Insulin signaling pathways potentially involved in regulation of skeletal muscle glycogen synthase were compared in dif- ferentiated human muscle cell cultures from nondiabetic and type 2 diabetic patients. Insulin stimulation of glycogen syn- thase activity as well as phosphorylation of MAPK, p70 S6 kinase, and protein kinase B (Akt) were blocked by the phos- phatidylinositol 3-kinase inhibitors wortmannin (50 nM) and LY294002 (10 M). In contrast to lean and obese nondiabetic subjects, where there were minimal effects (15-20% inhibi- tion), insulin stimulation of glycogen synthase in muscle cul- tures from diabetic subjects was greatly diminished (75%) by low concentrations of wortmannin (25 nM) or LY294002 (2 M). This increased sensitivity of diabetic muscle to impairment of insulin-stimulated glycogen synthase activity occurs together with diminished insulin-stimulation (by 40%) of IRS-1-associ- ated phosphatidylinositol 3-kinase activity in the same cells. Protein expression of IRS-1, p85, p110, Akt, p70 S6 kinase, and MAPK were normal in diabetic cells, as was insulin-stimulated phosphorylation of Akt, p70 S6 kinase, and MAPK. These stud- ies indicate that, despite prolonged growth and differentia- tion of diabetic muscle under normal metabolic culture con- ditions, defects of insulin-stimulated phosphatidylinositol 3-kinase and glycogen synthase activity in diabetic muscle persist, consistent with intrinsic (rather than acquired) de- fects of insulin action. (J Clin Endocrinol Metab 86: 4307- 4314, 2001) include: the ras/MAPK/p90rsk, phosphatidylinositol 3- kinase (PI 3-kinase)/protein kinase B (Akt), and PI 3-kinase/ mTOR/p70 S6 kinase pathways (10, 11-17). In several cell lines, structurally unrelated inhibitors of PI 3-kinase (wort- mannin and LY294002) inhibit insulin stimulation of GS (18 -22), suggesting that PI 3-kinase-dependent pathways might mediate this insulin response. Events downstream of PI 3-kinase and the relative importance of these pathways in mediating insulin stimulation of GS seem to be both tissue- and species-dependent. For example, constitutively active Akt has been shown to stimulate glycogen synthesis and elevate GS activity in L6 myotubes but not in 3T3L-1 adi-