A selective peroxisome proliferator-activated receptor-gamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists such as the thiazolidinediones are insulin sensitizers used in the treatment of type 2 diabetes. These compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans. We have identified a novel PPARgamma ligand, LG100641, that does not activate PPARgamma but selectively and competitively blocks thiazolidinedione-induced PPARgamma activation and adipocyte conversion. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. This novel PPARgamma antagonist does not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR), the heterodimeric partner for PPARgamma, or by a differentiation cocktail containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Surprisingly, LG100641, like the PPARgamma agonist rosiglitazone, increases glucose uptake in 3T3-L1 adipocytes. Such selective PPARgamma antagonists may help determine whether insulin sensitization by thiazolidinediones is mediated solely through PPARgamma activation, and whether there are PPARgamma-ligand-independent pathways for adipocyte differentiation. If selective PPARgamma modulators block adipogenesis in vivo, they may prevent obesity, lower insulin resistance, and delay the onset of type 2 diabetes.     

HRASLS3 is a PPARgamma-selective target gene that promotes adipocyte differentiation

The prevalence of obesity and its associated metabolic diseases worldwide has focused attention on understanding the mechanisms underlying adipogenesis. The nuclear receptor PPARgamma has emerged as a central regulator of adipose tissue function and formation. Despite the identification of numerous PPARgamma targets involved in a range of processes, from lipid droplet formation to adipokine secretion, information is still lacking on targets downstream of PPARgamma that directly affect fat cell differentiation. Here we identify HRASLS3 as a novel PPARgamma regulated gene with a role in adipogenesis. HRASLS3 expression increases during the differentiation of preadipocyte cell lines and is highly expressed in white and brown adipose tissue in mice. HRASLS3 expression is induced by PPARgamma ligands in preadipocyte cell lines as well in adipose tissue in vivo. We demonstrate that the HRASLS3 promoter contains a functional PPAR response element and is a direct target for regulation by PPARgamma/RXR heterodimers. Finally, we show that overexpression of HRASLS3 augments PPARgamma-driven lipid accumulation and adipogenesis, whereas siRNA-mediated knockdown of HRASLS3 expression decreases differentiation. Together, these results identify HRASLS3 as one of the downstream effectors of PPARgamma action in adipogenesis.     

The environmental obesogen tributyltin chloride acts via peroxisome proliferator activated receptor gamma to induce adipogenesis in murine 3T3-L1 preadipocytes

Obesogens are chemicals that predispose exposed individuals to weight gain and obesity by increasing the number of fat cells, storage of fats into existing cells, altering metabolic rates, or disturbing the regulation of appetite and satiety. Tributyltin exposure causes differentiation of multipotent stromal stem cells (MSCs) into adipocytes; prenatal TBT exposure leads to epigenetic changes in the stem cell compartment that favor the production of adipocytes at the expense of bone, in vivo. While it is known that TBT acts through peroxisome proliferator activated receptor gamma to induce adipogenesis in MSCs, the data in 3T3-L1 preadipocytes are controversial. Here we show that TBT can activate the RXR-PPARγ heterodimer even in the presence of the PPARγ antagonist GW9662. We found that GW9662 has a 10-fold shorter half-life in cell culture than do PPARγ activators such as rosiglitazone (ROSI), accounting for previous observations that GW9662 did not inhibit TBT-mediated adipogenesis. When the culture conditions are adjusted to compensate for the short half-life of GW9662, we found that TBT induces adipogenesis, triglyceride storage and the expression of adipogenic marker genes in 3T3-L1 cells in a PPARγ-dependent manner. Our results are broadly applicable to the study of obesogen action and indicate that ligand stability is an important consideration in the design and interpretation of adipogenesis assays.     

