Non-alcoholic fatty liver disease (NAFLD) affects ~25% of the world’s population and is a key factor in the pathogenesis of hepatic insulin resistance and type 2 diabetes (T2D). NAFLD is a predisposing factor for nonalcoholic steatohepatitis (NASH) and hepatocellular cancer and an independent risk factor for cardiovascular disease. However, there are currently no approved therapies to treat NAFLD. In order to address this unmet need, we plan to examine the cellular and molecular mechanisms by which glucagon alters hepatic mitochondrial fatty oxidation and hepatic anaplerotic fluxes in vivo and the potential role of the Inositol Triphosphate Receptor-1 (InsP3R-1) in mediating these effects. These questions will be addressed by a well-established team of interdisciplinary investigators using state-of-the-art nuclear magnetic resonance spectroscopy (NMR)-gas chromatographic-mass spectrometry (GC-MS) methodologies that we have recently developed to assess in vivo rates of hepatic mitochondrial fatty acid oxidation, hepatic glucose oxidation, and hepatic pyruvate carboxylase flux for the first time in awake liver-specific InsP3R-1 knockout mice. These methods will be applied to address the following Specific Aims: 1) To examine the role of InsP3R-1 in mediating glucagon’s effect to promote hepatic mitochondrial oxidation and hepatic gluconeogenesis. 2) To examine the role of IP3R-I in mediating exercise-induced increases in hepatic mitochondrial fat oxidation and reductions in NAFLD and NAFLD-associated hepatic insulin resistance. 3) To examine whether chronic stimulation of IP3R-I by glucagon will increase hepatic mitochondrial oxidation and reverse NAFLD and NAFLD-associated hepatic insulin resistance. Taken together, the studies proposed in this grant application are designed to generate a comprehensive mechanistic understanding of the impact of glucagon on hepatic mitochondrial fat oxidation and hepatic pyruvate carboxylase flux in vivo and on the role of InsP3R-I in mediating glucagon’s effects on hepatic mitochondrial function. Furthermore, it is anticipated that the results of these studies will provide important new insights into the mechanism by which novel glucagon agonists might reverse NAFLD and hepatic insulin resistance as well as potentially identify InsP3R-I as a novel therapeutic target for the treatment of NAFLD/NASH and T2D.