Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPARdelta ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPARdelta-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPARdelta-selective ligand and MIX significantly enhanced the early expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion, our results suggest that PPARdelta may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPARdelta in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.     

Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immunofluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor γ [PPARγ], CCAAT/enhancer-binding protein α, and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPARγ agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPARγ. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states.     

Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression

Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis.     

Prostaglandin reductase-3 negatively modulates adipogenesis through regulation of PPARγ activity

Adipocyte differentiation is a multistep program under regulation by several factors. Peroxisome proliferator-activated receptor γ (PPARγ) serves as a master regulator of adipogenesis. However, the endogenous ligand for PPARγ remained elusive until 15-keto-PGE₂ was identified recently as an endogenous PPARγ ligand. In this study, we demonstrate that zinc-containing alcohol dehydrogenase 2 (ZADH2; here termed prostaglandin reductase-3, PTGR-3) is a new member of prostaglandin reductase family that converts 15-keto-PGE₂ to 13,14-dihydro-15-keto-PGE₂. Adipogenesis is accelerated when endogenous PTGR-3 is silenced in 3T3-L1 preadipocytes, whereas forced expression of PTGR-3 significantly decreases adipogenesis. PTGR-3 expression decreased during adipocyte differentiation, accompanied by an increased level of 15-keto-PGE₂. 15-keto-PGE₂ exerts a potent proadipogenic effect by enhancing PPARγ activity, whereas overexpression of PTGR-3 in 3T3-L1 preadipocytes markedly suppressed the proadipogenic effect of 15-keto-PGE₂ by repressing PPARγ activity. Taken together, these findings demonstrate for the first time that PTGR-3 is a novel 15-oxoprostaglandin-Δ¹³-reductase and plays a critical role in modulation of normal adipocyte differentiation via regulation of PPARγ activity. Thus, modulation of PTGR-3 might provide a novel avenue for treating obesity and related metabolic disorders.     

Expression of p107 and p130 during human adipose-derived stem cell adipogenesis

Within the first 24 h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

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.     

Estrogen sulfotransferase inhibits adipocyte differentiation

The estrogen sulfotransferase (EST) is a phase II drug-metabolizing enzyme known to catalyze the sulfoconjugation of estrogens. EST is highly expressed in the white adipose tissue of male mice, but the role of EST in the development and function of adipocytes remains largely unknown. In this report, we showed that EST played an important role in adipocyte differentiation. EST was highly expressed in 3T3-L1 preadipocytes and primary mouse preadipocytes. The expression of EST was dramatically reduced in differentiated 3T3-L1 cells and mature primary adipocytes. Overexpression of EST in 3T3-L1 cells prevented adipocyte differentiation. In contrast, preadipocytes isolated from EST knockout (EST-/-) mice exhibited enhanced differentiation. The inhibitory effect of EST on adipogenesis likely resulted from the sustained activation of ERK1/2 MAPK and inhibition of insulin signaling, leading to a failure of switch from clonal expansion to differentiation. The enzymatic activity of EST was required for the inhibitory effect of EST on adipogenesis, because an enzyme-dead EST mutant failed to inhibit adipocyte differentiation. In vivo, overexpression of EST in the adipose tissue of female transgenic mice resulted in smaller adipocyte size. Taken together, our results suggest that EST functions as a negative regulator of adipogenesis.     

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.