulin levels (P < 0.05). The high dose LLAE also significantly decreased homeostasis model assessment of insulin resistance in obese rats (P < 0.05). Furthermore, the mRNA levels of PPARγ2 and GLUT4 in VAT of obese rats were significantly increased when compared with control rats, and were notably suppressed by LLAE intervention for 6 weeks (P < 0.05).

Conclusion: LLAE significantly reduces visceral fat mass and ameliorates insulin resistance in HFD-induced obese rats. These beneficial effects of LLAE may associate with its role in stimulating PPARγ2 expression in preadipocytes and subcutaneous adipocytes and suppressing PPARγ2 and GLUT4 expression in VAT.

Oleanolic Acid (olea europaea l.leaves)

Oleanolic acid is a triterpenoid compound that is widely present in vegetables, medicinal herbs, and other plants and has potent antioxidant and antiinflammatory properties. However, the potential of oleanolic acid to offset Obesity is not clear. This study tested the hypothesis that oleanolic acid Suppresses the Differentiation of 3T3-L1 Adipocytes by downregulating cellular induction of peroxisome proliferators-activated receptor γ (PPARγ) and cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein α (C/EBPα). The 3T3-L1 Adipocytes were cultured and differentiated in Dulbecco modified Eagle medium containing 10% fetal bovine serum for 6 to 8 days in the absence and presence of 1 to 25 μmol/L oleanolic acid according to differentiating protocols.

Nontoxic oleanolic acid, at 25 μmol/L or less, dose-dependently attenuated lipid accumulation in differentiated Adipocytes as evidenced by Oil Red O staining. Western blot analysis showed that the induction of PPARγ and C/EBPα was markedly attenuated in differentiated and oleanolic acid-treated Adipocytes at their transcriptional messenger RNA levels. Furthermore, this study examined whether oleanolic acid dampened the induction of visfatin, a proinflammatory and visceral fat-specific adipokine expressed in Adipocytes. Visfatin expression was Inhibited in differentiated Adipocytes exposed to a PPARγ Inhibit or GW9662.

In addition, the visfatin production was significantly repressed in 25 μmol/L oleanolic acid-treated Adipocytes, possibly through blocking PPARγ activation. These results demonstrate that oleanolic acid may be a promising agent to disturb Adipocyte Differentiation and suppress Obesity-associated Inflammation.

Oligonol (Lychee fruit Polyphenol )

Inhibition of Adipogenesis by Oligonol through Akt-mTOR Inhibition in 3T3-L1 Adipocytes

Polyphenols have recently become an important focus of study in Obesity research. Oligonol is an oligomerized Polyphenol , typically comprised of catechin-type Polyphenols from a variety of fruits, which has been found to exhibit better bioavailability and bioreactivity than natural Polyphenol compounds. Here, we demonstrated that Oligonol Inhibits 3T3-L1 Adipocyte Differentiation by reducing Adipogenic gene expression. During Adipogenesis, Oligonol downregulated the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and δ (C/EBPδ) in a dose-dependent manner and the expression of genes involved in lipid biosynthesis.

The AntiAdipogenic effect of Oligonol appears to originate from its ability to Inhibit the Akt and mammalian target of rapamycin (mTOR) signaling pathway by diminishing the phosphorylation of ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR and forkhead box protein O1 (Foxo1).

These results suggest that Oligonol may be a potent regulator of Obesity by repressing major Adipogenic genes through Inhibition of the Akt signaling pathway, which induces the Inhibition of lipid accumulation, ultimately Inhibiting Adipogenesis.

Orientin (Commelina communis L)

Vitexin, orientin and other Flavonoids from Spirodela polyrhiza Inhibit Adipogenesis in 3T3‐L1 cells

To investigate the Adipogenesis Inhibitory effect on lipid accumulation, 3T3‐L1 cells were treated with fractions and isolated Flavonoids of Spirodela polyrhiza. An ethanol extract of S. polyrhiza was fractionated into three fractions. The butanol soluble fraction (SPB) exhibited potent AntiAdipogenesis activity and decreased C/EBPα and PPARγ protein expression level in 3T3‐L1 cells without significant cytotoxicity. The Flavonoids were isolated from SPB and their chemical structures were identified as chrysoeriol (1), apigenin (2), luteolin (3), vitexin (4), cosmosin (5), orientin (6) and luteolin‐7‐O‐β‐d‐glucoside (7) by spectroscopic analysis.

Studies on the Adipogenesis and intracellular triglyceride accumulation Inhibitory effect showed that compounds 4 and 6 had the highest activity and decreased C/EBPα and PPARγ protein expression level in 3T3‐L1 cells. These results suggest that the Flavonoids isolated from SPB, especially compounds 4 and 6, contribute to the Inhibitory activity of S. polyrhiza in 3T3‐L1 cells.

Oroxylin A

Oroxylin A, a constituent of Oroxylum indicum Inhibits Adipogenesis and induces apoptosis in 3T3-L1 cells

Oroxylin A (OA) is a flavonoid found in Oroxylum indicum, a medicinal plant with multiple biological activities. This study was taken up to investigate the effect of OA, on Adipogenesis, Lipolysis and apoptosis in 3T3 L1 cells. Pre-Adipocytes were treated with 10–40 μM OA on various days of Adipogenesis treatment schedule. Mature Adipocytes were treated with OA for Lipolysis and apoptosis studies. In maturing Pre-Adipocytes, 10 μM OA suppressed intracellular lipid accumulation by 42.19% which was confirmed by lipidTox imaging of cells.

In addition, OA decreased the nuclear translocation of PPARγ and mRNA expression of its downstream genes (FAS and LPL) along with adiponectin secretion. In mature Adipocytes, 40 μM of OA decreased cell viability by 30% of control. Annexin V/PI staining showed induction of apoptosis which was further confirmed by enhanced levels of pro-apoptotic proteins Bax, cyt c, AIF and chromatin condensation. OA enhanced TNF-αsecretion, Lipolysis and decreased Akt phosphorylation in mature Adipocytes. Findings suggest that OA possibly exerts its Anti-Obesity effect by affecting Adipocyte life cycle at critical points of Differentiation and maturity.

When we compared the potency of OA with non-methoxylated Flavonoids morin, naringenin and kaempferol on Adipocyte life cycle OA was far more potent. Thus, study clearly indicates a new role for oroxylin A as regulator of Adipocyte life cycle. In addition, study also suggested a specific role of methoxylated group in exerting Lipolysis and cytotoxic effects in mature Adipocytes.

Phloretin

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and Diabetes. It is accepted that the regulation of Adipogenesis or adipokines expression, notably adiponectin, is able to Prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 Adipocyte Differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARγ and C/EBPα, the master Adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARγ target genes such as LPL, aP2, CD36 and LXRα are up-regulated by phloretin.

We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARγ transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate Adipocyte Differentiation and function.

Phloretin and phlorizin promote lipolysis and inhibit inflammation in mouse 3 T 3‐L 1 cells and in macrophage‐adipocyte co‐cultures

Scope: Previous studies found that phloretin (PT) and phlorizin (PZ) could inhibit glucose transport, with PT being a better inhibitor of lipid peroxidation. This study aimed to evaluate the antiobesity effects of PT and PZ in 3T3‐L1 cells and if they can modulate the relationship between adipocytes and macrophages.

Methods and results: Differentiated 3T3‐L1 cells were treated with PT or PZ. Subsequently, transcription factors of adipogenesis and lipolysis proteins were measured. In addition, RAW 264.7 macrophages treated with PT or PZ were cultured in differentiated media from 3T3‐L1 cells to analyze inflammatory mediators and signaling pathways. PT significantly enhanced glycerol release and inhibited the adipogenesis‐related transcription factors. PT also promoted phosphorylation of AMP‐activated protein kinase and increased activity of adipose triglyceride lipase and hormone‐sensitive lipase. PT suppressed the nuclear transcription factor kappa‐B and mitogen‐activated protein kinase pathways when RAW 264.7 cells were cultured in differentiated media from 3T3‐L1 cells. PZ improved lipolysis and inhibited the macrophage inflammatory response less effectively than PT.

Conclusion: This study suggests that PT is more effective than PZ at increasing lipolysis in adipocytes. In addition, PT also suppresses inflammatory response in macrophage that is stimulated by differentiated media from 3T3‐L1 cells.

Panicum miliaceum L.

Effects of Panicum miliaceum L. extract on Adipogenic transcription factors and fatty acid accumulation in 3T3-L1 Adipocytes

The dietary intake of whole grains is known to Reduce the incidence of chronic diseases such as Obesity, Diabetes, cardiovascular disease, and cancer. To investigate whether there are Anti-Adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals.

The results showed that treatment of 3T3-L1 Adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly Inhibited Adipocyte Differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest Anti-Adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-γ, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-α were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in Adipocytes, which is related to biological activity and cell characteristics.

These results suggest that some cereals efficiently suppress Adipogenesis in 3T3-L1 Adipocytes. In particular, the effect of P. miliaceum L. on Adipocyte Differentiation is associated with the downregulation of Adipogenic genes and fatty acid accumulation in Adipocytes.

Pelargonidin (Pelargonium hortorum )

Pelargonidin is a natural compound that exists widely in fruits, and exerts antioxidant, anti-atherosclerotic, anti-inflammatory, anti-hyperglycemic, and anti-Diabetic activities. However, there have not been any studies concerning its Anti-Obesity potential to date. Therefore, we evaluated the Anti-Obesity potential of pelargonidin via Inhibition of Adipogenesis in 3T3-L1 cells. The cellular oil droplet content was decreased to 68.14%, 56.75%, and 48.39% and triglyceride accumulation decreased to 74.53%, 61.54%, and 47.86% after incubation with 5 μM, 10 μM, and 20 μM pelargonidin, respectively, when compared with DMSO group.

Furthermore, pelargonidin treatment led to decrease in glucose consumption. Western blot assay illustrated that the expression of PPAR-γ was suppressed to 63.25%, 47.52%, and 21.23% after incubation with 5 μM, 10 μM, and 20 μM pelargonidin when compared with DMSO group. Then, we measured the expression of some target proteins of PPAR-γ, and found that pelargonidin decreased the expressions of HMGCR, LPL, Glut4, and A-FABP. Besides, the result of Luciferase Reporter Assay indicated that pelargonidin Inhibited PPAR-γ transcription activity. These results indicated that pelargonidin exerts Anti-Adipogenic activity in 3T3-L1 cells through Inhibition of PPAR-γ signaling pathway, and pelargonidin could be used as a potential Anti-Obesity agent.

Petalonia binghamiae ( brown algae)

We previously showed that an ethanolic extract of the edible brown algae Petalonia binghamiae promotes the Differentiation of 3T3-L1 preadipocytes and decreases hyperglycemia in streptozotocin-induced Diabetic mice. Here, we report that a water-soluble extract of P. binghamiae thalli, prepared by enzymatic digestion, Inhibits Preadipocyte Differentiation and Adipogenesis in a dose-dependent manner. In differentiating 3T3-L1 preadipocytes, the extract (designated PBEE) decreased the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding proteins α and β, and fatty acid-binding protein aP2.

It also Inhibited the mitotic clonal expansion process of Adipocyte Differentiation, and it Inhibited insulin-stimulated uptake of glucose into mature 3T3-L1 Adipocytes by reducing phosphorylation of insulin receptor substrate-1. In rats with high-fat diet (HFD)-induced Obesity, PBEE exhibited potent Anti-Obesity effects. In this animal model, increases in body weight and fat storage were suppressed by the addition of PBEE to the drinking water at 500 mg/L for 30 days.

PBEE supplementation Reduced serum levels of glutamic pyruvic and glutamic oxaloacetic transaminases and increased the serum level of high-density lipoprotein cholesterol. Moreover, it significantly decreased the accumulation of lipid droplets in liver tissue, suggesting a protective effect against HFD-induced hepatic steatosis. Taken together, these data demonstrate that PBEE Inhibits Preadipocyte Differentiation and Adipogenesis in cultured cells and in rodent models of Obesity.

Peucedanum japonicum Thunberg L.

Objectives: Partially purified hexane phase (HP) of Peucedanum japonicum Thunb (PJT) was identified as an AntiObesity candidate. However, the compound responsible for the AntiObesity activity remained unknown. Thus, in this study we isolated the active compound, to determine the mechanisms related to AntiObesity activity in vitro.

Methods: The HP was fractionated, and the effect on the triacylglycerol (TG) content was evaluated in 3T3-L1 preadipocytes and HepG2 hepatocytes. On the basis of comprehensive spectroscopic analyses, the structure of the active compound was identified as pteryxin, a known compound in PJT. However, to our knowledge, its biological activities against Obesity have not been reported previously. The dose-dependent effect on the TG content, and the gene expressions related to Adipogenesis, fatty acid catabolism, energy expenditure, Lipolysis, and lipogenesis due to pteryxin (10, 15, and 20 μg/mL) were examined in vitro.

Results: Pteryxin dose dependently suppressed TG content in both 3T3-L1 Adipocytes (by 52.7%, 53.8%, and 57.4%, respectively; P < 0.05) and HepG2 hepatocytes (by 25.2%, 34.1%, and 27.4%, respectively; P < 0.05). Sterol regulatory element-binding protein-1 (SREBP-1c), fatty acid synthase (FASN), and acetyl-coenzyme A carboxylase-1 (ACC1) were down-regulated in pteryxin-treated 3T3-L1 Adipocytes (by 18%, 36.1%, and 38.2%, P < 0.05) and HepG2 hepatocytes (by 72.3%, 62.9%, and 38.8%, respectively; P < 0.05). The Adipocyte size marker gene, paternally expressed gene1/mesoderm specific transcript (MEST) was down-regulated (by 42.8%; P < 0.05), and hormone-sensitive lipase, a lipid catabolizing gene was up-regulated (by 15.1%; P < 0.05) in pteryxin-treated Adipocytes. The uncoupling protein 2 (by 77.5%; P < 0.05) and adiponectin (by 76.3%; P > 0.05) were up-regulated due to pteryxin.

Conclusion: Our study demonstrated that pteryxin in PJT plays the key role in regulating the lipid metabolism-related gene network and improving energy production in vitro. Thus, the results suggest pteryxin as a new natural compound to be used as an AntiObesity drug in the pharmaceutical industry.

Phyllanthus emblica fruit

Background: Phyllanthus emblica L. (Indian gooseberry) is widely used in the Ayurveda for thousands of years to treat health complications including disorders of the immune system, Diabetes, and Obesity.

Purpose: For the first time, our study aims to demonstrate the molecular mechanisms of the fruit extract of Phyllanthus emblica (PEFE) involved in the promotion of fat cell apoptosis and alleviation of Adipogenesis.

Methods: The active constituents from PEFE were identified using high performance liquid chromatography-Mass spectrometry (HPLC-MS). We carried out the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effects of PEFE using 3T3-L1 Pre-Adipocytes. The colonogenic assay was carried out to determine the Inhibitory effect of 3T3-L1 Adipocytes after PEFE treatment. In addition, Inhibition of pancreatic lipase activity was performed and the lipolytic activity of PEFE and digallic acid was compared with the well-known standard drug orlistat. Besides, the molecular interaction and ligand optimization between digallic and Adipogenesis /apoptosis markers were also carried out. Furthermore, to confirm fat cell apoptosis we have used several detection methods that includes Hoechst staining, PI staining, Oil staining and qPCR respectively.

Results: Digallic acid was identified as a major component in the PEFE. The IC₅₀ values of digallic acid and PEFE were found to be 3.82 µg/ml and 21.85 µg/ml respectively. PEFE and digallic acid showed significant anti-lipolytic activity compared to the standard drug orlistat. In the mature Adipocytes, PEFE significantly decreased triglyceride accumulation by downregulating adiponectin, PPARγ, cEBPα, and FABP4 respectively. We further analyzed the expression of apoptosis related genes upon PEFE treatment. Apoptotic process initiated through upregulation of BAX and downregulation of BCL2 resulting in an increased caspase-3 activity. In addition, we have also confirmed the apoptosis and DNA fragmentation in 3T3-L1 cells using Hoechst, PI and TUNEL assays.

Conclusion: PEFE negatively regulates Adipogenesis by initiating fat cell apoptosis and therefore it can be considered as a potential herbal medicinal product for treating Obesity.

Piperine (Black Pepper)

Piperine Inhibits Adipogenesis by Antagonizing PPARγ Activity in 3T3-L1 cells

This study investigated the AntiAdipogenic activity of black pepper extract and its constituent piperine in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. Both black pepper extract and piperine, without affecting cytotoxicity, strongly Inhibited the Adipocyte Differentiation of 3T3-L1 cells. The mRNA expression of the master Adipogenic transcription factors, PPARγ , SREBP-1c, and C/EBPβ, was markedly decreased. Intriguingly, mRNA levels of PPARγ target genes were also down-regulated.

Moreover, a luciferase reporter assay indicated that pipierine significantly represses the rosiglitazone-induced PPARγ transcriptional activity. Finally, GST-pull down assays demonstrated that piperine disrupts the rosiglitazone-dependent interaction between PPARγ and coactivator CBP. Genome-wide analysis using microarray further supports the role of piperine in regulating genes associated with lipid metabolism.

Overall, these results suggest that piperine, a major component of black pepper, attenuates fat cell Differentiation by down-regulating PPARγ activity as well as suppressing PPARγ expression, thus leading to potential treatment for Obesity-related diseases.

Platycodin D

Platycodin D Inhibits Adipogenesis of 3T3‐L1 cells by modulating kruppel‐like factor 2 and peroxisome proliferator‐activated receptor γ

In this study, platycodin D was found to inhibit intracellular triglyceride accumulation in 3T3-L1 cells with an IC(50) of 7.1 microM. The expression levels of genes involved in lipid metabolism such as fatty-acid-binding protein 4 and lipoprotein lipase were significantly downregulated following treatment with platycodin D.

Treatment with platycodin D also resulted in a reduction of Peroxisome proliferator-activated receptor(PPAR)gamma expression and its binding to target DNA sequence. Among the various upstream regulators of PPARgamma, the expression of Kruppel-like factor(KLF)2, an anti-adipogenic factor, was significantly upregulated following platycodin D treatment. When the upregulation of KLF2 was inhibited by KLF2 siRNA, the expression and binding of PPARgamma to its target sequence were significantly recovered under these conditions.

The results of this study suggested that anti-adipogenic effect of platycodin D involves the upregulation of KLF2 and subsequent downregulation of PPARgamma.

Platycodin D, a novel activator of AMP-activated protein kinase, attenuates Obesity in db/db mice via regulation of Adipogenesis and thermogenesis

Background: Platycodi Radix (root of Platycodon grandiflorum) and its active compound platycodin D (PD) has been previously shown to possess anti-obesity properties, but the underlying mechanisms remain poorly understood.

Purpose: The present study was aimed to evaluate the anti-obese effect of PD and reveal its mechanism of action.

Study design/methods: Genetically obese db/db mice were orally treated with PD for 4 weeks, and body weight gain, adipose tissue weight, serum parameters were measured. Then, assays on adipogenic factors, thermogenic factors, and AMP-activated protein kinase (AMPK) pathway were performed in PD-treated 3T3-L1 murine adipocytes, human adipose-derived mesenchymal stem cells (hAMSCs), and primary cultured brown adipocytes.

Results: PD treatment attenuated body weight gain, suppressed white adipose tissue weight and improved obesity-related serum parameters in db/db mice. Two major adipogenic factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were decreased by PD treatment in WAT of db/db mice, 3T3-L1 adipocytes and hAMSCs. In BAT of db/db mice and primary cultured brown adipocytes, PD treatment elevated the expressions of uncoupled protein 1 (UCP1) and peroxisome proliferator-activated receptor γ coactivator 1 α (PCG1α), the key regulators of BAT-associated thermogenesis. In addition, PD activated AMPKα both in vivo and in vitro. However, when AMPK was inhibited by compound C, PD treatment failed to suppress adipogenic factors and increase thermogenic factors.

Conclusions: PD improved obesity in db/db mice by AMPK-associated decrease of adipogenic markers including PPARγ and C/EBPα. PD increased thermogenic factors such as UCP1 and PGC1α in db/db mice and primary cultured brown adipocytes. AMPK inhibition nullified the effects of PD, suggesting its anti-adipogenic and thermogenic actions were dependent on AMPK pathway activation.

Keywords: AMP-activated protein kinase pathway; Adipogenesis; Obesity; Platycodin D; Thermogenesis.

WNT/β-catenin pathway mediates theAnti-Adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum

Aims: This study was conducted to suggest the role of WNT/β-catenin pathway in the anti-adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum.

Main methods: Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate crucial role of β-catenin in the anti-adipogenic effects of platycodin D. Real-Time PCR and Western blot were used to analyze the expression levels of mRNAs and proteins in the WNT/β-catenin pathway.

Key findings: During the adipocyte differentiation of 3 T3-L1 cells, members of the WNT/β-catenin pathway were normally down-regulated, whereas platycodin D significantly reinstated the WNT/β-catenin pathway. The mRNA and protein expressions of disheveled (DVL) 2, which stabilize β-catenin, were increased by platycodin D treatment, but the protein level of AXIN, which induces the degradation of β-catenin, was decreased in platycodin D-treated cells. The nuclear level of β-catenin was normally down-regulated during adipogenesis, but platycodin D treatment led to the accumulation of β-catenin in the nucleus which resulted in the up-regulation of its target genes, cyclin D (CCND) 1 and peroxisome proliferator-activated receptor gamma (PPAR)γ. The anti-adipogenic effects of platycodin D were significantly attenuated in β-catenin siRNA-transfected cells compared with those of control siRNA-transfected cells. β-catenin siRNA transfection significantly recovered the levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and fatty acid binding protein (FABP)4 as well as intracellular lipid droplet formation, all of which were reduced by platycodin D treatment.

Significance: WNT/β-catenin pathway can be used as a therapeutic target of natural compounds for the regulation of adipogenesis.

Platyphylloside (Betula platyphylla)

LC/MS-based Analysis of Bioactive Compounds from the Bark of Betula platyphylla var. japonica and Their Effects on Regulation of Adipocyte and Osteoblast Differentiation

Betula platyphylla var. japonica (Betulaceae), also known as Asian white birch, is an endemic medicinal tree, the bark of which has been used in Chinese traditional medicine for the treatment of various inflammatory diseases. In our continuing search for bioactive compounds from Korean natural resources, a phytochemical investigation of the bark of B. platyphylla var. japonica led to the isolation of 7-oxo-β-sitosterol (1) and soyacerebroside I (2) from its ethanol extract as main components by liquid chromatography (LC)/Mass spectrometry (MS)-based analysis.

The structures of isolates were identified by comparison of ¹H and ¹³C nuclear magnetic resonance spectroscopic data and physical data with the previously reported values and LC/MS analyses. To the best of our knowledge, this is the first study to demonstrate that the isolated compounds, 7-oxo-β-sitosterol and soyacerebroside I, were isolated in B. platyphylla var. japonica.

We examined the effects of the isolates on the regulation of Adipocytes and osteoblast Differentiation. These isolates (1 and 2) produced fewer lipid droplets compared to the untreated negative control in Oil Red O staining of the mouse mesenchymal stem cell line without altering the amount of alkaline phosphatase staining. The results demonstrated that both compounds showed marginal Inhibitory effects on Adipocyte Differentiation but did not affect osteoblast Differentiation.

Pleurotus eryngii var. ferulae ‘Beesan No. 2’ (CEBT)

The Anti-Adipogenic Activity of a New Cultivar, Pleurotus eryngiivar. ferulae ‘Beesan No. 2’, through Down-Regulation of PPAR γ andC/EBP α in 3T3-L1 cells

Adipogenesis is one of the cellular processes and a highly controlled program. Nowadays,Inhibition of Adipogenesis has received attention as an effective way to regulate Obesity. In thecurrent study, we investigated the Inhibition effect of a chloroform extract of Pleurotus eryngiivar. ferulae ‘Beesan No. 2’ (CEBT) on Adipogenesis in 3T3-L1 murine preadipocytes.

Pleurotuseryngii var. ferulae is one of many varieties of King oyster mushroom andhas been reported tohave various biological activities, including antitumor and anti-Inflammation effects.Biological activities of ‘Beesan No. 2’, a new cultivar of Pleurotus eryngii var. ferulae, have notyet been reported. In this study, we found that CEBT suppressed Adipogenesis in 3T3-L1 cells through Inhibition of key Adipogenic transcription factors, such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Additionally, CEBT Reduced theexpression of the IRS/PI3K/Akt signaling pathway and its downstream factors, includingmammalian target of rapamycin and p70S6 kinase, which stimulate Adipogenesis.

Furthermore, β-catenin, a suppressor of Adipogenesis, was increased in CEBT-treated cells.These results indicate that Pleurotus eryngii var. ferulae ‘Beesan No. 2’ effectively Inhibited Adipogenesis, so this mushroom has potential as an Anti-Obesity food and drug.

Pluchea indica (L.) Less. Tea

The Effect of Pluchea indica (L.) Less. Tea on Adipogenesis in 3T3-L1 Adipocytes and Lipase Activity

Obesity and hyperlipidemia are a major problem in the world. Pluchea indica (L.) Less. tea (PIT) is a beverage that has various indications. This study focused on the effect of the PIT on Inhibiting Adipogenesis of 3T3-L1 cells and pancreatic lipase enzyme activity. The viability of 3T3-L1 cells was not significantly decreased after exposure to 200 to 1000 μg mL⁻¹ PIT compared to controls. The PIT at 750 to 1000 μg mL⁻¹ exhibited a significantly Reduced lipid accumulation compared to the control. The Inhibitory effects of the PIT at 250 to 1000 μg mL⁻¹ on lipase activity were significantly increased compared to control. The FTIR results showed that the integrated areas of lipids, proteins, nucleic acids, glycogen, and carbohydrates of the PIT-treated 3T3-L1 Adipocytes were significantly lower than the untreated 3T3-L1 Adipocytes.

These findings may indicate that the PIT is not only capable of Inhibiting lipids and carbohydrate accumulation in Adipocytes but also has a potential to Inhibit pancreatic lipase activity. So, the PIT may be further developed to the novel lipid-lowering herbal supplement for the management of overweight or Obesity.

Polygonum cuspidatum

Polygonum cuspidatum Inhibits pancreatic lipase activity and Adipogenesis via attenuation of lipid accumulation

Background: Obesity causes metabolic disease and is a serious health problem around the world. Polygonum cuspidatum (POCU1b) has been used clinically for the treatment of constipation, gallstones, hepatitis, and inflammation in East Asian countries. The principal aim of this study was to investigate for the first time whether the extract of Polygonum cuspidatum (POCU) biologically affects adipogenesis in 3 T3-L1 preadipocytes.

Methods: Fractions (n-hexan, ethyl acetate, n-butanol, and water) of POCU ethanol extract were evaluated in vitro for their inhibitory activities on pancreatic lipase. Of the fractions, the n-butanol of POCU ethanol extract (POCU1b) was examined anti-obesity activity in 3 T3-L1 preadipocytes. To examine the inhibitory effect of POCU1b on adipogenesis, 3 T3-L1 preadipocytes were treated every the other day with POCU1b at various concentrations (0 ~ 25 μg/mL) for twelve days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation. The expression of mRNA and proteins associated lipid accumulation was measured using RT-PCR and Western blotting analysis. We also examined the effect of POCU1b on level of phosphorylated AMP-activated protein kinase (pAMPK) in 3 T3-L1 preadipocytes with POCU1b at various concentrations during adipocyte differentiation.

