Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene in adipocytes

Peroxisome proliferator activated receptor-gamma (PPARgamma) and three CCAAT/enhancer binding proteins (C/EBPs) are transcription factors required for adipocyte differentiation. PPARgamma heterodimerizes with 9- cis retinoic acid receptor (RXR). Fatty acid metabolites and the newest class of type 2 diabetic medications are ligands. PPARgamma is implicated in several diseases including diabetes and cancer. In addition to their effects on adipocytes, C/EBPs regulate carbohydrate metabolism and immunity. C/EBPs and PPARgamma cooperatively activate adipogenesis; however, their precise mechanism(s) of action are unknown.; We study the transcriptional regulation of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene that controls alpha-glycerol-phosphate formation used in free fatty acid esterification in adipocytes. Dr. Beale previously identified an adipocyte-specific enhancer (ASE) upstream of the PEPCK gene, and found that PPARgamma regulates PEPCK induction in adipocytes through the ASE. PPARgamma binds two sites, gAF1/PCK1 and PCK2; however, only PCK2 is required for PEPCK gene expression in adipose tissue. Gene ablation studies showed that C/EBPbeta controls PEPCK transcription in adipocytes by an unknown mechanism. The present studies investigated the mechanism of C/EBP and PPARgamma transactivation of the PEPCK gene by characterizing the C/EBP and PPARgamma response elements.; We hypothesized that C/EBPbeta binding to the ASE was required for PEPCK induction. These studies demonstrated that C/EBPbeta transactivated the PEPCK gene via the ASE. We identified three functional C/EBP binding sites flanking PCK2; however, they were not required for C/EBP transactivation. We also found that PPARgamma/RXR and C/EBP interacted yielding synergic activation or inhibition of the PEPCK gene. The mechanism of this interaction and their roles are still under investigation.; We sought to identify the mechanism for the different functions of gAF1/PCK1 and PCK2. We hypothesized that gAF1/PCK1 and PCK2 bound different transcription factors in adipocytes. Chicken ovalbumin upstream promoter-transcription factor II inhibited PPARgamma/RXR activation through an intact gAF1/PCK1 element. Quite surprisingly, PPARgamma/RXR, is the predominant adipocyte binding species for both gAF1/PCK1 and PCK2. We then hypothesized that PPARgamma/RXR binding had site-selective effects. This was corroborated as ablating PCK2 abolished PPARgamma/RXRalpha transactivation, whereas ablating gAF1/PCK1 increased maximal PPARgamma/RXR activation. These studies provide a unique example of antagonism between two PPARgamma binding elements on a single promoter.