INSULIN INDUCTION OF GLUCOSE TRANSPORTER TRANSLOCATION

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The applicant proposes a program of research and study to prepare him for a career in the investigation of endocrine diseases. The phase 1 research will be conducted in the laboratory of Dr. Harvey F. Lodish at Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology. The applicant will study the mechanism by which insulin causes fat and muscle cells to take up and utilize glucose. This topic has broad implications for the pathophysiology of Type II Diabetes Mellitus, as well as for neuroendocrine hormone action, vasopressin regulation of renal water reabsorption, and control of cellular metabolism. In particular, the applicant is interested in elucidating the mechanism by which binding of insulin to its receptor induces fusion of vesicles containing the GLUT4 glucose transporter with the plasma membrane. Because of the similarities between vesicular trafficking in insulin-responsive tissues, nerve synapses, and yeast, the problem is approachable on several levels. The applicant will work with cultured 3T3-L1 adipocytes during phase I, and plans to characterize homologues of proteins known to be important for vesicle transport in other cell types, as well as novel genes that are strongly induced during adipocyte differentiation. Such genes include the small GTP-binding proteins Rab3A, Rab3D, Rab4 and Rab5, homologues of synaptophysin and synaptobrevin (VAMP), an isoform of syntaxin, and caveolin. The applicant will establish an assay for translocation of GLUT4 to the plasma membrane, and will construct a vector to drive adipocyte-specific gene expression in both transient transfections and permanent cell lines. These tools will allow him to overexpress various mutant genes or antisense RNAs, and to assess the effect of these interventions on insulin-stimulated glucose uptake. The applicant will also be able to analyze insulin-stimulated secretion of several proteins, including adipsin and a likely homologue of a C1q complement subunit protein. In a longer range project, the applicant proposes to use the tools he will have developed to search for novel genes important for insulin-triggered glucose uptake.