Anti-adipogenic activity of Cordyceps militaris in 3T3-L1 cells

Inhibition of adipocytes differentiation is suggested to be an important strategy for prevention and/or treatment of obesity. In our present study, Cordyceps militaris showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of cordycepin (1), guanosine (2) and tryptophan (3) as active compounds. All the three compounds were more effective in the prevention of early stage of adipogenesis than in lipolysis. In addition, combinational treatment of three compounds significantly increased anti-adipogenic activity.     

Anthraquinones from Morinda officinalis roots enhance adipocyte differentiation in 3T3-L1 cells

To search for anti-diabetic and insulin-sensitising natural products, the effect on adipocyte differentiation was investigated by assessing fat accumulation in 3T3-L1 preadipocytes using Oil Red O staining. Fractionation and separation of n-hexane and CHCl? fractions of Morinda officinalis (Rubiaceae) using several chromatographic methods led to the isolation of three anthraquinones, 1,2-dimethoxyanthraquinone (1), alizarin-2-methyl ether (2) and rubiadin-1-methyl ether (3). Among them, alizarin-2-methyl ether (2) showed the strongest enhancing activity, followed by rubiadin-1-methyl ether (3) and 1,2-dimethoxyanthraquinone (1). At a concentration of 100?μM, alizarin-2-methyl ether (2) enhanced adipocyte differentiation by up to 131% (compared to insulin-treated cells). Thus, these compounds could be beneficial in the treatment of diabetes.     

A quantified ginseng (Panax ginseng C.A. Meyer) extract influences lipid acquisition and increases adiponectin expression in 3T3-L1 cells

A Panax ginseng extract (PGE) with a quantified amount of ginsenosides was utilized to investigate its potential to inhibit proliferation, influence lipid acquisition and adiponectin expression in 3T3-L1 cells. Seven fingerprint ginsenosides were quantified using high performance liquid chromatography and their respective molecular weights were further confirmed via LC-ESI-MS analysis from four different extraction methods. Extraction using methanol under reflux produced significantly higher amounts of ginsenosides. The methanol extract consisted of Rg1 (47.40 ± 4.28 mg/g, dry weight of extract), Re (61.62 ± 5.10 mg/g), Rf (6.14 ± 0.28 mg/g), Rb1 (21.73 ± 1.29 mg/g), Rc (78.79 ± 4.15 mg/g), Rb2 (56.80 ± 3.79 mg/g), Rd (5.90 ± 0.41 mg/g). MTT analysis showed that PGE had a concentration-dependent cytotoxic effect on 3T3-L1 preadipocyte and the LC(50) value was calculated to be 18.2 ± 5 μg/mL. Cell cycle analysis showed minimal changes in all four phases. Differentiating adipocytes treated with ginseng extract had a visible decrease in lipid droplets formation measured by Oil red O staining. Consequently, triglycerides levels in media significantly (P < 0.05) decreased by 39.5% and 46.1% when treated at concentrations of 1 μg/mL and 10 μg/mL compared to untreated control cells. Western blot analysis showed that the adiponectin protein expression was significantly (P < 0.05) increased at 10 μg/mL, but not at 1 μg/mL. A quantified PGE reduced the growth of 3T3-L1 cells, down-regulated lipid accumulation and up-regulated adiponectin expression in the 3T3-L1 adipocyte cell model.     

Effect of Cortisol on Calpains in the C2C12 and 3T3-L1 Cells

The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.     

Effect of Cortisol on Caspases in the Co-cultured C2C12 and 3 T3-L1 Cells

The present study was carried out to understand the effect of cortisol on caspase expression in the C2C12 and 3 T3-L1 cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3 T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. A total of 10 μg/μl of cortisol was added to the medium. Following treatment of cortisol for 3 days, the cells in the lower well were harvested for analysis. Caspases such as caspase 3, caspase 7, and caspase 9 were selected for the analysis. qRT-PCR results indicated the significant increase in the mRNA expression of caspase 3, caspase 7, and caspase 9. Caspase 3, 7, and 9 activities were also increased in the mono- and co-cultured C2C12 and 3 T3-L1 cells. In addition, confocal microscopical investigation indicated that cortisol increases caspase expressions in the mono- and co-cultured C2C12 and 3 T3-L1 cells. Taking all these together, we concluded that the co-culture system reflects the exact effect of cortisol on caspase expression, which is quite distinct from one dimensional mono-cultured experiments.     

