Abstract 259: Activation of AMP-activated Protein Kinase by Metformin Is Mediated by Atypical Protein Kinase C-zeta
We have recently reported that metformin, one of the most widely used anti-diabetic drugs, improves vascular endothelial function by activating the AMP-activated kinase (AMPK). But how metformin activates AMPK is poorly defined. Here we report that metformin activates PKC-ζ leading to AMPK activation in endothelial cells.
Methods and Results: Incubation of Human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) with clinical relevant concentrations of metformin significantly increased the phosphorylation of both AMPK- Thr172 (control, 15.84±7.9; metformin, 168.3±25.6, P<001, n=4) and LKB1-Ser428 in parallel with increased translocation of PKC-ζ from cytosol to membrane, an essential step for PKC-ζ activation. Further, inhibition of PKC-ζ with pharmacological reagents (PKC-ζ specific pseudosubstrate) and genetic inhibitors (PKC-ζ dominant negative adenovirus) ablated metformin-induced phosphorylation of both AMPK-Thr172 and LKB1-Ser428. Moreover, inhibition of PKC-ζ abolished metformin-enhanced co-immunoprecipitation of LKB1 with both AMPK-alpha1 and AMPKalpha2, suggesting that PKC-ζ might enhance AMPK activation by increasing the association of AMPK-alpha with LKB1. Furthermore, metformin increased LKB1 translocation from nucleus into cytosol in BAEC. Further, metformin did not alter the levels of AMPK in LKB1-deficient Hela-S3 and A549 or in Hela-S3 overexpressing the kinase-dead LKB1 mutants. Conversely, metformin increased both AMPK activation and the association of AMPK with LKB in Hela-S3 overexpressing LKB1 wild type, suggesting the essential role of LKB1 in the activation of AMPK by metformin. Point mutation of either Ser 428 or Ser307 of LKB1 into alanine abolished metformin-enhanced AMPK-Thr172 phosphorylation, the translocation of LKB1 from cytosol into nucleus, as well as the association of LKB1 with AMPK. Finally, PKC-ζ phosphorylated both LKB1 and AMPK activation in in vitro kinase assays resulting in AMPK activation, supporting the essential role of PKC.
Conclusion: PKC-ζ is required for metformin-enhanced AMPK activation, likely by phosphorylating LKB1 at both ser428 and ser307 resulting in LKB1 translocation and its association with AMPK.