Project Summary/Abstract The production of glucose by the liver is a vital physiological process. Hepatic glucose production plays an important role in regulating normoglycemia during starvation, providing skeletal muscle with glucose during exercise, and contributing to hyperglycemia of diabetes. Gluconeogenesis, the process of converting the carbon in pyruvate, lactate, or amino acids into new glucose, plays an important role in glucose production by the liver. Gluconeogenesis requires the transport of pyruvate or amino acids across the impermeable inner mitochondrial membrane (IMM) and subsequent metabolism by enzymes exclusively localized to the mitochondrial matrix. Recent work from the laboratory of the applicant has demonstrated an important role for the mitochondrial pyruvate carrier (MPC) complex in gluconeogenesis from pyruvate/lactate. However, these studies also suggested an important role for pyruvate-alanine cycling as a compensatory mechanism when MPC activity was impaired. In addition, while amino acids like alanine are believed to be an important substrate for gluconeogenesis, many details regarding their metabolism are lacking. The significance of the proposed studies is that we will dissect the molecular mechanisms that mediate these processes in fasting, exercise, and diabetes. We hypothesize that the transcription factor ATF4 will regulate the expression of alanine transaminase 2 (ALT2) and that this enzyme will play an important role in regulating alanine-stimulated gluconeogenesis in diabetic liver (Specific Aim 1). We also hypothesize that that the effects of ALT2 deficiency on glucose production will be enhanced by concomitant loss of MPC activity to disrupt gluconeogenesis from both alanine and pyruvate. (Specific Aim 2). We also propose a third, exploratory Aim that has the potential for marked scientific advance. The transport of alanine across the impermeable IMM by a carrier-mediated process is required for alanine to be used for gluconeogenesis. However, the identity of the carrier that mediates this process has never been determined. We hypothesize that the yeast Avt5 and its mammalian homolog Slc38a10 serve as the mitochondrial alanine carrier and that these proteins are required for mitochondrial alanine metabolism (Specific Aim 3). We believe that the proposed studies will provide marked scientific advance towards our understanding of hepatic amino acid metabolism, which has been understudied to this point. In addition, these studies will provide insight into the effects of these metabolic pathways on hepatic gluconeogenesis and could impact pharmaceutical development of new drugs to treat diabetes.