Published Research on NR

New insights into the effects of NR on health and longevity are only beginning to be realized, with exciting findings publishing regularly in the scientific literature. These findings continually advance our knowledge of the potential effects and health benefits of maintaining healthy NAD levels through diet and supplementation with NR.

Below you will find the most recent published research on NR directly from PubMed. If you click on the links provided you will be directed outside of AboutNAD.com.

NAD precursor increases aerobic performance in mice
Crisol BM, Veiga CB, Braga RR, Lenhare L, Baptista IL, Gaspar RC, Muñoz VR, Cordeiro AV, da Silva ASR, Cintra DE, Moura LP, Pauli JR and Ropelle ER
Nicotinamide riboside (NR) acts as a potent NAD precursor and improves mitochondrial oxidative capacity and mitochondrial biogenesis in several organisms. However, the effects of NR supplementation on aerobic performance remain unclear. Here, we evaluated the effects of NR supplementation on the muscle metabolism and aerobic capacity of sedentary and trained mice.
Combined Treatment with L-Carnitine and Nicotinamide Riboside Improves Hepatic Metabolism and Attenuates Obesity and Liver Steatosis
Salic K, Gart E, Seidel F, Verschuren L, Caspers M, van Duyvenvoorde W, Wong KE, Keijer J, Bobeldijk-Pastorova I, Wielinga PY and Kleemann R
Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for β-oxidation and the TCA cycle, respectively. Ldlr -/-.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% /), nicotinamide riboside (NR; 0.3% /) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (-17%) and hepatic steatosis (-22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.
Genetic evidence for vitamin B3 acquisition from rice cells
Wilson RA, Fernandez J, Rocha RO, Marroquin-Guzman M and Wright JD
Following penetration, the devastating rice blast fungus , like some other important eukaryotic phytopathogens, grows in intimate contact with living plant cells before causing disease. Cell-to-cell growth during this biotrophic growth stage must involve nutrient acquisition, but experimental evidence for the internalization and metabolism of host-derived compounds is exceedingly sparse. This striking gap in our knowledge of the infection process undermines accurate conceptualization of the plant-fungal interaction. Here, through our general interest in metabolism and with a specific focus on the signalling and redox cofactor nicotinamide adenine dinucleotide (NAD), we deleted the gene encoding quinolinate phosphoribosyltransferase, catalyst of the last step in NAD biosynthesis from tryptophan. We show how is essential for axenic growth on minimal media lacking nicotinic acid (NA, an importable NAD precursor). However, Δ mutant strains were fully pathogenic, indicating NAD biosynthesis is dispensable for lesion expansion following invasive hyphal growth in leaf tissue. Because overcoming the loss of NAD biosynthesis can only occur if importable NAD precursors (which solely comprise the NA, nicotinamide and nicotinamide riboside forms of vitamin B3) are accessible, we unexpectedly but unequivocally demonstrate that vitamin B3 can be acquired from the host and assimilated into metabolism during growth in rice cells. Our results furnish a rare, experimentally determined example of host nutrient acquisition by a fungal plant pathogen and are significant in expanding our knowledge of events at the plant-fungus metabolic interface.
Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis
Pham TX, Bae M, Kim MB, Lee Y, Hu S, Kang H, Park YK and Lee JY
Liver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis.
A ribose-functionalized NAD with unexpected high activity and selectivity for protein poly-ADP-ribosylation
Zhang XN, Cheng Q, Chen J, Lam AT, Lu Y, Dai Z, Pei H, Evdokimov NM, Louie SG and Zhang Y
Nicotinamide adenine dinucleotide (NAD)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD analogue with high efficiency and yields. Notably, the generated 3'-azido NAD exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD provides an important tool for studying cellular PARylation.
Low Cellular NAD Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes
Yang K, Lauritzen KH, Olsen MB, Dahl TB, Ranheim T, Ahmed MS, Attramadal H, Aukrust P, Halvorsen B, Nyman TA, Sandanger Ø and Yndestad A
NAD is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD enhanced IL-1β release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD depletion, specifically TAK1, IKKβ, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.
