A peroxisome proliferator-activated receptor γ-retinoid X receptor heterodimer physically interacts with the transcriptional activator PAX6 to inhibit glucagon gene transcription (original) (raw)
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British Journal of Pharmacology, 2009
The transcription factor peroxisome proliferator-activated receptor g (PPARg) is essential for glucose homeostasis. PPARg ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARg have been found in several tissues including insulin-producing pancreatic islet b-cells. However, the role of PPARg at the insulin gene was unknown. Therefore, the effect of PPARg and PPARg ligands like rosiglitazone on insulin gene transcription was investigated. Experimental approach: Reporter gene assays were used in the b-cell line HIT and in primary mature pancreatic islets of transgenic mice. Mapping studies and internal mutations were carried out to locate PPARg-responsive promoter regions. Key results: Rosiglitazone caused a PPARg-dependent inhibition of insulin gene transcription in a b-cell line. This inhibition was concentration-dependent and had an EC50 similar to that for the activation of a reporter gene under the control of multimerized PPAR binding sites. Also in normal primary pancreatic islets of transgenic mice, known to express high levels of PPARg, rosiglitazone inhibited glucose-stimulated insulin gene transcription. Transactivation and mapping experiments suggest that, in contrast to the rat glucagon gene, the inhibition of the human insulin gene promoter by PPARg/rosiglitazone does not depend on promoter-bound Pax6 and is attributable to the proximal insulin gene promoter region around the transcription start site from -56 to +18. Conclusions and implications: The human insulin gene represents a novel PPARg target that may contribute to the action of thiazolidinediones in type 2 diabetes mellitus.
Molecular Endocrinology, 2006
Partial agonists of peroxisome proliferator-activated receptor-␥ (PPAR␥), also termed selective PPAR␥ modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPAR␥ ligands. In a cocrystal with PPAR␥ it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPAR␥-coactivator-1␣ (PGC1␣) to the receptor, a feature shared with other selective PPAR␥ modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1␣ but not its LXXLLdeficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNF␣ on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1␣ is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1␣ to favorable PPAR␥-target genes provides a possible molecular mechanism whereby partial PPAR␥ agonists dissociate TG accumulation from insulin signaling. (Molecular Endocrinology 20: 809-830, 2006) P EROXISOME PROLIFERATOR-ACTIVATED RE-CEPTOR-␥ (PPAR␥) (1) is a ligand-activated transcription factor of the nuclear receptor (NR) superfamily (1). PPAR␥ forms heterodimers with the retinoid X receptor (RXR) that transactivate PPAR-responsive elements (PPREs) of target genes involved in insulin signaling, lipid/glucose metabolism, immune response, cell cycle, and differentiation of epithelial or mesenchymal cells. The receptor is selectively activated by physiological fatty acid derivatives, such as 15-deoxy-⌬ 12,14 -prostaglandin J 2 , and by a panel of chemically diverse full agonists such as glitazars and thiazolidinediones (TZDs). The TZDs rosi-and pioglitazone are insulin-sensitizing drugs approved for therapy of type 2 diabetes mellitus in humans.
Journal of Biological Chemistry, 1997
Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a member of the nuclear hormone receptor superfamily, and is an important regulator of adipogenesis and adipocyte gene expression. PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, that differ only in their N termini. Both isoforms are activated by ligands that include the antidiabetic thiazoladinedione drugs and 15-deoxy-⌬ 12, 14prostaglandin J2, and potential differences in their function have yet to be described. We report that, in addition to a ligand-activated transcriptional activity, when studied under conditions of ligand depletion, intact PPAR␥ has a ligand-independent activation domain. To identify the basis for this ligand-independent activation, we used GAL4-PPAR␥ chimeric expression constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes. In both cell systems, isolated PPAR␥1 and PPAR␥2 N termini have activation domains, and the activation function of PPAR␥2 is 5-6-fold greater than that of PPAR␥1. Insulin enhances the transcriptional effect mediated by both PPAR␥1 and PPAR␥2 N-terminal domains. These data demonstrate that 1) PPAR␥ has an N-terminal (ligandindependent) activation domain; 2) PPAR␥1 and PPAR␥2 N termini have distinct activation capacities; and 3) insulin can potentiate the activity of the N-terminal domain of PPAR␥.
Endocrine-related Cancer, 2008
The peroxisome proliferator-activated receptor-g (PPARg) is a member of the nuclear hormone receptor superfamily. Ligand activation of PPARg is associated with differentiation and inhibition of proliferation in the normal and malignant cells. Herein, we studied the effects of PPARg and the PPARg agonists thiazolidinediones (TZDs) on the insulin receptor (IR), a cell membrane tyrosine kinase receptor protein, whose role is of paramount importance in mediating the metabolic and growth-promoting effects of the peptide hormone insulin. Overexpression of the PPARg1 in human hepatocellular (HepG2) cells was associated with decreased IR gene transcription and protein expression levels, and these reductions were more evident in the presence of TZDs. Since no PPARg response elements were identified on the IR promoter, we postulated that PPARg adversely affects the IR gene transcription by perturbing the assembly and stability of the transcriptionally active multiprotein-DNA complex identified previously, which includes the high-mobility group A1 protein, the ubiquitously expressed transcription factor (Sp1), the CAAT enhancer-binding protein (C/EBPb), and, in some cell lines, the developmentally regulated activator protein-2 (AP-2) transcription factor. Using glutathione S-transferase pull-down assays combined with electrophoretic mobility shift assay and chromatin immunoprecipitation, we demonstrated that by interacting with Sp1, C/EBPb, and AP-2, PPARg can prevent Sp1/AP-2 protein-protein association and inhibit binding of Sp1 and C/EBPb to DNA, thus reducing IR gene transcription. Our results demonstrate that IR is a new target gene of PPARg, and support a potential use of TZDs as anti-proliferative agents in selected neoplastic tissues overexpressing IRs. Endocrine
Journal of Biological Chemistry, 2008
We reported that peroxisome proliferator-activated receptor ␥ (PPAR␥) transcriptionally regulates the -cell differentiation factor pancreatic duodenal homeobox (PDX)-1 based on in vitro RNA interference studies. We have now studied mice depleted of PPAR␥ within the pancreas (PANC PPAR␥ ؊/؊) created by a Cre/loxP recombinase system, with Cre driven by the pdx-1 promoter. Male PANC PPAR␥ ؊/؊ mice were hyperglycemic at 8 weeks of age (8.1 ؎ 0.2 mM versus 6.4 ؎ 0.3 mM, p ؍ 0.009) with islet cytoarchitecture and pancreatic mass of islet -cells that were indistinguishable from the controls. Islet PDX-1 mRNA (p ؍ 0.001) and protein levels (p ؍ 0.003) were lowered 60 and 40%, respectively, in tandem with impaired glucose-induced insulin secretion and loss of thiazolidinedione-induced increase in PDX-1 expression. We next identified a putative PPAR-response element (PPRE) in the mouse pdx-1 promoter with substantial homology to the corresponding region of the human PDX-1 promoter. Electrophoretic mobility supershift assays with nuclear extracts from -cell lines and mouse islets, also in vitro translated PPAR␥ and retinoid X receptor, and chromatin immunoprecipitation analysis demonstrated specific binding of PPAR␥ and retinoid X receptor to the human and mouse pdx-1 ؋ PPREs. Transient transfection assays of -cells with reporter constructs of mutated PPREs showed dramatically reduced pdx-1 promoter activity. In summary, we have presented in vivo and in vitro evidence showing PPAR␥ regulation of pdx-1 transcription in -cells, plus our results support an important regulatory role for PPAR␥ in -cell physiology and thiazolidinedione pharmacology of type 2 diabetes.
Role of PPARg2 transcription factor in thiazolidinedione-induced insulin sensitization
Journal of Pharmacy and Pharmacology, 2011
Objectives Adipose tissue is the key regulator of energy balance, playing an active role in lipid storage and metabolism and may be a dynamic buffer to control fatty acid flux. Peroxisome proliferator-activated receptor gamma isoform-2 (PPARg2), an isoform of the nuclear hormone receptor superfamily, has been implicated in almost all aspects of human metabolic alterations such as obesity, insulin resistance, type-2 diabetes and dyslipidaemia. The PPARg2 isoform is highly present in adipose tissue where it functions as a thrifty phenotype, which promotes adipocyte differentiation and triglyceride storage. Thiazolidinediones, antidiabetic drugs, induce insulin sensitivity by controlling adipokines. The thiazolidinediones bind with PPARg2 in adipocytes and exert an agonist effect by enhancing adipogenesis and fatty acid uptake. Thiazolidinediones stimulate PPARg2, by which they down-regulate tumour necrosis factor-α, leptin, interleukin-6 and plasminogen and also enhance insulin sensit...
Diabetes, 2010
OBJECTIVE We previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARγ in GIP-R transcription. RESEARCH DESIGN AND METHODS Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARγ knockout mice (PANC PPARγ−/−), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS In vitro studies of INS-1 cells confirmed that PPAR-γ binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a ...
Journal of Biological Chemistry, 2003
The synthetic thiazolidinedione ligands of peroxisome proliferator-activated receptor-␥ (PPAR␥) improve insulin sensitivity in type II diabetes and induce GLUT4 mRNA expression in fat and muscle. However, the molecular mechanisms involved are still unclear. We studied the regulatory effects of PPAR␥ and its ligands on GLUT4 gene expression in primary rat adipocytes and CHO-K1 cells cotransfected with PPAR␥ and the GLUT4 promoter reporter. PPAR␥1 and PPAR␥2 repressed the activity of the GLUT4 promoter in a dose-dependent manner. Whereas this repression was augmented by the natural ligand 15⌬-prostaglandin J 2 , it was completely alleviated by rosiglitazone (Rg). Ligand binding-defective mutants PPAR␥1-L468A/E471A and PPAR␥2-L496A/ E499A retained the repression effect, which was unaffected by Rg, whereas the PPAR␥2-S112A mutant exhibited a 50% reduced capacity to repress GLUT4 promoter activity. The ؊66/؉163 bp GLUT4 promoter region was sufficient to mediate PPAR␥ inhibitory effects. The PPAR␥/retinoid X receptor-␣ heterodimer directly bound to this region, whereas binding was abolished in the presence of Rg. Thus, we show that PPAR␥ represses transcriptional activity of the GLUT4 promoter via direct and specific binding of PPAR␥/retinoid X receptor-␣ to the GLUT4 promoter. This effect requires an intact Ser 112 phosphorylation site on PPAR␥ and is completely alleviated by Rg, acting via its ligand-binding domain. These data suggest a novel mechanism by which Rg exerts its antidiabetic effects via detaching PPAR␥ from the GLUT4 gene promoter, thus leading to increased GLUT4 expression and enhanced insulin sensitivity.