Major diseases, obesity, the metabolic syndrome, and diabetes, are associated with impaired glucose homeo- stasis. Key to the maintenance of glucose homeostasis is the control of the subcellular distribution of the glucose transporter GLUT4 in muscle and fat cells. Under fasting conditions GLUT4 localizes mostly to intra- cellular vesicles (GLUT4 vesicles) and few GLUT4 are at the cell surface. This limits glucose uptake into muscle and fat cells and assures adequate glucose supply to the brain. Insulin stimulates, within minutes, movement, docking and fusion of GLUT4 vesicles with the plasma membrane (GLUT4 translocation). The increase of GLUT4 at the cell surface facilitates glucose disposal into muscle and fat cells, thereby normalizing circulating glucose levels after a meal. GLUT4 translocation is also crucial to increased glucose uptake in response to exercise. The molecular mechanism by which GLUT4 is retained under fasting conditions and by which insulin and exercise release GLUT4 to the cell surface in vivo in primary fat and muscle cells is unknown. Studies in cultured fat and muscle cells attribute a role in GLUT4 retention and release to the two Rab GTPase activating proteins (Rab GAPs), AS160 and Tbc1d1. Recent results with AS160 and Tbc1d1 knockout mice suggest that glucose uptake is regulated by AS160 in adipocytes, and by AS160 and Tbc1d1 in skeletal muscles. Our hypothesis is that AS160 and Tbc1d1 each alone control GLUT4 intracellular retention and release to the plasma membrane, and thus glucose uptake, in cells where only one of the two Rab GAPs is predominantly expressed (adipocytes and specific skeletal muscles). In cells in which both Rab GAPs are found (most skeletal muscles), we propose that they perform in a collaborative fashion or respond to different stimuli to regulate GLUT4 retention and release, and thus glucose uptake. The cell type- and signal-specific roles of AS160 and Tbc1d1 then differentially regulate whole body glucose homeostasis. Using single AS160 and Tbc1d1 knockout mice, and double AS160 and Tbc1d1 knockout mice, and adipocytes and skeletal muscles isolated from these mice, we will first determine which of the two Rab GAPs regulate glucose uptake and GLUT4 subcellular distribution in adipocytes and different skeletal muscles under fasting conditions and in response to insulin and exercise. We will then investigate whether the two Rab GAPs control the same or different steps in GLUT4 retention and release, and define mechanisms by which they perform their functions. For these studies GLUT4 trafficking will be monitored in live cells by total internal reflection fluorescence and confocal microscopy. Finally, we will establish the roles of AS160 and Tbc1d1 in whole body glucose homeostasis under fasting conditions and in response to insulin and exercise. Metabolic analysis of the mice will include measurements of insulin sensitivity and hyperinsulinemic-euglycemic clamps. This research will elucidate fundamental mechanisms that contribute to the maintenance of glucose homeostasis and provide crucial information towards validation of AS160 and Tbc1d1 as drug targets to improve glucose homeostasis.