Nicotinamide nucleotide transhydrogenase mutation analysis in Chinese patients with thyroid dysgenesis.

Thyroid dysgenesis (TD) accounts for 80% cases of congenital hypothyroidism, which is the most common neonatal disorder. Until now, the gene mutations have been reported associated with TD can only account for 5% cases, suggesting the genetic heterogeneity of the pathology. Nicotinamide nucleotide transhydrogenase (NNT) plays a crucial role in regulating redox homeostasis, patients carrying NNT mutations have been described with a clinical phenotype of hypothyroidism. As TD risk is increased in the context of several syndromes and redox homeostasis is vital for thyroid development and function, NNT might be a candidate gene involved in syndromic TD. Therefore, we performed target sequencing (TS) in 289 TD patients for causative mutations in NNT and conducted functional analysis of the gene mutations. TS and Sanger sequence were used to screen the novel mutations. For functional analysis, we performed western blot, measurement of NADPH/NADPtotal and H2 O2 generation, cell proliferation, and wounding healing assay. As a result, three presumably pathogenic mutations (c.811G > A, p.Ala271Ser; c.2078G > A, p.Arg693His; and c.2581G > A, p.Val861Met) in NNT had been identified. Our results showed the damaging effect of NNT mutations on stability and catalytic activity of proteins and redox balance of cells. In conclusion, our findings provided novel insights into the role of the NNT isotype in thyroid physiopathology and broaden the spectrum of pathogenic genes associated with TD. However, the pathogenic mechanism of NNT in TD is still need to be investigated in further study.

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through ß-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP- and low-ADP-stimulated respiration that accelerating flux through ß-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (∼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within ß-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.

LncRNA NNT-AS1 regulates proliferation, ECM accumulation and inflammation of human mesangial cells induced by high glucose through miR-214-5p/smad4.

BACKGROUND: LncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target. METHODS: Bioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis. RESULTS: Results from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. CONCLUSION: The data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells.

Loss of Nnt Increases Expression of Oxidative Phosphorylation Complexes in C57BL/6J Hearts.

Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains.

Undesirable effects of chemical inhibitors of NAD(P)+ transhydrogenase on mitochondrial respiratory function.

NAD(P)+ transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt-/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability.

Nicotinamide Nucleotide Transhydrogenase (Nnt) is Related to Obesity in Mice.

The C57BL/6J (B6J) mouse strain has been widely used as a control strain for the study of metabolic diseases and diet induced obesity (DIO). B6J mice carry a spontaneous deletion mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11, resulting in expression of a truncated form of Nnt, an enzyme that pumps protons across the inner mitochondrial membrane. It has been proposed that this mutation in B6J mice is associated with epigonadal fat mass and altered sensitivity to diet induced obesity. To define the role of Nnt in the development of diet induced obesity, we generated first backcross (BC1) hybrids of wild type Nnt C57BL/6NTac and mutated Nnt C57BL/6JRj [(C57BL/6NTac×C57BL/6JRj)F1×C57BL/6NTac]. Body weight gain and specific fat-pad depot mass were measured in BC1 hybrids under high fat diet conditions. Both sexes of BC1 hybrids indicate that mice with Nnt wild type allele are highly sensitive to DIO and exhibit higher relative fat mass. In summary, our data indicate that the Nnt mutation in mice is associated with sensitivity to DIO and fat mass.

Calcium Signaling and Reactive Oxygen Species in Mitochondria.

In heart failure, alterations of Na+ and Ca2+ handling, energetic deficit, and oxidative stress in cardiac myocytes are important pathophysiological hallmarks. Mitochondria are central to these processes because they are the main source for ATP, but also reactive oxygen species (ROS), and their function is critically controlled by Ca2+ During physiological variations of workload, mitochondrial Ca2+ uptake is required to match energy supply to demand but also to keep the antioxidative capacity in a reduced state to prevent excessive emission of ROS. Mitochondria take up Ca2+ via the mitochondrial Ca2+ uniporter, which exists in a multiprotein complex whose molecular components were identified only recently. In heart failure, deterioration of cytosolic Ca2+ and Na+ handling hampers mitochondrial Ca2+ uptake and the ensuing Krebs cycle-induced regeneration of the reduced forms of NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate), giving rise to energetic deficit and oxidative stress. ROS emission from mitochondria can trigger further ROS release from neighboring mitochondria termed ROS-induced ROS release, and cross talk between different ROS sources provides a spatially confined cellular network of redox signaling. Although low levels of ROS may serve physiological roles, higher levels interfere with excitation-contraction coupling, induce maladaptive cardiac remodeling through redox-sensitive kinases, and cell death through mitochondrial permeability transition. Targeting the dysregulated interplay between excitation-contraction coupling and mitochondrial energetics may ameliorate the progression of heart failure.

Long noncoding RNA NNT-AS1 functions as an oncogene in breast cancer via repressing ZFP36 expression.

