title: Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species.
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.
authors.affiliation: Vascular Research Laboratory, Graduate School of Medicine, University of Tennessee, Knoxville 37920, USA. mzou@mc.utmck.edu
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Zou
authors.forename: Stacy S
authors.initials: SS
authors.lastname: Kirkpatrick
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Davis
authors.forename: John S
authors.initials: JS
authors.lastname: Nelson
authors.forename: Walter G
authors.initials: WG
authors.lastname: Wiles
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Michael
authors.initials: M
authors.lastname: Brownlee
authors.forename: Michael B
authors.initials: MB
authors.lastname: Freeman
authors.forename: Mitch H
authors.initials: MH
authors.lastname: Goldman
mesh_terms: D055372:AMP-Activated Protein Kinases; D000818:Animals; D001011:Aorta; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D009097:Multienzyme Complexes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D017672:Nitrogen Compounds / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*
chemical_list: D007004:Hypoglycemic Agents; D009097:Multienzyme Complexes; D017672:Nitrogen Compounds; D008687:Metformin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases