INSULIN STIMULATION OF GLUCOSE TRANSPORT

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Our long-term goal is to understand how insulin causes an increase in glucose uptake by fat and muscle cells-specifically, to identify, characterize, and clone proteins that transduce the signal from the insulin-activated receptor protein-tyrosine kinase to the fusion of intracellular vesicles containing the GLUT-4 glucose transporter with the plasma membrane. We have cloned a new low-mw GTP binding protein, Rab3D, that is highly expressed in insulin-responsive tissues, and has many of the properties expected of a protein that regulates exocytosis of GLUT-4-rich vesicles. Indeed, we have generated a presumed dominant-negative Rab3D mutation, N135I, that cannot bind GTP; expression of this mutant but not the wild-type Rab3D in differentiated 3T3-L1 adipocytes inhibits the ability of insulin to increase glucose transport. Recently, we have cloned homologs of the synaptic vesicle proteins synaptophysin and synaptobrevin (VAMP) that, like Rab3D, are abundantly expressed in insulin responsive tissues and are induced during adipogenesis in vitro. We also showed that Rab3A, whose expression was thought confined to nerve and neuroendocrine cells, is also abundantly expressed in insulin responsive tissues and is induced during adipogenesis. We hypothesize that activation of the insulin receptor induces an exchange of GTP for GDP on Rab3A or 3D, much in the way that ras is activated by other receptor protein-tyrosine kinases, and that a Rab3GTP directly induces membrane fusion. Our specific goals are: (1) Elucidating the roles of Rab3D and Rab3A in exocytosis of GLUT4- and GLUT1- containing vesicles and in secretion of adipsin. This includes generation of other dominant-negative mutations in Rab3A and 3D and examining the effects on insulin-stimulation of glucose transport and translocation of GLUT1 and GLUT4 to the cell surface in transfected 3T3-L1 adipocytes; and examining the effects of GTPgammaS and synthetic peptides corresponding to the presumed effector domain of Rab3 on exocytosis of GLUT- 4 in two permeabilized adipocyte systems and also in permeabilized adipocytes expressing mutant Rab3 proteins; (2) Determining the subcellular localization of Rab3D, Rab3A, and the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated adipocytes; (3) Determining the roles of the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated glucose transport by generating clonal lies of 3T3-l1 adipocytes that express specific mutant forms of these proteins or that overexpress the wild-type protein, and examining them for insulin-stimulation of translocation of GLUT4 and GLUT1 to the cell surface and secretion of adipsin; and (4) Cloning Rab3D or RabA GDP-GTP exchange protein or GAP proteins that are induced during adipogenesis, using expression cloning, hybridization, or PCR and a suitable full-length subtractive, adipocyte-specific expression cDNA library. We will test whether these proteins are intermediates in the signal transduction pathway from the insulin receptor to exocytosis of glucose transporters.