Twenty-five years ago we showed that skeletal muscle in type 2 diabetes preferentially oxidizes carbohydrate over fat and exhibits what we termed “metabolic inflexibility”. This results in accumulation of fat in insulin sensitive tissues and leads to insulin resistance. Genetic activation of the pyruvate oxidation pathway through deletion of pyruvate dehydrogenase (PDH) kinase showed that an increase in glucose oxidation is sufficient to induce insulin resistance by this mechanism. Although many studies have described how the consequences of lipid accumulation lead to insulin resistance, little is known of the mechanisms causing metabolic inflexibility to begin with. In the past project period, we developed evidence for two potential mechanisms. The first of these derives from our findings that acetylation on lysine 23 of the mitochondrial solute carrier adenine nucleotide translocase 1 (ANT1) lowers the affinity of the protein for ADP. This is associated with a higher KmADP for respiration and ATP synthesis. Modeling predicts this leads to higher free ADP and AMP concentrations. Higher [ADP]f would enhance glycolytic rates, raise [pyruvate], and activate PDH via inactivation of PDH kinase, leading to higher rates of glucose oxidation. Second, we used a proteomics screen of livers of high fat fed mice and discovered an uncharacterized mitochondrial protein, KIAA0564 (VWA8), that dampens fat oxidation, is elevated in skeletal muscle of type 2 diabetic or morbidly obese patients undergoing bariatric surgery, and has genetic variants that are associated with obesity, diabetes, and abnormal plasma lipid levels. Given this, we believe it is timely to return to the question of what mechanisms drive elevated carbohydrate oxidation and metabolic inflexibility in muscle of patients with type 2 diabetes and morbid obesity. The overall goal of this proposal is to determine how acetylation of ANT1 at lysine 23 and expression of the novel mitochondrial protein VWA8 influence fuel selection in patients with Type 2 diabetes mellitus. We propose 1. To determine the mechanisms responsible for metabolic inflexibility in skeletal muscle of patients with type 2 diabetes mellitus, 2. To determine the mechanisms responsible for impaired control of respiration and higher resting carbohydrate oxidation in skeletal muscle of patients with type 2 diabetes mellitus, and 3. To further characterize the mechanisms by which VWA8 regulates fuel selection. !