Aging-dependent mitochondrial bioenergetic impairment in the skeletal muscle of NNT-deficient mice.

Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt-/-) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt-/- mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt-/- mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt-/- mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.

Lack of NAD(P)+ transhydrogenase activity in patients with primary adrenal insufficiency due to NNT variants.

BACKGROUND: Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment of the gonads. OBJECTIVE: Despite the description of different homozygous and compound heterozygous NNT variants in PAI patients, the extent to which the function and expression of the mature protein are compromised remains to be clarified. DESIGN: The activity and expression of mitochondrial NAD(P)+ transhydrogenase (NNT) were analyzed in blood samples obtained from patients diagnosed with PAI due to genetically confirmed variants of the NNT gene (n = 5), heterozygous carriers as their parents (n = 8), and healthy controls (n = 26). METHODS: NNT activity was assessed by a reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMCs). The enzymatic assay was validated in PBMC samples from a mouse model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blotting and reverse transcription quantitative polymerase chain reaction and for mitochondrial oxygen consumption. RESULTS: NNT activity was undetectable (<4% of that of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In patients' parents, NNT activity was approximately half that of the healthy controls. Mature NNT protein expression was lower in patients than in the control groups, while mRNA levels varied widely among genotypes. Moreover, pathogenic NNT variants did not impair mitochondrial bioenergetic function in PBMCs. CONCLUSIONS: The manifestation of PAI in NNT-mutated patients is associated with a complete lack of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT variants.

Surgical site occurrence after prophylactic use of mesh for prevention of incisional hernia in midline laparotomy: systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: The use of mesh is the standard for the prevention of incisional hernia (IH). However, the effect of surgical site occurrence (SSO) has never been compared. The aim of this meta-analysis was to evaluate the prevalence of SSO and measure its negative effect through the calculation of the number needed to treat for net effect (NNT net). METHODS: A meta-analysis was performed according to the PRISMA guidelines. The primary objective was to determine the prevalence of SSO and IH, and the secondary objective was to determine the NNT net as a metric to measure the combined benefits and harms. Only published clinical trials were included. The risk of bias was analyzed, and the random effects model was used to determine statistical significance. RESULTS: A total of 15 studies comparing 2344 patients were included. The incidence of IH was significantly lower in the mesh group than in the control group, with an OR of 0.29 (95% CI 0.16-0.49, p = 0.0001). The incidence of SSO was higher in the mesh group than in the control group, with an OR of 1.21 (95% CI 0.85-1.72, p = 0.0001) but without statistical significance. Therefore, the way to compare the benefits and risks of each of the studies was done with the calculation of the NNT net, which is the average number of patients who need to be treated to see the benefit exceeding the harm by one event, and the result was 5, which is the average number of patients who need to be treated to see the benefit exceeding the harm by one event. CONCLUSION: The use of mesh reduces the prevalence of IH and it does not increases the prevalence of SSO, the NNT net determined that the use of mesh continues to be beneficial for the patient.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

Drugs to Prevent Bronchopulmonary Dysplasia: Effect of Baseline Risk on the Number Needed to Treat.

Infants born very preterm have a variable baseline risk of bronchopulmonary dysplasia (BPD). Using the example of evidence-based drug therapies to prevent BPD, we designed a visual aid that displays the "number needed to treat" with CIs for caffeine, vitamin A, and hydrocortisone over a range of baseline risks.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

Facilitation of Ca2+ -induced opening of the mitochondrial permeability transition pore either by nicotinamide nucleotide transhydrogenase deficiency or statins treatment.

Mitochondrial redox imbalance and high Ca2+ uptake induce the opening of the permeability transition pore (PTP) that leads to disruption of energy-linked mitochondrial functions and triggers cell death in many disease states. In this review, we discuss the major results from our studies investigating the consequences of NAD(P)-transhydrogenase (NNT) deficiency, and of statins treatment for mitochondrial functions and susceptibility to Ca2+ -induced PTP. We highlight the aggravation of high fat diet-induced fatty liver disease in the context of NNT deficiency and the role of antioxidants in the prevention of statins toxicity to mitochondria.

Redox imbalance due to the loss of mitochondrial NAD(P)-transhydrogenase markedly aggravates high fat diet-induced fatty liver disease in mice.

The mechanisms by which a high fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunctions and redox imbalance. We hypothesized that a HFD would increase mitochondrial reliance on NAD(P)-transhydrogenase (NNT) as the source of NADPH for antioxidant systems that counteract NAFLD development. Therefore, we studied HFD-induced liver mitochondrial dysfunctions and NAFLD in C57Unib.B6 congenic mice with (Nnt+/+) or without (Nnt-/-) NNT activity; the spontaneously mutated allele (Nnt-/-) was inherited from the C57BL/6J mouse substrain. After 20 weeks on a HFD, Nnt-/- mice exhibited a higher prevalence of steatohepatitis and content of liver triglycerides compared to Nnt+/+ mice on an identical diet. Under a HFD, the aggravated NAFLD phenotype in the Nnt-/- mice was accompanied by an increased H2O2 release rate from mitochondria, decreased aconitase activity (a redox-sensitive mitochondrial enzyme) and higher susceptibility to Ca2+-induced mitochondrial permeability transition. In addition, HFD led to the phosphorylation (inhibition) of pyruvate dehydrogenase (PDH) and markedly reduced the ability of liver mitochondria to remove peroxide in Nnt-/- mice. Bypass or pharmacological reactivation of PDH by dichloroacetate restored the peroxide removal capability of mitochondria from Nnt-/- mice on a HFD. Noteworthy, compared to mice that were chow-fed, the HFD did not impair peroxide removal nor elicit redox imbalance in mitochondria from Nnt+/+ mice. Therefore, HFD interacted with Nnt mutation to generate PDH inhibition and further suppression of peroxide removal. We conclude that NNT plays a critical role in counteracting mitochondrial redox imbalance, PDH inhibition and advancement of NAFLD in mice fed a HFD. The present study provide seminal experimental evidence that redox imbalance in liver mitochondria potentiates the progression from simple steatosis to steatohepatitis following a HFD.

A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities.

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.