Nef inhibits glucose uptake in adipocytes and contributes to insulin resistance in human immunodeficiency virus type I infection.

Human immunodeficiency virus (HIV) infection is associated with insulin resistance. HIV type 1 Nef downregulates cell surface protein expression, alters signal transduction, and interacts with the cytoskeleton and proteins involved in actin polymerization. These functions are required for glucose uptake by insulin-stimulated adipocytes. We sought to determine whether Nef alters adipocyte glucose homeostasis. Using radiolabeled glucose, we found that adipocytes exposed to recombinant Nef took in 42% less glucose after insulin stimulation than did control cells. This reduction resulted from a Nef-dependent inhibition of glucose transporter 4 (GLUT4) trafficking, as assessed by means of immunofluorescence microscopy. Immunoblot analysis revealed a decrease in phosphorylation of signal transducing proteins after Nef treatment, and fluorescence microscopy showed a dramatic alteration in cortical actin organization. We conclude that Nef interferes with insulin-stimulated processes in adipocytes. We have identified HIV Nef, which is detectable and antigenic in serum samples from HIV-infected people, as a novel contributor to the development of insulin resistance.

Postreceptoral adipocyte insulin resistance induced by nelfinavir is caused by insensitivity of PKB/Akt to phosphatidylinositol-3,4,5-trisphosphate.

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.

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors.

Insulin recruits two transmembrane proteins, GLUT4 and IRAP, to the plasma membrane of muscle cells and adipocytes. The subcellular trafficking and localization of GLUT4, and to a lesser extent IRAP, have been intensely studied, yet the molecular mechanisms responsible for their insulin-responsive compartmentalization remain unknown. Herein we have investigated the endocytosis and recycling of IRAP from the cell surface back to the insulin-responsive compartment (IRC). Our results show that a key dileucine motif at position 76,77 (LL76,77), although required for the initial biosynthetic entry of IRAP into the IRC, is dispensable for entry into the IRC via the endosomal system. Indeed, we found that an AA76,77 mutant of IRAP is fully capable of undergoing endocytosis and is correctly routed back to the IRC. To verify that the AA76,77 mutant enters the bona fide IRC, we show that the internalized IRAP-AA76,77 construct is sequestered in an IRC that is insensitive to brefeldin A yet sensitive to a dominant-interfering mutant of AS160 (AS160-4P). In addition, we show that the GGA clathrin adaptors are not required for the re-entry of IRAP from the cell surface back into the IRC, whereas the Q-SNARE syntaxin 6 is required for this process.

Dual regulation of Rho and Rac by p120 catenin controls adipocyte plasma membrane trafficking.

During 3T3L1 adipogenesis there is a marked reduction in beta-catenin and N-cadherin expression with a relatively small decrease in p120 catenin protein levels. Cell fractionation demonstrated a predominant decrease in the particulate (membrane-bound) pool of p120 catenin with little effect on the soluble pool, resulting in a large redistribution from the plasma membrane to the cytosol. Reexpression of p120 catenin inhibited constitutive (transferrin receptor) and regulated mannose 6-phosphate receptor and GLUT4 trafficking to the plasma membrane. The inhibition of membrane trafficking was specific for p120 catenin function as this could be rescued by co-expression of N-cadherin. Moreover, overexpression of a p120 catenin deletion mutant (p120delta622-628) or splice variant (p120-4A), neither of which could regulate Rho or Rac activity, showed no significant effect. The inhibition of GLUT4 translocation was also observed upon the simultaneous expression of a constitutively active Rac mutant (Rac1/Val12) in combination with a dominant-interfering Rho mutant (RhoA/Asn19). This was recapitulated by expression of the Rho ADP-ribosylation factor (C3ADP) in combination with constitutively active Rac1/Val12. Moreover, siRNA-mediated knockdown of p120 catenin resulted in increased basal state accumulation of GLUT4 at the plasma membrane. Together, these data demonstrate that p120 catenin plays an important role in maintaining the basal tone of membrane protein trafficking in adipocytes through the dual regulation of Rho and Rac function and accounts for reports implicating Rho or Rac in the control of GLUT4 translocation.