More than 40% of HIV-infected patients on highly active antiretroviral therapy (HAART) experience fat redistribution (lipodystrophy), a syndrome associated with insulin resistance primarily affecting insulin-stimulated nonoxidative glucose metabolism (NOGM(ins)). Skeletal muscle biopsies, obtained from 18 lipodystrophic nondiabetic patients (LIPO) and 18 nondiabetic patients without lipodystrophy (NONLIPO) before and during hyperinsulinemic (40 mU.m(-2).min(-1))-euglycemic clamps, were analyzed for insulin signaling effectors. All patients were on HAART. Both LIPO and NONLIPO patients were normoglycemic (4.9 +/- 0.1 and 4.8 +/- 0.1 mmol/l, respectively); however, NOGM(ins) was reduced by 49% in LIPO patients (P <0.001). NOGM(ins) correlated positively with insulin-stimulated glycogen synthase activity (I-form, P <0.001, n = 36). Glycogen synthase activity (I-form) correlated inversely with phosphorylation of glycogen synthase sites 2+2a (P <0.001, n = 36) and sites 3a+b (P <0.001, n = 36) during clamp. Incremental glycogen synthase-kinase-3alpha and -3beta phosphorylation was attenuated in LIPO patients (Ps <0.05). Insulin-stimulated Akt Ser473 and Akt Thr308 phosphorylation was decreased in LIPO patients (P <0.05), whereas insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity increased significantly (P <0.001) and similarly (NS) in both groups during clamp. Thus, low glycogen synthase activity explained impaired NOGM(ins) in HIV lipodystrophy, and insulin signaling defects were downstream of PI 3-kinase at the level of Akt. These results suggest mechanisms for the insulin resistance greatly enhancing the risk of type 2 diabetes in HIV lipodystrophy.