 The molecular nature of insulin resistance in human muscle is still incompletely defined. Our data indicate that acetylation of mitochondrial proteins in humans is regulated by muscle contraction and is dysregulated in insulin resistance. Poor function of mitochondria in skeletal muscle is a hallmark of insulin resistance in skeletal muscle. We propose to use a combination of clinical research and mass spectrometry techniques to determine how the cytosolic and mitochondrial protein acetylation is regulated by muscle contraction in insulin sensitive and resistant human volunteers. We will test the hypothesis that mitochondrial protein acetylation is decreased to a greater degree following a bout of exercise in insulin sensitive than in insulin resistant human muscle. Using these techniques we also propose to determine how acetylation of mitochondrial adenine nucleotide translocase ANT1 at lysines 10, 23, and 92 regulates ANT1 structure and function. Finally, we propose 4 to use a combination of molecular modeling and in vitro assays together with the approach developed in Aim 3 to characterize the role of acetylation in other mitochondrial proteins. Protein targets for this aim will be prioritized based on the potential role of the protein in insulin resistance or mitochondrial function as well as dysregulation of its acetylation state in insulin resistant muscle. 