Effect of Coculturing on the Myogenic and Adipogenic Marker Gene Expression

The present experiment was carried out to evaluate the effect of coculturing on myogenic and adipogenic marker gene expressions with the use of C2C12 and 3 T3-L1 preadipocyte cells under the coculture system. C2C12 and 3 T3-L1 cells were cocultured using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3 T3-L1 cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates, and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. After coculture of the C2C12 and 3 T3-L1 cells for 48 and 72 h, the cells in the lower well were harvested for analysis, and this process was carried out for both cells. Myogenic markers such as myogenin, MyoD, Myf5, PAX3, and PAX7 mRNA expressions were analyzed in the cocultured C2C12 cells. Adipogenic markers such as fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activating receptor (PPARγ), CCAAT/enhancer-binding protein (CEBPA), adiponectin, lipoprotein lipase, and fatty acid synthase mRNA expressions were analyzed in the cocultured 3 T3-L1 cells. Myogenic and adipogenic marker gene mRNA expressions were significantly altered in the cocultured C2C12 and 3 T3-L1 cells when compared with the monocultured C2C12 and 3 T3-L1 cells.     

Cell-Cell Communication Between Fibroblast and 3T3-L1 Cells Under Co-culturing in Oxidative Stress Condition Induced by H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The present study was carried out to understand the interaction between fibroblast and 3T3-L1 preadipocyte cells under H₂O₂-induced oxidative stress condition. H₂O₂ (40 μM) was added in co-culture and monoculture of fibroblast and 3T3-L1 cell. The cells in the lower well were harvested for analysis and the process was carried out for both cells. The cell growth, oxidative stress markers, and antioxidant enzymes were analyzed. Additionally, the mRNA expressions of caspase-3 and caspase-7 were selected for analysis of apoptotic pathways and TNF-α and NF-κB were analyzed for inflammatory pathways. The adipogenic marker such as adiponectin and PPAR-γ and collagen synthesis markers such as LOX and BMP-1 were analyzed in the co-culture of fibroblast and 3T3-L1 cells. Cell viability and antioxidant enzymes were significantly increased in the co-culture compared to the monoculture under stress condition. The apoptotic, inflammatory, adipogenic, and collagen-synthesized markers were significantly altered in H₂O₂-induced co-culture of fibroblast and 3T3-L1 cells when compared with the monoculture of H₂O₂-induced fibroblast and 3T3-L1 cells. In addition, the confocal microscopical investigation indicated that the co-culture of H₂O₂-induced 3T3-L1 and fibroblast cells increases collagen type I and type III expression. From our results, we suggested that co-culture of fat cell (3T3-L1) and fibroblast cells may influence/regulate each other and made the cells able to withstand against oxidative stress and aging. It is conceivable that the same mechanism might have been occurring from cell to cell while animals are stressed by various environmental conditions.     

Screening of anti-obesity agent from herbal mixtures

Globally, one in three of the World's adults are overweight and one in 10 is obese. By 2015, World Health Organization (WHO) estimates the number of chubby adults will balloon to 2.3 billion--Equal to the combined populations of China, Europe and the United States. The discovery of bioactive compounds from herbs is one possible way to control obesity and to prevent or reduce the risks of developing various obesity-related diseases. In this study, we screened anti-obesity agents such as methyl gallate from the herbal composition known as HemoHIM that actively inhibits lipid formation as evidenced by Oil Red O staining and triglyceride (TG) contents in 3T3-L1 adipocytes, suggesting their use as an anti-obesity agent. Furthermore, the amount of glycerol released from cells into the medium had increased by treatment of methyl gallate in a concentration-dependent manner. The present study suggests that a promising anti-obesity agent like methyl gallate might be of therapeutic interest for the treatment of obesity.     

