The GLUT4 facilitative glucose transporter is the major insulin- responsive isoform and is primarily expressed in muscle and adipose tissue, the two tissue types responsible for the maintenance of normal glucose homeostasis in the post-prandial state. The regulation of the GLUT4 glucose transporter occurs at multiple levels, including transcription, translation, and intracellular vesicular trafficking. Alterations in various aspects of these processes directly lead to peripheral tissue insulin resistance diabetes results from an inability of insulin to stimulate glucose uptake and GLUT4 translocation from intracellular storage sites to the cell surface membrane, despite the presence of normal cells of GLUT4 protein. Based upon the central role of GLUT4 vesicle trafficking in the pathophysiology associated with insulin resistance, we have proposed a series of studies to specifically identify and characterize the functional role of a novel syntaxin 4 binding protein, Synip which is directly involved in the intracellular trafficking of the GLUT4 containing vesicles. This will be accomplished by determining the interaction of Synip with the other established syntaxin 4 binding proteins (Munc18c, SNAP23 and VAMP2) in regulation insulin- stimulated GLUT4 translocation. Since our preliminary data demonstrates that insulin rapidly induces the dissociation of the Synip-syntaxin 4 complex, we will examine the signal transduction pathways and potential allosteric modifications of Synip responsible for its reduction in syntaxin 4 binding affinity. To identify the cellular trafficking patterns of GLUT4 vesicles and regulation by Synip, we will take advantage of GLUT4- enhanced Green Fluorescent Protein (eGFP) fusions that can be used to observe the movement of GLUT4 containing vesicles in real time. Using this type of approach we have been able to distinguish between GLUT4 vesicle docking versus GLUT4 vesicle fusion with the plasma membrane. These events will be biochemically defined by performing a detailed analysis of plasma membrane GLUT4 association using intact cell immunofluorescence, immunoelectron microscopy, subcellular fractionation, plasma membrane sheet isolation and glucose transport activity. We will then use this system to distinguish between GLUT4 vesicle docking versus fusion and to assess the functional role of Synip in regulating these processes. In this manner, we hope to establish several of the key biochemical and cell biological mechanisms regulating the insulin-stimulated trafficking/docking/fusion of GLUT4 containing vesicles.
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