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.

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.