Elecampane (Inula helenium) Root Extract and Its Major Sesquiterpene Lactone,Alantolactone, Inhibit Adipogenesis of 3T3-L1 Preadipocytes

Recent studies have shown that Nur77 and AMPKα play an important role in regulating adipogenesis and isoalantolactone (ISO) dual-targeting AMPKα and Nur77 inhibits adipogenesis. In this study, we hypothesized that Inula helenium (elecampane) root extract (IHE), which contains two sesquiterpene lactones, alantolactone (ALA) and ISO, as major compounds, might inhibit adipogenesis. Here, we found that ALA and IHE simultaneously target AMPKα and Nur77 and inhibited adipogenic differentiation of 3T3-L1 cells, accompanied by the decreased expression of adipocyte markers. Further mechanistic studies demonstrated that IHE shares similar mechanisms of action with ISO that reduce mitotic clonal expansion during the early phase of adipogenic differentiation and decrease expression of cell cycle regulators. These results suggest that IHE inhibits adipogenesis, in part, through co-regulation of AMPKα and Nur77, and has potential as a therapeutic option for obesity and related metabolic dysfunction.     

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.     

Pyruvate dehydrogenase kinase 1 and 2 deficiency reduces high-fat diet-induced hypertrophic obesity and inhibits the differentiation of preadipocytes into mature adipocytes

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity.Subject terms: Mechanisms of disease, Obesity     

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.     

Syringin: A Phenylpropanoid Glycoside Compound in Cirsium brevicaule A. GRAY Root Modulates Adipogenesis

Cirsium brevicaule A. GRAY is a wild perennial herb, and its roots (CbR) have traditionally been used as both food and medicine on the Japanese islands of Okinawa and Amami. The present study evaluated the antiadipogenic effect of CbR using mouse embryonic fibroblast cell line 3T3-L1 from JCRB cell bank. Dried CbR powder was serially extracted with solvents of various polarities, and these crude extracts were tested for antiadipogenic activity. Treatment with the methanol extract of CbR showed a significant suppression of lipid accumulation in 3T3-L1 cells. Methanol extract of CbR was then fractionated and subjected to further activity analyses. The phenylpropanoid glycosidic molecule syringin was identified as an active compound. Syringin dose dependently suppressed lipid accumulation of 3T3-L1 cells without cytotoxicity, and significantly reduced the expressions of peroxisome proliferator-activated receptor gamma, the master regulator of adipogenesis, and other differentiation markers. It was demonstrated that syringin effectively enhanced the phosphorylation of the AMP-activated protein kinase and acetyl-CoA carboxylase. These results indicate that syringin attenuates adipocyte differentiation, adipogenesis, and promotes lipid metabolism; thus, syringin may potentially serve as a therapeutic candidate for treatment of obesity.     

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.     

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.     

Real-Time,Label-Free Monitoring of 3T3-L1 Preadipocyte Differentiation by a Bioelectrical Impedance Assay

Monitoring the differentiation of preadipocyte cell lines is critical for investigating the adipogenesis process and drug screening. In this study, a biosensor known as an electric cell-substrate impedance sensor was employed to monitor the entire process of 3T3-L1 preadipocyte differentiation. The magnitude of the impedance mirrors the differentiation status of cells in real-time. Before differentiation, 3T3-L1 preadipocytes were cultured on the surface of the microelectrode array until full confluence. Differentiation was then induced by hormone treatment, and the magnitude of the impedance of the differentiated cells significantly decreased. The differentiation process was identified by Oil Red O staining at the process end-point. We further employed a type of retinoic acid (TTNPB) that inhibits preadipocyte differentiation and assessed its effects on impedance changes. TTNPB was simultaneously added to the culture medium with the hormone inducer of differentiation. There was a good linear correlation between the reciprocal of the average impedance value and the end-point Oil Red O staining assay when different concentrations of TTNPB were added. Importantly, this is the first study to demonstrate that the inhibition of the dynamic morphological changes by TTNPB during preadipocyte differentiation is not a monotonic process, which presumably reflects the timing of intracellular molecular events.     

Ursolic Acid Lactone Obtained from Eucalyptus tereticornis Increases Glucose Uptake and Reduces Inflammatory Activity and Intracellular Neutral Fat: An In Vitro Study

Obesity has a strong relationship to insulin resistance and diabetes mellitus, a chronic metabolic disease that alters many physiological functions. Naturally derived drugs have aroused great interest in treating obesity, and triterpenoids are natural compounds with multiple biological activities and antidiabetic mechanisms. Here, we evaluated the bioactivity of ursolic acid lactone (UAL), a lesser-known triterpenoid, obtained from Eucalyptus tereticornis. We used different cell lines to show for the first time that this molecule exhibits anti-inflammatory properties in a macrophage model, increases glucose uptake in insulin-resistant muscle cells, and reduces triglyceride content in hepatocytes and adipocytes. In 3T3-L1 adipocytes, UAL inhibited the expression of genes involved in adipogenesis and lipogenesis, enhanced the expression of genes involved in fat oxidation, and increased AMP-activated protein kinase phosphorylation. The range of biological activities demonstrated in vitro indicates that UAL is a promising molecule for fighting diabetes.     

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.     

The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 µM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.     

Control of life cycle of mouse adipogenic 3T3-L1 cells by dietary lipids and metabolic factors

Adipocytes function not only as in the storage and mobilization of lipids but also as endocrine cells by secreting tumor necrosis factor-α (TNF-α), free fatty acids, and other cytokines. To study the effects of dietary lipids and metabolic factors on the control of the life cycle of adipocytes, we utilized mouse 3T3-L1 preadipocytes that could be induced to differentiate into adipocytes. To evaluate the role of endogenous prostaglandins (PGs) in the adipogenic changes, we examined the effect of specific inhibitors of cyclooxygenase (COX). SC-560, a specific COX-1 inhibitor, suppressed adipogenesis dose dependently, suggesting a role of constitutive COX-1 in the endogenous synthesis of PGs, including PGJ₂ derivatives formed by mature adipocytes with the ability to promote adipogenesis. NS-398, a COX-2 inhibitor, had little influence on the maturation processes. Both COX inhibitors were effective in stimulating apoptosis of preadipocytes induced by TNF-α, indicating that both PGE₂ and PGF_2α produced by preadipocytes through the action of both COX isoforms serve as survival factors. However, the effect of both inhibitors was negligible for the proliferation of preadipocytes. Moreover, conjugated linolenic acid from bitter gourd at lower concentrations that was without effects by itself synergistically stimulated TNF-α-induced apoptosis. Therefore, dietary lipid factors are capable of controlling the life cycle of adipocytes together with metabolic factors.     

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.     

Anti-Obesity Effects of a Mixture of Atractylodes macrocephala and Amomum villosum Extracts on 3T3-L1 Adipocytes and High-Fat Diet-Induced Obesity in Mice

Since the potential of (3:1) mixtures of Atractylodes macrocephala and Amomum villosum extracts has been proposed in the management of obesity, the purpose of present study was to investigate the effects of AME:AVE (3:1) mixture on weight loss, obesity-related biochemical parameters, adipogenesis and lipogenesis related proteins in 3T3-L1 cells and HFD-induced obesity in a mouse model. Treatment with AME:AVE (3:1) mixture inhibited lipid accumulation. Furthermore, the treatment with 75 and 150 mg/kg of AME:AVE (3:1) significantly decreased the body weight gain, white adipose tissue (WAT) weight, and plasma glucose level in HFD-induced obese mice. Moreover, treatment with 75 and 150 mg/kg AME:AVE (3:1) also significantly lowered the size of adipocytes in adipose tissue and reduced the lipid accumulation in liver. AME:AVE (3:1) treatment significantly decreased the expression of proteins related to adipogenesis and lipogenesis in 3T3-L1 adipocytes and WAT of HFD-induced obese mice. These results suggest that the AME:AVE herbal mixture (3:1) has anti-obesity effects, which may be elicited by regulating the expression of adipogenesis and lipogenesis-related proteins in adipocytes and WAT in HFD-induced obesity in mice.     

The p-Phthalates Terephthalic Acid and Dimethyl Terephthalate Used in the Manufacture of PET Induce In Vitro Adipocytes Dysfunction by Altering Adipogenesis and Thermogenesis Mechanisms

Public health concerns associated with the potential leaching of substances from Polyethylene terephthalate (PET) packaging have been raised due to the role of phthalates as endocrine-disrupting chemicals or obesogens. In particular, changes in the environment such as pH, temperature, and irradiation can improve contaminant migration from PET food packaging. In this study, the in vitro effects of p-phthalates terephthalic acid (TPA) and dimethyl terephthalate (DMT) on murine adipocytes (3T3-L1) were evaluated using concentrations that might be obtained in adult humans exposed to contaminated sources. TPA and, in particular, DMT exposure during 3T3-L1 differentiation increased the cellular lipid content and induced adipogenic markers PPAR-γ, C/EBPß, FABP4, and FASN, starting from low nanomolar concentrations. Interestingly, the adipogenic action of TPA- and DMT-induced PPAR-γ was reverted by ICI 182,780, a specific antagonist of the estrogen receptor. Furthermore, TPA and DMT affected adipocytes’ thermogenic program, reducing pAMPK and PGC-1α levels, and induced the NF-κB proinflammatory pathway. Given the observed effects of biologically relevant chronic concentrations of these p-phthalates and taking into account humans’ close and constant contact with plastics, it seems appropriate that ascertaining safe levels of TPA and DMT exposure is considered a high priority.