N-Caffeoyltryptophan enhances adipogenic differentiation in preadipocytes and improves glucose tolerance in mice

Coffee consumption has been shown to reduce the risk of developing type 2 diabetes mellitus (T2DM) in humans; however, the exact mechanism is not completely understood. Here, we demonstrate that N-caffeoyltryptophan (CTP), an ingredient of coffee, enhances adipogenic differentiation and promotes glucose uptake into adipocytes. CTP increased lipid accumulation and adipogenic markers (PPARγ, C/EBPα, and FABP4) expression in mouse 3T3-L1 preadipocyte cell lines and primary preadipocytes. In addition, CTP promoted glucose uptake in 3T3-L1 cells. In the oral glucose tolerance test, daily administration of CTP (30 mg/kg/day, i.p.) for a week reduced blood glucose levels in mice. In 3T3-L1 cells, adipogenic differentiation and increased adipogenic markers expression induced by CTP were inhibited by U0126, a selective MEK1/2 inhibitor. Furthermore, mRNA induction of Pparg by CTP was abrogated in SIRT1 siRNA-transfected 3T3-L1 cells. These results suggest the involvement of the MEK/ERK signaling and SIRT1 in the mechanism of adipogenic function of CTP. Taken together, CTP might contribute to the reduction in postprandial glycemia and a subsequent reduction in onset risk for T2DM.     

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.     

Endothelin-1 inhibits adipogenic differentiation of 3T3-L1 preadipocytes

The effect of endothelin (ET)-1 on the adipogenic differentiation of 3T3-L1 preadipocytes was examined. Cellular morphology and lipoprotein lipase activity were used as differentiation markers. ET-1 inhibited the hormone-induced adipogenic differentiation of 3T3-L1 preadipocytes morphologically and biochemically in a dose-dependent manner. These findings promote ET-1 as a potent inhibitor of adipogenic differentiation, playing an important role in cellular differentiation of preadipocytes and making it a significant regulator of lipid metabolism.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.     

Type I collagen inhibits adipogenic differentiation via YAP activation in vitro

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3-L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3-L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3-L1 cell migration via stimulating the nuclear translocation of yes-associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high-fat diet fed mice. Increased expression of YAP in the cells cultured on col I-coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small-interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro.     

miR-128-3p regulates 3T3-L1 adipogenesis and lipolysis by targeting <Emphasis Type="Italic">Pparg</Emphasis> and <Emphasis Type="Italic">Sertad2</Emphasis>

Differentiation of adipocytes and their aggregation to adipose tissue are critical for mammalian growth and development. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play important roles in adipogenesis and lipid metabolism. miR-128-3p may contribute to adipose tissue development according to the previous studies. However, the role of miR-128-3p in the process of preadipocyte differentiation and lipid metabolism is not yet understood. The purpose of this research was to investigate the biological function and molecular mechanism of miR-128-3p in 3T3-L1 cells. In the present study, we found that miR-128-3p was downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-128-3p obstructed the expressions of adipogenic marker genes as well as the lipid droplets accumulation and triglyceride content, suggesting the importance of miR-128-3p for adipogenesis. Moreover, miR-128-3p could lead to the retardation of cell proliferation in 3T3-L1 preadipocytes. Further evidences showed that, as a negative regulator of adipogenesis, miR-128-3p could directly target peroxisome proliferator-activated receptor γ (Pparg) which resulted in the suppression of 3T3-L1 preadipocyte differentiation, and miR-128-3p could also bind with SERTA domain containing 2 (Sertad2) which drove triglyceride hydrolysis and lipolysis. In addition, inhibition of Sertad2 with siRNA displayed the same effects as overexpression of miR-128-3p. Our research demonstrated that miR-128-3p impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis. Taken together, this study offers profound insight into the mechanism of miRNA-mediated adipogenesis and lipid metabolism.     

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling,down-regulation of PPARγ and induction of iNOS

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC₅₀ of 10 μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for 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.     

Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes

Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5–7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone.