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CELLULAR METABOLISM

Ca²⁺/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by -lipoic acid in C2C12 myotubes

¹Department of Animal Science and Interdepartmental Molecular and Cellular Life Science Program and ²Division of Pharmaceutical Science and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming

Submitted 23 March 2007 ; accepted in final form 30 July 2007

--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 -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.

skeletal muscle

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