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Postreceptoral Adipocyte Insulin Resistance Induced by Nelfinavir Is Caused by Insensitivity of PKB/Akt to Phosphatidylinositol-3,4,5-Trisphosphate

Departments of Clinical Biochemistry (I.K., A.M., L.M., R.B.-R., N.B., A.R.) and Chemical Engineering (I.K.), The International Center for Health and Nutrition (A.R.), The Leslie and Susan Gonda (Goldschmied) Center for Diabetes Research and Education (N.B., A.R.), Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84103, Israel; Department of Pharmacological Sciences (R.T.W., J.C.H.), Stony Brook University, Stony Brook, New York 11794; and Department of Medicine (J.E.P.), Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Assaf Rudich, M.D., Ph.D., Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel. E-mail: rudich{at}bgu.ac.il.

Adipocyte insulin resistance can be caused by proximal insulin signaling defects but also from postreceptor mechanisms, which in large are poorly characterized. Adipocytes exposed for 18 h to the HIV protease inhibitor nelfinavir manifest insulin resistance characterized by normal insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate proteins, preserved in vitro phosphatidylinositol 3-kinase (PI 3-kinase) assay activity but impaired activation of PKB/Akt and stimulation of glucose uptake. Here we aimed to assess whether impaired PKB/Akt activation is indeed rate limiting for insulin signaling propagation in response to nelfinavir and the mechanism for defective PKB/Akt activation. Nelfinavir treatment of 3T3-L1 adipocytes impaired the insulin-stimulated translocation and membrane fusion of myc-glucose transporter (GLUT)-4-green fluorescent protein (GFP) reporter. Phosphorylation of PKB/Akt substrates including glycogen synthase kinase-3 and AS160 decreased in response to nelfinavir, and this remained true, even in cells with forced generation of phosphatidylinositol-3,4,5-trisphohphate (PIP₃) by a membrane-targeted active PI 3-kinase, confirming that impaired PKB/Akt activation was rate limiting for insulin signal propagation. Cells expressing a GFP-tagged pleckstrin homology domain of general receptors for phosphoinositides 1, which binds PIP₃, revealed intact PIP₃-mediated plasma membrane translocation of this reporter in nelfinavir-treated cells. However, expression of a membrane-targeted catalytic subunit of PI 3-kinase failed to induce myc-GLUT4-GFP translocation in the absence of insulin, as it did in control cells. Conversely, a membrane-targeted and constitutively active PKB/Akt mutant was normally phosphorylated on S473 and T308, confirming intact PKB/Akt kinases activity, and induced myc-GLUT4-GFP translocation. Collectively, nelfinavir uncovers a postreceptor mechanism for insulin resistance, caused by interference with the sensing of PIP₃ by PKB/Akt, leading to impaired GLUT4 translocation and membrane fusion.