title: Selective inhibition of deactivated mitochondrial complex I by biguanides.
abstract: Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O₂(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O₂(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O₂(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Matsuzaki
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Humphries
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D007511:Ischemia / Q000201:enzymology; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry; D008297:Male; D008687:Metformin / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000201:enzymology; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology; D010629:Phenformin / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D013367:Submitochondrial Particles / Q000187:drug effects / Q000201:enzymology; D013481:Superoxides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D015636:Magnesium Chloride; D013481:Superoxides; D008687:Metformin; D010629:Phenformin; D042967:Electron Transport Complex I