Results: POCU1b exhibited the most pronounced inhibitory effects on pancreatic lipase activity. We found that POCU1b inhibited adipocyte differentiation in 3 T3-L1 preadipocytes in a dose-dependent manner, as evidenced by the reduced formation of lipid droplets and decreased glycerol-3-phosphate dehydrogenase (GPDH) activity. We also showed that the expression levels of adipocyte differentiation-related protein (ADRP) and perilipin (a protein that coats lipid droplets in adipocytes) were both reduced after POCU1b treatment. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and CCAAT/enhancer-binding protein-alpha (C/EBP-α) proteins, both major adipogenic transcription factors, were markedly reduced by POCU1b. Moreover, ADRP, perilipin, C/EBP-α, and PPAR-γ mRNA levels were also reduced by POCU1b. Levels of phosphorylated AMP-activated protein kinase (pAMPK) were elevated after POCU1b treatment (5, 10, and 25) in a dose-dependent manner.

Conclusions: Taken together, these results suggest that the anti-obesity effects of POCU1b involve the inhibition of pancreatic lipase activity and adipogenesis via the down-regulation of lipid accumulation.

Porphyra yezoensis

The micosporine-like amino acids-rich aqueous methanol extract of laver (Porphyra yezoensis) Inhibits Adipogenesis and induces apoptosis in 3T3-L1 Adipocytes

Results of our study demonstrated that treatment with the laver extract caused Inhibition of Adipogenesis, a decrease in proliferation of preadipocytes, and an increase in the apoptosis of mature Adipocytes. It appears that these effects were caused by increasing oxidative stress, as demonstrated by the depletion and oxidation of the cellular glutathione pool in the extract-treated Adipocytes. Our results suggest that a prooxidant role of laver extract is associated with its AntiAdipogenic and proapoptotic effects.

Prieurianin

Prieurianin causes weight loss in diet-induced obese mice and Inhibits Adipogenesis in cultured preadipocytes

The global increase in the incidence of obesity has emerged as one of the most serious public health risks in recent years. Despite the enormity of the obesity pandemic, there are currently only two FDA-approved therapies for its treatment and these drugs exhibit modest efficacy and have limiting side effects. Prieurianin is a plant limonoid product that deters feeding in insect larvae. We investigated in this study the effects of prieurianin on weight loss and adipogenesis.

Our results showed that prieurianin causes weight loss by reducing energy intake in obese mice on high-calorie diet. We also found that prieurianin is anti-adipogenic in cultured preadipocytes and adipocytes by inhibiting proliferation and differentiation of preadipocytes into adipocytes, and induces either dedifferentiation or delipidation of mature adipocytes. Whether prieurianin can potentially be used for obesity treatment in human warrants further investigation.

Characterization of the Anti–Obesity and Anti-Adipogenic Effects of the Limonoid Prieurianin

Phospholipid transfer protein (PLTP) is critically important for reverse cholesterol transport (RCT), and its expression level and activity increase when mice are fed a high fat diet. RCT is the process by which accumulated cholesterol from the blood vessel walls, peripheral tissues and macrophages is transported back to the liver for excretion. Interestingly, topoisomerase I inhibitors used in chemotherapy have been shown in our laboratory to dose dependently induced PLTP expression in both in vivo and in vitro studies. Since PLTP transports phospholipid as well as cholesterol into high density lipoprotein (HDL), we asked whether elevated PLTP levels might increase the transfer of drugs into HDL via RCT, thus increasing tumor cells resistance to the drug.

However, we found that camptothecin, topoisomerase I inhibitor, does not accumulate in HDL or in other lipoprotein subfractions, thus ruling out the possibility of PLTP mediating the transfer of camptothecin into HDL for liver metabolism. The limonoid prieurianin, like topoisomerase I inhibitors, has also been shown to dose dependently increase PLTP mRNA and proteins levels, and here we show that prieurianin causes weight loss by reducing food intake in morbidly obese mice and in mice on high-calorie diet. Additionally, prieurianin is anti-adipogenic and (i) inhibits the proliferation and differentiation of preadipocytes into adipocytes, and (ii) induces either dedifferentiation or delipidation of mature adipocytes.

Gene expression profiling showed that prieurianin suppresses the expression of a number of genes involved in fat metabolism, and inhibits the transcriptional activity of the adipogenesis master regulators including the CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator-activated receptor gamma (PPARγ).

Procyanidin B2 (grape seed)

Procyanidin Effects on Adipocyte -Related Pathologies

Procyanidins, a class of Flavonoids , have clear and well-defined beneficial effects against several pathologies including cardiovascular heart disease. Now, studies in vivo are revealing the effects of procyanidins against Obesity, where they Prevent Weight Gain and Adipose Tissue Mass increase, and against Diabetes and insulin resistance, where they act as antihiperglycemic agents. Several mechanisms may be responsible for these effects. One of these, due to the key role of Adipose Tissue in the development of Obesity and insulin resistance, is their effect on Adipocytes.

In this review we compile the studies that indicate a protective role for procyanidins in Obesity and insulin resistance, focusing on their effects on the Adipocyte, where procyanidins modify lipid synthesis, lipid degradation, glucose uptake, and adipose Differentiation.

Pterostilbene (Blueberry)

Inhibitory effects of garcinol and pterostilbene on cell proliferation and Adipogenesis in 3T3-L1 cells

Moreover, garcinol and pterostilbene caused an Inhibition of lipid accumulation in the 3T3-L1 Adipocyte Differentiation phase. Garcinol and pterostilbene also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin, resistin, and fatty acid synthase (FAS) in 3T3-L1 Adipocyte Differentiation. In 3T3-L1 Adipocytes , garcinol significantly down-regulated the protein expressions of PPARγ and FAS as well as up-regulated the protein expressions of adipose triglyceride lipase (ATGL) and adiponectin. Garcinol also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin and FAS. These results suggest that garcinol and pterostilbene have Anti-Adipogenic effects on preadipocytes and Adipocytes.

Punicalagin

Pomegranate husk extract, punicalagin and ellagic acid Inhibit fatty acid synthase and Adipogenesis of 3T3-L1 Adipocyte

Fatty acid synthase (FAS) has been recognized as a potential therapeutic target for obesity. In this study, for the first time, the inhibitory effect of pomegranate husk extract, punicalagin and ellagic acid on FAS was investigated. We found them potently inhibiting the activity of FAS with half-inhibitory concentration values (IC₅₀) of 4.1 μg/ml (pomegranate husk extract), 4.2 μg/ml (4.50 μM, punicalagin) and 1.31 μg/ml (4.34 μM, ellagic acid), respectively. Moreover, they all exhibited time-dependent inactivation of FAS. Punicalagin and ellagic acid inhibited FAS with different mechanisms compared to previously reported inhibitors, through inactivating acetyl/malonyl transferase and β-ketoacyl synthase domains, respectively.

Additionally, 100 μg/ml pomegranate husk extract, 5.24 μg/ml (5 μM) punicalagin and 4.5 μg/ml (15 μM) ellagic acid effectively reduced lipid accumulation inside FAS over-expressed 3T3-L1 adipocytes. Since FAS plays a key role in the biosynthesis pathway of fatty acid, these findings suggest that pomegranate husk extract, punicalagin and ellagic acid have potential in the prevention and treatment of obesity.

Pomegranate polyphenols and urolithin A inhibit α-glucosidase, dipeptidyl peptidase-4, lipase, triglyceride accumulation and adipogenesis related genes in 3T3-L1 adipocyte-like cells

Ethnopharmacological relevance: Pomegranate fruit is considered an antidiabetic medicine in certain systems of traditional medicine. In addition, pomegranate polyphenols are known as powerful antioxidants with beneficial effects such as the reduction of oxidative / inflammatory stress and the increase of protective signalling such as antioxidant enzymes, neurotrophic factors and cytoprotective proteins.

Aim of the study: This work evaluates the effects of pomegranate juice, its main polyphenols known as ellagic acid and punicalagin, as well as its main metabolite urolithin A, on physiological and pharmacological targets of metabolic diseases such as obesity and diabetes.

Materials and methods: For this purpose, enzyme inhibition bioassays of lipase, α-glucosidase and dipeptidyl peptidase-4 were carried out in cell-free systems. Similarly, adipocytes derived from 3T3-L1 cells were employed to study the effects of ellagic acid, punicalagin and urolithin A on adipocyte differentiation and triglyceride (TG) accumulation.

Results: Pomegranate juice, ellagic acid, punicalagin and urolithin A were able to inhibit lipase, α-glucosidase and dipeptidyl peptidase-4. Furthermore, all tested compounds but significantly the metabolite urolithin A displayed anti-adipogenic properties in a dose-dependent manner as they significantly reduced TG accumulation and gene expression related to adipocyte formation such as adiponectin, PPARγ, GLUT4, and FABP4 in 3T3-L1 adipocytes.

Conclusion: These results may explain from a molecular perspective the beneficial effects and traditional use of pomegranate in the prevention of metabolic-associated disorders such as obesity, diabetes and related complications.

Punicalagin, a Pomegranate‐Derived Ellagitannin, Suppresses Obesity and Obesity ‐Induced Inflammatory Responses Via the Nrf2/Keap1 Signaling Pathway

Scope: Punicalagin (PCG) is one of the most abundant phytochemicals found in pomegranates. The effects and mechanistic action of PCG on obesity and obesity-induced inflammatory and oxidant responses are investigated in vitro and in vivo.

Methods and results: The effect of PCG on adipogenesis is examined using Oil red O staining. The effects and mechanism of action of PCG on inflammatory responses are determined in adipocyte-conditioned medium (ACM)-cultured macrophages, a cell-to-cell contact system, and a transwell system. The effects of PCG on obesity and obesity-induced inflammatory/oxidant responses are examined in high-fat diet (HFD)-fed mice. PCG effectively suppresses lipid accumulation in adipocytes and adipocyte-induced inflammatory responses in adipocyte-macrophage co-culture systems. Small interfering RNA (siRNA) transfection indicates that the PCG-mediated anti-inflammatory effect is exerted via the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1(Nrf2/Keap1) pathway. PCG administration results in a significant reduction in body and white adipose tissue (WAT) weights. PCG favorably regulates pro- and anti-inflammatory cytokines, downregulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Immunohistochemical (IHC) analysis demonstrates that PCG differentially modulates the distribution of complement component 3 receptor 4 subunit (CD11c) and cluster of differentiation 206 (CD206). PCG regulates the level of antioxidant and oxidant molecules by activating Nrf2/Keap1 signaling.

Conclusions: PCG ameliorates obesity and obesity-induced inflammatory responses via activation of Nrf2/Keap1 signaling, suggesting that PCG has potential as an oral agent to control obesity-mediated diseases.

Keywords: HFD-fed mice; Nrf2/Keap1 signaling; co-culture; obesity; obesity-induced inflammatory response; punicalagin.

Beneficial effects of pomegranate peel extract and probiotics on pre-Adipocyte Differentiation

The beneficial effects of pomegranate are due to the ellagitannins and anthocyanins content, which are protective toward a wide variety of diseases including inflammatory diseases. Many investigators have reported that pomegranate waste (peel and seeds) extracts, made from waste product of industrial processing, show free radical scavenger and a potent antioxidant capacity. Pomegranate extracts (PEs) were also reported to possess noteworty antibacterial, antiviral, hypolipidemic, and anti-inflammatory bioactivities thanks to the polyphenolic compounds content, which includes punicalagins, gallic acid, and ellagic acid derivatives.

The focus of the present manuscript was to study the prebiotic potentiality of a PE, soluble in water, and characterized through HPLC–PDA–ESI/MSⁿ for its phenolic content. Moreover, since it has been reported that pomegranate extracts decreased the level of lipids in the blood and that a number of probiotic strains have been shown to affect adipogenesis in cell culture, this study was also performed to test the in vitro effects of PE and probiotic L. rhamnosus GG ATCC 53103 strain (LGG) on 3T3-L1 cell line.

PE and probiotics substantially reduced the triglyceride content and intracellular lipid increase, compared to the control group. However, the combination treatment of PE and LGG filtered spent broth (SB) was the most effective in reducing triglyceride content and intracellular lipid accumulation. The mRNA expression levels of the main transcriptional factors implicated in adipocyte differentiation were substantially lower in 3T3-L1 cells treated with PE and LGG filtered SB. These results evidenced that a synergistic effect of probiotics and polyphenols contained in PE may affect in vitro adipogenesis and may contribute in development of new nutraceutical/probiotic-based remedies to prevent and to treat obesity.

Quercetin

Quercetin‐rich onion peel extract Suppresses Adipogenesis by down‐regulating Adipogenic transcription factors and gene expression in 3T3‐L1 Adip ocytes

Background: Onion peel contains a high amount of quercetin, which has been reported to have anti-cholesterol, antithrombotic and insulin-sensitizing properties. This study aimed to elucidate the anti-adipogenic effects of quercetin-rich onion peel extract (OPE) and to compare it with commercially available quercetin using 3T3-L1 preadipocytes.

Results: Without affecting cell viability, both OPE and quercetin averted adipogenesis, as characterized by dose-dependent decreases in intracellular triglyceride content and glycerol 3-phosphate dehydrogenase activity, but the effect was more pronounced with OPE than with quercetin. The mRNA expression levels of key adipogenic genes such as PPARγ, C/EBPα, FABP4, aP2 and LPL were decreased in a dose-dependent manner by both OPE and quercetin.

Conclusion: The results indicate that OPE treatment significantly prevents intracellular lipid accumulation via hyperactivation of genes regulating lipolysis as compared with quercetin alone.

Keywords: 3T3-L1 cells; adipogenesis; glycerol 3-phosphate dehydrogenase (GPDH); onion peel extract (OPE); quercetin.

Enhanced Inhibition of Adipogenesis and induction of apoptosis in 3T3-L1 Adipocytes with combinations of resveratrol and quercetin

Certain flavonoids have been shown to have specific effects on biochemical and metabolic functions of adipocytes. In this study, we investigated the effects of combinations of resveratrol and quercetin on adipogenesis and apoptosis in 3T3-L1 cells. In maturing preadipocytes resveratrol and quercetin at 25 microM individually suppressed intracellular lipid accumulation by 9.4+/-3.9% (p<0.01) and 15.9+/-2.5%, respectively, (p<0.001). The combination of resveratrol and quercetin at the same dose, however, decreased lipid accumulation by 68.6+/-0.7% (p<0.001). In addition, combinations of resveratrol and quercetin at 25 microM significantly decreased the expression of peroxisome proliferators-activated receptor gamma (PPAR gamma) and CCAAT/enhancer-binding protein (C/EBP)alpha, both of which act as key transcription factors.

In mature adipocytes resveratrol and quercetin at 100 microM individually decreased viability by 18.1+/-0.6% (p<0.001) and 15.8+/-1% (p<0.001) and increased apoptosis (100 microM) by 120.5+/-8.3% (p<0.001) and 85.3+/-10% (p<0.001) at 48 h, respectively. Combinations of resveratrol and quercetin further decreased viability (73.5+/-0.9%, p<0.001) and increased apoptosis (310.3+/-9.6%, p<0.001) more than single compounds alone. The combination of resveratrol and quercetin at 100 muM increased release of cytochrome c from mitochondria to cytosol and decreased ERK 1/2 phosphorylation. Taken together, our data indicate that combinations of resveratrol and quercetin can exert potential anti-obesity effects by inhibiting differentiation of preadipocytes and inducing apoptosis of mature adipocytes.

Cigarette smoke extract-induced Adipogenesis in Graves’ orbital fibroblasts is Inhibited by quercetin via reduction in oxidative stress

Cigarette smoking is known to aggravate Graves’ orbitopathy (GO) severity by enhancing adipogenesis. We investigated the effect of quercetin, an antioxidant, on adipocyte differentiation induced by cigarette smoke extract (CSE) in primary cultured orbital fibroblasts (OFs) from GO patients. Freshly prepared CSE was added to the cells and H(2)O(2) was used as a positive control. Intracellular reactive oxygen species (ROS) generation and adipogenesis were measured. The expressions of proteins peroxisome proliferator-activated receptor (PPAR) γ, CCAAT-enhancer-binding proteins (C/EBP) α and β, and heme oxygenase-1 (HO-1), an antioxidant enzyme, were examined during adipogenic differentiation. In result, CSE and H(2)O(2) dose-dependently stimulated intracellular ROS production in normal and Graves’ OFs. The effect of 2% CSE was similar to that of 10 μM H(2)O(2); both concentrations were noncytotoxic and were used throughout the experiment.

Quercetin pretreatment reduced the ROS generation stimulated by either CSE or H(2)O(2) in preadipocyte OFs. CSE and H(2)O(2) stimulated adipocyte differentiation in cultured OFs. The addition of quercetin (50 or 100 μM) suppressed adipogenesis. Quercetin also suppressed ROS generation in differentiating OFs during adipogenesis stimulated by CSE and H(2)O(2). Additionally, the expressions of PPARγ, C/EBPα, and C/EBPβ proteins were reduced in the quercetin-treated OFs. Quercetin also reduced the CSE- and H(2)O(2)-induced upregulation of ROS and HO-1 protein in differentiated OFs and preadipocyte OFs. As shown in this study, quercetin inhibited adipogenesis by reducing ROS in vitro, supporting the use of quercetin in the treatment of GO.

Quercetin prevents Adipogenesis by regulation of transcriptional factors and lipases in OP9 cells

With the industrialization of society, the increase in the prevalence of obesity and metabolic disorders has become an important health concern in a number of countries. Quercetin (3,30,40,5,7-pentahydroxyflavone) is well known as a bioactive flavonoid in a variety of biological resources. The aim of the present study was to explore the machanisms responsible for the anti-adipogenic activity of quercetin and its effects on the lipolysis in OP9 mouse stromal cells which rapidly differentiate into adipocytes. The differentiation of OP9 cells into adipocytes was evaluated by the measurement of lipid accumulation by Oil Red O (ORO) staining; lipid accumulation was significantly impaired by treatment with quercetin. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to measure the expression levels of CCAAT/enhancer binding protein α (C/EBPα), proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein-1 (SREBP-1) and fatty acid synthase (FAS).

The mRNA expression levels of lipases, such as adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) were also measured by RT-PCR. Quercetin significantly decreased the expression of transcription factors, including C/EBPα, PPARγ and SREBP-1c both at the protein and mRNA level. The results from the present study demonstrate that quercetin prevents adipogenesis by upregulating ATGL and HSL expression and downregulating FAS, LPL and adipocyte fatty acid-binding protein (aP2) expression, as well as the expression of transcription factors. Our data suggest that quercetin has therapeutic potential by regulating the expression of transcriptional factors and enzymes associated with adipogenesis.

Combined effects of genistein, quercetin, and resveratrol in human and 3T3-L1 Adipocytes

The natural compounds genistein (G), quercetin (Q), and resveratrol (R) have been reported to each exhibit anti-adipogenic activities in adipocytes and antiproliferative and pro-apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on adipogenesis and apoptosis in primary human adipocytes (HAs) and 3T3-L1 murine adipocyte (MAs). Combined treatment with 6.25 microM G, 12.5 microM Q, and 12.5 microM R during the 14-day differentiation period caused an enhanced inhibition of lipid accumulation in maturing HAs that was greater than the responses to individual compounds and to the calculated additive response. Glycerol 3-phosphate dehydrogenase activity, a marker of late adipocyte differentiation, was decreased markedly in HAs treated with the combination of G+Q+R. In addition, combined treatment with 50 microM G, 100 microM Q, and 100 microM R for 3 days decreased cell viability and induced apoptosis in early- and mid- phase maturing and lipid-filled mature HAs. In contrast, no compound alone induced apoptosis. Oil Red O stain and Hoechst 33342 stain were performed to confirm the effects on lipid accumulation and apoptosis, respectively.

We also determined whether MAs responded to the combination treatment similarly to HAs. As in HAs, G+Q+R treatment decreased lipid accumulation in maturing MAs and increased apoptosis in pre- and lipid-filled mature MAs more than the responses to G, Q, and R when used separately. These results show that lower concentrations of combined treatments with several natural compounds may be useful for treatments for obesity through the suppression of adipogenesis and enhanced adipocyte apoptosis.

Quercetin attenuates adipose hypertrophy, in part through activation of Adipogenesis in rats fed a high-fat diet

An impaired capacity of adipose tissue expansion leads to adipocyte hypertrophy, inflammation and insulin resistance (IR) under positive energy balance. We previously showed that a grape pomace extract, rich in flavonoids including quercetin (Q), attenuates adipose hypertrophy. This study investigated whether dietary Q supplementation promotes adipogenesis in the epididymal white adipose tissue (eWAT) of rats consuming a high-fat diet, characterizing key adipogenic regulators in 3T3-L1 pre-adipocytes.

Consumption of a high-fat diet for 6 weeks caused IR, increased plasma TNFα concentrations, eWAT weight, adipocyte size and the eWAT/brown adipose tissue (BAT) ratio. These changes were accompanied by decreased levels of proteins involved in angiogenesis, VEGF-A and its receptor 2 (VEGF-R2), and of two central adipogenic regulators, i.e. PPARγ and C/EBPα, and proteins involved in mature adipocyte formation, i.e. fatty acid synthase (FAS) and adiponectin. Q significantly reduced adipocyte size and enhanced angiogenesis and adipogenesis without changes in eWAT weight and attenuated systemic IR and inflammation. In addition, high-fat diet consumption increased eWAT hypoxia inducible factor-1 alpha (HIF-1α) levels and those of proteins involved in adipose inflammation (TLR-4, CD68, MCP-1, JNK) and activation of endoplasmic reticulum (ER) stress, i.e. ATF-6 and XBP-1. Q mitigated all these events. Q and quercetin 3-glucoronide prevented TNFα-mediated downregulation of adipogenesis during 3T3-L1 pre-adipocytes early differentiation. Together, Q capacity to promote a healthy adipose expansion enhancing angiogenesis and adipogenesis may contribute to reduced adipose hypertrophy, inflammation and IR. Consumption of diets rich in Q could be useful to counteract the adverse effects of high-fat diet-induced adipose dysfunction.

The Inhibitory effects of quercetin on Obesity and Obesity -induced Inflammation by regulation of MAPK signaling

Quercetin is a flavonoid found in fruits, vegetables, leaves and grains. It has inhibitory, antiviral, antiasthma, anticancer and antiinflammatory effects. Research has suggested that obesity is linked to metabolic disorders. In this study, we examined the inhibitory effect of quercetin on lipid accumulation and obesity-induced inflammation using 3T3-L1, RAW264.7, zebrafish and mouse models. Quercetin suppressed protein levels of the key adipogenic factors C/EBPβ, C/EBPα, PPARγ and FABP4 and the TG-synthesis enzymes lipin1, DGAT1 and LPAATθ. Activation of m-TOR and p70S6K, which are related to insulin and adipogenesis, was down-regulated during adipogenesis in 3T3-L1 cells. Recent research suggested that MAPK signaling factors were involved in adipogenesis and inflammation and that the adipokines MCP-1 and TNF-α attracted macrophages into adipose tissue. Our data showed that quercetin inhibited the MAPK signaling factors ERK1/2, JNK and p38MAPK and MCP-1 and TNF-α in adipocytes and macrophages. Quercetin also inhibited secretion of the inflammatory cytokines IL-1β and IL-6 and stimulated that of IL-10, an antiinflammatory cytokine.

In this study, we confirmed the inhibitory effects of quercetin in adipogenesis and inflammation using a mouse model. In mice, quercetin reduced body weight (almost 40%) and suppressed expression of adipogenic, lipogenic and inflammation-related cytokines. Our data demonstrated that quercetin inhibits lipid accumulation and obesity-induced inflammation in the cell and animal models. Our study suggested that quercetin may represent a potential therapeutic agent for other metabolic disorders by regulating obesity and obesity-induced inflammation.

Raspberry ketone

Heme oxygenase-1 mediates anti–Adipogenesis effect of raspberry ketone in 3T3-L1 cells

Background: Obesity is caused by excessive accumulation of body fat and is closely related to complex metabolic diseases. Raspberry ketone (RK), a major aromatic compound in red raspberry, was recently reported to possess anti-obesity effects. However, its mechanisms are unclear.

Aim: Adipogenesis plays a critical role in obesity and, therefore, this study aimed to investigate the effect and mechanisms of action of RK on adipogenesis in 3T3-L1 preadipocytes.

Materials and methods: 3T3-L1 preadipocytes were differentiated in medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Adipocyte lipid contents were determined using oil-red O staining while adipogenic transcription factor and lipogenic protein expressions were determined using western blotting.

Results: RK (300–400 µM) strongly inhibited lipid accumulation during 3T3-L1 preadipocyte differentiation into adipocytes. RK reduced the CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferation-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) expressions and increased heme oxygenase-1 (HO-1), Wnt10b, and β-catenin expressions in 3T3-L1 adipocytes. Additionally, RK inhibited lipid accumulation, and adipogenic transcription factor and lipogenic protein expressions were all decreased by inhibiting HO-1 or β-catenin using tin protoporphyrin (SnPP) or β-catenin short-interfering RNA (siRNA), respectively. Furthermore, Wnt10b and β-catenin expressions were negatively regulation by SnPP.

Conclusion: RK may exert anti-adipogenic effects through modulation of the HO-1/Wnt/beta-catenin signaling pathway.

Raspberry ketone, a naturally occurring phenolic compound, Inhibits Adipogenic and lipogenic gene expression in 3T3-L1 Adipocytes

Context: Raspberry ketone (RK) is a natural phenolic compound of red raspberry. The dietary intake of RK has been reported to exert anti-obese actions and alter the lipid metabolism in vivo and human studies.

Objective: To elucidate a possible mechanism for anti-obese actions of RK, the effects of RK on the adipogenic and lipogenic gene expression in 3T3-L1 adipocytes were investigated.

Materials and methods: 3T3-L1 maturing pre-adipocytes were treated from day 2 to day 8 of differentiation and mature adipocytes for 24 h on day 12 with 1, 10, 20, and 50 μM of RK. Triacylglycerols were assessed by spectrophotometry and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR).

Results: Treatment of adipocytes with RK suppressed adipocyte differentiation and fat accumulation in a concentration-dependent manner. RK suppressed the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT enhancer binding protein-α (C/EBPα), which led to further down-regulation of adipocyte fatty acid-binding protein-2 (aP2). In addition, treatment with 10 μM of RK also reduced mRNA levels of lipogenic genes such as acetyl-CoA carboxylase-1 (ACC1), fatty acid synthase (FASN), and stearoyl-CoA desaturase-1 (SCD1). In mature adipocytes, RK increased the transcriptional activities of genes involved in lipolysis and the oxidative pathways including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and carnitine palmitoyl transferase-1B (CPT1B).

Discussion and conclusion: These findings suggest that RK holds great promise for an herbal medicine with the biological activities altering the lipid metabolism in 3T3-L1 adipocytes.

Keywords: Adipogenesis; herbal medicine; lipid metabolism; lipogenesis.

Raspberry ketone Reduced lipid accumulation in 3T3-L1 cells and ovariectomy-induced Obesity in wistar rats by regulating autophagy mechanisms

This study aimed to determine the antiobesity effects of raspberry ketone (RK), one of the major aromatic compounds contained in raspberry, and its underlying mechanisms. During adipogenesis of 3T3-L1 cells, RK (300 μM) significantly reduced lipid accumulation and downregulated the expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferation-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS). RK also reduced the expression of light chain 3B (LC3B), autophagy-related protein 12 (Atg12), sirtuin 1 (SIRT1), and phosphorylated-tuberous sclerosis complex 2 (TSC2), whereas it increased the level of p62 and phosphorylated-mammalian target of rapamycin (mTOR). Daily administration of RK decreased the body weight (ovariectomy [Ovx] + RK, 352.6 ± 5 vs Ovx, 386 ± 5.8 g; P < 0.05), fat mass (Ovx + RK, 3.2 ± 0.05 vs Ovx, 5.0 ± 0.4 g; P < 0.05), and fat cell size (Ovx + RK, 6.4 ± 0.6 vs Ovx, 11.1 ± 0.7 × 10³ μm²; P < 0.05) in Ovx-induced obesity in rats.

The expression of PPARγ, C/EBPα, FAS, and FABP4 was significantly reduced in the Ovx + RK group compared with that in the Ovx group. Similar patterns were observed in autophagy-related proteins and endoplasmic reticulum stress proteins. These results suggest that RK inhibited lipid accumulation by regulating autophagy in 3T3-L1 cells and Ovx-induced obese rats.

Keywords: adipogenesis; autophagy; obesity; ovariectomy; raspberry ketone.

Raspberry ketone induces brown-like Adipocyte formation through suppression of autophagy in Adipocytes and Adipose Tissue

Promoting white adipose tissue (WAT) to acquire brown-like characteristics is a promising approach for obesity treatment. Although raspberry ketone (RK) has been reported to possess antiobesity activity, its effects on the formation of brown-like adipocytes remain unclear. Therefore, we investigated the effects and underlying mechanism of RK on WAT browning in 3T3-L1 adipocytes and rats with ovariectomy (Ovx)-induced obesity. RK (100 μM) significantly induced browning of 3T3-L1 cells by increasing mitochondrial biogenesis and the expression of browning-specific proteins (PR domain containing 16, PRDM16; peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC-1α; uncoupling protein-1, UCP-1) and lipolytic enzymes (hormone-sensitive lipase and adipose triglyceride lipase). RK significantly reduced the expression of the autophagy-related protein Atg12 and increased the expression of p62 and heme oxygenase 1 (HO-1).

Additionally, these effects of RK were reversed by the HO-1 inhibitor SnPP (20 μM). In addition, RK (160 mg/kg, gavage, for 8 weeks) significantly reduced body weight gain (Ovx+RK, 191.8 ± 4.6 g vs. Ovx, 223.6 ± 5.9; P < .05), food intake, the amount of inguinal adipose tissue (Ovx+RK, 9.05 ± 1.1 g vs Ovx, 12.9 ± 0.92 g; P < .05) and the size of white adipocytes in Ovx rats. Moreover, compared to expression in the Ovx group, the levels of browning-specific proteins were significantly higher and the levels of autophagy-related proteins were significantly lower in the Ovx+RK group. Therefore, this study elucidated the mechanism associated with RK-induced WAT browning and thus provides evidence to support the clinical use of RK for obesity treatment.

Keywords: 3T3-L1 Adipocyte; Beige adipocyte; Obesity; Ovariectomy; Raspberry ketone.

Raspberry ketone promotes the Differentiation of C3H10T1/2 stem cells into osteoblasts

The decrease in the bone mass associated with osteoporosis caused by ovariectomy, aging, and other conditions is accompanied by an increase in bone marrow adipose tissue. The balance between osteoblasts and adipocytes is influenced by a reciprocal relationship. The development of modalities to promote local/systemic bone formation by inhibiting bone marrow adipose tissue is important in the treatment of fractures or metabolic bone diseases such as osteoporosis. In this study, we examined whether raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK], which is one of the major aromatic compounds of red raspberry and exhibits anti-obesity action, could promote osteoblast differentiation in C3H10T1/2 stem cells. Confluent C3H10T1/2 stem cells were treated for 6 days with 10-100 μg/mL of RK in culture medium containing 10 nM all-trans-retinoic acid (ATRA) or 300 ng/mL recombinant human bone morphogenetic protein (rhBMP)-2 protein as an osteoblast-differentiating agent.

RK in the presence of ATRA increased alkaline phosphatase (ALP) activity in a dose-dependent manner. RK in the presence of rhBMP-2 also increased ALP activity. RK in the presence of ATRA also increased the levels of mRNAs of osteocalcin, α1(I) collagen, and TGF-βs (TGF-β1, TGF-β2, and TGF-β3) compared with ATRA only. RK promoted the differentiation of C3H10T1/2 stem cells into osteoblasts. However, RK did not affect the inhibition of early-stage adipocyte differentiation. Our results suggest that RK enhances the differentiation of C3H10T1/2 stem cells into osteoblasts, and it may promote bone formation by an action unrelated to adipocyte differentiation.

Raspberry ketone attenuates high-fat diet-induced Obesity by improving metabolic homeostasis in rats

Objective: To investigate the molecular mechanisms of the anti-obese effect of raspberry ketone against high-fat diet fed rats.

Methods: Fifty adult male rats were randomly assigned to receive a standard diet, a high fat diet, and the high-fat diet and 0.5%, 1% or 2% raspberry ketone. Body weight, biochemical parameters and gene expression of CCAAT enhancer-binding protein (C/EBP)δ, fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), peroxisome proliferator-activated receptor alpha (PPAR-α), hormone-sensitive lipase (HSL) and hepatic carnitine palmitoyltransferase 1 A (CPT1A) were investigated.

Results: Body weight, blood glucose, insulin, total lipids, triacylglycerols, total cholesterol and low-density lipoprotein cholesterol were increased in high-fat diet fed rats. These high fat diet-induced changes were attenuated by treatment with raspberry ketone. High-density lipoprotein cholesterol was decreased in highfat diet fed rats but increased in rats treated with raspberry ketone. Molecular investigations showed induction of gene expression of C/EBP-δ, FAS, ACC, CPT1A and inhibition of gene expression of PPAR-α and HSL in high-fat diet fed rats as compared with control. Raspberry ketone treament reversed these changes except CPT1A.

Conclusions: Raspberry ketone can prevent obesity induced by a high-fat diet in rats by induction of the expression of enzymes, controlling lipolysis and fatty acids β oxidation as well as inhibition of gene expressions of adipogenic factors.

Keywords: Raspberry ketone; Obesity; Molecular mechanism

Inhibitory effect of raspberry ketone on α-glucosidase: Docking simulation integrating Inhibition kinetics

Inhibition of α-glucosidase is directly associated with treatment of type 2 diabetes. In this regard, we conducted enzyme kinetics integrated with computational docking simulation to assess the inhibitory effect of raspberry ketone (RK) on α-glucosidase. RK bound to the active site of α-glucosidase and interacted with several key residues such as ASP68, TYR71, HIS111, PHE157, PHE158, PHE177, GLN181, ASP214, THR215, ASP349, ASP408, and ARG439, as detected by protein-ligand docking simulation. Subsequently, we confirmed the action of RK on α-glucosidase as the non-competitive type of inhibition in a reversible and rapidly binding manner. The relevant kinetic parameters were IC₅₀=6.17±0.46mM and K_i=7.939±0.211mM.

Regarding the structure-activity relationship, the higher concentration of RK induced slight modulation of the shape of the active site as monitored by hydrophobic exposure. The tertiary conformational change was linked to RK inhibition, and mostly involved regional changes of the active site. Our study provides insight into the functional role of RK due to its structural property of a hydroxyphenyl ring that interacts with the active site of α-glucosidase. We suggest that similar hydroxyphenyl ring compounds targeting the key residues of the active site might be potential α-glucosidase inhibitors.

Keywords: Inhibition; Raspberry ketone; α-Glucosidase.

Influence of Diet‐Induced Obesity on the Bioavailability and Metabolism of Raspberry Ketone (4‐(4‐Hydroxyphenyl)‐2‐Butanone) in Mice

Objectives: Raspberry ketone (RK) is the primary aroma compound in red raspberries and a dietary supplement for weight loss. This work aims to 1) compare RK bioavailability in male versus female, normal-weight versus obese mice; 2) characterize RK metabolic pathways.

Methods: Study 1: C57BL/6J male and female mice fed a low-fat diet (LFD; 10% fat) receive a single oral gavage dose of RK (200 mg kg^-1 ). Blood, brain, and white adipose tissue (WAT) are collected over 12 h. Study 2: Male mice are fed a LFD or high-fat diet (45% fat) for 8 weeks before RK dosing. Samples collected are analyzed by UPLC-MS/MS for RK and its metabolites.

Results: RK is rapidly absorbed (T_max ≈ 15 min), and bioconverted into diverse metabolites in mice. Total bioavailability (AUC_{0-12 h} ) is slightly lower in females than males (566 vs 675 nmol mL^-1 min^-1 ). Total bioavailability in obese mice is almost doubled that of control mice (1197 vs 679 nmol mL^-1 min^-1 ), while peaking times and elimination half-lives are delayed. Higher levels of RK and major metabolites are found in WAT of the obese than normal-weight animals.

Conclusions: RK is highly bioavailable, rapidly metabolized, and exhibits significantly different pharmacokinetic behaviors between obese and control mice. Lipid-rich tissues, especially WAT, can be a direct target of RK.

Keywords: bioavailability; metabolism; obesity; raspberry ketones.

Rehmannia glutinosa

Rehmannia Inhibits Adipocyte Differentiation and Adipogenesis

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPβ, as well as C/EBPα and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPβ to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.

Anti-Adipogenic effects of the traditional herbal formula Dohongsamul-tang in 3T3-L1 Adipocytes

Background: Blood stasis syndrome (BSS) is a general pattern identification and refers to pathological stagnation of blood circulation, dysfunction of endothelial cells or metabolic disorder in traditional Korean medicine (TKM). Dohongsamul-Tang (DHSMT) is a well-known traditional herbal formula which used for treatment and prevention of BSS by promoting blood circulation in TKM.

Methods: Cytotoxicity of DHSMT was examined by cell counting kit-8 (CCK-8). We also investigated the anti-adipogenesis effect of DHSMT by using Oil Red O staining, intracellular triglyceride assay leptin ELISA and western blot analysis in 3T3-L1 adipocytes. In addition, the accumulation of adiponectin, resistin and plasminogen activator inhibitor-1 (PAI-1) were measured by magnetic bead panel kit.

Results: Oil Red O staining showed that DHSMT markedly reduced fat accumulation without affecting cell cytotoxicity. DHSMT also significantly decreased accumulation of triglyceride and adipokines such as leptin, adiponectin, resistin and PAI-1 compared with fully differentiated adipocytes. Furthermore, our results found that DHSMT significantly suppressed the adipocyte differentiation by downregulating adipogenic-specific transcriptional factors such as peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα) and fatty acid binding protein 4 (FABP4) in adipocytes.

Conclusions: Taken together, our findings provide that DHSMT has potential for treatment and prevention of obesity or MS related to BSS.

Keywords: Dohongsamul-tang, Blood stasis syndrome, Anti-adipogenesis, Metabolic syndrome, Herbal medicine

Resveratrol

Resveratrol potentiates genistein’s AntiAdipogenic and proapoptotic effects in 3T3-L1 Adipocytes

Genistein (G) and resveratrol (R) individually inhibit adipogenesis in 3T3-L1 adipocytes and induce apoptosis in cancer cells. We investigated whether the combination of G and R resulted in enhanced effects on adipogenesis, lipolysis, and apoptosis in 3T3-L1 cells. Preadipocytes and mature adipocytes were treated with G and R individually at 50 and 100 micromol/L (G100; R100) and in combination. Both in preadipocytes and mature adipocytes, G and R individually decreased cell viability dose-dependently, but G100 + R100 further decreased viability by 59 +/- 0.97% (P < 0.001) and 69.7 +/- 1.2% (P < 0.001) after 48 h compared with G100 and R100, respectively. G100 + R100 induced apoptosis 242 +/- 8.7% (P < 0.001) more than the control after 48 h, whereas G100 and R100 individually increased apoptosis only 46 +/- 9.2 and 46 +/- 7.9%, respectively. G and R did not modulate mitogen-activated protein kinase expression by themselves, but G100 + R100 increased Jun-N-terminal kinase phosphorylation by 38.8 +/- 4.4% (P < 0.001) and decreased extracellular signal-regulating kinase phosphorylation by 48 +/- 3.4% (P < 0.001). Individually, G and R at 25 micromol/L (G25; R25) decreased lipid accumulation by 30 +/- 1.7% and 20.07 +/- 4.27%, respectively (P < 0.001).

However, G25 + R25 decreased lipid accumulation by 77.9 +/- 3.4% (P < 0.001). Lipolysis assay revealed that neither G25 nor R25 induced lipolysis, whereas G25 + R25 significantly increased lipolysis by 25.5 +/- 4.6%. The adipocyte-specific proteins PPARgamma and CCAAT/enhancer binding protein-alpha were downregulated after treatment with G + R, but no effect was observed with individual compounds. These results indicate that G and R in combination produce enhanced effects on inhibiting adipogenesis, inducing apoptosis, and promoting lipolysis in 3T3-L1 adipocytes. Thus, the combination of G and R is more potent in exerting antiobesity effects than the individual compounds.

Resveratrol exerts anti–Obesity effects via mechanisms involving down-regulation of Adipogenic and inflammatory processes in mice

Resveratrol is a natural polyphenolic stilbene derivative found in a variety of edible fruits, including nuts, berries, and grape skin. Although resveratrol has been suggested to improve thermogenesis in the brown adipose tissues of obese animals, there have been no reports on the anti-adipogenic and anti-inflammatory effects of resveratrol in the white adipose tissues of obese animals. The primary aim of this study was to investigate whether resveratrol attenuates high-fat diet (HFD)-induced adipogenesis and inflammation in the epididymal fat tissues of mice and to explore the underlying mechanisms involved in this attenuation. In comparison with HFD-fed mice, mice fed with a 0.4% resveratrol-supplemented diet (RSD) showed significantly lower body weight gain (-48%), visceral fat-pad weights (-58%), and plasma levels of triglyceride, FFA, total cholesterol, glucose, tumor necrosis factor (TNF) α, and monocyte chemoattractant protein-1 (MCP1).

Resveratrol significantly reversed the HFD-induced up-regulation of galanin-mediated signaling molecules (GalR1/2, PKCδ, Cyc-D, E2F1, and p-ERK) and key adipogenic genes (PPARγ2, C/EBPα, SREBP-1c, FAS, LPL, aP2, and leptin) in the epididymal adipose tissues of mice. Furthermore, resveratrol significantly attenuated the HFD-induced up-regulation of pro-inflammatory cytokines (TNFα, IFNα, IFNβ, and IL-6) and their upstream signaling molecules (TLR2/4, MyD88, Tirap, TRIF, TRAF6, IRF5, p-IRF3, and NF-κB) in the adipose tissues of mice. The results of this study suggest that resveratrol inhibits visceral adipogenesis by suppressing the galanin-mediated adipogenesis signaling cascade. It may also attenuate cytokine production in the adipose tissue by repressing the TLR2- and TLR4-mediated pro-inflammatory signaling cascades in HFD-fed mice.

Potential miRNA involvement in the Anti-Adipogenic effect of resveratrol and its metabolites

Objective: Scientific research is constantly striving to find molecules which are effective against excessive body fat and its associated complications. Taking into account the beneficial effects that resveratrol exerts on other pathologies through miRNA, the aim of the present work was to analyze the possible involvement of miRNAs in the regulation of adipogenic transcription factors peroxisome proliferator-activated receptor γ (pparγ), CCAAT enhancer-binding proteins α and β (cebpβ and cebpα) induced by resveratrol and its metabolites.

Methods: 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 μM of trans-resveratrol (RSV), trans-resveratrol-3-O-sulfate (3S), trans-resveratrol-3′-O-glucuronide (3G) and trans-resveratrol-4′-O-glucuronide (4G). After computational prediction and bibliographic search of miRNAs targeting pparγ, cebpβ and cebpα, the expression of microRNA-130b-3p (miR-130b-3p), microRNA-155-5p (miR-155-5p), microRNA-27b-3p (miR-27b-3p), microRNA-31-5p (miR-31-5p), microRNA-326-3p (miR-326-3p), microRNA-27a-3p (miR-27a-3p), microRNA-144-3p (miR-144-3p), microRNA-205-5p (miR-205-5p) and microRNA-224-3p (miR-224-3p) was analyzed. Moreover, other adipogenic mediators such as sterol regulatory element binding transcription factor 1 (srebf1), krüppel-like factor 5 (klf5), liver x receptor α (lxrα) and cAMP responding element binding protein 1 (creb1), were measured by Real Time RT-PCR. As a confirmatory assay, cells treated with RSV were transfected with anti-miR-155 in order to measure cebpβ gene and protein expressions.

Results: Of the miRNAs analyzed only miR-155 was modified after resveratrol and glucuronide metabolite treatment. In transfected cells with anti-miR-155, RSV did not reduce cebpβ gene and protein expression. 3S decreased gene expression of creb1, klf5, srebf1 and lxrα.

Conclusions: While RSV and glucuronide metabolites exert their inhibitory effect on adipogenesis through miR-155 up-regulation, the anti-adipogenic effect of 3S is not mediated via miRNAs.

An Inhibitory effect of resveratrol in the mitotic clonal expansion and insulin signaling pathway in the early phase of Adipogenesis

Resveratrol is known as a potent antiobesity compound that acts partly through inhibition of adipogenesis. However, the direct targets responsible for its antiadipogenic action are unclear. Our hypothesis is that resveratrol inhibits adipogenesis through modulation of mitotic clonal expansion (MCE) and cell signaling pathways in the early phase of differentiation. To test this, we examined the effects of resveratrol on MCE and insulin signaling pathway in the early phase of adipogenesis in murine preadipocytes. We observed that the antiadipogenic action of resveratrol is largely limited to the early phase of adipogenesis. Specifically, the presence of resveratrol in the first 24 hours of adipogenesis was required for its antiadipogenic effect. During the first 24 hours of adipogenesis, resveratrol impaired the progression of MCE by suppressing the cell cycle entry of preadipocytes to G2/M phase, and expression of cell cycle regulators cyclin A and cyclin-dependent kinase 2.

Concomitantly, resveratrol inhibited insulin signaling pathway in the early phase of adipogenesis. Furthermore, we revealed an inhibitory effect of resveratrol on insulin receptor (IR) activity, and this is likely through a direct physical interaction between resveratrol and IR. The antiadipogenic effect of resveratrol is through inhibition of the MCE and IR-dependent insulin signaling pathway in the early phase of adipogenesis.

Resveratrol induces apoptosis and Inhibits Adipogenesis in 3T3‐L1 Adipocytes

Resveratrol, a phytoallexin, has recently been reported to slow aging by acting as a sirtuin activator. Resveratrol also has a wide range of pharmacological effects on adipocytes. In this study, we investigated the effects of resveratrol on adipogenesis and apoptosis using 3T3-L1 cells. In mature adipocytes, 100 and 200 microM resveratrol decreased cell viability dose-dependently by 23 +/- 2.7%, and 75.3 +/- 2.8% (p < 0.0001), respectively, after 48 h treatment, and 100 microM resveratrol increased apoptosis by 76 +/- 8.7% (p < 0.0001). Resveratrol at 25 and 50 microM decreased lipid accumulation in maturing preadipocytes significantly by 43 +/- 1.27% and 94.3 +/- 0.3% (p < 0.0001) and decreased cell viability by 25 +/- 1.3% and 70.4 +/- 1.6% (p < 0.0001), respectively.

In order to understand the anti-adipogenic effects of resveratrol, maturing 3T3-L1 preadipocytes were treated with 25 microM resveratrol and the change in the expression of several adipogenic transcription factors and enzymes was investigated using real-time RT-PCR. Resveratrol down-regulated the expression of PPAR gamma, C/EBP alpha, SREBP-1c, FAS, HSL, LPL and up-regulated the expression of genes regulating mitochondrial activity (SIRT3, UCP1 and Mfn2). These results indicate that resveratrol may alter fat mass by directly affecting cell viability and adipogenesis in maturing preadipocytes and inducing apoptosis in adipocytes and thus may have applications for the treatment of obesity.

Resveratrol: anti–Obesity mechanisms of action

Resveratrol is a non-flavonoid polyphenol which belongs to the stilbenes group and is produced naturally in several plants in response to injury or fungal attack. Resveratrol has been recently reported as preventing obesity. The present review aims to compile the evidence concerning the potential mechanisms of action which underlie the anti-obesity effects of resveratrol, obtained either in cultured cells lines and animal models. Published studies demonstrate that resveratrol has an anti-adipogenic effect. A good consensus concerning the involvement of a down-regulation of C/EBPα and PPARγ in this effect has been reached. Also, in vitro studies have demonstrated that resveratrol can increase apoptosis in mature adipocytes.

Furthermore, different metabolic pathways involved in triacylglycerol metabolism in white adipose tissue have been shown to be targets for resveratrol. Both the inhibition of de novo lipogenesis and adipose tissue fatty acid uptake mediated by lipoprotein lipase play a role in explaining the reduction in body fat which resveratrol induces. As far as lipolysis is concerned, although this compound per se seems to be unable to induce lipolysis, it increases lipid mobilization stimulated by β-adrenergic agents.

The increase in brown adipose tissue thermogenesis, and consequently the associated energy dissipation, can contribute to explaining the body-fat lowering effect of resveratrol. In addition to its effects on adipose tissue, resveratrol can also acts on other organs and tissues. Thus, it increases mitochondriogenesis and consequently fatty acid oxidation in skeletal muscle and liver. This effect can also contribute to the body-fat lowering effect of this molecule.

Combination of low dose of the Anti-Adipogenic agents resveratrol and phenelzine in drinking water is not sufficient to Prevent Obesity in very-high-fat diet-fed mice

Purpose: Resveratrol inhibits lipid accumulation but suffers from limited bioavailability. The anti-depressive agent phenelzine limits adipogenesis in various models of cultured preadipocytes, and this hydrazine derivative also inhibits de novo lipogenesis in mature adipocytes. It was therefore tested whether resveratrol effects on adiposity reduction and glucose tolerance improvement could be reinforced by co-administration with phenelzine.

Methods: Mice fed a very-high-fat diet (VHFD, 60% calories as fat) were subjected to drinking solution containing low dose of resveratrol (0.003%) and/or 0.02% phenelzine for 12 weeks. Body fat content, glucose tolerance, food and water consumption were checked during treatment while fat depot mass was determined at the end of supplementation. Direct influence of the agents on lipogenesis and glucose uptake was tested in adipocytes.

Results: Epididymal fat depots were reduced in mice drinking phenelzine alone or with resveratrol. No limitation of body weight gain or body fat content was observed in the groups drinking resveratrol or phenelzine, separately or in combination. The altered glucose tolerance and the increased fat body composition of VHFD-fed mice were not reversed by resveratrol and/or phenelzine. Such lack of potentiation between resveratrol and phenelzine prompted us to verify in vitro their direct effects on mouse adipocytes. Both molecules inhibited de novo lipogenesis, but did not potentiate each other at 10 or 100 μM. Only resveratrol inhibited hexose uptake in a manner that was not improved by phenelzine.

Conclusions: Phenelzine has no interest to be combined with low doses of resveratrol for treating/preventing obesity, when considering the VHFD mouse model.

Enhanced Inhibition of Adipogenesis and induction of apoptosis in 3T3-L1 Adipocytes with combinations of resveratrol and quercetin

Combinations of resveratrol and quercetin further decreased viability (73.5+/-0.9%, p<0.001) and increased apoptosis (310.3+/-9.6%, p<0.001) more than single compounds alone. The combination of resveratrol and quercetin at 100 muM increased release of cytochrome c from mitochondria to cytosol and decreased ERK 1/2 phosphorylation. Taken together, our data indicate that combinations of resveratrol and quercetin can exert potential anti-obesity effects by inhibiting differentiation of preadipocytes and inducing apoptosis of mature adipocytes.

AntiAdipogenic effects and mechanisms of combinations of genistein, epigallocatechin-3-gallate, and/or resveratrol in preadipocytes

Natural bioactive compounds are considered an excellent alternative strategy for developing effective, safe, and cost-effective antiobesity agents. The aim of this study was to investigate if combinations of soy bean genistein (G), green tea epigallocatechin-3-gallate (E), and/or grape resveratrol (R) at low dosages synergistically inhibit preadipocyte differentiation both in 3T3-L1 cells and human primary preadipocytes (HPAs). Our results show that combinations of G, E, and/or R additively inhibited preadipocyte differentiation (39-56% of control) both in 3T3-L1 cells at 30 μM and HPAs at 15 μM, while the individual compounds have no antiadipogenic effect at the selected concentrations. We also observed similar patterns that combinations of G, E, and/or R additively reduced protein expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT binding proteins alpha (C/EBP-α), the two key preadipocyte differentiation regulators, both in differentiated 3T3-L1 cells and HPAs.

Moreover, combined G, E, and/or R attenuated protein expressions of fatty acid binding protein 4 and perilipin, two PPAR-γ/C/EBP-α downstream molecules in fat drop development in a very similar pattern, in inhibiting differentiation in preadipocytes. This combined antiadipogenic effect of G + E + R is additive, not synergistic according to our results and the Median-Effect Principle. In addition, we found that a lower concentration (15 μM) of G, E, and/or R is required in HPAs than the concentration (30 μM) needed in 3T3-L1 cells, to exert the combined antiadipogenic effect. These data suggest that combinations of G, E, and/or R intake or soy bean, green tea, and/or grape simultaneous consumption may prevent obesity in human being.

Keywords: EGCG; antiadipogenic; combinations; genistein; preadipocytes; resveratrol.

Retinoic Acid

Retinoic acid blocks Adipogenesis by Inhibiting C/EBPbeta-mediated transcription.

Adipocyte differentiation is thought to involve sequential induction of the transcription factors C/EBPbeta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBPalpha. C/EBPalpha expression is both necessary and sufficient for adipocyte differentiation. Here we report that ectopic expression of either C/EBPalpha or C/EBPbeta induces PPARgamma expression and adipogenesis and that retinoic acid (RA) completely inhibits adipogenesis by either form of C/EBP.

In studies of normal preadipocytes, RA does not prevent C/EBPbeta induction but blocks induction of PPARgamma, C/EBPalpha, and adipogenesis. In transient transfection studies, liganded RA receptor (RAR) specifically blocks transcriptional activation by either C/EBPalpha or C/EBPbeta. These results strongly suggest that C/EBPalpha substitutes for C/EBPbeta to induce adipocyte differentiation and that liganded RAR inhibits adipogenesis by blocking C/EBPbeta-mediated induction of downstream genes.

Retinoic acid Inhibits Adipogenesis via activation of Wnt signaling pathway in 3T3-L1 preadipocytes

Although retinoic acid (RA) is well known to inhibit the differentiation of 3T3-L1 cells into adipocytes both in vivo and in vitro, its molecular mechanism is not fully understood. In this report, we investigate the inhibitory mechanism of adipocyte differentiation by RA in 3T3-L1 cells. Because both RA and Wnt are known to inhibit adipogenesis at a common step involving the inhibition of PPAR-γ expression, we focused on the crosstalk between these two signaling pathways. We found that RA treatment resulted in a dramatic inhibition of adipogenesis, especially at an early phase of differentiation, and led to increased β-catenin protein expression. Moreover, RA enhances the transcriptional activity of β-catenin as well as Wnt gene expression during adipogenesis.

Taken together, the present study demonstrated that Wnt/β-catenin signaling may be associated with the RA-induced suppression of adipogenesis and may cooperatively inhibit adipocyte differentiation.

Rhamnetin (seabuckthorn)

Rhamnetin-induced suppression of clonal expansion during early stage of Adipogenesis

Adipocyte Differentiation plays a pivotal role in the progression of Obesity which is a major risk factor for several diseases such as Diabetes, hypertension and coronary heart disease. In this study, the Inhibitory effect of rhamnetin, a flavonoid compound, on Adipogenesis in 3T3-L1 cells was investigated. Rhamnetin decreased the accumulation of lipid droplets, and Inhibited the elevation of triglyceride content in the Adipocytes (IC₅₀ = 17.3 μM). The expressions of PPARγ, C/EBPα, and perilipin, Adipocyte Differentiation markers, were significantly Reduced by rhamnetin.

