title: Postreceptoral adipocyte insulin resistance induced by nelfinavir is caused by insensitivity of PKB/Akt to phosphatidylinositol-3,4,5-trisphosphate.
abstract: 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 (PIP(3)) 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 PIP(3), revealed intact PIP(3)-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 PIP(3) by PKB/Akt, leading to impaired GLUT4 translocation and membrane fusion.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva, Israel.
authors.forename: Ilana
authors.initials: I
authors.lastname: Kachko
authors.forename: Adva
authors.initials: A
authors.lastname: Maissel
authors.forename: Livnat
authors.initials: L
authors.lastname: Mazor
authors.forename: Ronit
authors.initials: R
authors.lastname: Ben-Romano
authors.forename: Robert T
authors.initials: RT
authors.lastname: Watson
authors.forename: June C
authors.initials: JC
authors.lastname: Hou
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Pessin
authors.forename: Nava
authors.initials: N
authors.lastname: Bashan
authors.forename: Assaf
authors.initials: A
authors.lastname: Rudich
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004195:Disease Models, Animal; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D007333:Insulin Resistance / Q000502:physiology*; D051379:Mice; D019888:Nelfinavir / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects* / Q000378:metabolism; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D051275:Glucose Transporter Type 4; D017320:HIV Protease Inhibitors; D007328:Insulin; D018129:Phosphatidylinositol Phosphates; C495693:Slc2a4 protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D019888:Nelfinavir; D005947:Glucose