Regulation of glucose transporter Glut4 by insulin

Insulin initiates a signaling cascade that results in the translocation of the insulin-regulated glucose transporter Glut4 from a unique intracellular storage compartment to the plasma membrane, thereby dramatically increasing the cellular capacity for glucose uptake. A critical rate-limiting step for whole body glucose disposal is at the level of glucose transport, and thus, defects in insulin-stimulated glucose transport mediated by Glut4 contribute to the pathogenesis of insulin resistance and type 2 diabetes. This dissertation attempts to address fundamental issues regarding the physiologic role and regulation of Glut4 by converging on this molecule from two vantage points.;The first line of investigation stems from clinical findings in patients infected with the human immunodeficiency virus (HIV). The use of HIV protease inhibitors (PIs) in antiretroviral therapy is associated with serious metabolic side effects that include central adiposity, hyperlipidemia, and insulin resistance. We have demonstrated that PIs unexpectedly act as relatively selective inhibitors of the transport function of Glut4. The inhibition of Glut4 by the PI indinavir is rapid and reversible. Kinetic experiments measuring zero-trans sugar uptake suggest a non-competitive mode of inhibition. Indinavir infusion in rats can acutely and reversibly induce a state of insulin resistance. We hypothesize that direct inhibition of Glut4 by PIs can contribute to the insulin resistance observed in patients receiving this class of drugs.;The second experimental approach is an effort directed towards developing a cell-free assay that reconstitutes key aspects of the insulin signaling pathway leading to Glut4 translocation. By recombining subcellular fractions of 3T3-L1 adipocytes in a test tube, we have been successful in reconstituting early events in the insulin signaling pathway up to and including the phosphorylation and activation of the key effector kinase Akt. Experimental access to cellular components afforded by this system was exploited to examine the regulation of Akt by phosphorylation. A major conclusion drawn from these studies is that the kinase activity that phosphorylates Akt on serine 473 appears to be distinct from phosphoinositide-dependent kinase 1 (PDK1) which phosphorylates Akt on threonine 308. The reconstitution of insulin-dependent Glut4 trafficking as well as other events downstream of Akt remains a long term goal.