Flavonol acylglycosides from flower of Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells

Obesity is a serious health problem worldwide. We investigated the anti-obesity effect of the flower of Albizia julibrissin DURAZZ. (Leguminosae). A 90% EtOH extract of the flower inhibited adipogenesis in 3T3-L1 preadipocytes, as well as the activity of glycerol-3-phosphate dehydrogenase (GPDH) activity. New flavonol acylglycosides (1-4) and eighteen known compounds (5-22) were isolated by bioassay-directed fractionation. These new glycosides were elucidated to be 3″-(E)-p-coumaroylquercitrin (1), 3″-(E)-feruloylquercitrin (2), 3″-(E)-cinnamoylquercitrin (3), and 2″-(E)-cinnamoylquercitrin (4) on the basis of spectroscopic and chemical analysis. These compounds inhibited adipogenesis in 3T3-L1 preadipocytes. In particular, 2 exhibited potent inhibitory effects on triglyceride accumulation. Furthermore, GPDH activity was inhibited by 2. Additionally, 2 inhibited glucose uptake in 3T3-L1 adipocytes. These results indicate that the 90% EtOH extract and compounds isolated from the flower of A. julibrissin inhibit adipogenesis in 3T3-L1 preadipocytes and may have anti-obesity effect through the inhibition of preadipocyte differentiation.     

In-silico therapeutic investigations of arjunic acid and arjungenin as an FXR agonist and validation in 3T3-L1 adipocytes

The present study was to illustrate the agonistic property of arjungenin and arjunic acid towards farnesoid X receptor protein (FXR).The pharmacokinetic properties like molecular interactions, absorption, distribution, metabolism, elimination and toxicity (ADMET) of the ligands were checked through in-silico studies. Protein-ligand docking was carried out using autodock software. Molecular docking analysis confirmed strong binding energy and interaction of arjungenin and arjunic acid with the target protein and the ADMET profiles identified for both compounds were promising.Further in vitro studies were performed in 3T3-L1 adipocyte to verify the agonistic property of arjungenin and arjunic acid. Oil red O staining was done to check differentiation induction. Adiponectin, leptin, triglycerides and total cholesterol levels were quantified. The mRNA expression of FXR, Cyp7a1, PPAR-γ and SREBP-1c were quantified using fluorescent real-time PCR. Cytotoxicity assay was confirmed that up to 150 μM concentration there is no significant cell death on treatment with arjunic acid and arjungenin. Treatment with arjungenin and arjunic acid confirms increased differentiation of the cells with significant (P < 0.05) increase in adiponectin (118.07% and 132.92%) and leptin (133.52% and 149.74%) protein levels compared to the negative control group. After treatment with arjungenin and arjunic acid in 3T3-L1 preadipocytes the mRNA expression of FXR, PPAR-γ and SREBP-1c were significantly (P < 0.01) increased and cyp7a1 was significantly (P < 0.01) decreased when compared with the negative control group. Overall, our results suggest that arjungenin and arjunic acid acts as an FXR agonist and may be useful for rational therapeutic strategies as a novel drug to treat cholesterol mediated metabolic syndrome and insulin resistance.     

Investigation of Coculture of Human Adipose-Derived Stem Cells and Mature Adipocytes

The purpose of this study was to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into adipocytes by coculturing them with human mature adipocytes. The transwell culture system was utilized for indirect coculture of hADSCs and human mature adipocytes at four different hADSCs-to-mature adipocytes ratios, i.e., 1:5, 1:1, 2:1, and 5:1. After 8?days of coculture, the Oil Red O and Trypan Blue stainings were performed for the evaluation of adipogenic differentiation of hADSCs. In addition, flow cytometric analysis and Hoechst 33342/PI double staining were performed after 20?days of coculture. The Oil Red O and Trypan Blue stainings showed that hADSCs with high viability could not differentiate into mature adipocytes after 8 or 20?days of coculture. However, flow cytometric analysis indicated that CD105 expression of hADSCs decreased after 20?days of coculture. These results indicated that hADSCs cocultured with human adult adipocytes could not successfully differentiate into adipocytes.     

miR-370-3p Regulates Adipogenesis through Targeting Mknk1

Excessive fat accumulation can lead to obesity, diabetes, hyperlipidemia, atherosclerosis, and other diseases. MicroRNAs are a class of microRNAs that regulate gene expression and are highly conserved in function among species. microRNAs have been shown to act as regulatory factors to inhibit fat accumulation in the body. We found that miR-370-3p was expressed at lower levels in the fat mass of mice on a high-fat diet than in mice on a normal control diet. Furthermore, our data showed that the overexpression of miR-370-3p significantly suppressed the mRNA expression levels of adipogenic markers. Thus, miR-370-3p overexpression reduced lipid accumulation. Conversely, the inhibition of miR-370-3p suppressed 3T3-L1 preadipocyte proliferation and promoted preadipocyte differentiation. In addition, Mknk1, a target gene of miR-370-3p, plays an opposing role in preadipocyte proliferation and differentiation. Moreover, consistent results from in vitro as well as in vivo experiments suggest that the inhibition of fat accumulation by miR-370-3p may result from the inhibition of saturated fatty acids that promote the accumulation of polyunsaturated fatty acids. In conclusion, these results suggest that miR-370-3p plays an important role in adipogenesis and fatty acid metabolism through the regulation of Mknk1.     

Inhibitory Effect of Acer truncatum Bunge Seed Coat Extract on Fatty Acid Synthase,Differentiation and Lipid Accumulation in 3T3-L1 Adipocytes

Acer truncatum Bunge is now widely cultivated throughout the world. Fatty acid synthase (FAS) is a potential target in the treatment of both obesity and cancer. Only a few FAS inhibitors have been reported. In this study, the inhibitory effect of A. truncatum seed coat (ESA) on FAS and the inhibition mechanisms were investigated using a FAS activity assay and an enzyme kinetics study. The main chemicals of ESA were analyzed with UPLC-MS/MS. The effects of ESA on 3T3-L1 adipocyte differentiation and lipid accumulation were investigated using Oil red O staining. We first identified seven main compounds (quinic acid, malic acid, gentisic acid, procyanidin dimer, procyanidin trimer, catechin, and quercetin) from 50% ethanol extracts of seed coats of A. truncatum (ESAs), which were then found to inhibit 3T3-L1 adipocyte differentiation at the concentration of 50 μg/mL. ESA obviously reduced the visible triglyceride droplets accumulation, and dramatically decreased the number of the adipocytes at a comparatively high concentration. It is suggested that the effects are due to the inhibition of FAS by ESA; FAS activity is inhibited by ESA at a half inhibition concentration (IC₅₀) of 0.57 μg/mL, which is lower than that of classically known FAS inhibitors. Meanwhile, ESA displayed different inhibition kinetics and reacting sites for FAS. These results provide new clues for the development of novel products for obesity treatment and a scientific basis for the full use of byproducts for future industrial production of vegetable oil.     

Chemical genetic identification of the histamine H1 receptor as a stimulator of insulin-induced adipogenesis

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.     

Kahweol inhibits adipogenesis of 3T3-L1 adipocytes through downregulation of PPARγ

Kahweol, a compound from Coffea arabica, possesses antioxidant, anti-inflammatory, and antitumour properties. However, an anti-adipogenic effect has not yet been reported. In this study, we have shown that kahweol has an anti-adipogenic effect on 3T3-L1 adipocytes. Kahweol significantly inhibited the differentiation of intracellular lipid accumulation in 3T3-L1 adipocytes, without being cytotoxic. It also downregulated the expression of adipogenesis-related gene, including an adipocytokine, adiponectin. This anti-adipogenic effect stems from an ability to inhibit key adipogenic regulators, including PPARγ and C/EBPα. These results demonstrate that kahweol significantly inhibits the differentiation of 3T3-L1 cells, and suggest that it has potential as a novel anti-obesity treatment.     

The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.