Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Attenuated Hepatic Triglyceride Accumulation in Alcoholic Fatty Liver Disease in Mice
Poly(ADP-ribose) polymerase (PARP) is an NAD-consuming enzyme and its specific role in the pathogenesis of alcoholic fatty liver disease (AFLD) remains elusive. In this study, we applied PJ34 [-(5,6-dihydro-6-oxo-2-phenanthridinyl)-2-acetamide hydrochloride] to inhibit hepatic PARP activity to examine the corresponding pathologic alteration in AFLD in mice and the underlying molecular mechanism. We found that PJ34 decreased the intracellular triglyceride (TG) content in hepatocytes. Moreover, PJ34 suppressed the gene expression of diglyceride acyltransferases DGAT1 and DGAT2 and elevated intracellular NADlevels in hepatocytes. These mechanistic observations were validated in alcohol-fed mice injected with PJ34 intraperitoneally. Our results indicate that the PJ34 injection attenuated hepatic TG accumulation in alcohol-fed mice. Furthermore, PJ34 injection lowered the gene expression of hepatic sterol regulatory element binding protein 1c, DGAT1, and DGAT2, whereas PJ34 injection augmented hepatic NADlevels in alcohol-fed mice. Finally, nicotinamide riboside supplementation alleviated hepatic TG accumulation in alcohol-fed mice. These data indicate that applying PARP-specific inhibitor PJ34 by intraperitoneal injection attenuated hepatic NADdepletion and TG accumulation in alcohol-fed mice and may be a potential candidate for use in AFLD therapy.
NADIntermediates: The Biology and Therapeutic Potential of NMN and NR
Research on the biology of NADhas been gaining momentum, providing many critical insights into the pathogenesis of age-associated functional decline and diseases. In particular, two key NADintermediates, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), have been extensively studied over the past several years. Supplementing these NADintermediates has shown preventive and therapeutic effects, ameliorating age-associated pathophysiologies and disease conditions. Although the pharmacokinetics and metabolic fates of NMN and NR are still under intensive investigation, these NADintermediates can exhibit distinct behavior, and their fates appear to depend on the tissue distribution and expression levels of NADbiosynthetic enzymes, nucleotidases, and presumptive transporters for each. A comprehensive concept that connects NADmetabolism to the control of aging and longevity in mammals has been proposed, and the stage is now set to test whether these exciting preclinical results can be translated to improve human health.
Directed Evolution of Membrane Transport Using Synthetic Selections
Understanding and engineering solute transporters is important for metabolic engineering and the development of therapeutics. However, limited available experimental data on membrane transporters makes sequence-function relationships complex to predict. Here we apply ligand-responsive biosensor systems that enable selective growth of E. coli cells only if they functionally express an importer that is specific to the biosensor ligand. Using this system in a directed evolution framework, we successfully engineer the specificity of nicotinamide riboside transporters, PnuC, to accept thiamine as a substrate. Our results provide insight into the molecular determinants of substrate recognition of the PnuC transporter family and demonstrate how synthetic biology can be deployed to engineer the substrate spectrum of small molecule transporters.
Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence
Nicotinamide adenine dinucleotide (NAD), the cell's hydrogen carrier for redox enzymes, is well known for its role in redox reactions. More recently, it has emerged as a signaling molecule. By modulating NAD-sensing enzymes, NADcontrols hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NADlevels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NADlevels in old or diseased animals can promote health and extend lifespan, prompting a search for safe and efficacious NAD-boosting molecules that hold the promise of increasing the body's resilience, not just to one disease, but to many, thereby extending healthy human lifespan.
A functional link between NADhomeostasis and N-terminal protein acetylation in
Nicotinamide adenine dinucleotide (NAD) is an essential metabolite participating in cellular redox chemistry and signaling, and the complex regulation of NADmetabolism is not yet fully understood. To investigate this, we established a NAD-intermediate specific reporter system to identify factors required for salvage of metabolically linked nicotinamide (NAM) and nicotinic acid (NA). Mutants lacking components of the NatB complex,andappeared as hits in this screen. NatB is an N-terminal acetyltransferase responsible for acetylation of the N terminus of specific Met-retained peptides. In NatB mutants, increased NA/NAM levels were concomitant with decreased NADWe identified the vacuolar pool of nicotinamide riboside (NR) as the source of this increased NA/NAM. This NR pool is increased by nitrogen starvation, suggesting NADand related metabolites may be trafficked to the vacuole for recycling. Supporting this, increased NA/NAM release in NatB mutants was abolished by deleting the autophagy proteinWe next examined Tpm1 (tropomyosin), whose function is regulated by NatB-mediated acetylation, and Tpm1 overexpression () was shown to restore some NatB mutant defects. Interestingly, althoughlargely suppressed NA/NAM release in NatB mutants, it did not restore NADlevels. We showed that decreased nicotinamide mononucleotide adenylyltransferase (Nma1/Nma2) levels probably caused the NADdefects, andwas sufficient to restore NADNatB-mediated N-terminal acetylation of Nma1 and Nma2 appears essential for maintaining NADlevels. In summary, our results support a connection between NatB-mediated protein acetylation and NADhomeostasis. Our findings may contribute to understanding the molecular basis and regulation of NADmetabolism.
Simultaneous Measurement of NAD Metabolome in Aged Mice Tissue Using Liquid Chromatography Tandem-Mass Spectrometry (LC/MS/MS)
Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels declined with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on a LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH by performing a single sample preparation. Further, we validate our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase of in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.
NADsupplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency
Emerging findings suggest that compromised cellular bioenergetics and DNA repair contribute to the pathogenesis of Alzheimer's disease (AD), but their role in disease-defining pathology is unclear. We developed a DNA repair-deficient 3xTgAD/Polβmouse that exacerbates major features of human AD including phosphorylated Tau (pTau) pathologies, synaptic dysfunction, neuronal death, and cognitive impairment. Here we report that 3xTgAD/Polβmice have a reduced cerebral NAD/NADH ratio indicating impaired cerebral energy metabolism, which is normalized by nicotinamide riboside (NR) treatment. NR lessened pTau pathology in both 3xTgAD and 3xTgAD/Polβmice but had no impact on amyloid β peptide (Aβ) accumulation. NR-treated 3xTgAD/Polβmice exhibited reduced DNA damage, neuroinflammation, and apoptosis of hippocampal neurons and increased activity of SIRT3 in the brain. NR improved cognitive function in multiple behavioral tests and restored hippocampal synaptic plasticity in 3xTgAD mice and 3xTgAD/Polβmice. In general, the deficits between genotypes and the benefits of NR were greater in 3xTgAD/Polβmice than in 3xTgAD mice. Our findings suggest a pivotal role for cellular NADdepletion upstream of neuroinflammation, pTau, DNA damage, synaptic dysfunction, and neuronal degeneration in AD. Interventions that bolster neuronal NADlevels therefore have therapeutic potential for AD.
Deficiency of the Mitochondrial NAD Kinase Causes Stress-Induced Hepatic Steatosis in Mice
The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice.
Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy
-Myocardial metabolic impairment is a major feature in chronic heart failure (HF). As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, NADis emerging as a metabolic target in a number of diseases including HF. Little is known on mechanisms regulating homeostasis of NADin the failing heart.-To explore possible alterations of NADhomeostasis in the failing heart, we quantified expression of NADbiosynthetic enzymes in human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by SRF transcription factor depletion in the heart (SRF) or of cardiac hypertrophy triggered by transverse aorta constriction (TAC). We studied the impact of NADprecursor supplementation on cardiac function in both mouse models.-We observed a 30% loss in levels of NADin the murine failing heart of both DCM and TAC mice that was accompanied by a decrease in expression of the NAMPT enzyme that recycles the nicotinamide (NAM) precursor whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside (NR) precursor is increased, to a higher level in the DCM (40 fold) than in TAC (4 fold). This shift was also observed in human failing heart biopsies compared to non-failing controls. We show that thegene is an AMPK and PPARalpha responsive gene that is activated by energy stress and NADdepletion in isolated rat cardiomyocytes. NR efficiently rescues NADsynthesis in response to FK866-mediated inhibition of NAMPT and stimulates glycolysis in cardiomyocytes. Accordingly, we show that NR supplementation in food attenuates the development of HF in mice, more robustly in DCM, and partially after TAC, by stabilizing myocardial NADlevels in the failing heart. NR treatment also robustly increases the myocardial levels of three metabolites, nicotinic acid adenine dinucleotide, methyl-NAM and N1-Methyl-4-pyridone-5-carboxamide, which can be used as validation biomarkers for the treatment.-The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of HF notably in the context of DCM, a disease with few therapeutic options.
Nicotinamide riboside, a form of vitamin B, protects against excitotoxicity-induced axonal degeneration
NADdepletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NADwas shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NADto NR prompted us to probe the effects of NADand NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NADreduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NADMoreover, the stronger effect of NR compared to NADdepends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NADand NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NADin excitotoxicity-induced AxD and that axonal protection involves defending intracellular NADhomeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B, protects against excitotoxicity-induced axonal degeneration.
Synthesis of β-Nicotinamide Riboside Using an Efficient Two-Step Methodology
A two-step chemical method for the synthesis of β-nicotinamide riboside (NR) is described. NR has achieved wide use as an NADprecursor (vitamin B3) and can significantly increase central metabolite NADconcentrations in mammalian cells. β-NR can be prepared with an efficient two-step procedure. The synthesis is initiated via coupling of commercially available 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose with ethyl nicotinate in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf).H NMR showed that the product was formed with complete stereoselectivity to produce only the β-isomer in high yield (>90% versus starting sugar). The clean stereochemical result suggests that the coupling proceeds via a cationic cis-1,2-acyloxonium-sugar intermediate, which controls addition by nucleophiles to generate predominantly β-stereochemistry. The subsequent deprotection of esters in methanolic ammonia generates the desired product in 85% overall yield versus sugar. © 2017 by John Wiley & Sons, Inc.
Emerging Potential Benefits of Modulating NADMetabolism in Cardiovascular Disease
Nicotinamide adenine dinucleotide (NAD) and related metabolites are central mediators of fuel oxidation and bioenergetics within cardiomyocytes. Additionally, NADis required for the activity of multifunctional enzymes including sirtuins and poly(ADP-ribose) polymerases (PARPs) that regulate post-translational modifications, DNA damage responses, and calcium signaling. Recent research indicates that NADparticipates in a multitude of processes dysregulated in cardiovascular diseases. Therefore, supplementation of NADprecursors including nicotinamide riboside (NR) that boost or replete the NADmetabolome may be cardioprotective. This review examines the molecular physiology and preclinical data with respect to NADprecursors in heart failure related cardiac remodeling, ischemic/reperfusion injury, and arrhythmias. In addition, alternative NADboosting-strategies and potential systemic effects of NADsupplementation with implications on cardiovascular health and disease are surveyed.
Perturbations of NAD salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle
Nicotinamide adenine dinucleotide (NAD) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT for maintaining NADlevels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (shNampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express cre recombinase in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2C12 myoblasts, Nampt and NADlevels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55% and 2-deoxyglucose uptake increased by 25% in shNampt KD cells. Treatment with the NADprecursor nicotinamide riboside restored NADlevels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and shNampt KD cells, respectively. Expression of cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NADlevels by 38% and 43%, respectively. Glucose uptake increased by 40% and mitochondrial complex IV respiration was compromised by 20%. HIF1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known SIRT6 target, were increased in shNampt KD cells. Thus, we propose that the shift towards glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NADlevels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.
