title: A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities.
abstract: NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP(+) ratio. This enzyme catalyzes the reduction of NADP(+) at the expense of NADH oxidation and H(+) reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-Nnt(MUT)) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-Nnt(MUT) mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-Nnt(W)) and from B6J-Nnt(MUT) mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-Nnt(MUT) mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca(2+)-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-Nnt(MUT) mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-Nnt(W) mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-Nnt(MUT) mice in biomedical research should not be overlooked.
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, Campinas, SP 13083-887, Brazil.
authors.forename: Juliana A
authors.initials: JA
authors.lastname: Ronchi
authors.forename: Tiago R
authors.initials: TR
authors.lastname: Figueira
authors.forename: Felipe G
authors.initials: FG
authors.lastname: Ravagnani
authors.forename: Helena C F
authors.initials: HC
authors.lastname: Oliveira
authors.forename: Anibal E
authors.initials: AE
authors.lastname: Vercesi
authors.forename: Roger F
authors.initials: RF
authors.lastname: Castilho
mesh_terms: D000818:Animals; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects
chemical_list: D009249:NADP; D006861:Hydrogen Peroxide; D009250:NADP Transhydrogenases; D005978:Glutathione
keywords: 3-OHB; 3-OHBDH; 3-acetylpyridine adenine dinucleotide; 3-hydroxybutyrate; 3-hydroxybutyrate dehydrogenase; AA; APAD; AcAc; B6J-Nnt(MUT) mice; B6JUnib-Nnt(W) mice; C57BL/6/JUnib mice carrying wild-type Nnt alleles; C57BL/6J mice carrying mutated Nnt alleles; Calcium; FCCP; GPx; GR; GSH; GSSG; Glutathione; IDH2; IDH3; Isoc; Krebs cycle intermediates; ME3; MPT; Mitochondrial NADPH; N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride.; NAD; NAD(+); NADH; NADP; NADP(+); NADPH; NNT; PCoA; ROS; Reactive oxygen species; SOD2; TMPD; Transhydrogenation; acetoacetate; antimycin A; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; glutathione; glutathione disulfide; glutathione peroxidase; glutathione reductase; isocitrate; mitochondrial NAD-dependent isocitrate dehydrogenase; mitochondrial NADP-dependent isocitrate dehydrogenase; mitochondrial NADP-dependent malic enzyme; mitochondrial permeability transition; mitochondrial superoxide dismutase; nicotinamide nucleotide transhydrogenase; oxidized form of NAD; oxidized form of NADP; palmitoyl coenzyme A; reactive oxygen species; reduced form of NAD; reduced form of NADP; t-BOH; t-BOOH; tert-butyl alcohol; tert-butyl hydroperoxide; β-nicotinamide adenine dinucleotide; β-nicotinamide adenine dinucleotide phosphate