Keir J. Menzies

NAD+ metabolism and the control of energy homeostasis - a balancing act between mitochondria and the nucleus

NAD(+) has emerged as a vital cofactor that can rewire metabolism, activate sirtuins, and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD(+) metabolism revived interest in NAD(+)-boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD(+) metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited.

Mitochondrial function and apoptotic susceptibility in aging skeletal muscle.

During aging, skeletal muscle undergoes sarcopenia, a condition characterized by a loss of muscle cell mass and alterations in contractile function. The origin of these decrements is unknown, but evidence suggests that they can be partly attributed to mitochondrial dysfunction. To characterize the nature of this dysfunction, we investigated skeletal muscle contractile properties, subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial biogenesis and function, as well as apoptotic susceptibility in young (6 months old) and senescent (36 months old) Fischer 344 Brown Norway rats. Muscle mass and maximal force production were significantly lower in the 36‐month group, which is indicative of a sarcopenic phenotype. Furthermore, contractile activity in situ revealed greater fatigability in the 36‐month compared to the 6‐month animals. This decrement could be partially accounted for by a 30% lower mitochondrial content in fast‐twitch muscle from 36‐month animals, as well as lower protein levels of the transcriptional coactivator peroxisome proliferator‐activated receptor γ coactivator‐1α. Enzyme activities and glutamate‐induced oxygen consumption rates in isolated SS and IMF mitochondria were similar between age groups. However, mitochondrial reactive oxygen species (ROS) production during state 3 respiration was ~1.7‐fold greater in mitochondria isolated from 36‐month compared to 6‐month animals, and was accompanied by a 1.8‐fold increase in the DNA repair enzyme 8‐oxoguanine glycosylase 1 in fast‐twitch muscle. Basal rates of release of cytochrome c and endonuclease G in SS mitochondria were 3.5‐ to 7‐fold higher from senescent animals. These data suggest that the age‐related sarcopenia and muscle fatigability are associated with enhanced ROS production, increased mitochondrial apoptotic susceptibility and reduced transcriptional drive for mitochondrial biogenesis.

NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice

A dietary supplement protects aging muscle The oxidized form of cellular nicotinamide adenine dinucleotide (NAD+) is critical for mitochondrial function, and its supplementation can lead to increased longevity. Zhang et al. found that feeding the NAD+ precursor nicotinamide riboside (NR) to aging mice protected them from muscle degeneration (see the Perspective by Guarente). NR treatment enhanced muscle function and also protected mice from the loss of muscle stem cells. The treatment was similarly protective of neural and melanocyte stem cells, which may have contributed to the extended life span of the NR-treated animals. Science, this issue p. 1436; see also p. 1396 A dietary supplement protects muscle stem cells and increases mouse longevity. Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD+) and its effect on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD+ precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the mdx (C57BL/10ScSn-Dmdmdx/J) mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs and melanocyte SCs and increases mouse life span. Strategies that conserve cellular NAD+ may reprogram dysfunctional SCs and improve life span in mammals.

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Sirtuin 1-mediated Effects of Exercise and Resveratrol on Mitochondrial Biogenesis

Background: SirT1 regulates mitochondrial biogenesis in various tissues. Results: Exercise combined with resveratrol has a SirT1-dependent synergistic effect on mitochondrial biogenesis, despite individual treatments being SirT1-independent. Conclusion: SirT1 is important for maintaining muscle mitochondrial content and function. Significance: The dependence of muscle mitochondrial biogenesis on SirT1 depends on the metabolic state of the muscle. The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.

Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe−/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204)

Protein acetylation in metabolism - metabolites and cofactors.

Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis.

Eliciting the mitochondrial unfolded protein response via NAD+ repletion reverses fatty liver disease

With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe−/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204)

Relationship between Sirt1 expression and mitochondrial proteins during conditions of chronic muscle use and disuse

Sirt1 is a NAD(+)-dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1alpha and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1alpha existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1alpha levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30-50% compared with the control muscle, whereas Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1alpha expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation

NAD+ treatment can reverse the functional decline in degenerating muscles. Making muscle work better Degenerating muscle—whether from muscular dystrophies, myopathies, or other diseases—loses its mitochondria (the energy supply) and an essential cofactor nicotinamide adenine dinucleotide (NAD+), while gaining an extra load of enzymes that use up NAD+, as reported by Ryu and colleagues. The resulting loss of NAD+ is exacerbated by a drop in NAD+ biosynthetic enzymes, such as NAMPT. Restoration of NAD+ levels in either mice or worms with disease-like degenerating muscles improved muscle function, a consequence of more mitochondria, more muscle structural proteins, and a decrease in inflammation. The authors suggest that NAD+ repletion may be a successful therapeutic approach for a number of muscle-wasting diseases. Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5′-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.