Triglyceride biosynthesis and clonal expansion of Adipocytes were completely Inhibited during the early stage by rhamnetin. Additionally, rhamnetin significantly decreased the expression of C/EBPβ, an early stage marker. Our results indicate that suppression of clonal expansion during the early stage of Adipogenesis by rhamnetin may be associated with Inhibition of the C/EBPβ, C/EBPα, and PPARγ pathways.

Rhein

Rhein, an Inhibit or of Adipocyte Differentiation and Adipogenesis

Rhein (RH), a compound purified from Radix et Rhizoma Rhei, has been used to alleviate liver and kidney damage. It is found that RH inhibited the differentiation of 3T3-L1 preadipocytes induced by differentiation medium in a time- and dose-dependent manner. It was revealed that RH downregulated the expression of adipogenesis-specific transcription factors PPARγ and C/EBPα, as well as their upstream regulator, C/EBPβ. Furthermore, the PPARγ target genes that are involved in adipocyte differentiation, such as CD36, aP2, acyl CoA oxidase, uncoupled protein 2, acetyl-CoA carboxylase, and fatty acid synthase, were reduced after to RH. In addition, high-fat diet-induced weight gain and adiposity were reversed by RH in C57BL/6 mice.

Consistent with the cells’ results, RH downregulated the mRNA levels of PPARγ and C/EBPα, and their downstream target genes in C57BL/6 mice. Taken together, adipocyte differentiation and adipogenesis were inhibited by RH in cultured cells and in rodent models of obesity. The evidence implied that RH was a potential candidate for preventing metabolic disorders.

Rhein protects against Obesity and related metabolic disorders through liver X receptor-mediated uncoupling protein 1 upregulation in brown Adipose Tissue

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs.

The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.

Keywords: LXR, diet-induced obesity, UCP1, antagonist, rhein.

Rhein: a review of pharmacological activities

Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) is a lipophilic anthraquinone extensively found in medicinal herbs, such as Rheum palmatum L., Cassia tora L., Polygonum multiflorum Thunb., and Aloe barbadensis Miller, which have been used medicinally in China for more than 1,000 years.

Its biological activities related to human health are being explored actively. Emerging evidence suggests that rhein has many pharmacological effects, including hepatoprotective, nephroprotective, anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The present review provides a comprehensive summary and analysis of the pharmacological properties of rhein, supporting the potential uses of rhein as a medicinal agent.

Effect of Rhein on 3T3-L1 Preadipocyte Differentiation and Related Gene Expression

Objective: The effect of rhein(1,8-dihydroxy-3-carboxyanthraquinone,Rhein,Rh) on the proliferation and differentiation of 3T3-L1 adipocyte and the related gene mRNA expression was investigated.

Method: MTT assay was used to detect 3T3-L1 cell proliferation by rhein(5,10,20,40,80,160 μM),rhein intervention 3T3-L1 adipocytes induced differentiation,oil red O staining and colorimetric analysis of the impact of rhein on 3T3-L1 adipocyte differentiation process of the accumulation of lipid droplets in the cytoplasm,biochemical detected rhein 3T3-L1 adipocytes triglyceride(TG). The expression of CCAAT enhancer binding proteins(C/EBPα),fatty acid synthetase(FAS),peroxisome proliferators activated receptor γ(PPARγ) genes was measured by real-time PCR.

Results: The results showed that rhein(20,40,80 μM)could inhibit adipocyte differentiation and reduce intracellular TG content in a dose-dependent manner. Furthermore,rhein down-regulated the mRNA expression of C/EBPα,FAS,and PPARγ genes.

Conclusion: Rhein can inhibit the differentiation of 3T3-L1 adipocytes, and the mechanism of action may be related to reducing adipose differentiation-related gene PPARγ,C/EBPα and FAS expression.

Rhizoma Polygonati

Negative regulation of Adipogenesis by kaempferol, a component of Rhizoma Polygonati falcatum in 3T3-L1 cells

Rhizoma Polygonati falcatum (RPF) has been used as a traditional herbal medicine in Asia, because of its anti-hyperglycemic, anti-triglycemic, and anti-tumor activity. In this study, we determined the anti-adipogenic potential of RPF extract and its component kaempferol in 3T3-L1 adipocytes, and the underlying molecular mechanism(s) using microarray analysis. Adipocyte differentiation of 3T3-L1 cells was significantly impaired by RPF extract and kaempferol as monitored by Oil Red O staining and quantitative measurement of lipid accumulation.

Finally, co-transfection assays using luciferase reporter gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis using peroxisome proliferator-activated receptor-γ (PPARγ) target genes indicated that kaempferol significantly repressed rosiglitazone-induced PPARγ transcriptional activity. Overall, our data suggests that kaempferol, a major component of RPF, may be beneficial in obesity, by reducing adipogenesis and balancing lipid homeostasis partly through the down-regulation of PPARγ.

Anti-Adipogenic effect of kaempferol, a component of Polygonati rhizoma

Objective: It has been reported that Polygonati rhizoma (Pr) has anti-hyperglycemia, anti-triglycemia, anti-diabetic, and anti-tumor activity. Total extract of Pr was tested to identify anti-adipogenic activity in 3T3-L1 differentiation and molecular mechanism of Pr in 3T3-L1 differentiation. Methods: Differentiation of 3T3-L1 pre-adipocyte was induced in the presence of Pr extract and kaempferol. The level of lipid accumulation was measured by Oil Red O staining. The expression of genes associated with adipocyte differentiation was measured by RT-PCR. Results: Extract of Pr and its component kaempferol reduced lipid accumulation in 3T3-L1 during adipogenesis and also reduced mRNA levels of genes associated with adipogenesis, such as adipsin, aP2, LPL, SERBP-1c and $P P A R γ$ . Conclusions: In this study, we showed that the molecular mechanism of Pr and kaempferol activity is related to regulation of $P P A R γ$ expression and activation.

Rosmarinic acid (Rosemary)

Radiation is a promising technique for improving the safety and shelf-life of processed foods. In the present investigation, the degradation mechanism and bioactivity improvement of rosmarinic acid (RA) were studied in response to various gamma irradiation doses (10, 20, and 50 kGy). RA exposed to gamma irradiation at 50 kGy was completely degraded and showed an increased Inhibitory effect against 3 T3-L1 Preadipocyte compare to the parent compound. Structures of the newly generated compounds 2–4 from irradiated RA at 50 kGy were elucidated based on spectroscopic methods, including ¹H nuclear magnetic resonance (NMR) and Mass spectrometry (MS). Interestingly, compounds 2 and 5 exhibited significantly enhanced Anti-Adipogenic properties in 3 T3-L1 cells compared to the original compound.

These results provide evidence that structural changes in RA induced by gamma irradiation might enhance biological efficacy.

Sida rhomboidea. Roxb leaf

Sida rhomboidea. Roxb Leaf Extract Down-Regulates Expression of PPARγ2 and Leptin Genes in High Fat Diet Fed C57BL/6J Mice and Retards in Vitro 3T3L1 Pre-Adipocyte Differentiation

Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract.

Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

S-Petasin (Petasites japonicus)

S-Petasin isolated from Petasites japonicus exerts Anti-Adipogenic activity in the 3T3-L1 cell line by Inhibiting PPAR-γ pathway signaling

Petasites japonicus is an edible and medicinal plant with a good flavor, and it is a rich source of bioactive compounds. S-Petasin has been isolated from Petasites hybridus (L.), Petasites officinalis (L.) and Petasites formosanus, but not from Petasites japonicus. In this study, we found that hexane extracts of Petasites japonicus Inhibited Adipogenesis in 3T3-L1 cells . After this we isolated s-petasin from Petasites japonicus. Subsequently, the 3T3-L1 Pre-Adipocytes were used to test whether s-petasin exerts an Anti-Adipogenic effect.

The results showed that s-petasin presented strong Anti-Adipogenic activity. Further studies illustrated that s-petasin Reduced glucose uptake. Moreover, results showed that triglyceride accumulation was Inhibited by s-petasin in differentiated 3T3-L1 cells . Western blot assay indicated that s-petasin down-regulated the expression of PPAR-γ and its target genes in a dose dependent manner. In conclusion, we isolated s-petasin from Petasites japonicus and found that it exerted Anti-Adipogenic activity against 3T3-L1 cell Differentiation through Inhibition of the expression of PPAR-γ pathway signaling.

Salicortin (Salix pseudo-lasiogyne Twigs)

Anti-Adipogenic Activities of Alnus incana and Populus balsamifera Bark Extracts, Part II: Bioassay-guided Identification of Actives Salicortin and Oregonin

Among modern day metabolic diseases, Obesity has reached epidemic proportions worldwide and novel therapeutic support strategies are urgently needed. Adipocytes are interesting targets in this context. Using ethnobotanical and bioassay screening techniques, we have identified two Boreal Forest plants used by the James Bay Cree that potently Inhibit Adipogenesis, namely Alnus incana ssp. rugosa (Speckled Alder) and Populus balsamifera (Balsam Poplar). The mode of action of this Inhibitory activity was reported in a companion paper.

The current study report the results of a classical bioassay-guided fractionation approach aimed at identifying the active principles responsible for the Inhibition of Adipogenesis, as measured using triglyceride accumulation in the 3T3-L1 Adipocyte model cell line. The glycosides oregonin and salicortin were isolated and identified as the respective active principles for Alnus incana and Populus balsamifera. These compounds thus offer promise as novel agents to mitigate the incidence or the progression of Obesity.

Sargassum micracanthum

Effect of Sargassum micracanthum extract on Lipid Accumulation and Reactive Oxygen Species (ROS) Production during Differentiation of 3T3-L1 preadipocytes

Obesity , a strong risk factor for the development of chronic diseases, is characterized by an increase in the number and size of Adipocytes differentiated from precursor cells, preadipocytes . Recent research suggests that increased reactive oxygen species (ROS) production in 3T3-L1 Adipocyte facilitates Adipocyte Differentiation and Fat Accumulation . This study was to investigate whether Reduced ROS production by Sargassum micracanthum extract (SME) could protect the development of Obesity through Inhibition of Adipogenesis . 3T3-L1 preadipocytes were treated SME for up to 8 days following standard induction of Differentiation . The extent of Differentiation reflected by amount of lipid accumulation and ROS production was determined by Oil red O staining and nitroblue tetrazolium (NBT) assay.

Treatment of SME significantly Inhibited ROS production and Adipocyte Differentiation that is depend on down regulation of NADPH oxidase 4 (NOX4), a major ROS generator, and peroxisome proliferator-activated receptor gamma ( $P P A R γ$ ) and CCAAT/enhancer-binding protein alpha ( $C / E B P α$ ), a key Adipogenic transcription factor. These results indicate that SME can Inhibit Adipogenesis through a Reduced ROS level that involves down-regulation of NOX4 expression or via modulation of Adipogenic transcription factor.

Sargassum serratifolium

Ethanolic extract of Sargassum serratifolium Inhibits Adipogenesis in 3T3-L1 preadipocytes by cell cycle arrest

The brown alga, Sargassum serratifolium C. Agardh, has been reported to have high levels of meroterpenoids having anti-inflammatory, antioxidant, and anti-Diabetic activities. This study investigated the effect of ethanolic extract of S. serratifolium (ESS) on the Inhibition of Adipogenesis and lipid accumulation in 3T3-L1 preadipocytes. ESS suppressed the accumulation of lipid droplets and triglycerides (TG), while enhancing the Lipolysis of intracellular TG. We found that ESS induced cell cycle arrest at the G1/S phase via suppressing protein expression of cyclin-dependent kinases (CDK) 2, CDK4, CDK6, and cyclin E.

Moreover, ESS Reduced the expression of CCAAT/enhancer-binding protein (C/EBP) β during the early stage of Adipogenesis , leading to suppressed expression of both messenger RNA (mRNA) and protein of C/EBPα and peroxisome proliferator-activated receptor γ. As a result, ESS Reduced mRNA and protein levels of lipogenic transcription factors such as retinoid X receptor, and sterol regulatory element-binding protein 1c and their target genes, such as fatty acid synthase and stearoyl-CoA desaturase (SCD) 1. Overall, ESS Inhibited Adipocyte Differentiation in early stage and lipid accumulation during Adipogenesis through downregulation of Adipogenic and lipogenic transcription factors, providing the Anti-Obesity mechanism of ESS.

Saururus chinensis

Inhibition of Adipogenesis in 3T3-L1 Adipocytes with Ethanol Extracts of Saururus chinensis

Obesity increases the risk of many adult diseases, such as atherosclerosis, Diabetes, hypertension, ischemic heart disease and breast cancer. Inhibition of Adipogenesis is an effective way in the Anti-Obesity management. Because of main components of Saururus chinensis is flavonoid, it has been showed some improvement by its antioxidant effects on the atherosclerosis, heart disease and Diabetic hyperlipidemia. But mechanism of Anti-Obesity effect of S. chinensis is not clear. We investigated the effects of ethanol extracts of S. chinensis on Adipogenesis in the 3T3-L1 pre-Adipocyte. The 3T3-L1 cell line is commonly used to study Adipogenesis in vitro.

In this study, ethanol extracts of S. chinensis significantly decrease the lipid accumulation in the 3T3-L1 cells proved by measuring triglyceride contents and Oil red O staining. The proposed mechanism of Inhibition of Adipogenesis in the 3T3-L1 cells with ethanol extracts of S. chinensis is down-regulation of transcriptional factors and Adipocyte-specific genes such CCAAT/enhancer binding protein $α$ ( $C / E B P α$ ) and Peroxisome proliferator activated receptor $γ$ ( $P P A R γ$ ) in concentration dependent pattern. These results suggest that ethanol extracts of S. chinensis Inhibits adipognesis in the 3T3-L1 cells and can be used as a safe and efficient natural substance to manage Anti-Obesity.

Sanggenol F

Sanggenol F exerts anti-Diabetic effects via promoting Adipocyte Differentiation and modifying adipokines expression

Adipose Tissue is not only a lipid storage site, but also a well-known endocrine organ. Dysfunction of Adipose Tissue is associated with irregular lipid metabolism, ectopic lipid accumulation and insulin resistance. It is proposed that modulating on Adipose Tissue is a reasonable way to ameliorate glucose and lipid metabolism. (±)-sanggenol F (SGF, purity >98.5%) was synthesized as a racemic mixture of natural (+)-sanggenol F. In this study, SGF was found to promote Adipocyte Differentiation, enhance insulin sensitivity, and upregulate beneficial adipokines expression in 3T3-L1 cells.

Furthermore, in vivo study showed that treatment with SGF for 4 weeks improved glucose metabolism, by decreasing fasting blood glucose and enhancing insulin sensitivity. It also improved lipid metabolism, with Reduced serum lipid level and ameliorated hepatic steatosis in db/db mice. During the process of target finding, we found that SGF had multiple activities of protein tyrosine phosphatase 1B Inhibition, peroxisome proliferator-activated receptor γ and peroxisome proliferator-activated receptor α agonism. These results showed the potential of SGF as a candidate for the therapy of type 2 Diabetes.

Scallop shell

Scallop shell extract Inhibits 3T3-L1 Preadipocyte Differentiation

In a previous study, we examined the in vitro and in vivo effects of organic components isolated from scallop shells (scallop shell extract) and found that rats fed with powdered scallop shell showed a reduction in white adipose tissue weight compared to rats fed a control diet. To clarify the mechanism of action, we have investigated the effects of scallop shell extract on the differentiation of 3T3-L1 preadipocyte cells. Scallop shell extract reduced lipid accumulation in 3T3-L1 cells during differentiation in a dose-dependent manner.

Glycerol-3-phosphate dehydrogenase activity also decreased following treatment with scallop shell extract, suggesting that the extract inhibits adipocyte differentiation. In addition, scallop shell extract suppressed the expression of adipogenic transcription factors (peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein-α) and inhibited differentiation-associated mitotic clonal expansion.

These results support the possibility that scallop shell extract may work as an anti-obesity agent via the inhibition of adipocyte differentiation.

Glycoproteins isolated from scallop shells Inhibit Differentiation of 3T3-L1 Preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells.

In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.

Securigera securidaca

Effects of Securigera securidaca Extract on Lipolysis and Adipogenesis in Diabetic Rats

Diabetes mellitus is associated with dysregulation of Adipose Tissue metabolism and increased level of serum lipids. In our previous work we found that Securigera securidaca decreases cholesterol level in blood of Diabetic rats. The present study was carried out to further investigate the effects of this plant on lipid metabolism, Lipolysis, and Adipogenesis, in Diabetic rats. Female Wistar rats were rendered Diabetic by intraperitoneal injection of streptozotocin. Retroperitoneal Adipose Tissue was removed from Diabetic animals after seven days of streptozotocin injection. Effect of hydroalcoholic extract of S. securidaca seeds (100–800 𝜇g/mL) on Adipose Tissue Lipolysis was evaluated in ex vivo condition. Also, to evaluate Adipogenesis , preadipocytes were isolated from Adipose Tissue and differentiated to Adipocytes in the presence of the extract.

The extract at concentration of 800 𝜇g/mL decreased both basal and catecholamine-stimulated Lipolysis (𝑃 < 0.05). Incubation of differentiating preadipocytes with 800 𝜇g/mL of S. securidaca extract decreased intracellular lipid droplet accumulation as evaluated with Oil Red O staining (𝑃 < 0.001).

The extract even at high concentrations had no effect on viability of preadipocytes. In conclusion, S. securidaca decreases Lipolysis and Adipogenesis without cytotoxicity, which makes it a good candidate for management of dyslipidemia and reduction of cardiovascular risks in Diabetes.

sesamol

Sesamol Ameliorates diet‐induced Obesity in C57BL/6J mice and Suppresses Adipogenesis in 3T3‐L1 cells via regulating mitochondria‐lipid metabolism

Scope: The aim of the current study was to investigate the effect of sesamol, a natural powerful antioxidant and anti-inflammatory phenol derivative of sesame oil, on adiposity and adiposity-related metabolic disturbances in mice fed with western diet, and the potential underlying mechanisms focusing on the mitochondria-lipid metabolism.

Methods and results: In the experimental model that consisted of 3-month-old C57BL/6J mice divided into three groups with/without sesamol in the drinking water including standard diet, high fat and high fructose diet (HFFD), and HFFD with sesamol. Results demonstrated that sesamol mitigated bodyweight gain, development of insulin resistance induced by HFFD. Sesamol was found partially normalized serum and hepatic lipid contents, as well as suppressed HFFD-induced lipogenesis in liver via regulating mitochondria-related triglyceride/cholesterol metabolism genes expressions. Importantly, sesamol decreased mass and adipocyte sizes of white adipose tissues and brown adipose tissues by improving mitochondria-related genes expressions including Pgc1a and Ucp1. Moreover, sesamol was also found to reduce differentiation and mitochondrial metabolic inhibitors (oligomycin and antimycin A) stimulated lipid accumulation in 3T3-L1 adipocytes.

Conclusion: Taken together, this study provides compelling evidence that sesamol supplementation reduced adipocyte size and adipogenesis of diet-induced obesity by regulating mitochondria lipid metabolism.

Keywords: Adipocyte; Diet-induced obesity; Lipid Metabolism; Mitochondria; Sesamol.

Anti-Adipogenic effects of sesamol on human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) from adult bone marrow are able to differentiate into adipocytes, osteoblasts, chondrocytes and neuronal cells. Adipocytes in bone marrow are primarily responsible for the maintenance of bone structure by maintaining cell number balance with other stromal cells. However, the number of adipocytes in the bone marrow increases with age, leading to an imbalance of the bone marrow microenvironment, which results in a disruption of bone structure.

In addition, the excessive number of adipocytes in bone marrow can cause diseases, such as osteoporosis or anemia. In this study, we investigated the effect of sesamol, a major natural phenolic compound of sesame oil, on the adipogenic differentiation of hMSCs. Numerous studies have reported the anti-oxidant property of sesamol, but its effect on cell differentiation has not yet been shown. We first found that sesamol treatment during adipogenic differentiation of hMSCs reduced intracellular lipid accumulation, which was unrelated to lipolysis. Interestingly, sesamol diminished the expression of genes responsible for adipogenesis, but increased the expression of osteogenic genes.

In addition, sesamol decreased the expression of genes necessary for adipocyte maturation without affecting the expression of hMSC-specific genes. Studies concerning intracellular signaling in hMSCs showed that the extracellular signal-regulated kinase 1/2 (ERK1/2) was decreased by sesamol, which was similar with the effect of an ERK1/2 inhibitor.

Overall, this study demonstrates that sesamol can attenuate the adipogenic differentiation of hMSCs without affecting its characteristics through the inhibition of ERK1/2 pathway. Herein, this study reports for the first time the effect of sesamol on hMSC differentiation and suggests the possibility of using sesamol as a therapeutic agent to treat intraosseous disruption triggered by the excessive adipogenesis of hMSCs.

Sesamol Increases Ucp1 Expression in White Adipose Tissues and Stimulates Energy Expenditure in High-Fat Diet-Fed Obese Mice

Sesamol found in sesame oil has been shown to ameliorate obesity by regulating lipid metabolism. However, its effects on energy expenditure and the underlying molecular mechanism have not been clearly elucidated. In this study, we show that sesamol increased the uncoupling protein 1 (Ucp1) expression in adipocytes. The administration of sesamol in high-fat diet (HFD)-fed mice prevented weight gain and improved metabolic derangements. The three-week sesamol treatment of HFD-fed mice, when the body weights were not different between the sesamol and control groups, increased energy expenditure, suggesting that an induced energy expenditure is a primary contributing factor for sesamol’s anti-obese effects. Consistently, sesamol induced the expression of energy-dissipating thermogenic genes, including Ucp1, in white adipose tissues. The microarray analysis showed that sesamol dramatically increased the Nrf2 target genes such as Hmox1 and Atf3 in adipocytes.

Moreover, 76% (60/79 genes) of the sesamol-induced genes were also regulated by tert-butylhydroquinone (tBHQ), a known Nrf2 activator. We further verified that sesamol directly activated the Nrf2-mediated transcription. In addition, the Hmox1 and Ucp1 induction by sesamol was compromised in Nrf2-deleted cells, indicating the necessity of Nrf2 in the sesamol-mediated Ucp1 induction.

Together, these findings demonstrate the effects of sesamol in inducing Ucp1 and in increasing energy expenditure, further highlighting the use of the Nrf2 activation in stimulating thermogenic adipocytes and in increasing energy expenditure in obesity and its related metabolic diseases.

Keywords: sesamol, uncoupling protein 1, Nrf2, energy expenditure, obesity, metabolic diseases

Sesamol intervention Ameliorates Obesity -associated metabolic disorders by regulating hepatic lipid metabolism in high-fat diet-induced obese mice

Background: Obesity has currently become a serious social problem to be solved. Sesamol, a natural bioactive substance extracted from sesame oil, has shown multiple physiological functions, and it might have an effect on the treatment of obesity.

Objective: This study was conducted to investigate the therapeutic effect and potential mechanisms of sesamol on the treatment of obesity and metabolic disorders in high-fat diet (HFD)-induced obese mice.

Methods: C57BL/6J male mice were fed HFD for 8 weeks to induce obesity, followed by supplementation with sesamol (100 mg/kg body weight [b.w.]/day [d] by gavage) for another 4 weeks. Hematoxylin and eosin staining was used to observe lipid accumulation in adipose tissues and liver. Chemistry reagent kits were used to measure serum lipids, hepatic lipids, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. ELISA kits were used to determine the serum insulin and free fatty acid (FFA) levels. Western blotting was used to detect the protein levels involved in lipid metabolism in the liver.

Results: Sesamol significantly reduced the body weight gain of obese mice and suppressed lipid accumulation in adipose tissue and liver. Sesamol also improved serum and hepatic lipid profiles, and increased insulin sensitivity. In the sesamol-treated group, the levels of serum ALT and AST decreased significantly. Furthermore, after sesamol treatment, the hepatic sterol regulatory element binding protein-1 (SREBP-1c) decreased, while the phosphorylated hormone sensitive lipase (p-HSL), the carnitine palmitoyltransferase 1α (CPT1α), and the peroxisome proliferator-activated receptor coactivator-1α (PGC1α) increased, which were responsible for the fatty acid synthesis, lipolysis, and fatty acid β-oxidation, respectively.

Conclusions: Sesamol had a positive effect on anti-obesity and ameliorated the metabolic disorders of obese mice. The possible mechanism of sesamol might be the regulation of lipid metabolism in the liver.

Keywords: sesamol, obesity, liver, lipid metabolism

In vitro Anti-Obesity effects of sesamol mediated by adenosine monophosphate-activated protein kinase and mitogen-activated protein kinase signaling in 3T3-L1 …

Sesamol is a phenol derivative of sesame oil and a potent anti-oxidant, anti-inflammatory, anti-hepatotoxic, and anti-aging compound. We investigated the effects of sesamol on the molecular mechanisms of adipogenesis in 3T3-L1 preadipocytes. The intracellular lipid accumulation accompanied by increased extracellular release of free glycerol was decreased during differentiation on treating 3T3-L1 with sesamol. Sesamol treatment on 3T3-L1 inhibited adipogenic differentiation by down-regulating adipogenesis-related factors (C/EBPα, PPARγ, and SREBP-1). Lipid accumulation was repressed by decreasing fatty acid synthase and by up-regulating lipolysis-response genes (HSL and LPL).

The molecular mechanisms of sesamol-induced inhibition in adipogenesis were mediated by increased levels of phosphorylated adenosine monophosphate-activated protein kinase and its substrate acetyl-CoA carboxylase. Sesamol treatment, in turn, modulated the different members of the mitogenactivated protein kinase family by suppressing phosphorylation of ERK 1/2 and JNK and by increasing the phosphorylation of p38. In summary, sesamol inhibits adipogenic differentiation by reducing phosphorylation levels of ERK 1/2 and JNK while inducing lipolysis by activating p38 and AMPK. Our results demonstrate that the molecular mechanisms of in vitro anti-obesity effects of sesamol are due to the combined effects of preventing both lipid accumulation and adipogenesis.

Keywords: sesamol, adipogenesis, anti-obesity, AMPK, lipolysis, MAPK

Sesamol Alleviates Obesity -Related Hepatic Steatosis via Activating Hepatic PKA Pathway

This study aimed to investigate the effect of sesamol (SEM) on the protein kinase A (PKA) pathway in obesity-related hepatic steatosis treatment by using high-fat diet (HFD)-induced obese mice and a palmitic acid (PA)-treated HepG2 cell line. SEM reduced the body weight gain of obese mice and alleviated related metabolic disorders such as insulin resistance, hyperlipidemia, and systemic inflammation. Furthermore, lipid accumulation in the liver and HepG2 cells was reduced by SEM. SEM downregulated the gene and protein levels of lipogenic regulator factors, and upregulated the gene and protein levels of the regulator factors responsible for lipolysis and fatty acid β-oxidation. Meanwhile, SEM activated AMP-activated protein kinase (AMPK), which might explain the regulatory effect of SEM on fatty acid β-oxidation and lipogenesis.

Additionally, the PKA-C and phospho-PKA substrate levels were higher after SEM treatment. Further research found that after pretreatment with the PKA inhibitor, H89, lipid accumulation was increased even with SEM administration in HepG2 cells, and the effect of SEM on lipid metabolism-related regulator factors was abolished by H89. In conclusion, SEM has a positive therapeutic effect on obesity and obesity-related hepatic steatosis by regulating the hepatic lipid metabolism mediated by the PKA pathway.

Development and evaluation of sesamol as an antiaging agent

Aging comprises various changes that occur in living organisms with the passage of time, leading to increased system entropy, loss of homeostasis and eventually death. Of the various theories proposed, the free radical theory of aging states that it is the shift in antioxidant/pro-oxidant balance that leads to increased oxidative stress, dysregulation of cellular function and aging. It has been proved that accumulated damage by free radicals produced by exposure to ultraviolet (UV) radiation results in extensive damage to the soft skin tissues, which is commonly known as “photoaging”.

