A fundamental knowledge of the molecular events which mediate insulin's intracellular signaling pathways will lead to a better understanding of the mechanisms of insulin action and the disease states in which insulin action is abnormal. Thus, the overall goals of this research proposal are to elucidate the molecular and biochemical events which occur after insulin binds to its cell surface receptor. 1) We have recently developed a single cell microinjection technique in 3T3-L1 cells which allows us to measure GLUT4 translocation in single cells by immunofluorescent microscopy of GLUT4 using anti-GLUT4 antibodies. We propose to utilize this technique to determine the signaling events that mediate GLUT4 translocation. These experiments will utilize native 3T3-L1 cells as well as 3T3-L1 cells expressing epitope tagged GLUT4 proteins. Other strategies to probe signaling mechanisms for GLUT4 translocation will include viral mediated gene transfer, as well as coinfection techniques employing epitope tagged GLUT4 cDNA. 2) We propose a series of new hypotheses to further explore the detailed post-receptor molecular and cellular mechanisms of insulin's mitogenic signaling pathway. These studies will utilize microinjection of a variety of stimulatory and inhibitory reagents involving antibodies, dominant interfering recombinant proteins, phosphonopeptides, promoter/reporter plasmid constructs, and ribozymes. 3) An additional method to introduce recombinant proteins into cells is retroviral gene transfer and several hypotheses will be tested using transient biologic assays of cells infected with cDNAs carried by the retroviral system. 4) Over the past couple of years, the PIs laboratory has provided evidence for additional components of the insulin signaling system, and has achieved preliminary purification of a potential new signaling protein. We will use the two hybrid yeast based capture system, as well as other purification and cloning strategies,to identify new signaling molecules directly related to insulin and growth factor action. In summary, the experiments proposed in this application will apply new molecular and cell biologic techniques to provide mechanistic information and test novel hypotheses, which will further our understanding of the cellular actions of insulin.
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