Abstract
Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-α that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.
Highlights
This article has been withdrawn by the authors
The authors were able to provide to the Journal data from repeat experiments for Fig. 4A performed at the time of the original work, which they state confirm the results
The authors offered to publish substitute figures based on the repeated experiments and corrected figures, alternatively, offered to repeat the experiments
Summary
This article has been withdrawn by the authors. The Journal raised questions that the AMPK immunoblot in Fig. 3C was reused in Fig. 4A as ACC, lanes 1 and 4 of the AMPK-P immunoblot in Fig. 4A were duplicated, and lanes 3 and 4 of the AMPK-P immunoblot in Fig. 4F were reused in Fig. 5A as ACC-P. Withdrawal: Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo : Role of mitochondrial reactive nitrogen species. This article has been withdrawn by the authors.
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