The overall objective of these proposed studies is to provide insight into the detailed molecular mechanisms underlying glucose transport regulation by insulin. Insulin-sensitive cells such as muscle and fat express one predominant glucose transporter isoform, GLUT4, but also contain a HepG2/brain-type (GLUT1) and perhaps others. It is established that a major action of insulin is to increase the levels of glucose transporters as well as certain receptors in the cell surface membrane at the expense of those in intracellular membranes. During the previous grant period, we have obtained substantial data indicating that the intrinsic activity of GLUT1, and perhaps GLUT4 can also be markedly regulated. In this proposal, we seek to investigate these two potential modes by which transporters are regulated in 3T3-L1 adipocytes which contain both GLUT4 and GLUT1. Our experiments shall address three key questions related to these mechanisms: 1. Does GLUT4 translocation to the cell surface solely account for the effect of insulin on glucose uptake? We have shown that expression of the human GLUT1 protein in transfected mouse 3T3-L1 cells leads to increased basal but no changes in insulin-stimulated glucose transport. We shall perform similar transfections with rat GLUT4 cDNA and a number of chimeric GLUT1/GLUT4 constructs we have engineered to directly determine the contribution of GLUT4 (insulin) using [2-3H]BMPA and a novel anti-exofacial peptide antibody we have produced that recognizes GLUT4 on the surface of intact cells. 2. Do the GLUT1 and GLUT4 transporters continuously recycle between intracellular and cell surface membranes? During the previous grant period we demonstrated that both the endocytic rate and exocytotic rate of transferrin receptor recycling are regulated processes. We propose to identify the step or steps in the trafficking pathway of the transporter proteins that are regulated by insulin. We shall study the cell surface residency times, coated vesicle localization, endocytic rates, and exocytotic rates for these transporters. These experiments will be conducted using the anti-exofacial GLUT4 antibody, [2-3H]BMPA, and metabolic labeling of transporters in pulse-chase studies, in conjunction with specific immunoprecipitations of GLUT1 and GLUT4 using protocols we have established. 3. How is the intrinsic activity of GLUT1 (and GLUT4?) regulated in intact rate fat and 3T3-L1 adipocytes? We have established that plasma membranes from 3T3-L1 adipocytes treated with protein synthesis inhibitors exhibit 7 fold elevations in glucose transport activity without a change in GLUT1 or GLUT4 levels. Reconstitution experiments will be performed to determine whether transporter inhibitory activity can be observed in extracts of control membranes. Affinity-columns will be prepared using large quantities of GLUT1 and GLUT4 obtained from baculovirus infected Sf9 cells that we have established. In these studies, we shall attempt to absorb and isolate transporter regulator proteins that suppress transport activity in control adipocytes.
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