Activation of the AMP-activated Protein Kinase by the Anti-diabetic Drug Metformin in Vivo: ROLE OF MITOCHONDRIAL REACTIVE NITROGEN SPECIES

Ming-Hui Zou,Stacy S Kirkpatrick,Bradley J Davis,John S Nelson,Walter G Wiles,Uwe Schlattner,Dietbert Neumann,Diertbert Neumann,Michael Brownlee,Michael B Freeman,Mitch H Goldman

doi:10.1074/jbc.m404421200

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-alpha 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

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This novel activation scheme may be implicated during hypoxia-reoxygenation, where we found that AMPK activation depends on ONOOϪ formation, as well as activation of c-Src and PI3K [28]
We demonstrate that metformin inhibits the complex I of the respiratory chain to generate mitochondrial O2. and ONOOϪ, which leads to AMPK activation via a c-Src and PI3K-dependent pathway

Summary

Introduction

ThMe tertefoaTrtmmhieinsn,taoornfteitcyolpfeethhIeIadmsiaobbsteetceeonsm, wwmaoistnhrlyedcuresanetwdlyndfrobuugynsdtfhotore asuertTvhheeod ArhseMt.ePTr-oahtcretiimvJaeotreupdrropntreoaitnleircnoankissiinesatdisneg(AofMthPrKe)e1 is a well subunits, con␣, ␤, exert itsqtuheersatpioeuntsictehffaetcttsh, aet AleMastPiKn pimarmt, buynaoctbivloatt- inaFndig␥., 3eaCchwofawshricehuhsaes adt ilenasFtitgw.o4isAofoarms sA(1C–C6,). Several studies suggest that there is a second AMPKK isoform that is not AMP-dependent (18, 24 –26) None of these studies have established the mechanism by which metformin activates AMPK. We further characterize that ONOOϪ activates AMPK via a c-Src and PI3K-dependent mechanism without a change in cellular AMP or ATP content [28]. Administration of metformin, which increased 3-nitrotyrosine, a stable maker for reactive nitrogen species (RNS) such as ONOOϪ, in the hearts of C57BL6, resulted in parallel activation of AMPK and ACCSer phosphorylation in the aorta and hearts of C57BL6 mice but not in those of eNOS knockout mice (eNOSϪ/Ϫ) (lacking NO, which is required for RNS formation). We conclude that activation of AMPK by metformin is mediated by mitochondrial RNS and PI3K pathway

Methods

Results

Conclusion