In vivo and in vitro studies of a functional peroxisome proliferator-activated receptor gamma response element in the mouse pdx-1 promoter.
We reported that peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptionally regulates the beta-cell differentiation factor pancreatic duodenal homeobox (PDX)-1 based on in vitro RNA interference studies. We have now studied mice depleted of PPARgamma within the pancreas (PANC PPARgamma(-/-)) created by a Cre/loxP recombinase system, with Cre driven by the pdx-1 promoter. Male PANC PPARgamma(-/-) 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 beta-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 beta-cell lines and mouse islets, also in vitro translated PPARgamma and retinoid X receptor, and chromatin immunoprecipitation analysis demonstrated specific binding of PPARgamma and retinoid X receptor to the human and mouse pdx-1 x PPREs. Transient transfection assays of beta-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 PPARgamma regulation of pdx-1 transcription in beta-cells, plus our results support an important regulatory role for PPARgamma in beta-cell physiology and thiazolidinedione pharmacology of type 2 diabetes.
Gupta
Dhananjay
D
Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Vermont, Burlington, Vermont 05405, USA.
Jetton
Thomas L
TL
Mortensen
Richard M
RM
Duan
Sheng Zhong
SZ
Peshavaria
Mina
M
Leahy
Jack L
JL
D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics; D066298:In Vitro Techniques; D050417:Insulin-Secreting Cells; D007515:Islets of Langerhans / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D047495:PPAR gamma / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020218:Response Elements; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics*
D018398:Homeodomain Proteins; D047495:PPAR gamma; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein