Type II diabetes mellitus is characterized by """"""""insulin resistance,"""""""" a condition where normal concentrations of serum insulin fail to adequately clear blood glucose. Although type II diabetes is a heterogeneous disease caused by several different biochemical defects, it is already clear that a common cause is failure of insulin to enhance glucose uptake, primarily into muscle. The mechanism by which insulin enhances glucose uptake into its main target tissues, muscle and fat, is to cause a movement or recruitment of tissue-specific glucose transporter, Glut4, from an intracellular storage pool to the cell surface, where it can function. It is this movement that is comprised in many type II diabetics, and therefore, it is the long-term goal of this work to determine the mechanistic details of the insulin-dependent translocation process. The intracellular storage pool of Glut4 consists of two or more vesicular compartments containing a number of additional cargo protein besides Glut4 itself. The first specific aim is to determine the composition of the major Glut4 containing compartments, endosomes and insulin-responsive vesicles (IRVs). These will be separated from one another by physical and immunological procedures, and the biochemical features that determine their uniqueness, vis a vis one another, will be identified. The second specific aim will address the nature of the regulation of IRV transit to the cell movement in the absence of insulin. Thus, the cytoplasmic portion of an aminopeptidase that completely co-localizes with Glut4 will be used as """"""""bait"""""""" in the yeast 2-hybrid systems, and in biochemical interaction assays, to identify cytosolic and/or cytoskeletal proteins that interact with Glut4 vesicles. The third and last specific aim will exploit the development of insulin-responsive vesicular trafficking in cultured fat murine cells as they differentiate from fibroblast into adipocytes. Immunological and genetic techniques will be used to compare genes expressed just before and just after the onset of insulin-sensitive vesicular traffic, e.g., those mediating the formation of the IRVs. The investigators expect these aims will identify novel proteins involved in aspects of insulin- regulated, and these may provide opportune for therapeutic intervention in type II ( and type I) diabetes mellitus.
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