The role of SIRT1 and adipogenic transcription factors in regulating adiponectin secretion and adipocyte functions

Adipose tissue plays a central role in controlling energy balance and insulin sensitivity through regulating metabolic homeostasis and secretion of endocrine factors. This study was designed to understand the mechanisms by which the two master regulators of adipogenesis, Peroxisome Proliferator Activated Receptor gamma (PPARgamma) and CCAAT/Enhancer Binding Protein alpha (C/EBPalpha), regulate adipocyte functions. The initial studies involved expressing mutants of C/EBPalpha with and without PPARgamma in mouse fibroblasts to search for novel C/EBPalpha target genes. The data demonstrate that individual domains of C/EBPalpha have different functions in activating gene expression. Specifically, Transactivation Element III (TE-III) domain regulates PPARgamma expression, while TE-II regulates adiponectin expression. During these studies, western blot analysis of 3T3-L1 adipocyte proteins detected a novel 24kD polypeptide. Mass spectrometric sequencing of this protein following its purification by high performance liquid chromatography and two-dimension gel electrophoresis identified it as glutathione S transferase zeta 1/maleylacetoacetate isomerase (GSTzeta1/MAAI). Its expression was found to be induced during terminal differentiation in a C/EBPalpha and PPARgamma dependent manner.; Adiponectin is produced exclusively in adipocytes in response to metabolic effectors to sensitize the liver and muscle to insulin. Reduced circulating adiponectin that usually accompany obesity contribute to insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. Food intake or increased plasma glucose increase circulating adiponectin without altering its synthesis. Additional studies in demonstrate that the endoplasmic reticulum (ER) oxidoreductase ERO1-Lalpha and effectors modulating PPARgamma and SIRT1 activity regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and ERO1-Lalpha expression are induced during the early phase of adipogenesis, then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPARgamma enhances ERO1-Lalpha expression and stimulates secretion of high molecular weight complexes of adiponectin in adipocytes. Moreover, ectopic expression of Ero1-Lalpha in 3T3 fibroblasts stimulates the secretion of adiponectin during their differentiation into adipocytes and knockdown of Ero1-Lalpha in 3T3-L1 adipocytes results in decreased adiponectin secretion. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to varying metabolic states.