A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet

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.     

Fisetin regulates obesity by targeting mTORC1 signaling

Fisetin, a flavonol present in vegetables and fruits, possesses antioxidative and anti-inflammatory properties. In this study, we have demonstrated that fisetin prevents diet-induced obesity through regulation of the signaling of mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth, cellular proliferation and lipid biosynthesis. To evaluate whether fisetin regulates mTORC1 signaling, we investigated the phosphorylation and kinase activity of the 70-kDa ribosomal protein S6 kinase 1 (S6K1) and mTORC1 in 3T3-L1 preadipocytes. Fisetin treatment of preadipocytes reduced the phosphorylation of S6K1 and mTORC1 in a time- and concentration-dependent manner. To further our understanding of how fisetin negatively regulates mTORC1 signaling, we analyzed the phosphorylation of S6K1, mTOR and Akt in fisetin-treated TSC2-knockdown cells. The results suggested that fisetin treatment inhibits mTORC1 activity in an Akt-dependent manner. Recent studies have shown that adipocyte differentiation is dependent on mTORC1 activity. Fisetin treatment inhibited adipocyte differentiation, consistent with the negative effect of fisetin on mTOR. The inhibitory effect of fisetin on adipogenesis is dependent of mTOR activity, suggesting that fisetin inhibits adipogenesis and the accumulation of intracellular triglycerides during adipocyte differentiation by targeting mTORC1 signaling. Fisetin supplementation in mice fed a high-fat diet (HFD) significantly attenuated HFD-induced increases in body weight and white adipose tissue. We also observed that fisetin efficiently suppressed the phosphorylation of Akt, S6K1 and mTORC1 in adipose tissue. Collectively, these results suggest that inhibition of mTORC1 signaling by fisetin prevents adipocyte differentiation of 3T3-L1 preadipocytes and obesity in HFD-fed mice. Therefore, fisetin may be a useful phytochemical agent for attenuating diet-induced obesity.     

Caudatin suppresses adipogenesis in 3T3-L1 adipocytes and reduces body weight gain in high-fat diet-fed mice through activation of hedgehog signaling

BackgroundThe regulative effects of caudatin, a C-21 steroid that is identified from Cynanchum bungee roots, on adipogenesis and obesity have not been studied. Many studies have demonstrated that the activation of hedgehog (Hh) signaling can help prevent obesity. Therefore, we hypothesized that caudatin can inhibit adipogenesis and obesity via activating the Hh signaling pathway.MethodsTo investigate the effects of caudatin on adipogenesis in 3T3-L1 preadipocytes and high-fat diet induced obesity in C57BL/6 mice, in vitro and in vivo experiments were performed. For in vitro evaluation, Oil red O staining were used to represent lipid accumulation in differentiated 3T3-L1 adipocytes. For in vivo assessment, male 5 week-old C57BL/6 mice were fed with standard chow diet, high-fat diet (HFD), HFD with 25 mg/kg caudatin, HFD with 1mg/kg purmorpharmine for 10 weeks, respectively. Hh signaling and key adipogenic marker involved in adipogenesis were evaluated by real-time PCR and western blot. The adipocyte size of white adopose tissue and lipid storage of liver were visualized by hematoxylin and eosin staining. In addition, the expression of Gli1 and peroxisome proliferator-activated receptor γ (PPARγ) in white adipose tissue were investigated by immunohistochemistry staining.ResultsCaudatin suppressed the accumulation of lipid droplets and downregulated the expression of key adipogenic factors, i.e., peroxisome proliferator-activated receptor γ PPARγ and CCAAT-enhancer binding protein α (C/EBPα), through activating Hh signaling in differentiated 3T3-L1 cells. Furthermore, caudatin and the Hh activator purmorpharmine significantly decreased body weight gain and white adipose tissue (WAT) weight in HFD-induced mice and affected adipogenic markers and Hh signaling mediators in WAT, which were in line with the in vitro experimental results.ConclusionTo our best knowledge, it is the first report to demonstrate that caudatin downregulated adipocyte differentiation and suppressed HFD-induced body weight gain through activating the Hh signaling pathway, suggesting that caudatin can potentially counteract obesity.     

Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/β-catenin signaling

Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/β-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo.     

