| GLUT4 is an insulin-regulated glucose transporter. GLUT4 storage vesicles (GSVs) represent the principal insulin-responsive compartment in muscle and adipose tissues. GSVs contain the following major proteins: GLUT4, insulin-responsive aminopeptidase (IRAP), sortilin, and a recently discovered component, LDL receptor related protein 1 (LRP1). In this thesis, we have explored the role of Glut4, IRAP, sortilin, and sortilin-associated protein, acyl-CoA synthetase long chain 1 (ACSL1), in the biogenesis and insulin responsiveness of GSVs in 3T3-L1 cells.;We have found that GLUT4 stably expressed in undifferentiated preadipocytes is not localized in GSVs and has a low insulin responsiveness regardless of the levels of its expression. At the same time, sortilin stably expressed in pre-adipocytes is localized in insulin-responsive vesicles and is markedly translocated to the plasma membrane in response to insulin stimulation. In pre-adipocytes double transfected with sortilin and GLUT4 cDNAs, the transporter is localized in GSVs and demonstrates insulin responsiveness similar to that observed in differentiated adipocytes. Moreover, endogenous TRAP interacts with and is recruited to GSVs by Glut4 and sortilin. Thus, sortilin may contribute not only to the biogenesis but, also, to the acquisition of insulin responsiveness of GSVs.;In order to further explain the function of sortilin in this compartment, we have identified several sortilin-binding proteins. One of the proteins that can interact with sortilin is ACSL1. Depletion of ACSL1 from 3T3-L1 adipocytes decreases protein levels of IRAP and GLUT4 and reduces the amount of GSVs suggesting that ACSL1 is involved in the process of GSVs formation.;Finally, we have explored the role of phosphatidylinositol 4 kinase (PI4K) type IIalpha in vesicle biogenesis. According to the previous reports, this enzyme is localized in a population of ubiquitous transport vesicles and may thus be involved in the early steps of the "GLUT4 pathway" that precede formation of GSVs. Although knock down of PI4K type IIalpha does not affect formation of these transport vesicles, it is still possible that PI4K type IIalpha may generate phosphatidylinositol phosphates required for the biogenesis of GSVs. Based on our results, we propose a model of "self-assembly" of GSVs from the individual protein components in adipose cells. |