The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD⁺ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice
Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, exhibits beneficial effects on metabolic syndrome. Sustained inflammation plays a crucial role in the pathogenesis of metabolic syndrome. Here we explored the effects of PSPC on high-fat diet (HFD)-induced hepatic inflammation and the mechanisms underlying these effects. Mice were divided into four groups: Control group, HFD group, HFD + PSPC group, and PSPC group. PSPC was administered by daily oral gavage at doses of 700 mg/kg/day for 20 weeks. Nicotinamide riboside (NR) was used to increase NAD⁺ levels. Our results showed that PSPC effectively ameliorated obesity and liver injuries in HFD-fed mice. Moreover, PSPC notably blocked hepatic oxidative stress in HFD-treated mice. Furthermore, PSPC dramatically restored NAD⁺ level to abate endoplasmic reticulum stress (ER stress) in HFD-treated mouse livers, which was confirmed by NR treatment. Consequently, PSPC remarkably suppressed the nuclear factor-κB (NF-κB) p65 nuclear translocation and nucleotide oligomerization domain protein1/2 (NOD1/2) signaling in HFD-treated mouse livers. Thereby, PSPC markedly diminished the NLR family, pyrin domain containing 3 (NLRP3) inflammasome activation, ultimately lowering the expressions of inflammation-related genes in HFD-treated mouse livers. In summary, PSPC protected against HFD-induced hepatic inflammation by boosting NAD⁺ level to inhibit NLRP3 inflammasome activation.
NADDeficits in Age-Related Diseases and Cancer
The phenomenon of aging has gained widespread attention in recent times. Although significant advances have been made to better understand aging and its related pathologies including cancer, there is not yet a clear mechanism explaining why diseases and cancer are inherent parts of the aging process. Finding a unifying equation that could bridge aging and its related diseases would allow therapeutic development and solve an immense human health problem to live longer and better. In this review, we discuss NADreduction as the central mechanism that may connect aging to its related pathologies and cancer. NADboosters would ensure and ameliorate health quality during aging.
1 H NMR-based metabolomics study for identifying urinary biomarkers and perturbed metabolic pathways associated with severity of IgA nephropathy: a pilot study
The severity of IgA nephropathy (IgAN), the most common primary glomerulonephritis, is judged on the basis of histologic and clinical features. A limited number of studies have considered molecular signature of IgAN for this issue, and no reliable biomarkers have been presented non-invasively for use in patient evaluations. This study aims to identify metabolite markers excreted in the urine and impaired pathways that are associated with a known marker of severity (proteinuria) to predict mild and severe stages of IgAN. Urine samples were analysed using nuclear magnetic resonance from biopsy-proven IgAN patients at mild and severe stages. Multivariate statistical analysis and pathway analysis were performed. The most changed metabolites were acetoacetate, hypotaurine, homocysteine, L-kynurenine and phenylalanine. Nine metabolites were positively correlated with proteinuria, including mesaconic acid, trans-cinnamic acid, fumaric acid, 5-thymidylic acid, anthranilic acid, indole, deoxyguanosine triphosphate, 13-cis-retinoic acid and nicotinamide riboside, while three metabolites were negatively correlated with proteinuria including acetoacetate, hypotaurine and hexanal. 'Phenylalanine metabolism' was the most significant pathway which was impaired in severe stage in comparison to mild stage of IgAN. This study indicates that nuclear magnetic resonance is a versatile technique that is capable of detecting metabolite biomarkers in combination with advanced multivariate statistical analysis. Copyright © 2017 John Wiley & Sons, Ltd.
Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells
Augmenting nicotinamide adenine dinucleotide (NAD) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NADprecursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NADfrom NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.
Circadian and Feeding Rhythms Orchestrate the Diurnal Liver Acetylome
Lysine acetylation is involved in various biological processes and is considered a key reversible post-translational modification in the regulation of gene expression, enzyme activity, and subcellular localization. This post-translational modification is therefore highly relevant in the context of circadian biology, but its characterization on the proteome-wide scale and its circadian clock dependence are still poorly described. Here, we provide a comprehensive and rhythmic acetylome map of the mouse liver. Rhythmic acetylated proteins showed subcellular localization-specific phases that correlated with the related metabolites in the regulated pathways. Mitochondrial proteins were over-represented among the rhythmically acetylated proteins and were highly correlated with SIRT3-dependent deacetylation. SIRT3 activity being nicotinamide adenine dinucleotide (NAD)level-dependent, we show that NADis orchestrated by both feeding rhythms and the circadian clock through the NADsalvage pathway but also via the nicotinamide riboside pathway. Hence, the diurnal acetylome relies on a functional circadian clock and affects important diurnal metabolic pathways in the mouse liver.
Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet
Metabolic flexibility is the ability to switch metabolism between carbohydrate oxidation (CHO) and fatty acid oxidation (FAO) and is a biomarker for metabolic health. The effect on metabolic health of nicotinamide riboside (NR) as an exclusive source of vitamin B3 is unknown and is examined here for a wide range of NR.
Targeting NAD+ in Metabolic Disease: New Insights Into an Old Molecule
Nicotinamide adenine dinucleotide (NAD+) is an established cofactor for enzymes serving cellular metabolic reactions. More recent research identified NAD+ as a signaling molecule and substrate for sirtuins and poly-adenosine 5'-diphosphate polymerases; enzymes that regulate protein deacetylation and DNA repair, and translate changes in energy status into metabolic adaptations. Deranged NAD+ homeostasis and concurrent alterations in mitochondrial function are intrinsic in metabolic disorders, such as type 2 diabetes, nonalcoholic fatty liver, and age-related diseases. Contemporary NAD+ precursors show promise as nutraceuticals to restore target tissue NAD+ and have demonstrated the ability to improve mitochondrial function and sirtuin-dependent signaling. This review discusses the accumulating evidence for targeting NAD+ metabolism in metabolic disease, maps the different strategies for NAD+ boosting, and addresses the challenges and open questions in the field. The health potential of targeting NAD+ homeostasis will inform clinical study design to identify nutraceutical approaches for combating metabolic disease and the unwanted effects of aging.
Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease
The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown.
The NAD+/PARP1/SIRT1 Axis in Aging
NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.
Nicotinamide riboside, a form of vitamin B3 and NAD+ precursor, relieves the nociceptive and aversive dimensions of paclitaxel-induced peripheral neuropathy in female rats
Injury to sensory afferents may contribute to the peripheral neuropathies that develop after administration of chemotherapeutic agents. Manipulations that increase levels of nicotinamide adenine dinucleotide (NAD) can protect against neuronal injury. This study examined whether nicotinamide riboside (NR), a third form of vitamin B3 and precursor of NAD, diminishes tactile hypersensitivity and place escape-avoidance behaviors in a rodent model of paclitaxel-induced peripheral neuropathy. Female Sprague-Dawley rats received 3 intravenous injections of 6.6 mg/kg paclitaxel over 5 days. Daily oral administration of 200 mg/kg NR beginning 7 days before paclitaxel treatment and continuing for another 24 days prevented the development of tactile hypersensitivity and blunted place escape-avoidance behaviors. These effects were sustained after a 2-week washout period. This dose of NR increased blood levels of NAD by 50%, did not interfere with the myelosuppressive effects of paclitaxel, and did not produce adverse locomotor effects. Treatment with 200 mg/kg NR for 3 weeks after paclitaxel reversed the well-established tactile hypersensitivity in a subset of rats and blunted escape-avoidance behaviors. Pretreatment with 100 mg/kg oral acetyl-L-carnitine (ALCAR) did not prevent paclitaxel-induced tactile hypersensitivity or blunt escape-avoidance behaviors. ALCAR by itself produced tactile hypersensitivity. These findings suggest that agents that increase NAD, a critical cofactor for mitochondrial oxidative phosphorylation systems and cellular redox systems involved with fuel utilization and energy metabolism, represent a novel therapeutic approach for relief of chemotherapy-induced peripheral neuropathies. Because NR is a vitamin B3 precursor of NAD and a nutritional supplement, clinical tests of this hypothesis may be accelerated.
Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay
Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.
The NADprecursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation
NADis an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NADconsuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NADlevels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NADlevels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NADconcentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NADlevels may be limiting under conditions of genotoxic stress and that boosting the NADsystem with nicotinic acid can improve genomic stability.
Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs
RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels. The boronyl groups inside the gel form relatively stable complexes with 1,2-cis diols, occurring naturally at the 3'-end of RNA, and also in the nicotinamide riboside of NAD-modified RNA at the 5'-end. The transient formation of diesters between the immobilized boronic acid and the diols causes lower mobility of the modified RNAs, compared to unmodified RNAs, resulting in two distinct bands for one RNA sequence. We used APB affinity gel electrophoresis to preparatively purify in vitro transcribed NAD-RNA from triphosphorylated RNA, to study the enzyme kinetics of the NAD-RNA decapping enzyme NudC, and to determine the NAD modification ratios of various cellular sRNAs. In summary, APB affinity gels can be used to study cofactor-modified RNAs with low amounts of material, and to rapidly screen for their occurrence in total RNA while avoiding complex sample treatments.
Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration
The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.
Nicotinamide riboside or IL-17A signaling blockers to prevent liver disorders
NADmetabolism: Bioenergetics, signaling and manipulation for therapy
We survey the historical development of scientific knowledge surrounding Vitamin B3, and describe the active metabolite forms of Vitamin B3, the pyridine dinucleotides NADand NADPwhich are essential to cellular processes of energy metabolism, cell protection and biosynthesis. The study of NADhas become reinvigorated by new understandings that dynamics within NADmetabolism trigger major signaling processes coupled to effectors (sirtuins, PARPs, and CD38) that reprogram cellular metabolism using NADas an effector substrate. Cellular adaptations include stimulation of mitochondrial biogenesis, a process fundamental to adjusting cellular and tissue physiology to reduced nutrient availability and/or increased energy demand. Several mammalian metabolic pathways converge to NAD, including tryptophan-derived de novo pathways, nicotinamide salvage pathways, nicotinic acid salvage and nucleoside salvage pathways incorporating nicotinamide riboside and nicotinic acid riboside. Key discoveries highlight a therapeutic potential for targeting NADbiosynthetic pathways for treatment of human diseases. A recent emergence of understanding that NADhomeostasis is vulnerable to aging and disease processes has stimulated testing to determine if replenishment or augmentation of cellular or tissue NADcan have ameliorative effects on aging or disease phenotypes. This experimental approach has provided several proofs of concept successes demonstrating that replenishment or augmentation of NADconcentrations can provide ameliorative or curative benefits. Thus NADmetabolic pathways can provide key biomarkers and parameters for assessing and modulating organism health.
Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism
Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.
