Insulin stimulates glucose uptake in its target tissues (fat, heart, and skeletal muscle) mainly or entirely via translocation of glucose transporting activity from an intracellular pool to the cell surface. The protein responsible for the insulin-sensitive glucose transport, GLUT4, has been identified and cloned. The mechanism of the translocation process remains unknown. It is crucially important to find an answer to this question, because it will explain the fundamental principles of insulin action, and, in addition, will shed light on the molecular bases of insulin resistance in diabetes mellitus. To approach this problem, we started with characterization of the intracellular GLUT4-containing membrane compartment (vesicles) in basal and in insulin-treated rat adipocytes. We have shown that, besides GLUT4, this compartment incorporates the IGF-II/Man-6-P receptor, the transferrin receptor, the novel aminopeptidase gpl60 and a number of minor components. Thus, several proteins which (may) participate in cellular nutrition and metabolism are co-localized in the same intracellular membrane vesicles. All these proteins recycle between GLUT4-vesicles and their functional site, the plasma membrane, in an insulin dependent fashion. Their hormone-sensitive recruitment to the cell surface represents the major mechanism hat regulates intracellular uptake of glucose, iron, and other metabolites. The question arises of whether insulin stimulates translocation of the whole pre-existing compartment (""""""""GLUT4-vesicles""""""""), or different component proteins of GLUT4-vesicles recycle separately. In the last case, insulin is likely to cause formation of new transport vesicles which deliver their individual cargo to the cell surface via unidentified pathways. To discriminate between these two possibilities is the first major goal of the project. Besides the cell surface translocation of GLUT4 and co-localized proteins, insulin administration induces other major changes in adipocyte vesicular traffic. For example, it stimulates fluid-phase endocytosis, internalization of insulin receptors and, likely, lysosomal targeting of the endocytosed ligands. Thus, the second major goal is to provide a comparative study of different trafficking events in order to determine their common biochemical requirements as well as possible differences. Eventually, this work will provide us with a detailed picture of intracellular protein traffic in adipocytes.
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