Although the human body has built-in antioxidant mechanisms to suppress uncontrolled free radicals, these mechanisms may at times fail due to overwhelming production of toxic radicals. This is where supplemental antioxidants can play a vital role in restoring the balance, lack of which produces visible wrinkling and premature aging. Sesamol, a highly acclaimed antioxidant, was selected as a suitable antioxidant to investigate in this study, and is for the first time shown to counter UV-induced oxidative stress (in terms of both biochemical and histopathological changes) in mouse skin.

The source of radiation was a 300-W UV lamp, and mice of the lacca strain were chosen as an animal model for this study. Free radical levels generated were estimated using the Ohkawa method for estimation of lipid peroxidation in animal tissues. The results from biochemical and histopathological investigations clearly confirm that the sesamol formulation is effective in preventing photodamage (lesions, ulcers and changes in skin integrity) due to chronic UV exposure.

SH21B is an Anti-Obesity composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner.

an Anti-Obesity herbal composition, Inhibits Fat Accumulation in 3T3-L1 Adipocytes and high fat diet-induced obese mice through the modulation of the Adipogenesis

SH21B is an anti-obesity composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner. It has been used for the treatment of obesity in traditional medical clinics in Korea, but its anti-obesity effects and mechanism of action have not been studied until now.

Aim of the study: This study was conducted to confirm the anti-obesity effects of SH21B and to elucidate its mechanism of action.

Materials and methods: 3T3-L1 adipocytes and high fat diet-induced obese mice were treated with SH21B, and its effect on gene expression was analyzed using microarray technology, real-time PCR and western blotting experiments.

Results: SH21B significantly inhibited fat accumulation in 3T3-L1 adipocytes and reduced adipose tissue and serum triglyceride levels in high fat diet-induced obese mice. Microarray analyses showed that SH21B affected more genes in the adipogenesis pathway than any other pathway studied. SH21B significantly decreased the expression of major transcription factors of the adipogenesis pathway and resulted in the down-regulation of lipid metabolizing enzymes involved in the transport, uptake and synthesis of lipids.

Conclusions: SH21B inhibits fat accumulation by down-regulating the expression of genes involved in the adipogenesis pathway.

The WNT/β-catenin pathway mediates the anti-Adipogenic mechanism of SH21B, a traditional herbal medicine for the treatment of Obesity

Aim of the study: This study was conducted to elucidate the molecular mechanisms of SH21B, a traditional Korean herbal medicine commonly used for the treatment of obesity.

Materials and methods: 3T3-L1 preadipocytes were differentiated into adipocytes in the presence or absence of SH21B. Changes in mRNA or protein levels were analyzed using microarray, real-time polymerase chain reaction and western blotting analyses. Small interference (si)RNA transfection experiments were conducted to elucidate the essential role of β-catenin.

Results: Microarray analyses showed that components of the WNT/β-catenin pathway including β-catenin, cyclin D1 and dishevelled 2 were up-regulated more than two-fold as a result of SH21B treatment during adipogenesis, which were confirmed by real-time PCR and western blotting. Modulation of the WNT/β-catenin pathway by SH21B resulted in the nuclear accumulation of β-catenin. Both intracellular lipid droplet formation and expressions of adipogenic genes including PPARγ, C/EBPα, FABP4 and LPL, which were inhibited by SH21B, were significantly recovered by β-catenin siRNA transfection.

Conclusions: SH21B modulates components of the WNT/β-catenin pathway during adipogenesis, and β-catenin plays a crucial role in the anti-adipogenic mechanism of SH21B.

Transcriptome Analyses for the Anti-Adipogenic Mechanism of an Herbal Composition

SH21B is a natural composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). In our previous study, we reported that SH21B inhibited adipogenesis and fat accumulation in 3T3-L1 cells through modulation of various regulators in the adipogenesis pathway. The aim of this study was to analyze the transcriptome profiles for the anti-adipogenic effects of SH21B in 3T3-L1 cells. Total RNAs from SH21B-treated 3T3-L1 cells were reverse-transcribed into cDNAs and hybridized to Affymetrix Mouse Gene 1.0 ST array.

From microarray analyses, we identified 2,568 genes of which expressions were changed more than two-fold by SH21B, and the clustering analyses of these genes resulted in 9 clusters. Three clusters among the 9 showed down-regulation by SH21B (cluster 4, cluster 6 and cluster 9), and two clusters showed up-regulation by SH21B (cluster 7 and cluster 8) during the adipogenesis of 3T3-L1 cells. It was found that many genes related to cell proliferation and adipogenesis were included in these clusters.

Clusters 4, 6 and 9 included genes which were related with adipogenesis induction and cell cycle arrest. Clusters 7 and 8 included genes related to cell proliferation as well as adipogenesis inhibition. These results suggest that the mechanisms of the anti-adipogenic effects of SH21B may be the modulation of genes involved in cell proliferation and adipogenesis.

shikonin, a natural compound isolated from Lithospermun erythrorhizon

Shikonin stimulates glucose uptake in 3T3-L1 Adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor.

However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.

Shikonin Suppresses ERK 1/2 phosphorylation during the early stages of Adipocyte Differentiation in 3T3-L1 cells

Background: The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.

Methods: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.

Results: Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

Conclusions: These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

Shikonin Inhibits Fat Accumulation in 3T3‐L1 Adipocytes

Shikonin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, is the main ingredient of Lithospermum erythrorhizon Sieb. et Zucc, and was reported to have various biological activities including antiinflammatory, anticancer, antimicrobial and others. This study aimed to elucidate, for the first time, the antiobesity activity of shikonin and its mechanism of action. Shikonin was found to inhibit fat droplet formation and triglyceride accumulation in 3T3‐L1 adipocytes.

The half inhibitory concentration, IC₅₀, for the inhibition of triglyceride accumulation was found to be 1.1 μM. The expression of genes involved in lipid metabolism, such as FABP4 and LPL, were significantly inhibited following shikonin treatment. Shikonin also inhibited the ability of PPARγ and C/EBPα, the major transcription factors of adipogenesis, to bind to their target DNA sequences. The expressions of mRNA and protein of PPARγ and C/EBPa were significantly down‐regulated following shikonin treatment. Among the upstream regulators of adipogenesis, only SREBP1C was found to be down‐regulated by shikonin.

The results of this study suggest that shikonin down‐regulates the expression of SREBP1C and subsequently the expression of PPARγ and C/EBPα. Together, these changes result in the down‐regulation of lipid metabolizing enzymes and reduced fat accumulation.

Shikonin Inhibits Adipogenesis by modulation of the WNT/β-catenin pathway

Aim: Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin.

Main methods: Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of β-catenin in the anti-adipogenic effects of shikonin.

Key findings: Shikonin prevented the down-regulation of β-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. β-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of β-catenin. Among the genes located in the WNT/β-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment.

Significance: This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/β-catenin pathway in vitro, and also suggests that WNT/β-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.

Shikonin Inhibits Adipogenic Differentiation via regulation of mir-34a-FKBP1B

Shikonin is a naturally occurring naphthoquinone pigment and a major constituent present in Lithospermum erythrorhizon. Since microRNAs (miRNAs) are one of the key post-transcriptional regulators of adipogenesis, their manipulation represents a potential new strategy to inhibit adipogenesis. Our aim was to investigate shikonin-dependent inhibition of adipogenesis with an emphasis on miRNA-related processes. Mir-34a increased during induced adipogenesis, and this was suppressed in the presence of shikonin. mRNA expression of FKBP1B, a suggested target of mir-34a according to bioinformatics studies, decreased during adipogenesis, but was recovered by shikonin treatment, which reversely correlated with mir-34a expression. A mir-34a inhibitor suppressed MDI-induced adipogenesis by blocking PPARγ and C/EBPα expression, while suppression of mir-34a recovered MDI-induced down-regulation of FKBP1B expression. A mir-34a mimic decreased FKBP1B mRNA expression in 3T3-L1 preadipocytes.

We also observed that mir-34a bound directly to the 3′-untranslated region of FKBP1B. Finally, FKBP1B overexpression attenuated MDI-induced adipogenesis, PPARγ, and C/EBPα expression. These results suggest that mir-34a regulates adipogenesis by targeting FKBP1B expression. Our findings reveal that shikonin prevents adipogenesis by blocking the mir-34a-FKBP1B pathway which represents a promising potential target for preventing obesity.

Shikonin protects against Obesity through the modulation of Adipogenesis , lipogenesis, and β-oxidation in vivo

Shikonin, a napthoquinone pigment in the L. erythrorhizon (Lithospermum erythrorhizon) plant, is used as a functional food to promote blood circulation and detoxification in Asia. We investigated the effect and molecular mechanisms of shikonin on high-fat diet-induced obesity in mice. C57BL/6J mice were fed a normal diet, a high-fat diet, or a high-fat diet supplemented with shikonin for 8 weeks. We measured body weight change, adipose tissue weights, and biochemical markers. To elucidate the molecular mechanism of shikonin on diet-induced obesity, western blotting and quantitative real time PCR were performed. Shikonin reduced high-fat diet-induced increases in body weight, white adipose tissue mass, serum triglyceride, and total cholesterol levels.

Blood insulin and leptin levels were significantly decreased by shikonin supplementation. Shikonin supplementation reduced protein content and mRNA expression of adipogenesis-related genes in white adipose tissue and lipogenesis-related genes in the liver, along with hepatic lipid content. Moreover, shikonin increased mRNA expression of the β-oxidation genes PPAR-α, PGC-1α, and ACOX1 in liver and skeletal muscle. Shikonin prevents high-fat diet-induced obesity in mice and may be a novel therapeutic approach for obesity prevention by modulating adipogenesis, lipogenesis, and fatty acid oxidation.

Sibiraea angustata

Inhibition of Adipocyte Differentiation and Adipogenesis by the traditional Chinese herb Sibiraea angustata

Obesity has become a major health concern due to its strong association with the metabolic syndrome. Inhibition of adipocyte differentiation represents a key strategy to inhibit obesity. Sibiraea angustata (SA), a traditional Chinese herb, has a wide range of pharmacological effects, such as improving digestive functions. Here, we report a novel antiadipogenic effect of SA. By using the SA water extract (SAW), SA acetic ether extract (SAA) and the 3T3-L1 model of adipocyte differentiation and adipogenesis, we showed that both SAW and SAA impaired the proliferation and adipo-differentiation of 3T3-L1 in a dose- and time-dependent manner. At the molecular level, treatment of 3T3-L1 cells with SAW or SAA inhibited the expression of the key adipocyte differentiation regulator CCAAT enhancer binding protein β (C/EBPβ), as well as peroxisome proliferator activated receptor γ, adipocyte protein-2, lipoprotein lipase and glucose transporter 4. Cell cycle analysis showed that both SAW and SAA blocked cell cycle at the G1-S transition phase, causing cells to remain in the preadipocyte state. The expression of CyclinA and cyclin-dependent kinase 2 was also inhibited by SAW and SAA.

Treatment with SAW also prevented the localization of C/EBPβ to the centromeres. Taken together, our results show that SA has a potent antiadipogenic effect in 3T3-L1 cells due to the inhibition of adipocyte differentiation and adipogenesis. We propose that SA may be used as a safe and effective neutraceutical to manage obesity.

Effects and mechanism of Sibiraea angustata on lipid metabolism in hight-fatted rats

To investigate the effect and mechanism of Sibiraea angustata on lipid metabolism in hight-fatted SD rats.After the obese model was built,Sibiraea angustata was administrated intragastrically to obese rats for 8 weeks. Peeled off fat around kidneys and made pathological tissue sections. The number and size of adipocytes were detected. The levels of adiponectin, adipoR2, AMPK, and PPARgamma mRNA in adipose tissue were detected by RT-PCR.

AMPK protein expression in adipose tissue were detected by Western Blot.Compared with the model group, the diameter of adipocytes were reduced while the number increased after adminiseration of Sibiraea angustata for 8 weeks. The levels of adiponectin, adipoR2, AMPK and PPARgamma mRNA were increased siginficantly. The expression of AMPK protein was also up-regulated significantly.Sibiraea angustata has anti-obesity effect. The mechanism may be related to the adiponectin signal transduction pathway.

Silibinin (Milk thistle)

Silibinin attenuates Adipogenesis in 3T3-L1 preadipocytes through a potential upregulation of the insig pathway

In the past few decades, the use of silibinin, a plant flavonoid extracted from the milk thistle, as a hepatoprotective and chemopreventive agent has gained much attention. In this study, we investigated the effects of silibinin on Adipogenesis. Treatment with silibinin suppressed terminal Differentiation of 3T3-L1 cells into Adipocytes as evidenced by Oil red O staining and TG assay results. Real-time RT-PCR analysis revealed that silibinin decreased the expression of Adipogenesis-related genes such as CAAT/enhancer binding protein-α, fatty acid synthase, sterol response element binding protein 1c, Adipocyte -specific lipid binding protein, peroxisome proliferator-activated receptor γ and lipoprotein lipase, and increased the expression of Preadipocyte factor-1, a Preadipocyte marker gene.

The Anti-Adipogenic effect of silibinin was associated with the up-regulation of insig-1 and insig-2. Collectively, these results suggest that silibinin Inhibits Adipocyte Differentiation through a potential up-regulation of insig-1 and insig-2 at an early phase in Adipocyte Differentiation.

Siphonaxanthin (Bryopsis corticulan)

The Green Algal Carotenoid Siphonaxanthin Inhibits Adipogenesis in 3T3-L1 preadipocytes and the Accumulation of Lipids in White Adipose Tissue of KK-Ay Mice

Background: Siphonaxanthin, a xanthophyll present in green algae, has been shown to possess antiangiogenic and apoptosis-inducing activities.

Objective: We evaluated the AntiObesity effects of siphonaxanthin by using a 3T3-L1 cell culture system and in Diabetic KK-Ay mice.

Methods:3T3-L1 cells were differentiated with or without 5 μmol/L siphonaxanthin, and lipid accumulation and critical gene expressions for Adipogenesis were examined. In vivo, 4-wk-old male KK-Ay mice were administered daily oral treatment of 1.3 mg siphonaxanthin for 6 wk and body weight, visceral fat weight, serum variables, and gene expressions involved in lipid metabolism were evaluated.

Results: Compared with the other carotenoids evaluated, siphonaxanthin potently Inhibited Adipocyte Differentiation. Siphonaxanthin significantly suppressed lipid accumulation at noncytotoxic concentrations of 2.5 and 5 μmol/L by 29% and 43%, respectively. The effects of siphonaxanthin were largely limited to the early stages of Adipogenesis. Siphonaxanthin significantly Inhibited protein kinase B phosphorylation by 48% and 72% at 90 and 120 min, respectively. The expressions of key Adipogenesis genes, including CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator activated receptor γ (Pparg), fatty acid binding protein 4 (Fabp4), and stearoyl coenzyme A desaturase 1 (Scd1), were elevated by 1.6- to 166-fold during Adipogenesis. After 8 d of Adipocyte Differentiation, siphonaxanthin significantly lowered gene expression of Cebpa, Pparg, Fabp4, and Scd1 by 94%, 83%, 95%, and 90%, respectively. Moreover, oral administration of siphonaxanthin to KK-Ay mice significantly Reduced the total weight of white Adipose Tissue (WAT) by 13%, especially the mesenteric WAT by 28%. Furthermore, siphonaxanthin administration Reduced lipogenesis and enhanced fatty acid oxidation in Adipose Tissue. Siphonaxanthin was observed to highly accumulate in mesenteric WAT, and the accumulation in the mesenteric WAT was almost 2- and 3-fold that in epididymal (P = 0.14) and perirenal (P < 0.05) WAT, respectively.

Conclusion: These results provide evidence that siphonaxanthin may effectively regulate Adipogenesis in 3T3-L1 cells and Diabetic KK-Ay mice.

Soyasaponin Ab

Soyasaponin Ab alleviates postmenopausal Obesity through browning of white adipose tissue

Postmenopausal obesity is becoming a serious global health concern. Soyasaponin Ab (SA) inhibits adipocyte differentiation; however, the preventive and therapeutic potential of SA in postmenopausal obesity has not been clearly established. Here, we investigated the anti-obesity effects of SA in postmenopausal obesity in vitro and in vivo. SA inhibited adipocytic differentiation of 3T3-L1 cells and reduced lipid accumulation and lipid droplet formation.

Moreover, SA significantly decreased weight gain, adipocyte area, serum metabolic profiles in OVX mice. SA also inhibited the lipid metabolism-related factors but increased the thermoregulatory factors in both in vitro and in vivo. SA treatment further increased energy expenditure through beige fat activation. Overall, we found that SA effectively accelerated energy consumption by stimulating beige fat activation, even under estrogen-deficient conditions. Thus, SA treatment may be a promising strategy for the prevention of postmenopausal obesity by promoting weight loss, reducing fat accumulation, and improving obesity-related metabolic disorders.

Soyasaponins Aa and Ab exert an anti‐Obesity effect in 3T3‐L1 Adipocytes through downregulation of PPARγ

Saponins are a diverse group of biologically functional products in plants. Soyasaponins are usually glycosylated, which give rise to a wide diversity of structures and functions. In this study, we investigated the effects and molecular mechanism of soyasaponins Aa and Ab in regulating adipocyte differentiation and expression of adipogenic marker genes in 3T3-L1 adipocytes. Soyasaponins Aa and Ab dose-dependently inhibited the accumulation of lipids and the expression of adiponectin, adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c, adipocyte fatty acid-binding protein 2, fatty acid synthase, and resistin in 3T3-L1 adipocytes.

In addition, soyasaponins Aa and Ab suppressed the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in HEK 293T cells. Furthermore, we confirmed that the expression of PPARγ and of CCAAT-enhancer-binding protein α (C/EBPα) was suppressed at both the mRNA and protein levels in 3T3-L1 adipocytes by treatment with soyasaponins Aa and Ab. Taken together, these findings indicate that soyasaponin Aa and Ab markedly inhibit adipocyte differentiation and expression of various adipogenic marker genes through the downregulation of the adipogenesis-related transcription factors PPARγ and C/EBPα in 3T3-L1 adipocytes.

Keywords: 3T3-L1 cells; adipogenesis; adipogenic marker genes; peroxisome proliferator-activated receptor gamma (PPARγ); soyasaponins.

stearidonic acid (SDA)

Suppression ofAdipocyte Differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells

Long-chain ω-3 PUFAs, mainly EPA and DHA, have been found to Reduce lipid accumulation in 3T3-L1 Adipocytes through the regulation of Adipogenic gene expression [22, 3T3-L1 Adipocytes. Prostaglandins Other Lipid Mediat. SDA has been targeted as a potential biologically active surrogate for EPA as it can be effectively converted to EPA by the body and shares many beneficial properties with EPA [33]. In the present study, the effects of SDA on Adipogenesis and lipid accumulation in 3T3-L1 cells were studied.

Adipogenesis and increased lipid accumulation are key features in Obesity. Beneficial effects of EPA and DHA from fish oil against Adipose Tissue expansion and Adipocyte Differentiation were previously identified. Decreased TG content in 3T3-L1 Adipocytes was found by treatment with high concentrations of sea cucumber and blue mussel extracts, which are rich in EPA and DHA [3T3-L1 Adipocyte metabolism. Food Funct. and also by treatment with EPA and DHA directlyIn line with these findings, we also observed a significant reduction in TG accumulation by EPA and DHA, as well as by SDA (Fig. 3).

To our knowledge, this is the first study showing the influence of SDA on lipid accumulation in 3T3-L1 Adipocytes. One study published opposite results, in which they found EPA and DHA promoted the Differentiation of 3T3-L1 preadipocytes as evidenced by increased lipid content However, they treated the 3T3-L1 cells not only during Differentiation, but also 24-h pretreatment before Differentiation, so their results included both effects of EPA and DHA on Differentiation and proliferation In our study, ω-3 fatty acids had an Inhibitory effect on 3T3-L1 cell Differentiation. It is not clear as to the effect of these ω-3 fatty acids on 3T3-L1 cell proliferation. In addition, our cells appeared to be more differentiated than theirs. As shown in Fig. 3, there is a large difference between non-differentiated control group and differentiated control group. However, in their study, a smaller number of 3T3-L1 preadipocytes were differentiated into mature Adipocytes filled with lipid droplets based on their Bodipy staining and fluorescent images.

The Differentiation of preadipocytes is regulated by a complex network of transcription factors, mainly the C/EBP family and PPARγ. SREBP-1c is implicated in stimulating endogenous PPARγ ligand production [37]. Adipocytes from mice in which the C/EBPα gene was disrupted showed defects in lipid accumulation [38]. Transgenic mice lacking PPARγ specifically in Adipose Tissue exhibited greatly Reduced sized fat pads [39]. SDA has been shown to regulate gene expression In the present study, it was hypothesized that SDA exerted Anti-Adipogenic effect by Inhibiting the expression of these transcription factors and their target genes. As expected, dose-dependently down-regulation of C/EBPα, C/EBPβ, PPARγ, and SREBP-1c by SDA was observed (Fig. 4a). Consistent with the suppression of these Adipogenic transcription factors, the expression of their target genes, AP2, FAS, SCD-1, LPL, GLUT4, and PEPCK were also significantly decreased dose-dependently by SDA treatment (Fig. 4b).

The Adipogenic genes are often connected with insulin sensitivity and inflammatory cytokines. AP2 has been demonstrated to act as an adipokine for the development of insulin resistance in liver Adipose-specific deletion of SCD-1 was found to induce GLUT1 up-regulation and was associated with decreased adiponectin and increased TNFα production Adipocyte-derived LPL was shown to induce macrophage activation and monocyte adhesion Inflammation has been shown to Inhibit the expression of GLUT4 and PEPCK in 3T3-L1 Adipocytes EPA and DHA have been reported to have preventive properties in the development of insulin resistance and Inflammation Therefore, it is necessary to further investigate the effect of SDA on adipokine secretion and its role in insulin resistance and inflammatory response for future study.

SDA is the Δ6 desaturase product of ALA in the bioconversion of ALA to EPA. In humans, the conversion of ALA to EPA is in low amounts (less than 7%) and in even lower amounts to DHA (less than 1%) due to the rate-limiting enzyme [48]. Nutritional supplementation with ALA was not able to induce the accumulation of long-chain ω-3 PUFAs. In the present study, it was hypothesized that by skipping the rate-limiting step, the conversion of SDA to EPA would be more efficient than that of ALA to EPA. By LC-MS/MS analysis, it was shown that SDA increased EPA content in 3T3-L1 cells by 345% compared to the control group (Fig. 6a). The efficacy of EPA enrichment by SDA was about 3.5-fold greater than the comparable level of ALA and was about one-third of the EPA enrichment by EPA. For DHA enrichment, both ALA and SDA, even EPA was not able to increase DHA content extensively in 3T3-L1 cells, although statistical significance was observed in SDA and EPA groups.

This was probably due to another rate limiting step which converts docosapentaenoic acid to DHA [49]. Human studies have reported that consumption of SDA, as ethyl esters, echium oil, or SDA-soybean oil, significantly increased the EPA level in red blood cells [8], peripheral blood mononuclear cell [11], plasma [15], and neutrophils [13]. In animal studies, feeding with SDA was shown to increase the EPA content in many tissues, including red blood cells, plasma, liver, muscle, heart, brain, and ileum of dogs [50], sows and piglets [51], lambs [52], and rodents [53, 54]. In addition, SDA can lead to EPA enrichment in animal products, such as egg yolk, chicken meat [55], and milk of dairy cows [56]. Based on human studies, the efficacy of SDA on EPA enrichment in different tissues range from 17 to 85% as much as the efficacy of EPA treatment on EPA enrichment [8, Metabolic Syndrome. When compared to ALA, SDA was about 1.9 to 4.3-fold as effective as that of ALA on EPA enrichment [57, 61]. Most studies did not find significant change in DHA enrichment with SDA supplementation.

These results indicate that SDA consumption will be expected to confer the health benefits associated with the consumption of EPA, but not DHA. While the exact molecular mechanisms by which SDA decreases gene transcription is not apparent from this study, the downstream biosynthesis of lipid-derived eicosanoids via its conversion to EPA may be a potential mechanism through which gene transcription effects could be modulated [3T3-L1 Adipocytes. Prostaglandins Other Lipid Mediat. Inhibits Adipogenesis via an autocrine-mediated interleukin-11/glycoprotein 130/STAT1-dependent signaling cascade. J Cell Biochem. Inhibition of 3T3-L1 Preadipocyte Differentiation by prostaglandin F2alpha. Endocrinology.

This study has demonstrated that SDA was able to Inhibit Adipocyte Differentiation and Reduce lipid accumulation in 3T3-L1 Adipocytes through down-regulation of Adipogenic transcription factors and lipid accumulation genes (Fig. 7). These findings warrant further investigation that will develop SDA as a natural and effective agent for the prevention or treatment of Obesity.

Sulforaphene

Sulforaphene Inhibition of Adipogenesis via Hedgehog Signaling in 3T3-L1 Adipocytes

Obesity is a risk factor for numerous metabolic disorders. In this study, we investigated the effects of the isothiocyanates sulforaphane (SA) and sulforaphene (SE) on Adipogenesis in 3T3-L1 Adipocytes . SE, a compound that is abundant in radish, Inhibited Adipogenesis by suppressing the Adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ , 69.2 ± 2.4%, P < 0.05) and CCAAT/enhancer-binding protein α (C/EBPα , 36.1 ± 3.1%, P < 0.05), thereby reducing Fat Accumulation in 3T3-L1 Adipocytes (45.6 ± 2.7%, P < 0.05); SA was less effective. SE exerted these activities through the activation of the Hedgehog (Hh) signaling pathway by restoring Smo ((2.1 ± 0.2)-fold, P < 0.05) and Gli1 ((2.8 ± 0.1)-fold, P < 0.05) expression, which was suppressed by Adipogenic signals.

These effects of SE were abrogated by treatment with the Hh Inhibit or vismodegib. Thus, SE Inhibits Adipocyte Differentiation via Hh signaling and may be an effective natural agent for preventing Adipocyte hyperplasia and Obesity.

Stinging nettles (Utrica dioica L.)

Identification of plant extracts with potential AntiDiabetic properties: effect on human peroxisome proliferator‐activated receptor (PPAR), Adipocyte Differentiation and insulin‐stimulated glucose uptake

Thiazolidinediones (TZDs) are insulin sensitizing drugs used to treat type 2 Diabetes. The primary target of the TZDs is the peroxisome proliferator‐activated receptor (PPAR) γ, a key regulator of Adipogenesis and glucose homeostasis. Currently prescribed TZDs are full PPARγ agonists, and their use is associated with several side effects. Partial PPARγ agonists appear to be associated with fewer side effects but may still confer the desired insulin sensitizing action. Extracts from common medicinal/food plants were tested in a screening platform comprising a series of bioassays, including tests for PPARγ, α and δ transactivation, Adipocyte Differentiation and insulin‐stimulated glucose uptake, allowing identification of plants containing potentially interesting PPAR agonists.

Twenty‐two plant extracts out of 133 were found to increase insulin‐stimulated glucose uptake and 18 extracts were found to activate PPARγ, 3 to activate PPARα and γ, 6 to activate PPARδ and γ, and 9 to activate PPARγ, α and δ. Among the 24 different plant species tested in the platform, 50% were shown to contain compounds capable of activating PPARγ and stimulating insulin‐dependent glucose uptake with no or little effect on Adipocyte Differentiation warranting further studies and characterization.

