Pyruvate carboxylase (PC) is a mitochondrial enzyme which converts pyruvate to oxaloacetate, one of important intermediates in the tricarboxylic acid cycle. As oxaloacetate is utilized as the precursor for many biosynthetic pathways including gluconeogenesis in liver and kidney, lipogenesis in liver and adipose tissue, neurotransmitter synthesis in astrocytes, dysregulation of PC expression is associated with metabolic syndrome including obesity and type 2 diabetes. Understanding regulation of PC will unravel molecular mechanisms underlying the above metabolic disorders, and may lead to the prevention of the disease as well as the development of new anti-diagetic drugs. This enzyme is also involved in the synthesis of coupling factor which supports insulin secretion in pancreatic -cells. Here we dissected the molecular mechanisms underlying transcription control of PC in hepatocytes and in pancrease. Using AML12 hepatocyte cell line as the model for liver, we demonstrate that the proximal promoter of PC gene is directed by liver-enriched transcription factor, HNF4 which binds to the novel HNF4-specific binding motif (H4-SBM), allowing the precise control of this promoter in a liver-specific manner. Using rat insulinoma INS-1 832/13 cell line as the model for pancreatic islets, we deomonstrate that expression of PC in this cell type is regulated by complex glucose-responsive element (GRE), comprising multiple E-box located in the distal promoter of PC gene. Elevated concentration of glucose stimulates binding of three transcription factors, namely, the carbohydrate responsive element binding protein (ChREBP), Specificity protein-1 (Sp1) and upstream stimulatory factor-2 (USF2) to the GRE. Suppression of these three transcription factors impairs glucosemediated transcriptional induction of PC gene indicating the critical role of those proteins in regulation of PC expression. We also cloned and identified the distal promoter of the human PC gene and found that the cis-acting elements and the putative binding sites of general and tissue-specific transcription factors, as well as the GRE therein, are highly homologous to those of the rat PC gene. Metformin, an anti-diabetic drug which is thought to act by lowering elevated plasma glucose in type 2 diabetic patients did not show any inhitory effect on PC expression in human hepatocyte cell line, HepG2. Our result together with the recent reports by other investigators, confirm that metformin does not lower plasma glucose through inhibition of gluconeogenic genes.