Emerging therapeutic roles for NAD(+) metabolism in mitochondrial and age-related disorders
Nicotinamide adenine dinucleotide (NAD(+)) is a central metabolic cofactor in eukaryotic cells that plays a critical role in regulating cellular metabolism and energy homeostasis. NAD(+) in its reduced form (i.e. NADH) serves as the primary electron donor in mitochondrial respiratory chain, which involves adenosine triphosphate production by oxidative phosphorylation. The NAD(+)/NADH ratio also regulates the activity of various metabolic pathway enzymes such as those involved in glycolysis, Kreb's cycle, and fatty acid oxidation. Intracellular NAD(+) is synthesized de novo from L-tryptophan, although its main source of synthesis is through salvage pathways from dietary niacin as precursors. NAD(+) is utilized by various proteins including sirtuins, poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases. The NAD(+) pool is thus set by a critical balance between NAD(+) biosynthetic and NAD(+) consuming pathways. Raising cellular NAD(+) content by inducing its biosynthesis or inhibiting the activity of PARP and cADP-ribose synthases via genetic or pharmacological means lead to sirtuins activation. Sirtuins modulate distinct metabolic, energetic and stress response pathways, and through their activation, NAD(+) directly links the cellular redox state with signaling and transcriptional events. NAD(+) levels decline with mitochondrial dysfunction and reduced NAD(+)/NADH ratio is implicated in mitochondrial disorders, various age-related pathologies as well as during aging. Here, I will provide an overview of the current knowledge on NAD(+) metabolism including its biosynthesis, utilization, compartmentalization and role in the regulation of metabolic homoeostasis. I will further discuss how augmenting intracellular NAD(+) content increases oxidative metabolism to prevent bioenergetic and functional decline in multiple models of mitochondrial diseases and age-related disorders, and how this knowledge could be translated to the clinic for human relevance.
Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding
The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D.
NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells
NADis a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NADprecursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NADsynthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NADsynthesis from other NADprecursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NADsynthesis, explaining the overlapping metabolic effects observed with the two compounds.
Overcoming ATM Deficiency by Activating the NAD/SIRT1 Axis
In this issue, Fang et al. (2016) show that both the DNA repair defect and mitochondrial dysfunction in ATMcells or mice are mitigated by the anti-aging compound nicotinamide riboside or a SIRT1 activator. This broad suppression by activating the NAD/SIRT1 axis may generally apply to diseases and aging maladies.
NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation
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 NADin 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 NADand are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NADlevels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NADbiosynthesis. Replenishing NADstores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utrmice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NADrepletion 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 NADmay benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.
Nicotinamide riboside is uniquely and orally bioavailable in mice and humans
Nicotinamide riboside (NR) is in wide use as an NADprecursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NADmetabolism in humans. We report that human blood NADcan rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NADwith distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NADmetabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD, is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NADrepletion.
New strategies in sport nutrition to increase exercise performance
Despite over 50 years of research, the field of sports nutrition continues to grow at a rapid rate. Whilst the traditional research focus was one that centred on strategies to maximise competition performance, emerging data in the last decade has demonstrated how both macronutrient and micronutrient availability can play a prominent role in regulating those cell signalling pathways that modulate skeletal muscle adaptations to endurance and resistance training. Nonetheless, in the context of exercise performance, it is clear that carbohydrate (but not fat) still remains king and that carefully chosen ergogenic aids (e.g. caffeine, creatine, sodium bicarbonate, beta-alanine, nitrates) can all promote performance in the correct exercise setting. In relation to exercise training, however, it is now thought that strategic periods of reduced carbohydrate and elevated dietary protein intake may enhance training adaptations whereas high carbohydrate availability and antioxidant supplementation may actually attenuate training adaptation. Emerging evidence also suggests that vitamin D may play a regulatory role in muscle regeneration and subsequent hypertrophy following damaging forms of exercise. Finally, novel compounds (albeit largely examined in rodent models) such as epicatechins, nicotinamide riboside, resveratrol, β-hydroxy β-methylbutyrate, phosphatidic acid and ursolic acid may also promote or attenuate skeletal muscle adaptations to endurance and strength training. When taken together, it is clear that sports nutrition is very much at the heart of the Olympic motto, Citius, Altius, Fortius (faster, higher, stronger).
Hepatic NAD(+) deficiency as a therapeutic target for non-alcoholic fatty liver disease in ageing
Ageing is an important risk factor of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD(+) ), a ubiquitous coenzyme, links ageing with NAFLD.
Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle
NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.
NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice
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-Dmd(mdx)/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.
CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism
Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.
Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation
Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae.
Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829
The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.
Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk
Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown.
Nicotinamide Riboside Opposes Type 2 Diabetes and Neuropathy in Mice
Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.
Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)
Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.
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 (Sirt1(hep-/-) ), 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.
Safety assessment of nicotinamide riboside, a form of vitamin B3
Nicotinamide riboside (NR) is a naturally occurring form of vitamin Bpresent in trace amounts in some foods. Like niacin, it has been shown to be a precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). The safety of Niagen™, a synthetic form of NR, was determined using a bacterial reverse mutagenesis assay (Ames), an in vitro chromosome aberration assay, an in vivo micronucleus assay, and acute, 14-day and 90-day rat toxicology studies. NR was not genotoxic. There was no mortality at an oral dose of 5000 mg/kg. Based on the results of a 14-day study, a 90-day study was performed comparing NR at 300, 1000, and 3000 mg/kg/day to an equimolar dose of nicotinamide at 1260 mg/kg/day as a positive control. Results from the study show that NR had a similar toxicity profile to nicotinamide at the highest dose tested. Target organs of toxicity were liver, kidney, ovaries, and testes. The lowest observed adverse effect level for NR was 1000 mg/kg/day, and the no observed adverse effect level was 300 mg/kg/day.
Caloric restriction and exercise "mimetics'': Ready for prime time?
Exercise and diet are powerful interventions to prevent and ameliorate various pathologies. The development of pharmacological agents that confer exercise- or caloric restriction-like phenotypic effects is thus an appealing therapeutic strategy in diseases or even when used as life-style and longevity drugs. Such so-called exercise or caloric restriction "mimetics" have so far mostly been described in pre-clinical, experimental settings with limited translation into humans. Interestingly, many of these compounds activate related signaling pathways, most often postulated to act on the common downstream effector peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in skeletal muscle. In this review, resveratrol and other exercise- and caloric restriction "mimetics" are discussed with a special focus on feasibility, chances and limitations of using such compounds in patients as well as in healthy individuals.
Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model
Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.
Nicotinamide Riboside Ameliorates Hepatic Metaflammation by Modulating NLRP3 Inflammasome in a Rodent Model of Type 2 Diabetes
Low-grade chronic inflammation (metaflammation) is a major contributing factor for the onset and development of metabolic diseases, such as type 2 diabetes, obesity, and cardiovascular disease. Nicotinamide riboside (NR), which is present in milk and beer, is a functional vitamin B3 having advantageous effects on metabolic regulation. However, the anti-inflammatory capacity of NR is unknown. This study evaluated whether NR modulates hepatic nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Male, 8-week-old KK/HlJ mice were allocated to the control or NR group. NR (100 mg/kg/day) or vehicle (phosphate-buffered saline) was administrated by an osmotic pump for 7 days. Glucose control, lipid profiles, NLRP3 inflammasome, and inflammation markers were analyzed, and structural and histological analyses were conducted. NR treatment did not affect body weight gain, food intake, and liver function. Glucose control based on the oral glucose tolerance test and levels of serum insulin and adiponectin was improved by NR treatment. Among tested lipid profiles, NR lowered the total cholesterol concentration in the liver. Histological and structural analysis by hematoxylin and eosin staining and transmission electron microscopy, respectively, showed that NR rescued the disrupted cellular integrity of the mitochondria and nucleus in the livers of obese and diabetic KK mice. In addition, NR treatment significantly improved hepatic proinflammatory markers, including tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1. These ameliorations were accompanied by significant shifts of NLRP3 inflammasome components (NLRP3, ASC, and caspase1). These results demonstrate that NR attenuates hepatic metaflammation by modulating the NLRP3 inflammasome.
Ketosis may promote brain macroautophagy by activating Sirt1 and hypoxia-inducible factor-1
Ketogenic diets are markedly neuroprotective, but the basis of this effect is still poorly understood. Recent studies demonstrate that ketone bodies increase neuronal levels of hypoxia-inducible factor-1α (HIF-1α), possibly owing to succinate-mediated inhibition of prolyl hydroxylase activity. Moreover, there is reason to suspect that ketones can activate Sirt1 in neurons, in part by increasing cytoplasmic and nuclear levels of Sirt1's obligate cofactor NAD(+). Another recent study has observed reduced activity of mTORC1 in the hippocampus of rats fed a ketogenic diet - an effect plausibly attributable to Sirt1 activation. Increased activities of HIF-1 and Sirt1, and a decrease in mTORC1 activity, could be expected to collaborate in the induction of neuronal macroautophagy. Considerable evidence points to moderate up-regulation of neuronal autophagy as a rational strategy for prevention of neurodegenerative disorders; elimination of damaged mitochondria that overproduce superoxide, as well as clearance of protein aggregates that mediate neurodegeneration, presumably contribute to this protection. Hence, autophagy may mediate some of the neuroprotective benefits of ketogenic diets. Brain-permeable agents which activate AMP-activated kinase, such as metformin and berberine, as well as the Sirt1 activator nicotinamide riboside, can also boost neuronal autophagy, and may have potential for amplifying the impact of ketogenesis on this process. Since it might not be practical for most people to adhere to ketogenic diets continuously, alternative strategies are needed to harness the brain-protective potential of ketone bodies. These may include ingestion of medium-chain triglycerides or coconut oil, intermittent ketogenic dieting, and possibly the use of supplements that promote hepatic ketogenesis - notably carnitine and hydroxycitrate - in conjunction with dietary regimens characterized by long daily episodes of fasting or carbohydrate avoidance.
Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae
NAD is a necessary cofactor present in all living cells. Some bacteria cannot de novo synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in Streptococcus pneumoniae, specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in Str. pneumoniae TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a pnuC mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host.
Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells
NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.
Fasting and refeeding differentially regulate NLRP3 inflammasome activation in human subjects
Activation of the NLRP3 inflammasome is associated with metabolic dysfunction, and intermittent fasting has been shown to improve clinical presentation of NLRP3 inflammasome-linked diseases. As mitochondrial perturbations, which function as a damage-associated molecular pattern, exacerbate NLRP3 inflammasome activation, we investigated whether fasting blunts inflammasome activation via sirtuin-mediated augmentation of mitochondrial integrity.
Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart
Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.
Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters
Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.
Regulation of NAD biosynthetic enzymes modulates NAD-sensing processes to shape mammalian cell physiology under varying biological cues
In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NADhomeostasis and contributes to longevity
Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast,, and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD(nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NADprecursor) homeostasis. Maintaining adequate NADpools has been shown to play key roles in life span extension, but factors regulating NADmetabolism and homeostasis are not completely understood. Recently, NADmetabolism was also linked to the phosphate (Pi)-sensingpathway in yeast. Canonicalactivation requires Pi-starvation. Interestingly, NADdepletion without Pi-starvation was sufficient to induceactivation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel withsignaling components to regulate NR/NADhomeostasis. These studies suggest that NADmetabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of, PKA, TOR and Sch9 pathways was reported to potentially affect NADmetabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NADhomeostasis, and cross-talk among nutrient sensing pathways and NADhomeostasis.
Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts
Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement.
Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD+ homeostasis in Saccharomyces cerevisiae
Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD(+) homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD(+) homeostasis, partially ameliorating the NAD(+) deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD(+) homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD(+) metabolism, cellular life span, and diseases associated with NAD(+) deficiency and aging.
Calorie restriction-mediated replicative lifespan extension in yeast is non-cell autonomous
In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR) allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.
Reversing neurodegenerative hearing loss
Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism
The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.
Activation of SIRT3 by the NAD⁺ precursor nicotinamide riboside protects from noise-induced hearing loss
Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD(+)) exhibits axon-protective effects in cultured neurons; however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD(+) levels in mice by administering nicotinamide riboside (NR), a recently described NAD(+) precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD(+)-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD(+) biosynthetic enzymes. These findings reveal that administration of NR activates a NAD(+)-SIRT3 pathway that reduces neurite degeneration caused by noise exposure.
Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage
Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC.
Boosting NAD to spare hearing
Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3.
A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157
hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).
Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homolog
PnuC transporters catalyze cellular uptake of the NAD+ precursor nicotinamide riboside (NR) and belong to a large superfamily that includes the SWEET sugar transporters. We present a crystal structure of Neisseria mucosa PnuC, which adopts a highly symmetrical fold with 3+1+3 membrane topology not previously observed in any protein. The high symmetry of PnuC with a single NR bound in the center suggests a simple alternating-access translocation mechanism.
Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells
The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.
Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae
Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD(+) homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability.
NAD+ and sirtuins in aging and disease
Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.
Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3
Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy.
NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease
Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans.
Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content
NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.
Identification of yeast and human 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) transporters
5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation.
Identification of a gene set to evaluate the potential effects of loud sounds from seismic surveys on the ears of fishes: a study with Salmo salar
Functional genomic studies were carried out on the inner ear of Atlantic salmon Salmo salar following exposure to a seismic airgun. Microarray analyses revealed 79 unique transcripts (passing background threshold), with 42 reproducibly up-regulated and 37 reproducibly down-regulated in exposed v. control fish. Regarding the potential effects on cellular energetics and cellular respiration, altered transcripts included those with roles in oxygen transport, the glycolytic pathway, the Krebs cycle and the electron transport chain. Of these, a number of transcripts encoding haemoglobins that are important in oxygen transport were up-regulated and among the most highly expressed. Up-regulation of transcripts encoding nicotinamide riboside kinase 2, which is also important in energy production and linked to nerve cell damage, points to evidence of neuronal damage in the ear following noise exposure. Transcripts related to protein modification or degradation also indicated potential damaging effects of sound on ear tissues. Notable in this regard were transcripts associated with the proteasome-ubiquitin pathway, which is involved in protein degradation, with the transcript encoding ubiquitin family domain-containing protein 1 displaying the highest response to exposure. The differential expression of transcripts observed for some immune responses could potentially be linked to the rupture of cell membranes. Meanwhile, the altered expression of transcripts for cytoskeletal proteins that contribute to the structural integrity of the inner ear could point to repair or regeneration of ear tissues including auditory hair cells. Regarding potential effects on hormones and vitamins, the protein carrier for thyroxine and retinol (vitamin A), namely transthyretin, was altered at the transcript expression level and it has been suggested from studies in mammalian systems that retinoic acid may play a role in the regeneration of damaged hair cells. The microarray experiment identified the transcript encoding growth hormone I as up-regulated by loud sound, supporting previous evidence linking growth hormone to hair cell regeneration in fishes. Quantitative (q) reverse transcription (RT) polymerase chain reaction (qRT-PCR) analyses confirmed dysregulation of some microarray-identified transcripts and in some cases revealed a high level of biological variability in the exposed group. These results support the potential utility of molecular biomarkers to evaluate the effect of seismic surveys on fishes with studies on the ears being placed in a priority category for development of exposure-response relationships. Knowledge of such relationships is necessary for addressing the question of potential size of injury zones.
Salvaging hope: Is increasing NAD(+) a key to treating mitochondrial myopathy?
Mitochondrial diseases can arise from mutations either in mitochondrial DNA or in nuclear DNA encoding mitochondrially destined proteins. Currently, there is no cure for these diseases although treatments to ameliorate a subset of the symptoms are being developed. In this issue of EMBO Molecular Medicine, Khan et al (2014) use a mouse model to test the efficacy of a simple dietary supplement of nicotinamide riboside to treat and prevent mitochondrial myopathies.
YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae
NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD(+) metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD(+) levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD(+) homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.
Partial reversal of skeletal muscle aging by restoration of normal NAD⁺ levels
That some aging-associated phenotypes may be reversible is an emerging theme in contemporary aging research. Gomes et al. report that age-associated oxidative phosphorylation (OXPHOS) defects in murine skeletal muscle are biphasic. In the first phase, OXPHOS is decreased because of reduced expression of mitochondrially encoded genes. Treatment of moderately old mice (first-phase OXPHOS defects) with nicotinamide adenine dinucleotide (NAD⁺) precursor nicotinamide mononucleotide (NMN) for 1 week restores oxidative phosphorylation activity and other markers of mitochondrial function in skeletal muscle. However, muscle strength is not restored. In very old animals (second-phase OXPHOS defects), expression of OXPHOS genes from both the nucleus and mitochondria is reduced and mitochondrial DNA integrity is diminished. Gomes et al. propose a model linking decreased NAD⁺ to loss of nuclear SIRT1 activity to stabilization of the hypoxia-associated transcription factor hypoxia-inducible factor 1-alpha (HIF-1a). HIF-1a promotes an hypoxic-like (Warburg effect) state in the cell. The HIF-1a protein interacts with c-Myc, decreasing c-Myc-regulated transcription of the key mitochondrial regulator mitochondrial transcription factor A (TFAM). Low levels of TFAM lead to first-phase OXPHOS dysfunction. The transition to irreversible phase 2 dysfunction remains to be characterized, but may be related to increased reactive oxygen species (ROS) production. This model suggests that intervention in mitochondrial aging may be possible using appropriate NAD⁺ precursors such as nicotinamide riboside. Restoring NAD⁺ levels may be beneficial throughout the organism. For example, aging-associated disturbances in circadian rhythm are linked to diminished SIRT1 activity, and loss of hematopoietic stem cell function to reduced SIRT3. Work to elucidate other biphasic aging mechanisms is strongly encouraged.
New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD(+) salvage from nicotinamide
In an effort to reconstitute the NAD(+) synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD(+)de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD(+) de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD(+) metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD(+) biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD(+) salvage pathway from nicotinamide.
Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection
This review focuses upon the biology and metabolism of a trace component in foods called nicotinamide riboside. Nicotinamide riboside is a precursor of nicotinamide adenine dinucleotide (NAD), and is a source of Vitamin B3. Evidence indicates that nicotinamide riboside has unique properties as a Vitamin B3. We review knowledge of the metabolism of this substance, as well as recent work suggesting novel health benefits that might be associated with nicotinamide riboside taken in larger quantities than is found naturally in foods.
CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells
NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.
Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models
Nicotinamide adenine dinucleotide (NAD)(+), a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD(+) expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer's disease (AD). Nicotinamide riboside (NR) is a NAD(+) precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD(+) in the cerebral cortex; (2) application of NR to hippocampal slices (10 μM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting PGC-1α-mediated BACE1 ubiquitination and degradation, thus preventing Aβ production in the brain.
Identification of the NAD(P)H binding site of eukaryotic UDP-galactopyranose mutase
UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.
The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity
As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.
Crystal structure of perakine reductase, founding member of a novel aldo-keto reductase (AKR) subfamily that undergoes unique conformational changes during NADPH binding
Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His(6)-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His(6)-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α(8)/β(6) barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family.
The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism
Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.
Insights into the redox cycle of human quinone reductase 2
NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H(2)O(2), and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.
The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation
NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.
4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?
We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.
Crystal structure of human nicotinic acid phosphoribosyltransferase
Nicotinic acid phosphoribosyltransferase (EC 126.96.36.199) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent.
The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia
The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism.
An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers
The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels.
Boosting NAD(+) for the prevention and treatment of liver cancer
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide yet has limited therapeutic options. We recently demonstrated that inhibition of de novo nicotinamide adenine dinucleotide (NAD(+)) synthesis is responsible for DNA damage, thereby initiating hepatocarcinogenesis. We propose that boosting NAD(+) levels might be used as a prophylactic or therapeutic approach in HCC.
The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats
Nicotinamide adenine dinucleotide (NAD(+)) and its phosphorylated form (NADP(+)) are key molecules in ubiquitous bioenergetic and cellular signaling pathways, regulating cellular metabolism and homeostasis. Thus, supplementation with NAD(+) and NADP(+) precursors emerged as a promising strategy to gain many and multifaceted health benefits. In this proof-of-concept study, we sought to investigate whether chronic nicotinamide riboside administration (an NAD(+) precursor) affects exercise performance.
NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy
Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.
Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NADlevels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study
NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NADprecursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NADin whole blood demonstrated that NRPT significantly increases the concentration of NADin a dose-dependent manner. NADlevels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NADlevels was sustained throughout the entire 8-week trial. NADlevels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NADlevels sustainably.
Regulatory Effects of NADMetabolic Pathways on Sirtuin Activity
NADacts as a crucial regulator of cell physiology and as an integral participant in cellular metabolism. By virtue of a variety of signaling activities this central metabolite can exert profound effects on organism health status. Thus, while it serves as a well-known metabolic cofactor functioning as a redox-active substrate, it can also function as a substrate for signaling enzymes, such as sirtuins, poly (ADP-ribosyl) polymerases, mono (ADP-ribosyl) transferases, and CD38. Sirtuins function as NAD-dependent protein deacetylases (deacylases) and catalyze the reaction of NADwith acyllysine groups to remove the acyl modification from substrate proteins. This deacetylation provides a regulatory function and integrates cellular NADmetabolism into a large spectrum of cellular processes and outcomes, such as cell metabolism, cell survival, cell cycle, apoptosis, DNA repair, mitochondrial homeostasis and mitochondrial biogenesis, and even lifespan. Increased attention to how regulated and pharmacologic changes in NADconcentrations can impact sirtuin activities has motivated openings of new areas of research, including investigations of how NADlevels are regulated at the subcellular level, and searches for more potent NADprecursors typified by nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). This review describes current results and thinking of how NADmetabolic pathways regulate sirtuin activities and how regulated NADlevels can impact cell physiology. In addition, NADprecursors are discussed, with attention to how these might be harnessed to generate novel therapeutic options to treat the diseases of aging.
Mitochondrial protein acetylation as a cell-intrinsic, evolutionary driver of fat storage: chemical and metabolic logic of acetyl-lysine modifications
Hormone systems evolved over 500 million years of animal natural history to motivate feeding behavior and convert excess calories to fat. These systems produced vertebrates, including humans, who are famine-resistant but sensitive to obesity in environments of persistent overnutrition. We looked for cell-intrinsic metabolic features, which might have been subject to an evolutionary drive favoring lipogenesis. Mitochondrial protein acetylation appears to be such a system. Because mitochondrial acetyl-coA is the central mediator of fuel oxidation and is saturable, this metabolite is postulated to be the fundamental indicator of energy excess, which imprints a memory of nutritional imbalances by covalent modification. Fungal and invertebrate mitochondria have highly acetylated mitochondrial proteomes without an apparent mitochondrially targeted protein lysine acetyltransferase. Thus, mitochondrial acetylation is hypothesized to have evolved as a nonenzymatic phenomenon. Because the pKa of a nonperturbed Lys is 10.4 and linkage of a carbonyl carbon to an ε amino group cannot be formed with a protonated Lys, we hypothesize that acetylation occurs on residues with depressed pKa values, accounting for the propensity of acetylation to hit active sites and suggesting that regulatory Lys residues may have been under selective pressure to avoid or attract acetylation throughout animal evolution. In addition, a shortage of mitochondrial oxaloacetate under ketotic conditions can explain why macronutrient insufficiency also produces mitochondrial hyperacetylation. Reduced mitochondrial activity during times of overnutrition and undernutrition would improve fitness by virtue of resource conservation. Micronutrient insufficiency is predicted to exacerbate mitochondrial hyperacetylation. Nicotinamide riboside and Sirt3 activity are predicted to relieve mitochondrial inhibition.