Insulin- and mitogen-activated protein kinase-mediated phosphorylation and activation of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor (PPAR) gamma plays an important role in adipocyte differentiation and the regulation of adipocyte gene expression. Insulin also serves to promote adipogenesis. We report that insulin and a PPARgamma ligand (thiazolidinedione (TZD)) stimulate in a synergistic manner the expression of an adipocyte-specific gene (aP2) in rat adipocytes and 3T3-L1 cells. Potential cross-talk between insulin signaling and PPARgamma was studied in Chinese hamster ovary cells expressing insulin receptors (CHO.T), PPARgamma, and reporter genes. Both TZD and insulin independently stimulated PPARgamma-mediated transactivation of aP2 promoter-luciferase reporter genes; both agents combined resulted in a synergistic effect. Co-transfection of CHO.T cells with dominant-negative mitogen-activated protein (MAP) kinase-kinase (MKK1) abrogated both insulin- and TZD-mediated activation of PPARgamma; transactivation was markedly increased in cells co-transfected with constitutively active MKK1. Both insulin and constitutively active MKK1 also stimulated 32P incorporation into PPARgamma in vivo. The conclusions are: 1) Insulin synergizes with a PPARgamma ligand and can activate the receptor in a ligand-independent fashion. 2) PPARgamma is phosphorylated in vivo by insulin stimulation or activation of the MAP kinase pathway. 3) MAP kinase is an important mediator of cross-talk between insulin signal transduction pathways and PPARgamma function.

Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha.

Recent studies indicate that a peroxisome proliferator-activated receptor, PPAR gamma, functions as an important adipocyte determination factor. In contrast, tumor necrosis factor-alpha (TNF alpha) inhibits adipogenesis, causes dedifferentiation of mature adipocytes, and reduces the expression of several adipocyte-specific genes. Here, we report that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes. PPAR gamma mRNA levels were reduced by 95% with 3 nM TNF alpha treatment for 24 h. Half-maximal effects were seen after 3 h treatment with 3 nM TNF alpha or with 50 pM TNF alpha (24-h exposure). Parallel reductions in PPAR gamma protein levels were also observed after treatment of 3T3-L1 adipocytes with TNF alpha. Using a ribonuclease protection assay, both alternatively spliced PPAR gamma isoforms (gamma 1 and gamma 2) were shown to be negatively regulated by TNF alpha. The down-regulation of PPAR gamma by TNF-alpha preceded the diminution in expression of other adipocyte-specific genes including CCAAT/enhancer binding protein and adipocyte fatty acid-binding protein (aP2). The effect of TNF alpha was specific for the gamma-isoform of PPARs, since the expression of PPAR delta mRNA was not affected by treatment with TNF alpha. Low level constitutive expression of PPAR gamma in 3T3-L1 adipocytes (at levels approximately 2- to 3-fold higher than in preadipocytes) partially blocked the inhibitory effect of TNF alpha on aP2 and adipsin expression. These findings support the following conclusions: 1) PPAR gamma expression is necessary for the maintenance of the adipocyte phenotype. 2) PPAR gamma, but not PPAR delta, expression is sufficient to attenuate TNF alpha-mediated effects on adipocyte phenotype. 3) Reduced PPAR gamma gene expression is likely to represent an important component of the mechanism by which TNF alpha exerts its antiadipogenic effects.

Down-regulation of the expression of the obese gene by an antidiabetic thiazolidinedione in Zucker diabetic fatty rats and db/db mice.

Obese (ob) is a recently identified gene involved in the regulation of energy balance in the mouse. We report here that AD-5075, a potent thiazolidinedione which lowered plasma glucose and triglyceride in Zucker diabetic fatty (ZDF) rats and db/db mice, decreased the expression of the ob gene in these animal models of obesity and non-insulin-dependent diabetes mellitus. The level of adipose ob mRNA in ZDF rats was 3-fold greater than that detected in the Zucker lean littermates. Chronic treatment with AD-5075 elicited a 67 and 70% reduction of ob mRNA in ZDF and control lean rats, respectively. Furthermore, the amount of adipose ob mRNA in db/db mice was 7 times higher than that detected in lean littermates. Treatment of db/db mice with AD-5075 resulted in a 78% reduction of the level of ob mRNA with parallel changes in circulating level of the ob gene product, leptin. The reduction of the ob mRNA in the Zucker lean rats was accompanied by significantly greater food intake and weight gain. However, in ZDF rats and db/db mice, there was profound increase in body weight without hyperphagia. The results demonstrate that the expression of the ob gene is up-regulated in these two rodent models of diabetes compared to their lean counterparts and that such overexpression is attenuated by treatment with an agent that improves insulin sensitivity and glucose homeostasis in vivo.

Antidiabetic thiazolidinediones block the inhibitory effect of tumor necrosis factor-alpha on differentiation, insulin-stimulated glucose uptake, and gene expression in 3T3-L1 cells.

Tumor necrosis factor-alpha (TNF alpha) is a cytokine implicated in the development of septic shock, cachexia, and other pathological states. Recent studies indicated a direct role for adipose expression of TNF alpha in obesity-linked insulin resistance and diabetes. Pioglitazone, CP-86,325 (CP), AD-5075, CS-045, ciglitazone, and englitazone are members of a new class of insulin-sensitizing thiazolidinedione derivatives with in vivo antidiabetic activities. To test whether these agents antagonize the effect of TNF alpha, 3T3-L1 cells were induced to differentiate in the presence of TNF alpha with or without thiazolidinedione derivatives. Incubation of 3T3-L1 cells with TNF alpha alone completely inhibited adipocyte conversion and expression of fatty acid-binding protein messenger RNA (mRNA). However, coincubation of TNF alpha-treated cells with CP (1 microM), AD-5075 (1 microM), pioglitazone (10 microM), or CS-045 (10 microM) blocked these effects. Long term incubation of 3T3-L1 adipocytes with a low dose of TNF alpha (50 pM) significantly decreased the levels of the adipocyte/muscle-specific glucose transporter (GLUT4) and the CCAAT enhancer-binding protein mRNAs, but did not affect expression of the ubiquitously expressed glucose transporter (GLUT1) or lipoprotein lipase mRNAs. Incubation of 3T3-L1 adipocytes with TNF alpha also inhibited insulin-stimulated 2-deoxyglucose uptake as well as expression of GLUT4 protein. Furthermore, in 3T3-L1 adipocytes, incubation with TNF alpha attenuated the expression of fatty acid-binding protein mRNA in a time- and dose-dependent manner. These inhibitory effects were partially or completely blocked by coincubation of the cells with CP. These results implicate that the insulin-sensitizing agents may exert their antidiabetic activities by antagonizing the inhibitory effects of TNF alpha.