Our research in this project has focused on defining the changes in expression of nuclear encoded mitochondrial genes that predict changes in insulin sensitivity in skeletal muscle, with the goal of defining the molecular mechanisms underlying the connection between mitochondrial dysfunction and insulin resistance in skeletal muscle. We have found that lipid oversupply produced by a lipid infusion decreases mRNA expression of PGC-11, NRF-1, and nuclear encoded mitochondrial genes and induces insulin resistance. This was consistent with our original hypothesis that decreasing PGC-11 expression would result in lower expression of nuclear encoded mitochondrial genes and lead to mitochondrial dysfunction associated with insulin resistance. Additional studies showed a complex relationship between mitochondrial dysfunction, insulin sensitivity, and gene expression. Other data suggested that insulin resistant individuals could be exercise resistant. We also have shown that ATP synthase 2 is phosphorylated in vivo, and this may be regulated by insulin and altered in insulin resistance. Relatively little is known about changes in mitochondrial protein abundance or post-translational modification in insulin resistance, or their response to exercise or lipid oversupply. The overall goal of this proposal is to understand how underlying changes in regulation of mitochondrial respiration, protein abundance and phosphorylation contribute to mitochondrial dysfunction. To accomplish this goal, we will use glucose clamps, muscle biopsies, novel proteomics techniques for quantification of protein abundance changes, and in vitro mitochondrial respiration measurements using an energy clamp. We propose: 1. To determine whether mitochondrial isolated from insulin resistant human muscle have decreased respiration during conditions of increased energy demand simulated by a creatine kinase "energy clamp". 2. To determine how insulin resistance alters the pattern of abundance of mitochondrial proteins. 3. To determine how insulin resistance alters phosphorylation of proteins in the ETC. We will use immunoprecipitation and mass spectrometry analysis to quantify site-specific changes in phosphorylation of ETC proteins. 4. To determine whether insulin resistance is accompanied by "exercise resistance" with regard to mitochondrial biogenesis. 5. To determine whether experimental lipid oversupply decreases mitochondrial respiratory function. PUBLIC HEALTH RELEVANCE: Obesity and type 2 diabetes mellitus are increasing in epidemic proportion. Their complications account for up to forty percent of health care costs in the U.S. Despite this, the mechanisms responsible for their development remain unclear. This project will help to clarify these mechanisms on a molecular level. Given the clear evidence of a link to mitochondrial dysfunction in the etiology of obesity and insulin resistance, the studies are of high clinical significance and may provide useful new insights into the control of mitochondrial bioenergetics