Breast cancer is one of the most common cancers in women. This study focuses on the effects of Long non-coding RNAs (lncRNAs) NNT-AS1 on breast cancer cell growth and metastasis. Fifty-six pairs of breast cancer (BC) tissues and matched paracarcinoma tissues were obtained. The BC cell lines and normal human breast cell line were employed. NNT-AS1 in BC cells was knocked down by shRNA. Cell counting kit-8 assay (CCK-8), colony formation assay, cell cycle analysis, cell apoptosis analysis, cound healing assay, Transwell assay, cioinformatics analysis, Western blot analysis and Xenograft model were used. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that expression of NNT-AS1 was obviously upregulated in breast cancer tissues compared with adjacent tissues (n=56). Knockdown of NNT-AS1 could attenuate breast cancer cell viability, proliferation, invasion and migration, as well as promote cell apoptosis and induce cell cycle arrest at G0/G1 phase. ZFP36 was directly combined with NNT-AS1, and silencing of ZFP36 could rescue tumor suppression role by downregulating NNT-AS1 on cell proliferation and metastasis. Knockdown of NNT-AS1 could suppress cell growth and metastasis via interacting with ZFP36 in vivo. This study demonstrated that knockdown of NNT-AS1 had tumor-suppressive effect on breast cancer progression and metastasis via interacting with ZFP36 in vitro and in vivo, which provides a new insight into the treatment and prognosis evaluation of breast cancer.

Long noncoding RNA NNT-AS1 promotes gastric cancer proliferation and invasion by regulating microRNA-363 expression.

Increasing studies showed that long noncoding RNAs (lncRNAs) had crucial regulatory roles in various tumors, including gastric cancer (GC). Recent studies demonstrated that lncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) played an important role in several tumors. However, the role and expression of NNT-AS1 in GC progression remain unknown. In our study, we indicated that NNT-AS1 expression was upregulated in GC samples compared with the nontumor tissues. We also showed that NNT-AS1 expression was upregulated in the GC cell lines. Ectopic expression of NNT-AS1 promoted GC cell line HGC-27 cell proliferation, cell cycle progression, and invasion. In addition, we showed that NNT-AS1 acted as a sponge competing endogenous RNA for microRNA-363 (miR-363), which was downregulated in the GC samples and cell lines. miR-363 expression was negatively related with NNT-AS1 expression in GC samples. Upregulated expression of miR-363 suppressed GC cell growth, cycle, and invasion. Furthermore, we reported that elevated expression of NNT-AS1 promoted GC cell proliferation, cycle, and invasion partly by suppressing miR-363 expression. These results indicated that lncRNA NNT-AS1 acted as an oncogene in the development of GC partly by inhibiting miR-363 expression.

Effect of glucose deprivation on the expression of genes encoding glucocorticoid receptor and some related factors in ERN1-knockdown U87 glioma cells.

OBJECTIVE: The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation. METHODS: The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction. RESULTS: It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition. CONCLUSIONS: Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.

Nicotinamide nucleotide transhydrogenase expression analysis in multiple sclerosis patients.

Background: Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial redox-induced proton pump that links NADPH synthesis to the mitochondrial metabolic pathway. It also participates in the regulation of immune responses. A long non-coding RNA namely NNT-antisense 1 (NNT-AS1) has been shown to be transcribed from the same locus and exert anti-proliferative effects in some tissues. Methods: In the current study, we evaluated expression of NNT and NNT-AS1 in peripheral blood of 50 relapsing-remitting multiple sclerosis patients compared with healthy subjects. The difference in NNT expression was significant in only in male subjects aged over 50 when compared with the corresponding control subgroup. Results: For NNT-AS1, based on the results of Quantile regression and adjustment of the effects of age and sex as well as the interaction between sex and disease status, no significant difference was found between cases and controls. Moreover, NNT and NNT-AS1 expressions were correlated with age in controls and in female subjects respectively. Conclusion: Finally, we assessed correlations between expressions of these genes and detected significant pairwise correlations between transcript levels of NNT and NNT-AS1 genes in both cases and controls. The current study highlights a gender-specific role for NNT in the pathogenesis of MS.

Nicotinamide nucleotide transhydrogenase is required for brain mitochondrial redox balance under hampered energy substrate metabolism and high-fat diet.

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 H2 O2 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 H2 O2 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.

Raf kinase inhibitor protein mediates myocardial fibrosis under conditions of enhanced myocardial oxidative stress.

