Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes.
alpha-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 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) 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 Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). 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.
Department of Animal Science, Interdepartmental Molecular and Cellular Life Science Program, University of Wyoming, Laramie, WY 82071, USA.
Qingwu W
QW
Shen
Mei J
MJ
Zhu
Junfeng
J
Tong
Jun
J
Ren
Min
M
Du
D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D053644:Naphthalimides / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D008063:Thioctic Acid / Q000494:pharmacology*
D001562:Benzimidazoles; D002614:Chelating Agents; D005227:Fatty Acids; D007546:Isoquinolines; D009097:Multienzyme Complexes; D053644:Naphthalimides; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; C458525:STO 609; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000249:Adenosine Monophosphate; D004533:Egtazic Acid; D008063:Thioctic Acid; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase; D002118:Calcium