Dexamethasone reverses TGF-beta-mediated inhibition of primary rat preadipocyte differentiation

Dexamethasone and transforming growth factor-beta (TGF-beta) show contrary effects on differentiation of adipocytes. Dexamethasone stimulates adipocyte differentiation whereas TGF-beta inhibits it. In the present study, we investigated whether dexamethasone could reverse the TGF-beta-mediated inhibition of preadipocyte differentiation. Primary rat preadipocytes, obtained from Sprague-Dawley rats, were pretreated with dexamethasone in the presence or absence of TGF-beta, prior to the induction of differentiation. Co-treatment of dexamethasone and TGF-beta before inducing differentiation reversed the TGF-beta-mediated inhibition of preadipocyte differentiation. In order to elucidate the mechanism by which dexamethasone reversed the effect of TGF-beta on the inhibition of preadipocyte differentiation, the expression of CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) was examined. Dexamethasone increased C/EBPalpha and PPARgamma expression in the absence of TGF-beta and also recovered the TGF-beta-mediated suppression of C/EBPalpha expression in preadipocytes. Its effect was sustained in differentiated adipocytes as well. However, those effects were not observed in 3T3-L1 preadipocytes or differentiated adipocytes. These results indicate that dexamethasone reverses the TGF-beta-mediated suppression of adipocyte differentiation by regulating the expression of C/EBPalpha and PPARgamma, which is dependent on the cellular context.     

猪脂肪组织发育过程中Wnt/β-catenin信号通路相关基因及脂肪转录因子的时序表达

为研究Wnt/b-catenin信号通路对猪脂肪组织发育的影响, 并探讨其可能的作用机制, 以1~120日龄长白猪脂肪组织为试验材料, 采用SQ RT-PCR法检测Wnt信号通路中相关基因β-catenin、GSK3β、Fz1及主要的脂肪转录因子PPARγ、C/EBPα 和分化早期标志基因LPL mRNA的表达变化; 石蜡切片免疫组化方法定性检测β-catenin在脂肪组织发育中的时序表达变化。SQ RT-PCR结果显示, β-catenin mRNA在猪出生第1天有高水平表达, 随着个体日龄的增长, 其表达量逐渐降低, 60日龄后维持在一个较低水平。GSK3β和Fz1 mRNA的表达量也伴随脂肪组织的发育而逐渐降低。而LPL、PPARγ和C/EBPα的 mRNA表达量随着个体日龄的增长而逐渐升高, 60日龄后仍保持高水平的表达。石蜡切片免疫组化结果显示, 随着脂肪组织的发育, β-catenin蛋白的表达也随之降低, 并且β-catenin蛋白的表达部位逐渐由细胞核和细胞质共表达变为只在细胞质中表达。上述基因表达的时序变化规律提示, β-catenin在维持前体脂肪细胞的未分化状态, 抑制脂肪组织发育中具有重要作用, 其作用机制可能是通过对脂肪细胞转录因子PPARγ、C/EBPα及分化早期标志基因LPL mRNA的调控来进行的。     

C/EBPalpha-dependent induction of glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI) expression during the differentiation of mouse fibroblasts into adipocytes

Western blot analysis of 3T3-L1 adipocyte proteins using an anti-C/EBPalpha antibody detected a 24kD polypeptide in addition to the expected 42 and 30kD isoforms of C/EBPalpha. Mass spectrometric sequencing of the protein following its purification by HPLC and preparative 2D gel electrophoresis identified it as glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI). Expression of GSTzeta/MAAI mRNA and protein was induced during the terminal phase of adipogenesis in 3T3-L1 preadipocytes. Ectopic expression of PPARgamma2 in NIH-3T3 fibroblasts exposed to insulin and troglitazone-induced perilipin production, but was incapable of activating GSTzeta/MAAI unless C/EBPalpha was also expressed. Similarly, ectopic expression of C/EBPalpha in PPARgamma +/- or PPARgamma -/- MEFs demonstrated that the C/EBPalpha-dependent induction of GSTzeta/MAAI production was dependent on expression of endogenous PPARgamma. These data suggest a role for GSTzeta/MAAI in mature adipocytes that may be responsive to the thiazolidinedione class of insulin sensitizing PPARgamma ligands.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.     

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.     

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.     

黄芩素对猪前体脂肪细胞增殖分化的影响

研究黄芩素(BAI)对猪前体脂肪细胞增殖分化的影响,并探讨其可能的作用机制。原代培养猪前体脂肪细胞,采用油红O染色观察细胞分化的形态学变化;MTT检测细胞增殖状况;油红O染色提取定量分析细胞内脂肪生成及细胞分化程度;分光光度法测定脂肪酸合酶(FAS)的活性;逆转录-聚合酶链反应(RT-PCR)检测分化特异基因过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达变化。结果显示,前体脂肪细胞在分化成脂肪细胞的过程中,其形态由梭形变成椭圆形、圆形,细胞内充满大小不一的脂滴;BAI浓度在160～640μmol/L时显著抑制其增殖(P<0.05)、BAI浓度为40～320μmol/L时显著抑制PPARγ2mRNA表达和FAS的活性,并抑制细胞分化(P<0.05)。以上结果说明,BAI对前体脂肪细胞增殖分化均有一定抑制作用,BAI可能通过抑制PPARγ2mRNA表达和降低FAS活性,从而抑制猪前体脂肪细胞分化。     

Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes

We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.     

Expression of CCAAT/enhancer binding protein C/EBPalpha, beta and delta in rat adipose stromal-vascular cells in vitro.

Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBPalpha, beta and delta levels were evaluated for different growth conditions and at different times using Western blots. Immediately after isolation C/EBPalpha, beta and delta could not be detected in S-V cells. After seeding for 24 h in Dulbecco's modified Eagle's medium (DMEM) with FBS, C/EBPalpha, beta and delta could all be detected. Cells at day 1 of culture in insulin, transferrin, triiodothyronine and selenium (ITTS) had increased levels of C/EBPalpha and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBPalpha levels similar to ITTS cultures at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures. C/EBPbeta was expressed uniformly throughout the culture period in either DMEM or ITTS cultures while C/EBPdelta expression was higher with DMEM treatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increased levels of C/EBPbeta and delta but significantly reduced levels of C/EBPalpha. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. However, by day 6 of culture the percentage of cells staining positively for C/EBPalpha in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBPalpha and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBPalpha and enhanced lipid accumulation. Because C/EBPalpha, beta and delta expression occurs very early in cell culture and C/EBPalpha and delta expression continues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo before being placed in culture. Thus, a large fraction of S-V cells may be further along in the differentiation program than 3T3 cells are when they begin differentiation.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.