Insulin resistance is a major factor in the pathogenesis of type 2 diabetes mellitus and recent studies in humans and rodent models have strongly implicated a causative role for intracellular lipid metabolites in the pathogenesis of insulin resistance in liver and muscle. Furthermore recent in vivo nuclear magnetic resonance spectroscopy (NMR) studies by our group in humans have suggested that inherited defects in mitochondrial function in young lean insulin resistant offspring of parents with type 2 diabetes mellitus as well as acquired defects in lean healthy elderly subjects may be responsible for increased intracellular lipid accumulation and insulin resistance in these individuals. This grant will build on these findings and further explore the role of mitochondrial dysfunction and dysregulated intracellular fatty acid metabolism in causing insulin resistance in unique transgenic and knockout mouse models as well as in awake rats using a novel antisense oligonucleotide approach. The specific aims that will be addressed in this proposal are: 1) To examine the role of muscle specific PGC1a and PGC1-3 over and under expression on insulin-stimulated rates of muscle glucose metabolism and muscle mitochondrial function, 2) To examine the impact of long chain CoA dehydrogenase deficiency in the pathogenesis of liver and muscle insulin resistance, 3) To examine the role of mitochondrial oxidative stress and mitochondrial DNA damage on mitochondrial function and insulin action, 4) To examine the individual roles of diacylglycerol acyl transferase 1 and diacylglycerol acyl transferase 2 on fat-induced hepatic insulin resistance, 5) To examine the role of protein kinase Ce in the pathogenesis of hepatic insulin resistance. All of these aims will be addressed in vivo using state-of-the-art methods including 31P, 13C and 1H NMR spectroscopy, liquid chromatography-tandem mass spectrometry in combination with radioactive isotopic techniques to obtain an integrated picture of whole-body glucose and energy metabolism as well as organ specific rates of insulin-stimulated glucose metabolism and mitochondrial function. It is anticipated that the results from these studies will provide important new insights into the role of mitochondrial dysfunction and alterations in intracellular fat oxidation in the pathogenesis of insulin resistance as well as identify potential novel targets for the prevention and treatment of type 2 diabetes mellitus.