Abstract The regulation of glucose homeostasis is a complex process, which is disrupted in disease states such as type 2 diabetes. Insulin is the primary hormone that regulates glucose homeostasis. Insulin stimulates glucose uptake in muscle and fat by mobilizing intracellular vesicles containing GLUT4 glucose transporters, which fuse and insert GLUT4 at the cell surface. Impairment of this process results in insulin resistance and contributes to the development of diabetes. Therefore, to understand the pathogenesis of metabolic disease, it is necessary to understand the molecular mechanisms that control GLUT4 trafficking, and to understand how this trafficking is modulated by insulin and disrupted in insulin resistance. Previous work identified the TUG protein as a major regulator of GLUT4 trafficking and glucose uptake in muscle and fat cells. The data support a model in which TUG mediates the intracellular retention of GLUT4 in specific vesicles within unstimulated cells. Insulin triggers TUG endoproteolytic cleavage to mobilize these vesicles to the cell surface. TUG cleavage coordinates glucose uptake with other physiologic effects, resulting from the action of proteins that co-traffic with GLUT4, as well as from action of the TUG C-terminal product to modulate gene expression. In insulin resistant individuals, impairment of this mechanism may contribute to the metabolic syndrome and obesity. Yet, it remains unknown how this mechanism is affected in insulin resistance, whether attenuated TUG cleavage causes insulin resistance in muscle, or whether TUG cleavage participates in exercise-stimulated glucose uptake. As well, the molecular mechanisms by which intact TUG retains GLUT4 in an insulin- responsive pool of vesicles are not understood. To address these questions, two Aims will be undertaken. Aim 1 will characterize insulin resistance and glucose homeostasis in mice with muscle-specific disruption of TUG or of TUG endoproteolytic cleavage, and will study the potential role of this pathway in exercise-induced glucose uptake. Aim 2 will study molecular mechanisms by which TUG traps GLUT4-containing vesicles in an insulin-responsive pool, and by which this process may be altered in insulin-resistant states. We anticipate that, together, these studies will result in an improved understanding of glucose metabolism and energy expenditure, with implications for the prevention and treatment of diabetes and the metabolic syndrome.