5'AMP-activated protein kinase (AMPK) regulates several aspects of metabolism. Recently, A-769662 was shown to activate AMPK in skeletal muscle. However, no biological effects of AMPK activation by A-769662 in this tissue have been reported. We hypothesized that A-769662 would increase glucose uptake in skeletal muscle. We studied incubated soleus and extensor digitorum longus (EDL) muscles from 129S6/sv and C57BL/6 mice. Glucose uptake increased only in soleus from 129S6/sv when concentrations of A-769662 was 500 microM (~15%, p<0.05) and 1 mM (~60%, p<0.01). AMPK beta1- but not beta2-containing complexes were dose-dependently activated by A-769662 in muscles from both genotypes (~100% at 200 microM and 300-600% at 1 mM). The discrepancy between the A-769662-induced AMPK activation pattern and stimulation of glucose uptake suggested these effects were unrelated. A-769662 increased phosphorylation of Akt in both muscles from both genotypes, with phosphorylation of T308 being significantly higher in soleus than in EDL in 129S6/sv mice (p<0.01). In soleus from 129S6/sv mice, IRS-1-associated PI3 kinase activity was markedly increased with A-769662, and Akt phosphorylation and glucose uptake were inhibited by wortmannin while phosphorylation of Acetyl-CoA Carboxylase (S227) was unaffected. Thus, A-769662 activates beta1-containing AMPK complexes in skeletal muscle, but induces glucose uptake through a PI3 kinase-dependent pathway. Although development of A-769662 has constituted a step forward in the search for AMPK activators targeting specific AMPK trimers, our data suggest that in intact muscle A-769662 has off-target effects. This may limit use of A-769662 to study the role of AMPK in skeletal muscle metabolism. Key words: A769662, TBC1D1 and TBC1D4, AMPK trimer composition, EDL and soleus.