Styrax japonica fruit

Anti-Adipogenic effects in 3T3-L1 cells of acetone extracts and fractions from Styrax japonica fruit

Reduction in reactive oxygen species (ROS) production facilitated by Styrax japonica (SJ) extracts and fractions for Inhibition of Obesity development through Inhibition of Adipogenesis was investigated. Treatment of cells with SJ extracts and fractions did not result in changes in cell viability. SJ extracts and fractions Inhibited lipid accumulation in a dose-dependent manner during Adipogenesis. Expressions of PPARγ and C/EBPα, key Adipogenic markers, were decreased in cells treated with SJ extracts and fractions.

Inhibitory effects of SJ extracts and fractions were the most powerful in the early stages of Differentiation (days 0-2). Intracellular ROS production was decreased in cells treated with SJ extracts and fractions. SJ extracts and fractions can Inhibit Adipogenesis via Reduced ROS levels, which involves down-regulation of Adipogenic transcription factors.

Sulforaphene

Sulforaphene Inhibition of Adipogenesis via Hedgehog Signaling in 3T3-L1 Adipocytes

Obesity is a risk factor for numerous metabolic disorders. In this study, we investigated the effects of the isothiocyanates sulforaphane (SA) and sulforaphene (SE) on Adipogenesis in 3T3-L1 Adipocytes. SE, a compound that is abundant in radish, Inhibited Adipogenesis by suppressing the Adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ, 69.2 ± 2.4%, P < 0.05) and CCAAT/enhancer-binding protein α (C/EBPα, 36.1 ± 3.1%, P < 0.05), thereby reducing Fat Accumulation in 3T3-L1 Adipocytes (45.6 ± 2.7%, P < 0.05); SA was less effective. SE exerted these activities through the activation of the Hedgehog (Hh) signaling pathway by restoring Smo ((2.1 ± 0.2)-fold, P < 0.05) and Gli1 ((2.8 ± 0.1)-fold, P < 0.05) expression, which was suppressed by Adipogenic signals. These effects of SE were abrogated by treatment with the Hh Inhibit or vismodegib.

Thus, SE Inhibits Adipocyte Differentiation via Hh signaling and may be an effective natural agent for preventing Adipocyte hyperplasia and Obesity.

Sulfuretin (Rhus verniciflua Stokes)

Exploration of underlying mechanism of anti-Adipogenic activity of sulfuretin

Sulfuretin is a natural flavonoid found in the plant Rhus verniciflua STOKES. The plant has been traditionally used as medicinal agent for antiviral, cathartic, diaphoretic, anti-rheumatic and sedative activities in East Asia. In this study we isolated and identified sulfuretin from R. verniciflua and investigated its anti-adipogenic activity against 3T3-L1 preadipocytes cells. We evaluated the effects of sulfuretin on the adipogenic transcription factors like peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), Fabp4, adiponectin and zinc fingerprint protein (Zfp) 521 by gene expression (real-time QPCR) and Western blot analysis. Sulfuretin treatment at Day 0 and 2 showed significant reduction of lipid production in 3T3-L1 cells in concentration dependent manner. Gene expression analysis (real-time PCR) revealed that sulfuretin inhibited the both major adipogenic factors (C/EBPα, C/EBPβ and PPARγ) and minor adipogenic factors (sterol regulatory element-binding protein (SREBP1c), adiponectin, FAS, Fabp4, Zfp423, and Ebf1). Western blot analysis showed the increased expression of β-catenin and suppression of PPARγ after sulfuretin treatment. Overall, sulfuretin is a natural flavonoid having potent anti-adipogenic activity through the suppression of major adipogenic factors C/EBPα, C/EBPβ and PPARγ, which initiate adipogenesis.

Sulfuretin has therapeutic activity against acquired lymphedema by reducing Adipogenesis

Acquired lymphedema is a pathological condition associated with lymphatic dysfunction caused by surgical treatments for cancer. Although global estimates of the prevalence of acquired lymphedema have been rising, there are currently no effective therapeutics available. Since adipose tissue accumulation is a clinical hallmark of lymphedema, we hypothesized that regulation of adipogenesis in lymphedematous tissue could be used as a therapeutic intervention against lymphedema. Toward this, we investigated the possibility of anti-adipogenic 30% ethanol Rhus verniciflua Stokes (RVS) extract as a potential lymphedema treatment. Oral administration of RVS extract ameliorated volumetric symptoms of lymphedema in a mouse model. RVS administration also reduced adipose tissue accumulation in lymphedematous tissue and downregulated expression of adipocyte markers, including Pparγ and Fabp4. Sulfuretin was identified as a major bioactive compound in the 30% ethanol RVS extract in liquid chromatography-mass spectrometry analysis. Similar to the activities of RVS, sulfuretin inhibited adipocyte differentiation in 3T3-L1 preadipocytes. Moreover, treatment with sulfuretin on lymphedema-induced mice reduced lymphedema volume, decreased the expression of adipogenic markers, but induced the expression of markers associated with lymphangiogenesis. Taken together, our data raise the possibility that sulfuretin might be used in therapeutic interventions against acquired lymphedema.

Keywords: Acquired lymphedema; Adipogenesis; Oral administration; Rhus verniciflua extract; Sulfuretin.

Sulfuretin Prevents Obesity and Metabolic Diseases in Diet Induced Obese Mice

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT.

To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin’s but not resveratrol’s anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.

Keywords: Adipocyte; Atf3; Diabetes; Metabolic diseases; Obesity; Sulfuretin.

tannic acid (Chinese gallnut extract)

Tannic Acid Stimulates Glucose Transport and Inhibits Adipocyte Differentiation in 3T3-L1 cells

Obesity is a major risk factor for Syndrome X and type II Diabetes (T2D). However, most AntiDiabetic drugs that are hypoglycemic also promote Weight Gain, thus alleviating one symptom of T2D while aggravating a major risk factor that leads to T2D. Adipogenesis, the Differentiation and proliferation of Adipocytes, is a major mechanism leading to Weight Gain and Obesity. It is highly desirable to develop pharmaceuticals and treatments for T2D that Reduce blood glucose levels without inducing Adipogenesis in patients. Previously, we reported that an extract from Lagerstroemia speciosa L. (banaba) possessed activities that both stimulated glucose transport and Inhibited Adipocyte Differentiation in 3T3-L1 cells.

Using glucose uptake assays and Western/Northern blot analyses as major tools and 3T3-L1 cells as a model, we showed that the banaba extract (BE) with tannin removed was devoid of the 2 activities, and tannic acid (TA), a major component of tannins, had the same 2 activities as BE. Inhibitors known to abolish insulin-induced glucose transport also blocked TA-induced glucose transport. We further detected that TA induced phosphorylation of the insulin receptor (IR) and Akt, as well as translocation of glucose transporter 4 (GLUT 4), the protein factors involved in the signaling pathway of insulin-mediated glucose transport.

We also demonstrated that TA Inhibited the expression of key genes for Adipogenesis. Differences between samples with or without TA in all of the quantitative assays were significant (P < 0.05). These results suggest that TA may be useful for the prevention and treatment of T2D and its associated Obesity. TA may have the potential to become the lead compound in the development of new types of AntiDiabetic pharmaceuticals that are able to Reduce blood glucose levels without increasing Adiposity.

Taraxacum officinale (Dandelion)

Reduction of Adipogenesis and Lipid Accumulation by Taraxacum officinale (Dandelion) Extracts in 3T3L1 Adipocytes : An in vitro Study

In this in vitro study, we have investigated the ability of Taraxacum officinale (dandelion) to Inhibit Adipocyte Differentiation and lipogenesis in 3T3‐L1 preadipocytes. HPLC analysis of the three plant extracts used in this study—leaf and root extracts and a commercial root powder—identified caffeic and chlorogenic acids as the main phenolic constituents. Oil Red O staining and triglyceride levels analysis showed decreased lipid and triglyceride accumulation, respectively. Cytotoxicity was assessed with the MTT assay showing non‐toxic effect among the concentrations tested.

DNA microarray analysis showed that the extracts regulated the expression of a number of genes and long non‐coding RNAs that play a major role in the control of Adipogenesis. Taken together, our results indicate that the dandelion extracts used in this study may play a significant role during Adipogenesis and lipid metabolism, and thus, supporting their therapeutic interest as potential candidates for the treatment of Obesity.

Tocotrienol (palm oil )

Tocotrienol Suppresses Adipocyte Differentiation and Akt Phosphorylation in 3T3-L1 preadipocytes

In vivo studies show that α-tocotrienol and γ-tocotrienol accumulate in Adipose Tissue . Furthermore, a recent study reports that the oral administration of γ-tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on Adipocyte Differentiation in 3T3-L1 preadipocytes , which differentiated into Adipocytes in the presence of 1.8 μmol/L insulin. TRF suppressed the insulin-induced mRNA expression of Adipocyte -specific genes such as PPARγ, Adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein-α (C/EBPα) compared with the Differentiation of 3T3-L1 preadipocytes into Adipocytes only in the presence of insulin.

To confirm the suppressive effect of TRF, the major components of TRF, such as α-tocotrienol, γ-tocotrienol, and α-tocopherol, were investigated. α-Tocotrienol and γ-tocotrienol decreased the insulin-induced PPARγ mRNA expression by 55 and 90%, respectively, compared with insulin, whereas α-tocopherol increased the mRNA expression.

In addition, γ-tocotrienol suppressed the insulin-induced aP2 and C/EBPα mRNA expression, triglyceride accumulation, and PPARγ protein levels compared with insulin. The current results also revealed that γ-tocotrienol Inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes . Thus, the AntiAdipogenic effect of TRF depends on α-tocotrienol and γ-tocotrienol, and γ-tocotrienol may be a more potent Inhibit or of Adipogenesis than α-tocotrienol.

Therefore, the results of this study suggest that tocotrienol Suppresses insulin-induced Differentiation and Akt phosphorylation in 3T3-L1 preadipocytes . Furthermore, tocotrienol could act as an AntiAdipogenic vitamin in the nutrient-mediated regulation of body fat through its effects on Differentiation .

Tricin (Wheat Grain )

Oat hull ethyl acetate extract (50 μg/μL) significantly Inhibited triglyceride accumulation in 3T3-L1 Adipocytes without causing cytotoxicity. The major active compound in the extract was isolated and identified as 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone, tricin by Mass spectrometry and 1H and ¹³C NMR analyses. The tricin content in oat hull was approximately 18 mg/kg dry weight, and tricin constituted 9.6% of the total phenolic compounds in oat hull extract. The treatment of 3T3-L1 cells either with extract or purified tricin resulted in decreased lipid accumulation. The extract significantly decreased the activity of glycerol-3-phosphate dyhydrogenase, a marker of Adipocyte Differentiation in a dose dependent manner.

The expression of major transcription factors, such as peroxisome proliferator-activated receptor (PPAR)-γ, CCATT/enhancer binding protein-α (CEBP/α), sterol regulatory binding protein 1 (SREBP1) and fatty acid synthase (FAS) was also significantly suppressed by the extract. When commercial purified tricin was added to 3T3-L1 cells the expression of all transcription factors were significantly decreased as low as 1.5 μg/μL treatment. These results suggest that oat hull extract containing tricin possesses anti-adipogeneic effect.

Trigonella foenum

Anti–Obesity action of INDUS810, a natural compound from Trigonella foenum-graecum: AMPK-dependent Lipolysis effect in Adipocytes

Objective: We investigated the anti-obesity effect and underlying action mechanism of INDUS810 isolated from Trigonella foenum-graecum L. (Fabaceae), an annual herb commonly known as fenugreek and reported to have hypocholesterolemic, antidiabetic, anticancer and gastroprotective properties.

Methods: For obese animal study, 4-week old mice were fed with normal diet or high-fat diet together with or without intraperitoneal injection of INDUS810 (200mg/kg) twice per week for 15weeks. 3T3-L1 cells were used to study action mechanism of INDUS810 in adipocyte differentiation and lipid metabolism.

Results: We found that INDUS810 can reduce high-fat diet-induced weight increase in epididymal white adipose tissue, interscapular brown adipose tissue and liver, as well as serum levels of total cholesterol and low-density lipoprotein cholesterol. Moreover, the insulin sensitivity was significantly improved in INDUS810-treated obese mice. In 3T3-L1 adipocytes, we found that INDUS810 could inhibit lipid accumulation at either differentiating or mature stages, and increase lipolysis activity in mature adipocytes. Additionally, INDUS810 has no effects on cell viability nor the expressions of adipocyte differentiation markers like fatty acid synthase, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α. In contrast, INDUS810 can increase protein levels of peroxisome proliferator-activated receptor α, peroxisome-proliferator-activated receptor-γ co-activator 1β, sirtuin 1 and sirtuin 3. Of note, INDUS810 can activate adenosine monophosphate-activated protein kinase, which leads to the reduction of lipid contents in adipocytes.

Conclusion: Our in vitro and in vivo studies suggest that INDUS810 is a potential anti-obesity agent, and this action depends on activate adenosine monophosphate-activated protein kinase activation.

Keywords: AMPK; Fenugreek; Lipolysis; Obesity.

Polyphenol stilbenes from fenugreek (trigonella foenum-graecum L.) seeds improve insulin sensitivity and mitochondrial function in 3T3-L1 Adipocytes

Fenugreek (Trigonella foenum-graecum L.) is a well-known annual plant that is widely distributed worldwide and has possessed obvious hypoglycemic and hypercholesterolemia characteristics. In our previous study, three polyphenol stilbenes were separated from fenugreek seeds. Here, we investigated the effect of polyphenol stilbenes on adipogenesis and insulin resistance in 3T3-L1 adipocytes. Oil Red O staining and triglyceride assays showed that polyphenol stilbenes differently reduced lipid accumulation by suppressing the expression of adipocyte-specific proteins.

In addition, polyphenol stilbenes improved the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by promoting the phosphorylation of protein kinase B (AKT) and AMP-activated protein kinase (AMPK). In present studies, it was found that polyphenol stilbenes had the ability to scavenge reactive oxygen species (ROS).

Results from adenosine triphosphate (ATP) production and mitochondrial membrane potentials suggested that mitochondria play a critical role in insulin resistance and related signaling activation, such as AKT and AMPK. Rhaponticin, one of the stilbenes from fenugreek, had the strongest activity among the three compounds in vitro. Future studies will focus on mitochondrial biogenesis and function.

Triticum aestivum

Flavonoids from Triticum aestivum Inhibit Adipogenesis in 3T3-L1 cells by upregulating the insig pathway

The present study aimed to compare the potential Anti-Adipogenic effects and underlying mechanisms of the luteolin, isoscoparin and isoorientin Flavonoids, purified from Triticum aestivum sprout (TA) in 3T3‑L1 cells. The cells were treated with different concentrations of Flavonoids for 8 days and the lipid accumulation was assessed using Oil‑Red‑O staining. The expression levels of the transcription factors and the genes involved in Adipogenesis in the cells were assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting. The results demonstrated that 10 µM luteolin, isoscoparin or isoorientin Inhibited lipid deposition in the cells by 74, 63 and 65%, respectively.

The Flavonoids also significantly Inhibited the transcriptional regulators of Adipogenesis , including peroxisome proliferator‑activated receptor‑γ, CAAT/enhancer binding protein‑α and sterol regulatory element binding protein (SREBP)‑1c, compared with the control cells. Similarly, there was a significant downregulation of the Adipocyte specific markers associated with lipid metabolism, including activating protein‑2, fatty acid synthase, hormone‑sensitive lipase and lipoprotein lipase, in the flavonoid treated cells. Notably, the cells treated with the Flavonoids demonstrated increased expression levels of the insulin‑induced genes, insig‑1 and insig‑2, which may have Inhibited the activation of the Adipogenic transcription factor, SREBP, eventually leading to the Inhibition of Adipogenesis.

Taken together, these results revealed that the Flavonoids from TA possessed an Inhibitory effect on Adipogenesis through downregulation of Adipogenic transcription factors and genes associated with lipid metabolism, and the upregulation of insig 1 and 2, suggesting that the Flavonoids from TA may be potential therapeutic agents for the prevention and treatment of Obesity.

Tyrosol (from Rhodiola crenulata)

Rhodiola‐induced Inhibition of Adipogenesis involves antioxidant enzyme response associated with pentose phosphate pathway

The aim of this study was to investigate whether Rhodiola crenulata extract and tyrosol, a major bioactive phenolic compound present in Rhodiola, change the activities of endogenous antioxidant enzyme response (AER) and energy pathways linked to proline‐mediated pentose phosphate pathway (PPP) during Adipogenesis. Treatment with Rhodiola extracts Inhibited the activities of proline dehydrogenase (PDH) and glucose‐6‐phosphate dehydrogenase (G6PDH) as well as lipid accumulation and reactive oxygen species (ROS) production.

The Inhibition of PDH and G6PDH activities by Rhodiola likely prevented proline oxidation required for critical ATP generation that is coupled to AER via the PPP, leading to Inhibition of Adipogenesis. Rhodiola extracts dose‐dependently increased superoxide dismutase (SOD) activity, resulting in a Reduced ROS level during Adipogenesis. Moreover, the effects of tyrosol, a major bioactive compound in Rhodiola species, were directly correlated with all observed effects by Rhodiola extracts. These results indicate that the AntiAdipogenic effects of Rhodiola extracts can be attributed to a phenolic tyrosol that may potentially disrupt proline‐mediated energy generation and AER via PPP, resulting in the suppression of Adipogenesis and lipid accumulation. This further provides a biochemical rationale to identify the roles of phenolics that modulate the cellular redox environment and therefore have relevance for Obesity management.

Ulmus pumila

An active extract of Ulmus pumila Inhibits Adipogenesis through regulation of cell cycle progression in 3T3-L1 cells

Obesity and its associated metabolic disorders has become a major obstacle in improving the average life span. In this regard therapeutic approach using natural compounds are currently receiving much attention. Herbal compounds rich in triterpenes are well known to regulate glucose and lipid metabolism. Here, we have found that Ulmus pumila (UP) contained at least four different triterpenoids and Inhibited Adipogenesis of 3T3-L1 cells.

The cell viability was dose dependently decreased by UP showing the increase of cell accumulation in G1 phase while reducing in S and G2/M phase of cell cycle. UP treatment also significantly decreased the GPDH activity and intracellular lipid accumulation. In addition, UP Inhibited the mRNA levels of Adipogenic transcription factors and lipogenic genes such as PPARγ, C/EBPα, SREBP1c and FAS while showing no effects on C/EBP-β and C/EBP-δ. Importantly enough, treatment of cells with UP suppressed the TNF-α induced activation of NF-κB signaling. Collectively, our results indicate that UP extract effectively attenuated Adipogenesis by controlling cell cycle progression and down regulating Adipogenic gene expression.

Highlights

► Ulmus pumila (UP) contains at least four different triterpenoids. ► UP affect the viability of Adipocytes by causing G1 cell cycle arrest. ► UP down regulated lipid accumulation dose dependently in 3T3-L1 cell line. ► UP suppressed the expression of C/EBPα , PPARγ , SREBP-1c, FAS, C/EBPβ, C/EBPδ and NF-κβ. ► UP can be of interest as therapeutic agent for preventing or treating Obesity.

Ursolic Acid

Ursolic acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK pathway

Background: Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood.

Objective: As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes.

Methods and results: The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBPβ), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT element binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished.

Conclusions: Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.

Ursolic acid, a promising dietary bioactive compound of anti‐Obesity (1045.40)

Ursolic acid has recently been reported to possess a promising anti-obesity effect. A number of studies have been conducted to elucidate the underlying mechanisms, which include the inhibition of pancreatic lipase activity, promotion of muscle hypertrophy, increase of brown fat, thermogenesis, energy expenditure, and lipolysis. Our recent study in 3T3-L1 preadipocytes has demonstrated that ursolic acid inhibits cell differentiation and adipogenesis. This compound modulates the expression or activity of many proteins or enzymes involved in fat cell differentiation, fatty acid synthesis and oxidation. Further studies have shown that ursolic acid increases the phosphorylation and activity of AMPK and the protein expression of Sirt1.

The anti-adipogenic effect of UA can be reversed by AMPK siRNA but not Sirt1 inhibitor. Moreover, when LKB1 is silenced or inhibited, the effect of UA on AMPK activation diminishes. These results demonstrate that ursolic acid inhibits preadipocyte differentiation and adipogene.

Vaccinium floribundum

Antioxidant Capacity and in Vitro Inhibition of Adipogenesis and Inflammation by Phenolic Extracts of Vaccinium floribundum and Aristotelia chilensis

Furthermore, a proanthocyanidin-enriched fraction from V. floribundum significantly increased Pref-1 expression in preadipocytes. Phenolic extracts decreased the production of nitric oxide (3.7−25.5%) and prostaglandin E₂ (9.1−89.1%) and the expression of inducible nitric oxide synthase (9.8−61.8%) and cycloxygenase-2 (16.6−62.0%) in lipopolysaccharide-stimulated RAW 264.7 macrophages. V. floribundum and A. chilensis phytochemicals limit Adipogenesis and inflammatory pathways in vitro, warranting further in vivo studies.

Vanillic Acid

Vanillic acid attenuates Obesity via activation of the AMPK pathway and thermogenic factors in vivo and in vitro

Previous studies have provided much evidence on the availability of VA in regulating inflammatory or immune diseases. For instance, VA has hepatoprotective effects by suppressing immune‐mediated liver Inflammation (40). Another study suggested that VA Suppresses Inflammation in ulcerative colitis (19). Because excessive Obesity is associated with Inflammation (41), these previous reports suggest that VA may potentially play a role in Obesity control. Therefore, in this study, we investigated whether VA has any AntiObesity effects with in vivo and in vitro experiment models.Fat tissue is a major organ involved in the development of Obesity and consists of two types of Adipose Tissues in humans: WAT and BAT.

They have opposite roles in maintaining energy homeostasis (42). Adipogenesis is a regulated cellular Differentiation process in which preadipocytes are transformed into mature Adipocytes (43). During Adipogenesis of 3T3‐L1 cells, early stage‐expressed master regulators, C/EBPα and PPARγ, induce Differentiation of ectopic expression in non Adipogenic fibroblasts (44, 45). PPARγ, a member of the nuclear receptor family, has been detected both in humans and mice. It acts as one of the key factors regulating glucose metabolism and involves in pathologies of various diseases such as Obesity , cancer, and Inflammation (46, 47). C/EBPα, a member of the basic region leucine zipper family of transcription factors, is involved in Adipogenesis of the liver and Adipose Tissue. In the present study, VA treatment resulted in decreased expressions of PPARγ and C/EBPα in 3T3‐L1 cells. Similarly, in vivo results showed that such factors were decreased by VA in WATs of db/db and HFD‐fed mice. We also observed that VA treatment decreased the expressions of several adipokine and Adipogenesis ‐related genes in 3T3‐L1 Adipocytes , including Retn, Fabp4, AdipoQ, and Lipin1. Adipokines are water‐soluble substances related to Adipocytes (48), of which resistin, aP2 and adiponectin, are all well‐known representatives that play important roles in Obesity (49, 50).

These results support the notion that VA Inhibits Adipogenesis by suppressing Adipogenic factors.In addition to the Adipose Tissue s, the liver is also an energy‐storing organ involved in whole‐body metabolism (51). It plays a central role in metabolic homeostasis because it is a major site of synthesis and metabolism of carbohydrates, proteins, and lipids (52, 53). The rapid increase in Obesity worldwide is associated with the increase of lipid storage in the liver, leading to nonalcoholic fatty liver diseases, especially in the Western world (54,55). Our results show that VA attenuates hepatotoxicity and hepatitis while reducing Fat Accumulation and activating FA oxidation in vivo and in vitro, suggesting its possible use for nonalcoholic fatty liver disease‐related Obesity.

AMPK is a major regulator of body energy homeostasis because it regulates hormones such as leptin and adiponectin, which are substances related to appetite and metabolism (49). AMPK is activated under situations of ATP exhaustion such as metabolic stress or exercise and regulates the AMP/ATP ratio by suppressing FA and glucose consumption, which leads to ATP production. AMPK activation includes AMP‐mediated allosteric activation and phosphorylation of the threonine residue Thr172 located in the catalytic subunit of AMPK, which is regulated by upstream kinases like LKB1 or Ca²⁺/calmodulin‐dependent kinase II β (10,11, 56‐59). Activated AMPK causes phosphorylation and inactivation of ACC and 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, which are all required for ATP consumption. AMPK also Inhibits the expressions of genes related to lipid synthesis including FA synthase, pyruvate kinase, and ACC. The inactivated pACC results in a decreased level of malonyl‐CoA and an increased activity of carnitine palmitoyltransferase 1 (CPT1), leading to increased FA oxidation in β‐oxidation (12‐14). In addition to ACC, AMPK has been shown to activate HSL activity.

When HSL is phosphorylated in response to PKA activating signals, it actively removes FAs from TG (60). Overexpression of AMPKα by 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR) or metformin in the liver of normal‐group and Obesity‐group animals increases the level of β‐hydroxybutyrate, which is the growth indicator of lipid oxidation, and Reduces blood TG. In addition to its action on enzyme activities, activation of AMPK also affects the expression of a number of glycolytic or lipogenic enzymes in the liver and Adipose Tissue. Included in this list are the genes for fatty acid synthase, ACC, and sterol regulatory element binding proteins (SREBP), key regulators of numerous lipogenic enzymes (61‐63). This shows that AMPK mediates the balance of fat metabolism in the liver through Reduced fat synthesis and Lipolysis (64). In our study, pAMPK and pLKB1 were increased in the liver tissues of both HFD‐fed and db/db mice by VA. VA also suppressed Fat Accumulation in the liver tissue, and increased expressions of pAMPK and pLKB1 in the WAT of HFD‐fed and db/db mice. Similarly, in Adipocytes, VA treatment activated the AMPK pathway.

We then pretreated the Adipocytes with siAMPKα to investigate the underlying mechanism of VA. The Inhibition rates of PPARγ and C/EBPα, which were originally suppressed by VA treatment, were significantly Reduced in the siAMPKα‐treated cells. This shows that the AntiAdipogenic action of VA is due at least partially to activation of the AMPK signaling pathway.On the basis of several reports regarding the importance of AMPK in BAT activation, we then performed experiments on brown Adipocyte—specific thermogenesis with the expectation that VA could induce it. Our results revealed that VA treatment in HFD‐induced obese mice and genetically obese db/db mice resulted in increased UCP1 and PGC1α in BAT. Furthermore, mitochondria‐specific proteins as well as UCP1 and PGC1α in primary brown Adipocytes were also increased by VA in a dose‐dependent manner. These results indicate that the AntiObesity effect of VA was due to the increased metabolic energy consumption through mitochondrial action. AMPK activation increases the expressions of mitochondria‐related genes through PGC1α expression. By increasing cellular NAD⁺ levels, AMPK activates the NAD‐dependent deacetylase SIRT1, which results in deacetylation or activation of PGC1α (65‐67).

The reported relationship between AMPK and these brown Adipocyte‐related factors including PGC1α and mitochondria suggests that VA has thermogenic effects. BAT is responsible for consuming energy in the form of heat, a process called thermogenesis. In contrast to previous knowledge, recent studies have shown that BAT also exists in adult humans (68). Brown Adipocytes, consisting of BAT, have a large quantity of mitochondria and specifically express a brown color. The inner membranes of mitochondria in these Adipocytes possess the UCP1 protein (69). The thermogenic action of BAT is dependent on the activity of this particular protein, which responses to cold exposure (70, 71). Recent studies also identified that PGC1α is related to both the expression and action of UCP1 (72). Thermogenesis in BAT by UCP1 is an important part of energy consumption in rodents. According to a study by Baumruk et al. (73), UCP1 overexpression in WAT led to Reduced Obesity induced by an HFD.

Therefore, UCP1 has gained recognition as a novel target in the field of Obesity. We observed elevated skin temperature of the BAT region in VA‐fed mice. After a 6 h cold challenge test, VA‐fed mice rapidly recovered their core body temperature. Further analysis showing increase in mitochondrial and thermogenic factors by VA treatment suggests a possibility of VA as a thermogenic agent. However, further investigation is essential to confirm the detailed mechanism of VA on thermogenesis. Because the results of this study provide evidence for the AMPK‐linked action of VA, the goal of our next study is to reveal the precise mechanisms of VA in brown Adipocytes.

Our study demonstrates the antiobese action of VA in vivo and in vitro. VA significantly improved Obesity in HFD‐induced obese mice and db/db mice. In addition, VA treatment markedly decreased the expressions of Adipogenic markers in 3T3‐L1 cells, which was due to AMPK activation. Furthermore, VA increased the expressions of mitochondrial and thermogenic factors in both BAT and primary cultured brown Adipocytes. These data, taken together, indicate that VA possesses AntiObesity effects by increasing energy metabolism in white and brown Adipocytes, suggesting its potential use in the field of Obesity treatment.

Veratrum nigrum

Veratri nigri rhizoma et radix (Veratrum nigrum L.) and its constituent jervine Prevent Adipogenesis

In this study, we have evaluated the effects of VN and its constituent jervine on Obesity using the in vivo HFD-induced obese mouse model and the in vitro 3T3-L1 Adipocyte model, for the first time.

Obesity is a chronic metabolic disorder caused by an imbalanced energy intake-expenditure status [34]. The prevalence of Obesity is growing; in the year 2008, the worldwide Obesity has nearly doubled since 1980 [1]. Current medications for the treatment of Obesity include mixed noradrenergic-serotonergic agents (sibutramine) [35] and absorption-reducing agents (orlistat) [36]. However, these two drugs show adverse effects at high frequencies. For example, sibutramine is reported to cause cardiac arrhythmias, constipation, and headache with only minimum weight loss [35], and orlistat can show steatorrhea and lipid-soluble-vitamin-deficiency [36]. Due to the limits of currently available drugs, the necessity for new drugs for the treatment of Obesity is rapidly growing, and the interest in natural products especially is increasing.

Veratrum nigrum L. is a medicinal plant used in Traditional Chinese and Korean Medicine native to Asia and Europe. In the plant, mainly the stem and root of Veratrum nigrum L., Veratri Nigri Rhizoma et Radix, are administered internally as an emetic medicine in cases of strokes or epilepsies or also topically treated in order to kill parasites or to stop pruritus [37]. But because of its toxicology, Veratri Nigri Rhizoma et Radix is not widely used, as it is difficult to prepare a safe yet effective dose [38]. Therefore, only few reports on Veratri Nigri Rhizoma et Radix are currently published. Among those studies, none has reported the effects of VN on Obesity or Adipogenesis.

Jervine (C₂₇H₃₉O₃N), a steroidal alkaloid derived from the Veratrum genus [39], which is reported to have antitumor effects [40, 41], was detected as an active compound of VN by the HPLC analysis. Jervine and VN both successfully suppressed lipid accumulation and expressions of Adipogenic genes PPARγ and C/EBPα in 3T3-L1 Adipocytes. However, the AntiAdipogenic effects of VN were higher than jervine, and thus further investigations were performed in order to assess the effects of VN.

As the basic in vitro experiments preceded suggested positive effects on Obesity, an in vivo experiment was carried on using C57BL/6J mice. As expected, VN had beneficial effects on Obesity in the animal model, too. The Weight Gains and sWAT weights were significantly suppressed in the VN administered group. Serum analyses also confirmed the beneficial effects of VN on Obesity. On the other hand, ALT and creatinine, the barometers measuring liver and kidney toxicity, respectively, were not negatively affected but showed lower levels than the HFD group. These results are conflict to the formerly known toxicity of Veratrum nigrum [38, 42]. The in vivo results do not only prove the beneficial effects of VN in Obesity, but the toxicity-safe dosage of VN also shows potential application to human treatment as well, leading to expansion of the limited oral use of VN.

Based on the positive in vivo results on Obesity, we then performed more experiments back at the cell level, in order to find out which responsible mechanism was giving the beneficial effects. First, an Oil Red O staining assay showed suppressed lipid accumulation by VN treatment. In addition, the mRNA levels of Adipogenic genes including PPARγ, C/EBPα, aP2, adiponectin, resistin, and LIPIN1 were downregulated. The suppression of the genes suggested the Inhibiting effect on Adipogenesis by VN treatment. PPARγ and C/EBPα are well known as important regulators of Adipogenesis [5–7], while adipose-derived adipokines, aP2, adiponectin, and resistin possess their roles in Adipocytes in lipid signaling [9], glucose regulation [43], and insulin resistance [10], respectively. On the other hand, LIPIN1 is a candidate gene for lipodystrophy [11]. In addition to these genes, the level of SIRT1, the NAD⁺-dependent protein deacetylase [12], which is able to suppress Adipogenesis, was significantly upregulated by VN at the highest dose of 1 mg/mL. The elevated SIRT1 expression suggested the effects of VN on the SIRT1-AMPKα axis, which is a key factor in the etiology of Obesity.

Previous studies have reported the detailed role of SIRT1-AMPKα axis in Obesity. According to Ruderman et al., AMPKα is suggested to play a central role in Metabolic Syndromes [44]. Other numerous studies also link the phosphorylation of AMPK to Obesity in 3T3-L1 models [3, 8, 24, 45, 46]. Several genetic rodent models with a Metabolic Syndrome phenotype, such as ob/ob mice or fa/fa rats, show decreased AMPK activity [47], and when the decreased AMPK activity is restored by AICAR, they showed improved glucose homeostasis [48, 49]. Sirtuins, a group of histone/protein deacetylases, are regulated by the NAD⁺/NADH ratio. SIRT1 is the most well-known member of this family, which is reported to respond to changes in energy expenditure [47], which is similar to AMPK. Other studies revealed that SIRT1 can activate AMPK by deacetylating LKB1, the upstream kinase of AMPK [50, 51], and vice versa AMPK can activate SIRT1 by increasing the NAD⁺/NADH ratio [52]. Therefore, these previous reports suggest the important role of SIRT1-AMPKα axis, or circle, in Obesity.

The Real-Time RT-PCR result showing the upregulation of the AntiAdipogenic geneSIRT1 by VN treatment suggested the possible effects of VN on the SIRT1-AMPKαcircle. As we expected, a Western blot analysis confirmed the effect of VN on AMPKαphosphorylation, subsequently to the previous results. VN treatment also suppressed the expression of PPARγ and C/EBPα at the protein levels. Unlike AMPKα, however, the phosphorylation of ACC and LKB1, the upstream and downstream enzymes of AMPKα, respectively, were not upregulated as we expected. These results were conflict to our former researches, in which the protein expressions of p-ACC or p-LKB1 were successfully elevated by treatments showing antiobese features [8, 24]. LKB1, also known as serine/threonine kinase 11, is a protein kinase encoded from the LKB1 gene [53]. Originally known as a tumor suppressor, LKB1 is also related to Obesity due to its role as an upstream factor of the energy homeostasis regulator, AMPK [54]. The downstream target of AMPK, ACC, is dephosphorylated by AMPK Inhibition [55], and activation of AMPK leads to Inhibition of cholesterol synthesis by direct phosphorylation of ACC [56]. However, recent studies report that p-LKB1 [57] or p-ACC [58] is not essential in the cascade of AMPK phosphorylation.

In contrast to the p-ACC and p-LKB1 expressions, the effect of VN on AMPKαactivation was surely confirmed, as we coadministered the AMPK activator AICAR and the AMPK Inhibit or Compound C with VN. AICAR and Compound C were able to activate or attenuate the phosphorylation of AMPKα. In addition, the treatment of VN was able to restore the Inhibited AMPKα phosphorylation by Compound C, nearly up to the AMPK level by AICAR activation. These cotreatment results suggest the effect of VN on the LKB1-AMPKα-ACC axis, by solely affecting the AMPK activation only.

Our results on sole phosphorylation of AMPK in the LKB1-AMPK-ACC pathway suggest that the AntiAdipogenic features of VN resulted from direct activation of AMPK by VN and proceed through SIRT1 activation, which leads to Inhibition of PPARγ and C/EBPα. However, the detailed mechanism for how VN regulates Adipogenesis regarding sole activation of AMPK within the LKB1-AMPK-ACC axis requires further investigation.

In summary, our results demonstrated that VN contains jervine, and they both can attenuate lipid accumulation during 3T3-L1 Adipogenesis. VN showed beneficial effects on Obesity in a HFD-induced obese C57BL/6J mouse model. In 3T3-L1 Adipocytes, VN was able to attenuate Adipogenic factors and upregulate SIRT1 and AMPKαphosphorylation, suggesting its ability to activate the SIRT1-AMPKα circle. These results led to further investigations involving the LKB1-AMPKα-ACC axis. VN treatment was able to compensate for the action of the AMPK Inhibit or, Compound C. These results suggest the potential of VN as an AMPKα axis-modulating antiobese agent.

Viburnum opulus L.

Viburnum opulus L. Juice Phenolics Inhibit Mouse 3T3-L1 cells Adipogenesis and Pancreatic Lipase Activity

Viburnum opulus L. fruit is a rich source of phenolic compounds that may be involved in the prevention of metabolic diseases. The purpose of this study was to determine the effects of Viburnum opulus fresh juice (FJ) and juice purified by solid-phase extraction (PJ) on the Adipogenesis process with murine 3T3-L1 Preadipocyte cell line and pancreatic lipase activity in triolein emulsion, as well as their phenolic profiles by UPLC/Q-TOF-MS. Decrease of lipids and triacylglycerol accumulation in differentiated 3T3-L1 cells were in concordance with downregulation of the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPβ/α), and sterol regulatory element-binding protein 1c (SREBP-1c).

Furthermore, regulation of PPARγ-mediated β-lactamase expression by V. opulus components in reporter gene assay, as well as their binding affinity to ligand-binding domain of PPARγ, were tested. In addition, the levels of enzymes involved in lipid metabolism, like fatty acid synthase (FAS) or acetyl-CoA carboxylase (ACC), were decreased, along with inflammatory cytokines, like tumor necrosis factorα (TNFα), interleukin-6 (Il-6) and leptin. Moreover, FJ and PJ were able to Inhibit pancreatic lipase, which potentially could Reduce the fat absorption from the intestinal lumen and the storage of body fat in the Adipose Tissues.

Thirty-two phenolic compounds with chlorogenic acid as the dominant compound were identified in PJ which revealed significant biological activity. These data contribute to elucidate V. opulus juice phenolic compounds’ molecular mechanism in Adipogenesis regulation in 3T3-L1 cells and dietary fat Lipolysis.

Vitexilactone, a Constituent from Vitex trifolia L

The Rosiglitazone-Like Effects of Vitexilactone, a Constituent from Vitex trifolia L. in 3T3-L1 preadipocytes

We isolated and identified three compounds, vitexilactone (1), vitexicarpin (2) and oleanolic acid (3), from the ethyl acetate extract of Vitex trifolia L., which up-regulates lipid accumulation and Adipogenesis in 3T3-L1 preadipocytes. Among the isolated compounds, the rosiglitazone (ROS)-like effects of 1 were the strongest. Compound 1 and ROS increased lipid accumulation and the expression of adiponectin and GLUT4 in the cell membrane and miniaturized the Adipocytes and decreased the phosphorylation of IRS-1, ERK1/2 and JNK in 3T3-L1 cells.

Since ROS-like effects of 1 in 3T3-L1 cells was suppressed by the addition of bisphenol A diglycidyl ether (BADGE), a peroxisome proliferator-activated receptor γ (PPARγ) antagonist, 1 was considered to act on Adipocytes as an action selective PPARγ agonist. From the results in the present study, 1 is a potential candidate for use as a novel insulin sensitizer.

Vitexin from Mung Bean

Vitexin, orientin and other Flavonoids from Spirodela polyrhiza Inhibit Adipogenesis in 3T3‐L1 cells

The Flavonoids were isolated from SPB and their chemical structures were identified as chrysoeriol (1), apigenin (2), luteolin (3), vitexin (4), cosmosin (5), orientin (6) and luteolin‐7‐O‐β‐d‐glucoside (7) by spectroscopic analysis. Studies on the Adipogenesis and intracellular triglyceride accumulation Inhibitory effect showed that compounds 4 and 6 had the highest activity and decreased C/EBPα and PPARγ protein expression level in 3T3‐L1 cells. These results suggest that the Flavonoids isolated from SPB, especially compounds 4 and 6, contribute to the Inhibitory activity of S. polyrhiza in 3T3‐L1 cells.

Vitisin A from Iris lactea Pall. var. chinensis (Fisch.) Koidz

Vitisin A Inhibits Adipocyte Differentiation through cell cycle arrest in 3T3-L1 cells

Inhibition of Adipocyte Differentiation is one approach among the Anti-Obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, Inhibits Adipocyte Differentiation most effectively of 18 stilbenes tested. Fat Accumulation and PPARγ expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly Inhibited Preadipocyte proliferation and consequent Differentiation within the first 2 days of treatment, indicating that the Anti-Adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the Preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was Reduced. Therefore, vitisin A seems to induce G1 arrestthrough p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A Inhibits Adipocyte Differentiation through Preadipocyte cell cycle arrest.

Wasabi (Wasabia japonica Matsum.) leaves

This study investigated the effect of hot water extract of wasabi (Wasabia japonica Matsum.) leaves (WLE), without its specific pungent constituents, such as allyl isothiocyanate, on the Differentiation of 3T3-L1 preadipocytes. WLE suppressed the increase in glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride (TG) accumulation, markers of Adipogenesis, in a dose-dependent manner. Quantitative real time RT-PCR results showed that WLE significantly Reduced the mRNA expression levels of peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein (C/EBP) α, both key Adipogenic transcription factors, as subsequently were the mRNA expression levels of their target genes, such as Adipocyte fatty acid binding protein 2 (aP2).

Western blot analysis results showed that the protein expression levels of both PPARγ and C/EBPα were also Inhibited by WLE. Thus, WLE suppressed the Differentiation of 3T3-L1 preadipocytes, and the suppressive effect was mediated, in part, through the altered regulation of PPARγ, C/EBPα, and other specific genes, such as aP2. These results suggest that WLE may Prevent Obesity and insulin resistance by Inhibiting the Differentiation of preadipocytes.

Wedelolactone

Wedelolactone Inhibits Adipogenesis through the ERK pathway in human Adipose Tissue ‐derived mesenchymal stem cells

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as Anti‐Inflammatory, growth Inhibitory, and pro‐apoptotic, the effects of wedelolactone on stem cell Differentiation remain largely unknown. Here, we report that wedelolactone Inhibits the Adipogenic Differentiation of human Adipose Tissue‐derived mesenchymal stem cells (hAMSCs).

Wedelolactone Reduced the formation of lipid droplets and the expression of Adipogenesis ‐related proteins, such as CCAAT enhancer‐binding protein‐α (C/EBP‐α), peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), lipoprotein lipase (LPL), and Adipocyte fatty acid‐binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, Inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone‐mediated Inhibition of Adipogenic Differentiation. Taken together, these results indicate that wedelolactone Inhibits Adipogenic Differentiation through ERK pathway and suggest a novel Inhibitory effect of wedelolactone on Adipogenic Differentiation in hAMSCs.

Widdrol (lindera communis)

Widdrol Blocks 3T3-L1 preadipocytes Growth and Differentiation Due to Inhibition of Mitotic Clonal Expansion

Adipocyte Differentiation is strongly associated with Obesity, which causes metabolic disorders. In this study, we investigated the Inhibitory effects of widdrol on 3T3-L1 Preadipocyte growth and Differentiation. Widdrol decreased lipid droplet accumulation and down-regulated Adipogenic transcription factors such as C/EBPα, C/EBPβ,and PPARγ. Widdrol blocked Preadipocyte proliferation and Differentiation through the Inhibition of mitotic clonal expansion, which was accompanied by the failure of degradation of p21, a cyclin-dependent kinase Inhibit or cell-cycle analysis clearly indicated that widdrol actively induces cell-cycle arrest at the G1-S phage transition, causing cells to remain in the Preadipocyte state. Moreover, widdrol increased p21 expression and Inhibited Rbphosphorylation in Preadipocyte incubated in a hormonemedium.

Therefore, these findings clearly suggest thatwiddrol blocks Preadipocyte growth and Differentiation through the Inhibition of mitotic clonal expansion by p21-and Rb-dependent G1 arrest and can be developed as apotent Anti-Adipogenic agent for reducing Obesity.

Xanthohumol (hops extract)

Effect of xanthohumol and isoxanthohumol on 3T3-L1 cell apoptosis and Adipogenesis

Xanthohumol (XN), the chalcone from beer hops has several biological activities. XN has been shown to induce apoptosis in cancer cells and also has been reported to be involved in lipid metabolism. Based on these studies and our previous work with natural compounds, we hypothesized that XN and its isomeric flavanone, isoxanthohumol (IXN), would induce apoptosis in Adipocytes through the mitochondrial pathway and would Inhibit maturation of preadipocytes. Adipocytes were treated with various concentrations of XN or IXN. In mature Adipocytes both XN and IXN decreased viability, increased apoptosis and increased ROS production, XN being more effective.

Furthermore, the Antioxidants ascorbic acid and 2-mercaptoethanol prevented XN and IXN-induced ROS generation and apoptosis. Immunoblotting analysis showed an increase in the levels of cytoplasmic cytochrome c and cleaved poly (ADP-ribose) polymerase (PARP) by XN and IXN. Concomitantly, we observed activation of the effectors caspase-3/7. In maturing preadipocytes both XN and IXN were effective in reducing lipid content, XN being more potent. Moreover, the major Adipocyte marker proteins such as PPARγ, C/EBPα, and aP2 decreased after treatment with XN during the maturation period and that of DGAT1 decreased after treatment with XN and IXN. Taken together, our data indicate that both XN and IXN Inhibit Differentiation of preadipocytes , and induce apoptosis in mature Adipocytes, but XN is more potent.

zeaxanthin

Anti–Obesity effects of zeaxanthin on 3T3-L1 Preadipocyte and high fat induced obese mice

Zeaxanthin, a type of carotenoid, has been proven to exhibit anti-lipogenesis effect; however, the detailed mechanism of this effect is less known. Herein, we evaluated the effects of zeaxanthin on the inhibition of adipogenesis in 3T3-L1 adipocytes and obesity in high-fat diet fed C57BL/6J mice. Zeaxanthin significantly decreased the intracellular lipid content in a dose-dependent manner (5–15 μM) in adipocytes without causing cytotoxicity. In high-fat-diet-induced obese mice, oral administration of 20 mg kg⁻¹ zeaxanthin attenuated the progression of obesity and improved dyslipidemia. It exhibits an anti-adipogenic effect via down-regulating the transcriptional factors and adipocyte-specific genes involved in adipogenesis, both in vitro and in vivo.

Furthermore, zeaxanthin treatment reversed the MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1.0 μM dexamethasone, and 1.0 μg mL⁻¹ insulin) and HFD (high-fat diet)-induced inhibition of AMPK phosphorylation in adipocytes and epididymal adipose tissues, respectively, thereby modulating the energy metabolism. These results indicated that zeaxanthin plays anti-adipogenic and anti-obesity roles by inducing AMPK activation, inhibiting lipogenesis, and decreasing intracellular lipid content, adipocyte size, and adipose weight.

Zeaxanthin promotes mitochondrial biogenesis and Adipocyte browning via AMPKα1 activation

Zeaxanthin (ZEA), a type of oxygenated carotenoid with strong antioxidant activity, has previously been found to exhibit an anti-lipogenesis effect. In the present study, we investigated the effect of ZEA on brown-like adipocyte formation and mitochondrial biogenesis in 3T3-L1 adipocytes. Brown adipocyte-specific markers, mitochondrial biogenesis and oxidative stress, and the involvement of AMP-activated protein kinase (AMPK) α1 were assessed. ZEA treated adipocytes demonstrated a brown-like pattern, with upregulated expression of uncoupling protein 1 (UCP1) and other brown adipocyte markers. In addition, ZEA intervention induced a dramatic increase in mitochondrial DNA (mtDNA) content and in the mRNA levels of genes associated with mitochondrial biogenesis.

Furthermore, ZEA attenuated mitochondrial oxidative damage caused by lipid peroxidation in adipocytes, significantly improved the mitochondrial membrane potential (MMP), and scavenged intracellular reactive oxygen species (ROS) and mitochondrial superoxide. Finally, we concluded that AMPKα1 mediated the ZEA-caused inhibition of lipid accumulation and promotion of brown and beige adipocyte-biomarker expression, as the positive effects of ZEA were diminished by Prkaa1 (AMPKα1) knockdown. These findings demonstrated that ZEA promoted the expression of brown and beige adipogenesis markers and mitochondrial biogenesis, which involved AMPKα1 activation, thus contributing to the anti-obesity effects of ZEA.

Zizyphus jujube

Effect of Zizyphus jujuba Extract on the Inhibition of Adipogenesis in 3T3-L1 preadipocytes

Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. The Zizyphus jujuba fruit has been used as traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. We investigated the Anti-Obesity effect of Z. jujuba on Adipocyte Differentiation of 3T3-L1 preadipocytes and found that treatment with an extract of Z. jujuba suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability.

Further fractionation of the initial Z .jujuba extract with organic solvent revealed that the chloroform fraction (CHCl₃–F) elicited the most Inhibitory effect, which involved significant attenuation of the expression of key Adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)γ and CCAAT enhancer binding proteins (C/EBPs) at the protein level. These results suggest that CHCl₃–F may block Adipogenesis, at least in part, by decreasing the expression of PPARγ, C/EBPα and β.

α-Mangostin (Garcinia mangostana L)

α-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 cells via PPARγ, GLUT4, and Leptin Expressions

Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 Diabetes, and cancer. The development of Obesity is also accompanied by significant Differentiation of preadipocytes into Adipocytes. In this study, we investigated the activity of α-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and Adipocyte Differentiation of 3T3-L1 cells focusing on PPARγ, GLUT4, and leptin expressions. α-Mangostin was found to Inhibit cytoplasmic lipid accumulation and Adipogenic Differentiation.

Cells treated with 50 μM of α-mangostin Reduced intracellular Fat Accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[³H] glucose uptake activity showed that α-mangostin significantly improved the glucose uptake () with highest activity found at 25 μM. In addition, α-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50 μM of α-mangostin. qRT-PCR analysis showed Reduced lipid accumulation via Inhibition of PPARγ gene expression.

Induction of glucose uptake and free fatty acid release by α-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that α-mangostin might be possible candidate for the effective management of Obesity in future.

β-asarone (Acorus calamus essential oil)

Acorus calamus (Araceae) has been incorporated into traditional medicines and used in food supplements for hundreds of years. The aim of this study was to investigate the Inhibitory effects of A. calamus essential oil (calamus oil) on the Differentiation of 3T3-L1 preadipocytes into Adipocytes. The major Anti-Adipogenic component of calamus oil was purified and identified as β-asarone. β-Asarone significantly Inhibited intracellular lipid accumulation during Adipocyte Differentiation in a concentration-dependent manner.

In addition, the protein and mRNA expression levels of C/EBPβ, C/EBPα, and PPARγ were decreased in 3T3-L1 cells treated with β-asarone during Differentiation. Phosphorylation of ERK1/2, which is known to regulate the early phase of Adipogenesis, was attenuated by β-asarone treatment. These results suggest that β-asarone exerts Anti-Adipogenic activity, in part by suppressing the expression of Adipogenic transcription factors.

β-Cryptoxanthin (Orange peel)

β-Cryptoxanthin Suppresses the Adipogenesis of 3T3-L1 cells via RAR Activation

We recently reported that the oral intake of β-cryptoxanthin exerted Anti-Obesity effects by lowering visceral fat levels. In the present study, we characterized the molecular mechanisms underlying the lipid-lowering effects of β-cryptoxanthin on 3T3-L1 cells. Consistent with our previous findings, β-cryptoxanthin rapidly Reduced the level of intracellular lipids in 3T3-L1 cells as assessed by Oil red O staining. Using an in vitro nuclear receptor binding assay, we demonstrated the ability of β-cryptoxanthin to bind to and activate members of the retinoic acid receptor (RAR) family. Accordingly, treatment of cells with LE540, an RAR antagonist, abolished the β-cryptoxanthin-dependent suppression of 3T3-L1 Adipogenesis, suggesting that β-cryptoxanthin mediates its effects on 3T3-L1 cells via RAR activation.

In addition, real-time RT-PCR analysis revealed that β-cryptoxanthin down-regulates mRNA expression of PPARγ, a key regulator of Adipocyte Differentiation, and that this Inhibition was blocked by LE540 treatment. Taken together, these data indicate that RAR activation contributes to the molecular mechanism by which β-cryptoxanthin prevents Obesity.

13-Methylberberine

(Coptis chinensis)

13-Methylberberine, a Berberine Analogue With Strong Anti-Adipogenic Effects

Lipid metabolism modulation is a main focus of Metabolic Syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its Anti-Obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their Anti-Adipogenic effects on 3T3-L1 Adipocytes and found that 13-methylberberine exhibited the most potent activity. 13-Methylberberine down-regulated the expression of the main Adipocyte Differentiation transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ ) and CCAAT enhancer binding protein alpha (C/EBPα), as well as their target genes. PPARγ, C/EBPα, and sterol regulatory element binding protein 1 (SREBP-1) protein levels were Reduced, and this lipid-reducing effect was attenuated by an AMP-activated protein kinase (AMPK) Inhibit or, indicating that the effect of this compound requires the AMPK signaling pathway. Decreased Akt phosphorylation suggested Reduced de novo lipid synthesis. C-13 methyl substitution of berberine increased its accumulation in treated cells, suggesting that 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an Anti-Obesity drug.

►Coptis chinensis alkaloids exert Anti-Adipogenic activity on 3T3-L1 Adipocytes by downregulating C/EBP-α and PPAR-γ

►Berberine (BBR) Attenuated Palmitic Acid (PA)-Induced Lipotoxicity in Human HK-2 Cells by Promoting Peroxisome Proliferator-Activated Receptor a (PPAR-a )

►Berberine and coptisine are two major components of Chinese goldthread. Berberine exerts anticancer effects through modulating Mcl-1, Bcl-xL, COX-2, MDR, TNF-α, IL-6, iNOS, IL-12, intercellular adhesion molecule-1, ELAM-1, MCP-1, CINC-1, cyclin D1, AP-1, HIF-1α, PPAR-γ, topoisomerase II, and Inhibiting stress-induced mitogen-activated protein kinase activation.

►Anti-Adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways

►Coptis chinensis inflorescence Ameliorates hyperglycaemia in 3T3-L1 Preadipocyte and streptozotocin-induced Diabetic mice

►CCP increased AMP-dependent kinase (AMPK) phosphorylation in 3T3-L1 pre- and mature Adipocytes and improved impaired AMPK phosphorylation in the muscles and livers of HFD-induced Diabetic mice.

►Berberine increases adipose triglyceride lipase in 3T3-L1 Adipocytes through the AMPK pathway

►Berberine, an isoquinoline alkaloid, could suppress fat deposition in 3T3-L1 cells;

►►The intracellular total cholesterol (TC) and triglyceride (TG) contents in XKA treated 3T3-L1 cells were also evaluated.

►The design and synthesis of a novel compound of berberine and baicalein that Inhibits the efficacy of lipid accumulation in 3T3-L1 Adipocytes

►Identification of berbamine dihydrochloride from barberry as an Anti-Adipogenic agent by high-content imaging assay

►Anti‐Adipogenic Activity of Berberine is Not Mediated by the WNT/β‐Catenin Pathway

►For this study, we used extracts of Coptis chinensis, Mahonia aquifolium, Berberis vulgaris and Chelidonium majus containing BBR and other alkaloids and studied various processes related to Adipocyte functionality.