Fibrosis is a hallmark of maladaptive cardiac remodelling. Here we report that genome-wide quantitative trait locus (QTL) analyses in recombinant inbred mouse lines of C57BL/6 J and DBA2/J strains identified Raf Kinase Inhibitor Protein (RKIP) as genetic marker of fibrosis progression. C57BL/6 N-RKIP-/- mice demonstrated diminished fibrosis induced by transverse aortic constriction (TAC) or CCl4 (carbon tetrachloride) treatment compared with wild-type controls. TAC-induced expression of collagen Iα2 mRNA, Ki67+ fibroblasts and marker of oxidative stress 8-hydroxyguanosine (8-dOHG)+ fibroblasts as well as the number of fibrocytes in the peripheral blood and bone marrow were markedly reduced in C57BL/6 N-RKIP-/- mice. RKIP-deficient cardiac fibroblasts demonstrated decreased migration and fibronectin production. This was accompanied by a two-fold increase of the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), the main transcriptional activator of antioxidative proteins, and reduced expression of its inactivators. To test the importance of oxidative stress for this signaling, C57BL/6 J mice were studied. C57BL/6 J, but not the C57BL/6 N-strain, is protected from TAC-induced oxidative stress due to mutation of the nicotinamide nucleotide transhydrogenase gene (Nnt). After TAC surgery, the hearts of Nnt-deficient C57BL/6 J-RKIP-/- mice revealed diminished oxidative stress, increased left ventricular (LV) fibrosis and collagen Iα2 as well as enhanced basal nuclear expression of Nrf2. In human LV myocardium from both non-failing and failing hearts, RKIP-protein correlated negatively with the nuclear accumulation of Nrf2. In summary, under conditions of Nnt-dependent enhanced myocardial oxidative stress induced by TAC, RKIP plays a maladaptive role for fibrotic myocardial remodeling by suppressing the Nrf2-related beneficial effects.

Absence of Nicotinamide Nucleotide Transhydrogenase in C57BL/6J Mice Exacerbates Experimental Atherosclerosis.

BACKGROUND: Mitochondrial reactive oxygen species (ROS) contribute to inflammation and vascular remodeling during atherosclerotic plaque formation. C57BL/6N (6N) and C57BL/6J (6J) mice display distinct mitochondrial redox balance due to the absence of nicotinamide nucleotide transhydrogenase (NNT) in 6J mice. We hypothesize that differential NNT expression between these animals alters plaque development. METHODS: 6N and 6J mice were treated with AAV8-PCSK9 (adeno-associated virus serotype 8/proprotein convertase subtilisin/kexin type 9) virus leading to hypercholesterolemia, increased low-density lipoprotein, and atherosclerosis in mice fed a high-fat diet (HFD). Mice were co-treated with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO to assess the contribution of mitochondrial ROS to atherosclerosis. RESULTS: Baseline and HFD-induced vascular superoxide is increased in 6J compared to 6N mice. MitoTEMPO diminished superoxide in both groups demonstrating differential production of mitochondrial ROS among these strains. PCSK9 treatment and HFD led to similar increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of plaque formation. MitoTEMPO reduced plasma lipids but did not affect plaque formation in 6N mice. In contrast, MitoTEMPO surprisingly increased plaque formation in 6J mice. CONCLUSION: These data indicate that loss of NNT increases vascular ROS production and exacerbates atherosclerotic plaque development.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress.

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism's multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic-pituitary-adrenal axis, sympathetic adrenal-medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases.

Quantitative proteomics reveals EVA1A-related proteins involved in neuronal differentiation.

EVA1A is an autophagy-related protein, which plays an important role in embryonic neurogenesis. In this study, we found that loss of EVA1A could decrease neural differentiation in the brain of adult Eva1a-/- mice. To determine the mechanism underlying this phenotype, we performed label-free quantitative proteomics and bioinformatics analysis using the brains of Eva1a-/- and wild-type mice. We identified 11 proteins that were up-regulated and 17 that were down-regulated in the brains of the knockout mice compared to the wild-type counterparts. Bioinformatics analysis indicated that biological processes, including ATP synthesis, oxidative phosphorylation, and the TCA cycle, are involved in the EVA1A regulatory network. In addition, gene set enrichment analysis showed that neurodegenerative diseases, such as Alzheimer's disease and Huntington's disease, were strongly associated with Eva1a knockout. Western blot experiments showed changes in the expression of nicotinamide nucleotide transhydrogenase, an important mitochondrial enzyme involved in the TCA cycle, in the brains of Eva1a knockout mice. Our study provides valuable information on the molecular functions and regulatory network of the Eva1a gene, as well as new perspectives on the relationship between autography-related proteins and neural differentiation.

The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria.

The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism.

Three-Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence-Structure Analysis of its Disease-Causing Variations.

Defective mitochondrial proteins are emerging as major contributors to human disease. Nicotinamide nucleotide transhydrogenase (NNT), a widely expressed mitochondrial protein, has a crucial role in the defence against oxidative stress. NNT variations have recently been reported in patients with familial glucocorticoid deficiency (FGD) and in patients with heart failure. Moreover, knockout animal models suggest that NNT has a major role in diabetes mellitus and obesity. In this study, we used experimental structures of bacterial transhydrogenases to generate a structural model of human NNT (H-NNT). Structure-based analysis allowed the identification of H-NNT residues forming the NAD binding site, the proton canal and the large interaction site on the H-NNT dimer. In addition, we were able to identify key motifs that allow conformational changes adopted by domain III in relation to its functional status, such as the flexible linker between domains II and III and the salt bridge formed by H-NNT Arg882 and Asp830. Moreover, integration of sequence and structure data allowed us to study the structural and functional effect of deleterious amino acid substitutions causing FGD and left ventricular non-compaction cardiomyopathy. In conclusion, interpretation of the function-structure relationship of H-NNT contributes to our understanding of mitochondrial disorders.