Methotrexate enhances 3T3-L1 adipocytes hypertrophy

Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and ³H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 μM). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. ³H-Deoxyglucose (³H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and ³H-thymidine incorporation (P?<?0.05). MTX (0.1 μM) treatment increased lipid accumulation and basal ³H-DG uptake by adipocytes (P?<?0.05). In 0.1 μM MTX-treated adipocytes, insulin stimulation did not result in an increase of ³H-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX.     

Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity

This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity.     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.     

Garcinia cambogia extract ameliorates visceral adiposity in C57BL/6J mice fed on a high-fat diet

The aim of present study is to evaluate the effects of Garcinia cambogia on the mRNA levels of the various genes involved in adipogenesis, as well as on body weight gain, visceral fat accumulation, and other biochemical markers of obesity in obesity-prone C57BL/6J mice. Consumption of the Garcinia cambogia extract effectively lowered the body weight gain, visceral fat accumulation, blood and hepatic lipid concentrations, and plasma insulin and leptin levels in a high-fat diet (HFD)-induced obesity mouse model. The Garcinia cambogia extract reversed the HFD-induced changes in the expression pattern of such epididymal adipose tissue genes as adipocyte protein aP2 (aP2), sterol regulatory element-binding factor 1c (SREBP1c), peroxisome proliferator-activated receptor gamma2 (PPARgamma2), and CCAT/enhancer-binding protein alpha (C/EBPalpha). These findings suggest that the Garcinia cambogia extract ameliorated HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis, such as aP2, SREBP1c, PPARgamma2, and C/EBPalpha in the visceral fat tissue of mice.     

Vascular damage in obese female rats with hypoestrogenism

Increase in body weight and adiposity has deleterious consequences on health. The aim of this study was to compare morphological and metabolic changes in the arterial vessels of Wistar rats with conditions of obesity, hypoestrogenism, and hypoestrogenism plus obesity. Ovariectomized rats (hypoestrogenic condition) received 30 % sugar in drinking water plus standard diet during 10 weeks. The hypoestrogenic-obese (HE-OB) group presented increase in weight, blood pressure, hypertriglyceridemia, and hyperglycemia compared with other groups. The morphological study in aortic vessels from HE showed damage in endothelial smooth muscle tissue compared with the other groups. Adipose cells volume in HE-OB (59.33?±?2.38 μ³?×?10⁵) and obese (OB) (54.95?±?1.36 μ³?×?10⁵) groups were significantly larger than control group (36.38?±?0.98 μ³?×?10⁵). In the HE group adipocyte hyperplasia was observed, while in OB group adipocyte hypertrophy and hyperplasia was shown. The vascular reactivity in HE-OB and OB groups presented decrease in the relaxation to acetylcholine compared with control conditions (p?<?0.05), whereas the addition of N^G-nitro-l-arginine methyl ester resulted in a greater inhibition of relaxation in HE-OB and OB groups compared with control conditions (p?<?0.05). These findings suggest that the dysfunction in blood vessels observed in estrogen deficiency and obesity conditions contributes to early cardiovascular alterations.     

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.     

27-Hydroxycholesterol suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells

Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.     

Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity

Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences.     

RGS2 promotes adipocyte differentiation in the presence of ligand for peroxisome proliferator-activated receptor gamma.

The events at the earliest stage of adipocyte differentiation are yet to be fully elucidated. Previously, we cloned the genes that are induced at the beginning of the differentiation of mouse 3T3-L1 preadipocyte cells. We found that the gene expression of regulators of G protein signaling-2 (RGS2) rapidly increased after the addition of inducers and decreased at 3-12 h. The expression pattern of RGS2 mRNAs differed among growth-arrested and proliferating 3T3-L1 cells and NIH-3T3 cells, indicating a specificity for adipogenesis. Here we report that the ectopic expression of RGS2 using a retroviral system in mouse NIH-3T3 cells promotes adipogenesis only in the presence of BRL49653, which is a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). These results strongly suggest that RGS2 play a crucial role in the program of adipocyte differentiation and may contribute to the function of PPARgamma.     

Obestatin as a regulator of adipocyte metabolism and adipogenesis

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/β, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPβ, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.