Insulin resistance in skeletal muscle, a prodrome of type 2 diabetes mellitus, is associated with and a likely consequence of mitochondrial damage. Autophagy, a catabolic process that degrades aggregate proteins and damaged organelles, such as mitochondria, may be crucial in maintaining metabolic homeostasis. Atg6, a protein essential in initiating autophagy, interacts with and is inhibited by Bcl-2 family proteins that tether Atg6 to the mitochondrial membrane. We have recently utilized a multisystem approach to show: 1) overexpression of Vps30 (yeast homolog of Atg6) protects budding yeast cells against mitochondrial uncoupler CCCP-induced growth arrest; 2) both saturated fatty acid feeding and heterozygous Atg6 gene deletion lead to accumulation of damaged mitochondria in drosophila indirect flight muscles; and 3) Atg6+/- mice appear glucose intolerant compared to wild type littermates on high-fat diet. These findings strongly support conserved function of Atg6 in coping with mitochondrial damage and maintaining metabolic homeostasis ranging from yeast to mammals. My central hypothesis is that induction of autophagy through release of Atg6 from Bcl-2 plays a critical role in maintaining insulin sensitivity and mitochondrial health in skeletal muscle following high-fat diet in mice. To test this central hypothesis, I propose the following specific aims: Specific Aim 1. To ascertain whether Atg6 is sufficient and necessary to maintain a healthy mitochondrial population and prevent insulin resistance in skeletal muscle induced by high-fat diet in mice. Specific Aim 2. To determine if lipid overload-induced mitochondrial damage leads to release of Atg6 from mitochondrial Bcl-2 in skeletal muscle. Significance. These experiments will define the essential function and regulation of autophagy in maintaining a healthy mitochondrial population and therefore preserving skeletal muscle insulin sensitivity in response to mitochondrial damage; thereby, identifying potential new therapeutic targets to combat insulin resistance and diabetes. PUBLIC HEALTH RELEVANCE: Type 2 diabetes is a complex disease which originates with peripheral resistance to insulin, often the onset occurs at the skeletal muscle. This disease has been associated with the degeneration of mitochondria (the cell's powerplant), therefore targeting the removal of degenerated mitochondria by a process called autophagy, may ameliorate insulin resistance and type 2 diabetes. In these studies we will elucidate the role of mitochondrial stress-induced autophagy in the onset of insulin resistance.