AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor of intracellular energy. Activation of AMPK is associated with increased phosphorylation of the a-subunit at threonine 172 (T172) and decreased phosphorylation at serine 485 in AMPK alpha 1 and serine 491 in AMPK alpha 2 (S485/491). One potential mediator of AMPK phosphorylation is phosphatidylinositol 3-kinase (PI3K); however, the mechanism and the identities of the specific PI3K isoforms that regulate AMPK activation are not known. To determine whether PI3K p110 alpha regulated AMPK activation in muscle cells, C2C12 myoblasts were subjected to pharmacological inhibition of p110 alpha, siRNA directed against p110 alpha, or overexpression of constitutively-active or dominant negative p110 alpha. Chemical inhibition, siRNA, and expression of dominant-negative p110 alpha were all associated with increased AMPK T172 phosphorylation, whereas expression of constitutively-active p110 alpha reduced T172 phosphorylation. Conversely, pharmacological inhibition of p110 alpha reduced AMPK S485/491 phosphorylation, while constitutively-active p110 alpha increased AMPK S485/491 phosphorylation. This p110 alpha-mediated increase in AMPK S485/491 phosphorylation was eliminated in the presence of the Akt inhibitor MK2206, suggesting that p110 alpha-mediated phosphorylation of AMPK alpha at S485/491 is Akt-dependent. In response to oligomycin or serum starvation, AMPK T172 phosphorylation was elevated in p11 alpha-deficient myoblasts compared to control myoblasts. Overall, our findings identify PI3K p110a as a mediator of AMPK phosphorylation in myoblasts. (C) Published by Elsevier Inc.