-α-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 μM ALA increased the activity and phosphorylation of the AMPK α-subunit at Thr172. Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser79 was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca2+ concentration measured by fura-2 fluorescent microscopy ( P < 0.05), showing that ALA may activate AMPK through enhancing Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca2+ by loading cells with 25 μM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser79. Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser79. In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.
Alpha-lipoic acid decreases hepatic lipogenesis through adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent pathways