Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. We previously demonstrated that phosphorylation of Threonine-308 on Akt (pAkt-T308), Akt2 activity, and GS activity in muscle were positivity associated with insulin sensitivity. Now, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. 181 non-diabetic twins were examined with the euglycemic-hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing kinase activity assays and phospho-specific western blotting. The insulin-stimulated GS activity was positively associated with pAkt-T308 (P=0.01) and Akt2 activity (P=0.04), but not pAkt-S473 or IRS-1-PI3K activity. Furthermore, pAkt-T308 and Akt2 activity were negatively associated with NH(2)-terminal GS phosphorylation (P=0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P