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Ca2+/calmodulin‐dependent protein kinase kinases are mainly responsible for AMP‐activated protein kinase activation by α‐ lipoic acid in C2C12 myotubes
Author(s) -
Shen Qingwu W,
Ren Jun,
Du Min
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1204-d
Subject(s) - ampk , amp activated protein kinase , protein kinase a , chemistry , kinase , skeletal muscle , phosphorylation , glucose uptake , endocrinology , biochemistry , microbiology and biotechnology , medicine , biology , insulin
α‐Lipoic acid (ALA) increases glucose uptake in skeletal muscle and is used as an anti‐diabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by activation of AMP‐activated protein kinase (AMPK) in skeletal muscle. Here we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. ALA (0, 100, 200, and 500 μM) were used to treat C2C12 myotubes for 6 hours. Incubation of C2C12 cells with 200 and 500 μM of ALA increased (p < 0.05) phosphorylation of AMPK α subunit at Thr 172 . The phosphorylation of AMPK substrate, acetyl CoA carboxylase (ACC) at Ser 79 was also increased (p<0.05). No difference in ATP, AMP, and calculated AMP/ATP ratio was observed among different treatment groups, indicating that ALA did not alter energy level in cells. Thus, the upstream AMPK kinase, LKB1, might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased intracellular calcium concentration measured by fura‐2 fluorescent microscopy(P<0.05), showing that ALA may activate AMPK through enhancing calcium/calmodulin‐dependent protein kinase kinases (CaMKK). Indeed, chelation of intracellular free calcium by loading cells with 25 μM of BAPTA‐AM for 30 minutes abolished ALA‐induced activation of AMPK and in turn phosphorylation of ACC at Ser 79 . Furthermore, inhibition of CaMKK with its selective inhibitor, STO‐609, abolished ALA stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser 79 . Thus, CaMKK is the kinase responsible for the ALA‐induced AMPK activation. In summary, skeletal muscle cells possess a CaMKK dependent pathway for AMPK activation, which is mainly responsible for the AMPK activation stimulated by ALA.