Insulin inhibits AMPK activity and phosphorylates AMPK Ser485/491 through Akt in hepatocytes, myotubes and incubated rat skeletal muscle

• Insulin and IGF-1 stimulate Ser485/491 phosphorylation in hepatocytes and myotubes.
• Insulin-induced Ser485/491 p-AMPK is associated with reductions in AMPK activity.
• Inhibition of Akt attenuates insulin and IGF-1 stimulated p-AMPK on Ser485/491.
• Akt inhibition partially prevents suppression of AMPK activity by insulin.
• mTOR/p70S6K signaling is not involved in the phosphorylation of AMPK Ser485/491.

Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser485/491, on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser485/491 site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/− insulin for 10-min. Insulin significantly increased Ser485/491 p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr172. Akt inhibition both attenuated the increase in Ser485/491 p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser485/491 AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser485/491 p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser485/491 on its α-subunit.