►Berberine increases expression of GATA-2 and GATA-3 during Inhibition of Adipocyte Differentiation

18β-Glycyrrhetinic acid

(licorice root)

18β-Glycyrrhetinic acid Inhibits Adipogenic Differentiation and stimulates Lipolysis

18β-Glycyrrhetinic acid (18β-GA) obtained from the herb liquorice has various pharmacological properties including anti-inflammatory and anti-bacterial activities. However, potential biological Anti-Obesity activities are unclear. In this study, novel biological activities of 18β-GA in the Adipogenesis of 3T3-L1 preAdipocyte s and in Lipolysis of differentiated Adipocytes were identified. Mouse 3T3-L1 cells were used as an in vitro model of Adipogenesis and Lipolysis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce Differentiation. The amount of lipid droplet accumulation was determined by an AdipoRed assay. The expression of several Adipogenic transcription factors and enzymes was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. 18β-GA dose-dependently (1–40 μM) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10 μM of 18β-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α and adiponectin, which are markers of Adipogenic Differentiation via Akt phosphorylation. Also, in differentiated Adipocytes, 18β-GA increased the level of glycerol release and up-regulated the mRNA of hormone-sensitive lipase, adipose TG lipase and perilipin, as well as the phosphorylation of hormone-sensitive lipase at Serine 563. The results indicate that 18β-GA alters fat Mass by directly affecting Adipogenesis in maturing preadipocytes and Lipolysis in matured Adipocytes. Thus, 18β-GA may be useful for the treatment of Obesity.

Highlights

► 3T3-L1 preAdipocytes and stimulates Lipolysis from ScienceDirect’s AI-generated Lipolysis in differentiated Adipocytes.

► Anti-Adipogenic effect of 18β-GA is caused by down-regulation of PPARγ and inactivation of Akt signalling.

► Lipolytic effect of 18β-GA is mediated by up-regulation of HSL, ATGL and perilipin and activation of HSL.

18β‐glycyrrhetinic acid attenuates anandamide‐induced Adiposity and high‐fat diet induced Obesity

Scope: Previous reports suggest that licorice extract has various metabolically beneficial effects and may help to alleviate Adiposity and hyperlipidemia. However, underlying anti‐Obesity mechanisms still remain elusive. Moreover, it is unknown which single ingredient in licorice extract would mediate such effects. We aimed to demonstrate that licorice extract and its active ingredients can Inhibit Adipocyte Differentiation and Fat Accumulation.

Methods and results: 18β‐glycyrrhetinic acid (18β‐GA) alleviated the effects of CB 1R agonist, anandamide (AEA ) on CB 1R signaling in a concentration‐dependent manner. Consistently, 18β‐GA suppressed AEA ‐induced Adipocyte Differentiation in 3T 3‐L 1 cells through the downregulation of AEA ‐induced MAPK activation and expression of Adipogenic genes including C /EBP ‐α and PPAR‐γ. The protein levels of fatty acid synthase and stearoyl‐C oA desaturase 1 were also decreased and the phosphorylation of acetyl‐CoA carboxylase was increased in 18β‐GA pretreated cells. The supplementation of 18β‐GA significantly lowered body weight, fat weight, and plasma lipids levels in obese animal models.

Conclusion: These results may provide a novel insight into the molecular mechanism involved in anti‐Adipogenic and anti‐Obesity effects of 18β‐GA by suppressing the activation of CB 1R induced by AEA . Thus, 18β‐GA may exert beneficial effects against Obesity‐related metabolic disorders.

2,6-Dimethoxy-1,4-benzoquinone (vanillin)

2,6-Dimethoxy-1,4-benzoquinone Inhibits 3T3-L1 Adipocyte Differentiation via Regulation of AMPK and mTORC1

2,6-Dimethoxy-1,4-benzoquinone is a natural phytochemical present in fermented wheat germ. It has been reported to ex- hibit anti-inflammatory, antitumor, and antibacterial activ- ities. However, the Anti-Adipogenic effects of 2,6-dimethoxy- 1,4-benzoquinone and the mechanisms responsible have not previously been elucidated. Such findings may have ramifica- tions for the treatment of Obesity.

2,6-Dimethoxy-1,4-benzo- quinone (5 and 7.5 μM) significantly Reduced the expression of various Adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ and CCAAT/enhancer bind- ing protein α as well as Adipocyte protein 2 and fatty acid syn- thase. 2,6-Dimethoxy-1,4-benzoquinone upregulated AMP- dependent protein kinase phosphorylation and Inhibited the mature form of sterol regulatory element-binding protein 1c. Notably, 2,6-dimethoxy-1,4-benzoquinone attenuated mam- malian target of rapamycin complex 1 activity in 3T3-L1 and mouse embryonic fibroblast cells.

These findings highlight a potential role for 2,6-dimethoxy-1,4-benzoquinone in the suppression of Adipogenesis. Further studies to determine the Anti-Obesity effects of 2,6-dimethoxy-1,4-benzoquinone in animal models appear warranted.

25 Hydroxycholesterol

25-Hydroxycholesterol Inhibits Adipogenic Differentiation of C3H10T1/2 Pluripotent Stromal Cells

Understanding of Adipogenesis is important to find remedies for Obesity and related disorders. In addition, it is also critical in bone disorders because there is a reciprocal relationship between Adipogenesis and osteogenesis in bone micro-environment. Oxysterols are pro-osteogenic and Anti-Adipogenic molecules via hedgehog activation in pluripotent bone marrow stomal cells. However, no study has evaluated the role of specific oxysterols in C3H10T1/2 cells, which are a good cell model for studying osteogenesis and Adipogenesis in bone-marrows.

Thus, we investigated the effects of specific oxysterols on Adipogenesis and expression of Adipogenic transcripts in C3H10T1/2 cells. Treatment of cells with DMITro significantly induced mRNA expression of PPARγ. This induction was significantly Inhibited by 25-HC. The expression of C/cepα, Fabp4 and Lpl was also Inhibited by 25-HC. To determine the mechanism by which 25-HC Inhibits Adipogenesis, the effects of the hedgehog signalling pathway Inhibit or, cyclopamine and CUR61414, were evaluated. Treatment of C3H10T1/2 cells with DMITro + cyclopamine or DMITro + CUR61414 for 96h did not modulate Adipocyte Differentiation ; cyclopamine and CUR61414 did not reverse the Inhibitory effects of 25-HC, suggesting that the canonical hedgehog signalling may not play a role in the Anti-Adipogenic effects of 25-HC in C3H10T1/2 cells.

In addition, LXR agonist did not Inhibit Adipogenesis, but 25-HC strongly Inhibits Adipogenesis of C3H10T1/2 cells. Our observations showed that 25-HC was the most potent oxysterol in Inhibiting Adipogenesis and the expression of key Adipogenic transcripts in C3H10T1/2 cells among the tested oxysterols, suggesting its potential application in providing an intervention in osteoporosis and Obesity. We also report that the Inhibitory effects of 25-HC on Adipogenic Differentiation in C3H10T1/2 cells are not mediated by hedgehog signaling and LXR.

3-Chloro-4,5-dihydroxybenzaldehyde (Benzoic acid)

•CDB Inhibit of Adipogenesis in 3T3-L1 Adipocyte cells.

•CDB suppress Fat Accumulation by Inhibition of fatty acid synthesis in Adipocytes.

•CDB decreased Adipogenesis -specific proteins in 3T3-L1 Adipocyte cells.

•CDB increased levels of phosphorylated AMPK in Adipocytes.

Obesity is a serious health issue in many industrialized countries. It is a medical condition with excessive levels of fat accumulated in Adipocytes. The objective of the present study was to determine the Inhibitory effect of 3-chloro-4,5-dihydroxybenzaldehyde (CDB) on Adipogenesis in 3T3-L1 Adipocyte cells. CDB suppressed the Differentiation and decreased lipid accumulation and triglyceridescontents in 3T3-L1 Adipocytes. Its suppression effect on Fat Accumulation was mediated via expression of Adipogenesis factors (C/EBPα , SREBP-1c, PPARγ, and adiponectin) during Adipocyte Differentiation in white Adipocyte cells. CDB’s ability to supfpress Fat Accumulation was increased in a concentration-dependent manner.

It Inhibited fatty acid synthesis related proteins including FAS, FABP4, leptin, and perilipin. It also increased expression of phosphorylated AMPK in Adipocytes cells. These observations suggest that CDB has potential Anti-Obesity effect with ability to improve metabolic diseases.

3,5-Dicaffeoyl-Epi-Quinic Acid

(Halophyte Atriplex gmelinii)

3,5-Dicaffeoyl-Epi-Quinic Acid Isolated From Edible Halophyte Atriplex gmelinii Inhibits Adipogenesis via AMPK/MAPK Pathway in 3T3-L1 Adipocytes

Atriplex gmelinii is an edible halophyte that has been suggested to possess various health benefits. In the present study, 3,5-dicaffeoyl-epi-quinic acid (DEQA) isolated from A. gmelinii was tested for its ability to Prevent Adipogenesis in 3T3-L1 cells. Also, the molecular mechanisms by which DEQA affects Differentiation of 3T3-L1 cells were investigated. The introduction of DEQA to differentiating 3T3-L1 preAdipocytes resulted in suppressed Adipogenesis and lowered expression of Adipogenesis-related factors, PPARγ, C/EBPα, and SREBP-1c. Treatment of 3T3-L1 Adipocytes with DEQA notably decreased the levels of phosphorylated p38, ERK, and JNK.

In addition, presence of DEQA upregulated the levels of both inactive and phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylase (ACC). Taken together, current results indicated that DEQA exhibited a significant AntiAdipogenesis activity by activation of AMPK and downregulation of MAPK signal pathways in 3T3-L1 preAdipocytes.

3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF)

(naturally occurring polymethoxyflavone found in Tangerine peel)

Adipogenesis by 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone from Orange Peel in 3T3-L1 cells Suppression of Adipogenesis by 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone from Orange Peel in 3T3-L1 cells

We reported previously that hydroxylated polymethoxyflavones (HPMFs) effectively suppressed Obesity in high-fat-induced mouse. In this study, we further investigated the molecular mechanism of action of 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), one of major HPMFs in orange peel. Treatment of 5-OH-HxMF effectively Inhibited lipid accumulation by 55–60% in a dose-dependent manner. The 5-OH-HxMF attenuated Adipogenesis through downregulating Adipogenesis-related transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs), as well as downstream target fatty acid synthase and acetyl-CoA carboxylase (ACC). 5-OH-HxMF activated adenosine monophosphate-activated protein kinase signaling and silent mating type information regulation 1 (SIRTUIN 1 or SIRT1) in 3T3-L1 Adipocytes to decrease lipid accumulation.

In addition, the Inhibition rate of lipid accumulation was compared between 5-OH-HxMF and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HpMF). 5-OH-HxMF Inhibited lipid accumulation 15–20% more than HpMF did, indicating that hydroxyl group at position 5 can be a key factor in the suppression of Adipogenesis.

Heptamethoxyflavone Inhibits Adipogenesis via enhancing PKA signaling

3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), a naturally occurring polymethoxyflavone found in citrus peel, is known to have neuroprotective, anti-inflammatory, and immunomodulatory effects. However, the effects of HMF on Adipogenesis remain unclear. Here, we demonstrate that HMF Inhibits the early stage of Adipogenesis and maturation in 3T3-L1 Adipocytes. HMF treatment during Preadipocyte Differentiation for 8 days Reduced lipid accumulation in a dose-dependent manner, and the expression levels of key Adipogenic transcription factors (peroxisome proliferator-activated receptor gamma (PPARγ ) and CCAAT/enhancer-binding protein α (C/EBPα )) and the lipogenic transcription factor, sterol regulatory element-binding protein (SREBP1), were lower after the initial 4 days of the Differentiation.

Moreover, PPARγ expression level was lower even after the initial 2 days, but C/EBPα and SREBP1 expression was not. HMF upregulated the phosphorylation of protein kinase A catalytic subunit α (PKACα), AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in 3T3-L1 cells. The phosphorylation of ACC leads to the Inhibition of Adipogenesis.

Furthermore, the induction of phosphorylation of AMPK and ACC by HMF was abolished by RNA interference targeting PKACα. Taken together, our results suggest that HMF might Inhibit the early stage of Adipogenesis via the activation of PKA signaling in 3T3-L1 cells.

Effect of hydroxylated polymethoxyflavones and acetylated polymethoxyflavones on Adipogenesis in vivo and in vitro

Excess energy is a major cause of Obesity and it can increase the number and size of Adipocytes, eventually expanding the Adipose Tissue. Adipose Tissue can be deposited in the intra-abdominal area and it affects the function of other organs, such as the liver, pancreas, and skeletal muscle, therefore, it is a risk factor related to liver disease and type 2 Diabetes. Diet modification and the Inhibition of the cause of Obesity-related molecular mechanisms could be solutions for controlling the expansion of adipose Mass and decreasing the prevalence of Obesity . Polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones (HPMFs) such as nobiletin, tangeretin, 5-demethyltangeretin, and 5-demethylnobiletin are unique Flavonoids that almost exclusively exist in the peel of the citrus genus and have many health-beneficial effects. However, their lipophilic structure and characteristics give them poor aqueous solubility and low oral bioavailability.

In the current research, we prepared 5-demethyltangeretin (5-OH-Tan), 5-demethylnobiletin (5-OH-Nob), 5-acetyloxy-6,7,8,4′-tetramethoxyflavone(5-Ac-Tan), and 5-acetyloxy-6,7,8,3′,4′-pentamethoxyflavone (5-Ac-Nob) through chemical modification from nobiletin and tangeretin. Firstly, we found that 5-Ac-Nob had better anti-Adipogenesis activity than 5-OH-Nob, 5-OH-Tan, and its anti-Adipogenesis ability by increased the phosphorylated-LKB1 and AMPKα protein levels and decreases the transcriptional factor SREBP-1 and lipogenesis-related enzyme fatty acid synthase protein levels in a 3T3-L1 Preadipocyte model.

Therefore, we further investigated the anti-Adipogenesis effect of 5-Ac-Nob by using a diet with 45% calories from fat to induce Obesity in C57BL/6J male mice. We found that mice fed with 5-Ac-Nob had lower body weight; intra-abdominal fat, plasma and liver triacylglycerol levels, and plasma cholesterol level and it had potential to Prevent fatty liver by increased phosphorylated-LKB1 and AMPKα protein levels and increased the level of a lipogenesis-related enzyme, an inactive form of phosphorylated acetyl-CoA carboxylase protein, in the liver.

In addition, we also found that 5-OH-Nob could be a metabolite hydrolyzed from 5-Ac-Nob in plasma when the mouse were administered with 100 mg/ kg bw of 5-Ac-Nob by oral gavage. All these results showed that 5-Ac-Nob could be a 5-OH-Nob prodrug to alleviate lipid accumulation by activated AMPKα and then affect lipid synthesis in vivo and in vitro.

Prevention of Obesity and Hyperlipidemia by Heptamethoxyflavone in High-fat Diet-induced Rats

Polymethoxyflavones (PMFs) have been shown to Prevent Obesity, ameliorate type 2 Diabetes, and regulate lipid metabolism in vitro and in vivo. However, little is known about the contribution of 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF) to Prevent Obesity and regulate lipid metabolism in vivo. We aimed to investigate the potential efficacy of HMF on preventing Obesity and hyperlipidemia in rats fed a high-fat diet (HFD) and its underlying mechanisms. Male Sprague–Dawley rats were fed a normal diet or an HFD with or without HMF (0.02%, 0.04% and 0.08%, w/w) for 6 weeks. The supplementation of HMF not only significantly decreased body Weight Gain (HFD, 336.50 ± 18.84 g; LHMF, 309.43 ± 20.74 g; MHMF, 296.83 ± 13.88 g; HHMF, 265.71 ± 19.09 g; respectively, p< 0.05) and Adipose Tissues weight (p < 0.05), but also markedly lowered serum levels of total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol (p < 0.05) in the sixth week in a dose-dependent manner compared with the HFD group.

HMF also significantly alleviated hepatic steatosis in the liver (liver weight g/100 g body weight of HFD, 4.86 ± 0.11%; LHMF, 4.02 ± 0.33%; MHMF, 4.05 ± 0.31%; HHMF, 3.72 ± 0.34%; respectively, p < 0.05). Furthermore, transcriptome analysis and real-time quantitative RT-PCR demonstrated that HMF supplementation markedly downregulated hepatic genes related to Adipogenesis transcription and inflammatory responses, and significantly upregulated genes related to fatty acid oxidation and energy expenditure. These results indicated that HMF could effectively Prevent Obesity and hyperlipidemia by regulation of the expression of lipid metabolism-related and inflammatory response-related genes.

6-Gingerol (Ginger)

6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). In this study, we investigated the effects of 6-gingerol on the Inhibition of Adipogenesis Adipogenesis in 3T3-L1 from ScienceDirect’s AI-generated Topic Pages3T3-L1 cells. After treatment with 6-gingerol in Differentiation medium for 4 or 8 days, the 3T3-L1 cells were lysed for experimental analysis. Cells were stained with Oil-Red-O to detect oil droplets in Adipocyte from ScienceDirect’s AI-generated Topic PagesAdipocytes.

The 3T3-L1 cells were lysed and measured for triglyceride contents. The protein expression of Adipogenesis -related transcription factor was evaluated by Western blot analysis. 6-Gingerol suppressed oil droplet accumulation and Reduce d the droplet size in a concentration (5–15 μg/ml)- and time-dependent manner. Treatment of 3T3-L1 cells with 6-gingerol Reduce d the protein levels of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)α. Additionally, the protein levels of fatty acid synthase (FAS) and Adipocyte -specific fatty acid binding protein (aP2) decreased upon treatment with 6-gingerol.

Meanwhile, 6-gingerol diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9). These results suggest that 6-gingerol effectively Suppresses Adipogenesis and that it exerts its role mainly through the significant down-regulation of PPARγ and C/EBPα and subsequently Inhibits FAS and aP2 expression. 6-Gingerol also Inhibited Differentiation in 3T3-L1 cells by attenuating the Akt/GSK3β pathway. Our findings provide important insights into the mechanisms underlying the Anti-Adipogenic activity of 6-gingerol.

6,7,4′-Trihydroxyisoflavone

A metabolite of daidzein, 6,7,4′‐trihydroxyisoflavone, Suppresses Adipogenesis in 3T3‐L1 preadipocytes via ATP‐competitive Inhibition of PI3K

Scope: Daidzein is one of the major soy isoflavones. Following ingestion, daidzein is readily metabolized in the liver and converted into hydroxylated metabolites. One such metabolite is 6,7,4′‐trihydroxyisoflavone (6,7,4′‐THIF), which has been the focus of recent studies due to its various health benefits, however, its anti‐Adipogenic activity has not been investigated. Our objective was to determine the effects of 6,7,4′‐THIF on Adipogenesis in 3T3‐L1 preadipocytes and elucidate the mechanisms of action involved.

Methods and results: Adipogenesis was stimulated in 3T3‐L1 preadipocytes. Both 6,7,4′‐THIF and daidzein were treated in the presence and absence of mixture of isobutylmethylxanthine, dexamethasone, and insulin (MDI). We observed that 6,7,4′‐THIF, but not daidzein, Inhibited MDI‐induced Adipogenesis significantly at 40 and 80 μM, associated with decreased peroxisome proliferator‐activated receptor‐γ and C/EBP‐α protein expression. 6,7,4′‐THIF significantly suppressed MDI‐induced lipid accumulation in the early stage of Adipogenesis, attributable to a suppression of cell proliferation and the induction of cell cycle arrest. We also determined that 6,7,4′‐THIF, but not daidzein, attenuated phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathway. 6,7,4′‐THIF was found to Inhibit PI3K activity via direct binding in an ATP‐competitive manner.

Conclusion: Our results suggest that 6,7,4′‐THIF Suppresses Adipogenesis in 3T3‐L1 preadipocytes by directly targeting PI3K. Soy isoflavones like 6,7,4′‐THIF may have potential for development into novel treatment strategies for chronic Obesity.

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6 reviews for ANTI-ADIPOGENIC: Adipocyte Differentiation Inhibitor 200:1 – New!

Rated 5 out of 5
Rich Ryan – October 26, 2020
If you have trouble losing weight, or if you diet and then gain it all back in a week, then this is the blend you need.
I’ve lost up to 1.5 lbs each day while fasting with this blend (and exercise). And if I keep taking it, then I don’t gain all the weight back after breaking fast. It’ll help you burn your love handles away and keep you from gaining them right back!
It has a great appetite suppressing effect as well.
Amazing Blend!
For ultimate appetite suppression and fat burning, pair with AutoPhagy and ThermoGenesis, and do 22/2 Intermittent Fasting.
To keep your mood up during fasting, pair with Trinity. And for more energy pair with Original Blend.
Rated 5 out of 5
Boladale Abdulazeez – April 11, 2021
I was introduced to this blend by my sister who has been on this blend for years, she encouraged me to give anti adipogenic and autography a try but i decided to buy the march combo for a start after my discussion with Gavin.
I orded the blend on the 7th of March, came in on the 10th and I started taking it immediately.
I started with weight 265 kg on the 10th of March and by the end of March I already lost about 45lbs, my skin glows and I feel lot stronger and healthier.
I still have alot to work on me, but am work in progress and am glad I am able to focus clearer, eat with control and feel stronger….I will add another review by the end of April.
Thank you Gavin, this blend truly works, and your customer services is amazing 100%…. excellent.
Rated 5 out of 5
MS (verified owner) – February 22, 2022
I have to say this is a remarkable product. I have been using this blend for over 3 months now, along with several other blends. I have been trying to adhere to the 22/2 protocol, but there are days which I can not. Even though, I have manage to reduce my body fat percentage, while increasing muscle in the gym. I’ve gotta put it down to this blend. It does what it says on the tin! I’ve noticed since I’ve run out about a month ago, eating the same diet, my body feels “flabbier”. My BF percentage even went up by 1%. The only thing I’ve changed is not having this blend.
Sometimes it takes not having something to realize it’s true benefit and hence why I wanted to write this review today.
I’ve got to hand it to Gavin, the blends are incredible. When you’ve been using them as long as I have, you become used to a bubble of light surrounding you, protecting you from bullshit.
Thanks!
Rated 5 out of 5
Yvonne (verified owner) – August 14, 2022
So I found the Interstellar site while researching Dry Fasting. I’ve been wet fasting for several years & seen tremendous benefits. I started intermittent fasting/OMAD after my health went to hell 5 years ago. I had been health conscious for 10 years, plant based, organic eating, no junk, juices, sweets, etc., after a Hyperinsulinanemia diagnosis, plus fungal acne, severe depression mainly brought on by stress I was able to turn it around in a couple months eating very low insulin/fasting/OMAD all that Gavin is a proponent of but I had never tried dry fasting as I honestly had no information on it.
Last year I was diagnosed with Generalized Anxiety Disorder, ADD and TBI (from childhood) from brain scans I did @ the Amen clinics. Which explained the struggles with depression & mental health despite going to great lengths to heal holistically and naturally. Having trained in holistic nutrition and being a natural researcher, I dug into the Interstellar site & I loved how well researched & science based everything was. It’s hard to find any solid information online about dry Fasting so I came away with so much info + about the Interstellar blends too. I’m a big proponent of medicinal plants and herbs so I knew their power but then seeing the hundreds of testimonials and then the weight loss group and everyone who was able to dump a huge amount of weight in no time I was like no way! I had to see it to believe it. I was out of the US but went to great lengths to order and get the blends.
I ordered the April sampler. My biggest goal was to lose weight but also brain health (focus/attention)… I had crippling anxiety. I was going through a terrible time emotionally and within a week most of the anxiety was gone! As in my stomach was tight day in and out and POOF! GONE! I was training hard at the time and the Pre-Workout blends (Nebula, Thermo, Anti Adipogenic & Matcha in black coffee had me going like superwoman 🦸🏾‍♀. My trainer, who had won several strongman championships couldn’t understand!
I got back stateside last month and I just had to try Rewire… I have struggled with my weight yo-yo-ing due to emotional eating/binging despite being so health focused (food addiction) so couldn’t wait to give it a go. Got a couple samplers and started Rewire this month along with the other cognitive enhancement blends (Trinity, Autonomous, Seven Sages, Luteolin) along with fasting as I work to heal my brain. I am loving the blends thus far and Gavin and the entire Interstellar’s team commitment to outstanding service, integrity and healing and the support of the community he has created. Truly inspiring!
Rated 5 out of 5
Patty (verified owner) – August 19, 2022
I’ve been taking the blends for over a year. LET ME TELL YOU, these are by far the BEST thing I stumbled upon. I have always been into alternative medicine and am never willing to take what big Pharma is pushing on me. I am a retired Registered Nurse and have pushed back against Pharma my entire career…weird right but my eyes were opened.
I have suffered with arthritis, pain, low energy but feeling of impending doom, thinning hair, and high blood pressure. Let me tell you….I tried many supplements to try and reverse these symptoms and nothing worked. Again by the Grace of God, I came across Gavin and his life saving blends. What a game changer!!
Here’s my results. By taking Peel and Spice for Arthritis, I have function back in my wrists and pain is under control. Low Energy and impending doom? GONE with NEBULA and TRINITY…I have never felt so calm and let things pass in my entire life! I use to be high strung and everything effected me. After having what I think was COVID..(never got tested) I started loosing my hair. How disturbing to me. I have been taking the hair blend and is thankfully growing back. Last but not least, EKG has brought my blood pressure into normal range. At every annual physical, my blood pressure was so high sometimes they didn’t want me to leave the clinic. This is a game changer for me. So happy I’m not in the critical zone anymore.
I could go on and on about the blends. Purge, Glucose Blocker, Anti Adipogenic, Thermo, and Autophagy for loosing weight…OFF THE CHARTS. I lost 10 pounds and have kept it off for over a year.
This is getting long so just try them….you’ll never go back! Thank You Gavin so much for your life saving and beautiful blends. They are filled with Love.
Rated 5 out of 5
Patricia (verified owner) – August 19, 2022
I’ve been taking the blends for over a year. LET ME TELL YOU, these are by far the BEST thing I stumbled upon. I have always been into alternative medicine and am never willing to take what big Pharma is pushing on me. I am a retired Registered Nurse and have pushed back against Pharma my entire career…weird right but my eyes were opened.
I have suffered with arthritis, pain, low energy but feeling of impending doom, thinning hair, and high blood pressure. Let me tell you….I tried many supplements to try and reverse these symptoms and nothing worked. Again by the Grace of God, I came across Gavin and his life saving blends. What a game changer!!
Here’s my results. By taking Peel and Spice for Arthritis, I have function back in my wrists and pain is under control. Low Energy and impending doom? GONE with NEBULA and TRINITY…I have never felt so calm and let things pass in my entire life! I use to be high strung and everything effected me. After having what I think was COVID..(never got tested) I started loosing my hair. How disturbing to me. I have been taking the hair blend and is thankfully growing back. Last but not least, EKG has brought my blood pressure into normal range. At every annual physical, my blood pressure was so high sometimes they didn’t want me to leave the clinic. This is a game changer for me. So happy I’m not in the critical zone anymore.
I could go on and on about the blends. Purge, Glucose Blocker, Anti Adipogenic, Thermo, and Autophagy for loosing weight…OFF THE CHARTS. I lost 10 pounds and have kept it off for over a year.
This is getting long so just try them….you’ll never go back! Thank You Gavin so much for your life saving and beautiful blends. They are filled with Love.

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ANTI-ADIPOGENIC: Adipocyte Differentiation Inhibitor 200:1 – New!

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6 reviews for ANTI-ADIPOGENIC: Adipocyte Differentiation Inhibitor 200:1 – New!

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