Functional role of RXRs and PPARgamma in mature adipocytes

The peroxisome proliferator-activated receptor gamma (PPARgamma) is abundantly expressed in adipocytes, and plays an important role in adipocyte differentiation and fat accretion. It is a heterodimeric partner of the retinoid X receptors alpha, beta and gamma, which are also expressed in the adipose tissue. As lethality of PPARgamma(-/-) and RXRalpha(-/-) mouse fetuses precluded the analysis of PPARgamma and RXRalpha functions in mature adipocytes, we generated RXRalpha(ad-/-) and PPARgamma(ad-/-) mice, in which RXRalpha and PPARgamma are selectively ablated in adult adipocytes, respectively. Even though the adiposity of RXRalpha(ad-/-) mice is similar to that of control mice when fed a regular diet, they are resistant to chemically and dietary-induced obesity. However, mature adipocytes lacking either both RXRalpha and RXRgamma or PPARgamma die, and are replaced by newly formed adipocytes. Thus, in adipocytes, RXRalpha is essential for lipogenesis, but RXRgamma can functionally replace RXRalpha for the adipocyte vital functions exerted by PPARgamma/RXR heterodimers.     

Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity

In an effort to identify novel candidate regulators of adipogenesis, gene profiling of differentiating 3T3-L1 preadipocytes was analyzed using a novel algorithm. We report here the characterization of xanthine oxidoreductase (XOR) as a novel regulator of adipogenesis. XOR lies downstream of C/EBPbeta and upstream of PPARgamma, in the cascade of factors that control adipogenesis, and it regulates PPARgamma activity. In vitro, knockdown of XOR inhibits adipogenesis and PPARgamma activity while constitutive overexpression increases activity of the PPARgamma receptor in both adipocytes and preadipocytes. In vivo, XOR -/- mice demonstrate 50% reduction in adipose mass versus wild-type littermates while obese ob/ob mice exhibit increased concentrations of XOR mRNA and urate in the adipose tissue. We propose that XOR is a novel regulator of adipogenesis and of PPARgamma activity and essential for the regulation of fat accretion. Our results identify XOR as a potential therapeutic target for metabolic abnormalities beyond hyperuricemia.     

PPARγ变异与复杂疾病

过氧化物酶体增殖激活受体γ（PPARγ）是核激素受体超家族成员。PPARγ基因主要表达于脂肪组织,促进脂肪细胞分化,调控多种脂肪细胞分泌的蛋白质因子基因的表达。它也是糖尿病治疗药物噻唑烷二酮类化合物(TZDs)作用的靶分子。PPARγ的常见多态性影响胰腺β细胞功能,导致胰岛素分泌及外周组织对胰岛素敏感性的改变。它与2型糖尿病、肥胖、心血管疾病、癌症的发病风险相关联,阐明PPARγ的作用机制对复杂疾病的诊断、预防和治疗具有重要意义。     

Proteome analysis of adipogenesis

Adipose tissue plays a crucial endocrine role in controlling whole body glucose homeostasis and insulin sensitivity. Given the substantial rise in obesity and obesity-related diseases such as diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. Many studies have successfully exploited gene array technology to monitor changes in the profile of expressed genes during adipocyte differentiation, although this method only measures changes at the level of individual mRNA species. Using two-dimensional polyacrylamide gel electrophoresis, high-throughput image analysis, and candidate picking coupled with sequencing mass spectrometry, we have followed the changes in protein expression profile that occur during the differentiation of 3T3-L1 fibroblasts into adipocytes in response to dexamethasone, isobutyl methyl xanthine and insulin, or to the PPARgamma agonist, ciglitazone. Using this technique we have found alterations in the profile of over 2000 protein species during adipogenesis. Our studies reveal previously unknown alterations during adipogenesis in the expression or mobility (on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of coactosin, which promotes actin filament destabilization, several signalling molecules, including RhoGDI-1, RhoGDI-2 and EHD1, and NEDD5 a protein involved in cytokinesis.     

Activation of RXR–PPAR heterodimers by organotin environmental endocrine disruptors

The nuclear receptor retinoid X receptor‐α (RXR‐α)–peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR‐α and PPAR‐γ ligands, the mechanism by which these compounds bind to and activate the RXR‐α–PPAR‐γ heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR–PPAR‐α, ‐γ, ‐δ heterodimers, primarily through its interaction with RXR. In addition, the 1.9 Å resolution structure of the RXR‐α ligand‐binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR‐α ligand‐binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways.