Background:  Protease inhibitor (PI) therapy of HIV infection is linked to metabolic abnormalities, such as insulin resistance. But, patients not receiving PI also experience metabolic defects, suggesting a direct role for HIV itself. Adipocytes translocate glucose transporter 4 (Glut4) from intracellular storage sites to the cell membrane in response to insulin signaling, allowing glucose uptake and blood sugar homeostasis. HIV Nef disrupts proper protein trafficking along the secretory pathway, and Glut4 traverses this very pathway in response to insulin. We hypothesize that Nef disrupts Glut4 transport, inhibiting glucose uptake and contributing to insulin resistance in HIV-infected people.
Methods:  3T3L1 adipocytes were treated with 100 ng HIV or simian immunodeficiency virus (SIV) Nef and glucose uptake in response to 10 nM insulin was quantified by a ³H-glucose uptake assay. Cells were transfected with a Glut4-GFP or a Myc-Glut4-GFP construct and treated with 10 ng or 100 ng SIV Nef or 10 ng SIV Gag. Cells were then stimulated with 0 nM, 10 nM, or 100 nM of insulin and fixed. Myc-Glut4-GFP-transfected cells were stained with a Texas Red-conjugated Myc antibody. Cells were scored for Glut4 translocation and the degree of translocation was quantified by microscopy. Cell lysates were collected for immunoblot analysis of Akt phosphorylation to examine insulin signaling.

Results:  Glucose uptake by HIV Nef-treated cells was ~50% less than control cells in response to 10 nM insulin (4-fold increase of ³H-glucose uptake versus an 8-fold increase, respectively). Similarly, SIV Nef-treated cells took up ~30% less glucose than control cells. Glut4 translocation occurred in ~50% of control cells in response to 100 nM insulin, while only 18% of SIV Nef-treated cells were positive for Glut4 translocation (p = 0.001). No difference was observed between control and Gag-treated cells. There was a significant difference in fluorescence intensity between control and SIV Nef-treated cells stimulated with 10 nM of insulin (p ≤0.001). Insulin-stimulated Akt phosphorylation was equivalent between control and SIV Nef-treated cells.

Conclusions:  Nef decreases glucose uptake by insulin-stimulated adipocytes, and likely mediates this effect by inhibiting Glut4 translocation without altering the classical insulin signal transduction pathway. Hence, Nef may cause insulin resistance by inhibiting Glut4 trafficking to the plasma membrane, thereby decreasing glucose uptake into the cell and destabilizing glucose homeostasis.