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.

The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP؉ at the expense of NADH oxida- The coenzymes nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are soluble electron carriers that are reduced in oxidative reactions during the catabolism of energy substrates in the cytosol and mitochondria. Despite their structural similarity, reduced NAD and NADP are used to drive rather different processes in the cells (1). Given their roles as specific electron donors in diverse metabolic pathways, it is interesting that the redox states of NAD and NADP are linked to each other in the mitochondria of many organisms because the enzyme nicotinamide nucleotide transhydrogenase (NNT) 4 catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other (2).The academic and clinical interest in the understanding of the role of NNT in redox metabolism (3) has grown substantially because mutations in the gene (Nnt) encoding this protein are present in the most widely used experimental mouse substrain (i.e. C57BL/6J) (4) and in some humans (5). Homozygous mutations of Nnt that are linked to familiar glucocorticoid insufficiency have been documented in humans (5), whereas the C57BL/6J mouse substrains have been shown to exhibit metabolic abnormalities (6 -8) with major implications regarding their use as experimental models in basic research (9 -12). The phenotype of the Nnt mutation in heterozygotes is not clear in humans (13) or mice.NADP ϩ reduction at the expense of NADH oxidation in the mitochondrial matrix is the well known primary role of NNT. Although mitochondria possess three other enzymatic oxidative reactions linked to NADP ϩ reduction (i.e. the enzymes isocitrate dehydrogenase (IDH2), malic enzymes (MEs), and glutamate dehydrogenase (GDH)), as highlighted in Fig. 1

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BackgroundReactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects.MethodsWe cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitória (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample.ResultsGenotype frequencies in both samples were consistent with the Hardy- Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height2.7 than those carrying the CC genotype in Campinas (76.8 ± 1.6 vs 70.9 ± 1.4 g/m2.7; p = 0.009), and in Vitória (45.6 ± 1.9 vs 39.9 ± 1.4 g/m2.7; p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03).ConclusionsThe T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height2.7 and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.

Among mitochondrial NADP‐reducing enzymes, nicotinamide nucleotide transhydrogenase (NNT) establishes an elevated matrix NADPH/NADP+ by catalyzing the reduction of NADP+ at the expense of NADH oxidation coupled to inward proton translocation across the inner mitochondrial membrane. Here, we characterize NNT activity and mitochondrial redox balance in the brain using a congenic mouse model carrying the mutated Nnt gene from the C57BL/6J strain. The absence of NNT activity resulted in lower total NADPH sources activity in the brain mitochondria of young mice, an effect that was partially compensated in aged mice. Nonsynaptic mitochondria showed higher NNT activity than synaptic mitochondria. In the absence of NNT, an increased release of H2O2 from mitochondria was observed when the metabolism of respiratory substrates occurred with restricted flux through relevant mitochondrial NADPH sources or when respiratory complex I was inhibited. In accordance, mitochondria from Nnt‐/‐ brains were unable to sustain NADP in its reduced state when energized in the absence of carbon substrates, an effect aggravated after H2O2 bolus metabolism. These data indicate that the lack of NNT in brain mitochondria impairs peroxide detoxification, but peroxide detoxification can be partially counterbalanced by concurrent NADPH sources depending on substrate availability. Notably, only brain mitochondria from Nnt−/− mice chronically fed a high‐fat diet exhibited lower activity of the redox‐sensitive aconitase, suggesting that brain mitochondrial redox balance requires NNT under the metabolic stress of a high‐fat diet. Overall, the role of NNT in the brain mitochondria redox balance especially comes into play under mitochondrial respiratory defects or high‐fat diet.