Mitochondrial function in liver cells is resistant to perturbations in NAD salvage capacity
Dall M, Trammell SAJ, Asping M, Hassing AS, Agerholm M, Vienberg SG, Gillum MP, Larsen S and Treebak JT
Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD levels, but the metabolic phenotype of deficient hepatocytes in mice is unknown. Here, we assessed role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific knockout (HNKO) mice, having a 50% reduction of liver NAD levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD, suggesting that the mitochondrial NAD pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [N]glutamine, HNKO hepatocytes had a higher [N]NAD enrichment than WT hepatocytes, indicating that HNKO mice compensate through NAD synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD levels and that the knockout has only minor consequences for mitochondrial function in the mouse liver.
Roads to the Fountain of Youth? Rejuvenating Intestinal Stem Cells
Larrick JW and Mendelsohn AR
The intestinal stem cells (ISCs) of old mice and humans exhibit a reduced capacity for regeneration and repair. Compromised intestinal function may play a key role in systemic aging-related changes: not only in the affected gut, but also in the nervous and cardiovascular systems. For example, progression of age-related neurodegenerative diseases such as Alzheimer's and Parkinson's has been linked to increased inflammation from gut microbiota in old mammals, which, in turn, may be linked bidirectionally with reduced ISC function. Intestinal organoid formation has been used to dissect the mechanisms of decline of ISC function. Alterations of the Wnt pathway, including downregulation of Wnt ligands in ISCs and upregulation of Wnt ligand inhibitor Notum in Paneth cells, and dysregulation of mTORC1 contribute to the observed age-related decline. Short-term fasting, caloric restriction, and peroxisome proliferator-activated receptor delta agonists have been reported to increase ISC function in adult mice. Moreover, the mTOR inhibitor rapamycin, NAD+ precursor nicotinamide riboside, and ABC99, a small molecule Notum inhibitor, have all been reported to rejuvenate ISC function in old mice and thus may have promise in humans. However, there is some controversy over the key mechanisms involved in loss of function of ISCs, which likely results, in part, from differences in how the organoid assays are performed. Moreover, how the microbiome modulates the function of ISCs and vice versa remains to be elucidated.
Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures
Elhassan YS, Kluckova K, Fletcher RS, Schmidt MS, Garten A, Doig CL, Cartwright DM, Oakey L, Burley CV, Jenkinson N, Wilson M, Lucas SJE, Akerman I, Seabright A, Lai YC, Tennant DA, Nightingale P, Wallis GA, Manolopoulos KN, Brenner C, Philp A and Lavery GG
Nicotinamide adenine dinucleotide (NAD) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.
Effects of nicotinamide riboside on endocrine pancreatic function and incretin hormones in obese, non-diabetic men
Dollerup OL, Trammell SAJ, Hartmann B, Holst JJ, Christensen B, Møller N, Gillum MP, Treebak JT and Jessen N
Augmenting NAD+ metabolism through dietary provision of NAD+ precursor vitamins translate to improved glucose handling in rodent models of obesity and diabetes. Preclinical evidence suggests that the NAD+/SIRT1 axis may be implicated in modulating important gut-related aspects of glucose regulation. We aimed to test if NAD+ precursor supplementation with nicotinamide riboside (NR) affects β-cell function, α-cell function, and incretin hormone secretion as well as circulating bile acids levels in humans.
Nicotinamide adenine dinucleotide emerges as a therapeutic target in aging and ischemic conditions
Hosseini L, Vafaee MS, Mahmoudi J and Badalzadeh R
Nicotinamide adenine dinucleotide (NAD) has been described as central coenzyme of redox reactions and is a key regulator of stress resistance and longevity. Aging is a multifactorial and irreversible process that is characterized by a gradual diminution in physiological functions in an organism over time, leading to development of age-associated pathologies and eventually increasing the probability of death. Ischemia is the lack of nutritive blood flow that causes damage and mortality that mostly occurs in various organs during aging. During the process of aging and related ischemic conditions, NAD levels decline and lead to nuclear and mitochondrial dysfunctions, resulting in age-related pathologies. The majority of studies have shown that restoring of NAD using supplementation with intermediates such as nicotinamide mononucleotide and nicotinamide riboside can be a valuable strategy for recovery of ischemic injury and age-associated defects. This review summarizes the molecular mechanisms responsible for the reduction in NAD levels during ischemic disorders and aging, as well as a particular focus is given to the recent progress in the understanding of NAD precursor's effects on aging and ischemia.
Subcellular characterization of NAD+ biosynthesis in metastatic melanoma by using organelle-specific biosensors
Gaudino F, Manfredonia I, Managò A, Audrito V, Raffaelli N, Vaisitti T and Deaglio S
NAD+ plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described, genetically-encoded NAD+ biosensor (LigA-cpVENUS), which was targeted to the cytosol, mitochondria and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM).
Supplementation with Nicotinamide Riboside Reduces Brain Inflammation and Improves Cognitive Function in Diabetic Mice
Lee HJ and Yang SJ
The purpose of this study is to investigate whether nicotinamide riboside (NR) can improve inflammation and cognitive function in diabetic mice. ICR male mice were fed for 14 weeks with either high-fat chow diet (HF, 60% kcal fat) or standard chow diet (CON, 10% kcal fat). HF, streptozotocin, and nicotinamide were used to induce hyperglycemia. NR or vehicle was delivered via stomach gavage for six weeks. Oral glucose tolerance test, Y-maze test, and nest construction test were conducted before and after the NR treatment period. NR treatment induced down-regulation of NLRP3, ASC, and caspase-1. NR reduced IL-1 expression significantly by 50% in whole brains of hyperglycemic mice. Other inflammatory markers including TNF-α and IL-6 were also attenuated by NR. Brain expression of amyloid-β precursor protein and presenilin 1 were reduced by NR. In addition, NR induced significant reduction of amyloid-β in whole brains of diabetic mice. NR treatment restored hyperglycemia-induced increases in brain karyopyknosis to the levels of controls. Nest construction test showed that NR improved hippocampus functions. Spatial recognition memory and locomotor activity were also improved by NR supplementation. These findings suggest that NR may be useful for treating cognitive impairment by inhibiting amyloidogenesis and neuroinflammation.
Functions of aryl hydrocarbon receptor (AHR) and CD38 in NAD metabolism and nonalcoholic steatohepatitis (NASH)
Bock KW
Aryl hydrocarbon receptor (AHR), identified in studies of dioxin toxicity, has been characterized as ligand-activated transcription factor involved in diverse functions including microbial defense, cell proliferation, immunity and NAD metabolism. AHR targets of the latter function are PARPs/ARTs and CD38 that are regulating glucose and lipid metabolism via NAD-dependent sirtuins. Deregulation of these pathways may facilitate obesity and age-dependent pathologies. The present commentary is focused on AHR and CD38 signaling in liver. CD38 is functioning as ectoNADase and Ca mobilizing enzyme in endoplasmic reticulum and endolysosomal membranes. Deregulation of TCDD-activated AHR and CD38 may facilitate hepatic steatosis and inflammation. However, these proteins are also involved in protection against inflammation and CD38-mediated age-related decreased NAD levels that may be responsible for neurodegeneration. Further knowledge about the complexity of these pathways is needed to avoid pathologies. Therapeutic modulation of AHR and CD38 remains a challenging task.
The Potential Use of Metabolic Cofactors in Treatment of NAFLD
Mardinoglu A, Ural D, Zeybel M, Yuksel HH, Uhlén M and Borén J
Non-alcoholic fatty liver disease (NAFLD) is caused by the imbalance between lipid deposition and lipid removal from the liver, and its global prevalence continues to increase dramatically. NAFLD encompasses a spectrum of pathological conditions including simple steatosis and non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and liver cancer. Even though there is a multi-disciplinary effort for development of a treatment strategy for NAFLD, there is not an approved effective medication available. Single or combined metabolic cofactors can be supplemented to boost the metabolic processes altered in NAFLD. Here, we review the dosage and usage of metabolic cofactors including l-carnitine, Nicotinamide riboside (NR), l-serine, and -acetyl-l-cysteine (NAC) in human clinical studies to improve the altered biological functions associated with different human diseases. We also discuss the potential use of these substances in treatment of NAFLD and other metabolic diseases including neurodegenerative and cardiovascular diseases of which pathogenesis is linked to mitochondrial dysfunction.
Targeting NAMPT as a therapeutic strategy against stroke
Wang SN and Miao CY
Stroke is the second and the leading most common cause of death in the world and China, respectively, but with few effective therapies. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) salvage synthesis in mammals, thereby influencing NAD-dependent enzymes and constituting a strong endogenous defence system against various stresses. Accumulating in-vitro and in-vivo studies have demonstrated the neuroprotective effect of NAMPT in stroke. Here, we review the direct evidence of NAMPT as a promising target against stroke from five potential therapeutic strategies, including NAMPT overexpression, recombinant NAMPT, NAMPT activators, NAMPT enzymatic product nicotinamide mononucleotide (NMN), and NMN precursors nicotinamide riboside and nicotinamide, and describe the relevant mechanisms and limitations, providing a promising choice for developing novel and effective therapeutic interventions against ischaemic and haemorrhagic stroke.
Safety and Metabolism of Long-term Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy Overweight Adults
Conze D, Brenner C and Kruger CL
Nicotinamide riboside (NR) is a newly discovered nicotinamide adenine dinucleotide (NAD) precursor vitamin. A crystal form of NR chloride termed NIAGEN is generally recognized as safe (GRAS) for use in foods and the subject of two New Dietary Ingredient Notifications for use in dietary supplements. To evaluate the kinetics and dose-dependency of NR oral availability and safety in overweight, but otherwise healthy men and women, an 8-week randomized, double-blind, placebo-controlled clinical trial was conducted. Consumption of 100, 300 and 1000 mg NR dose-dependently and significantly increased whole blood NAD (i.e., 22%, 51% and 142%) and other NAD metabolites within 2 weeks. The increases were maintained throughout the remainder of the study. There were no reports of flushing and no significant differences in adverse events between the NR and placebo-treated groups or between groups at different NR doses. NR also did not elevate low density lipoprotein cholesterol or dysregulate 1-carbon metabolism. Together these data support the development of a tolerable upper intake limit for NR based on human data.
Nicotinamide riboside promotes autolysosome clearance in preventing doxorubicin-induced cardiotoxicity
Zheng D, Zhang Y, Zheng M, Cao T, Wang G, Zhang L, Ni R, Brockman J, Zhong H, Fan GC and Peng T
Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD), prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.
Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in d2-mdx mice
Spaulding HR, Quindry T, Hammer K, Quindry JC and Selsby JT
Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy, hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice, therefore it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-month-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed following treatment with Q, NR, and/or Lis for 7-months. To mimic typical pharmacology of DMD patients a group was treated with prednisolone (Pred) in combination with Q, NR and Lis. At 11-months of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, while fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest treatment with Q, NR, Lis and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage.
Aerobic and resistance exercise training reverses age-dependent decline in NAD salvage capacity in human skeletal muscle
de Guia RM, Agerholm M, Nielsen TS, Consitt LA, Søgaard D, Helge JW, Larsen S, Brandauer J, Houmard JA and Treebak JT
Aging decreases skeletal muscle mass and strength, but aerobic and resistance exercise training maintains skeletal muscle function. NAD is a coenzyme for ATP production and a required substrate for enzymes regulating cellular homeostasis. In skeletal muscle, NAD is mainly generated by the NAD salvage pathway in which nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting. NAMPT decreases with age in human skeletal muscle, and aerobic exercise training increases NAMPT levels in young men. However, whether distinct modes of exercise training increase NAMPT levels in both young and old people is unknown. We assessed the effects of 12 weeks of aerobic and resistance exercise training on skeletal muscle abundance of NAMPT, nicotinamide riboside kinase 2 (NRK2), and nicotinamide mononucleotide adenylyltransferase (NMNAT) 1 and 3 in young (≤35 years) and older (≥55 years) individuals. NAMPT in skeletal muscle correlated negatively with age (r  = 0.297, P < 0.001, n = 57), and VO peak was the best predictor of NAMPT levels. Moreover, aerobic exercise training increased NAMPT abundance 12% and 28% in young and older individuals, respectively, whereas resistance exercise training increased NAMPT abundance 25% and 30% in young and in older individuals, respectively. None of the other proteins changed with exercise training. In a separate cohort of young and old people, levels of NAMPT, NRK1, and NMNAT1/2 in abdominal subcutaneous adipose tissue were not affected by either age or 6 weeks of high-intensity interval training. Collectively, exercise training reverses the age-dependent decline in skeletal muscle NAMPT abundance, and our findings highlight the value of exercise training in ameliorating age-associated deterioration of skeletal muscle function.
NAD metabolites interfere with proliferation and functional properties of THP-1 cells
Petin K, Weiss R, Müller G, Garten A, Grahnert A, Sack U and Hauschildt S
Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.
Dihydronicotinamide riboside is a potent NAD concentration enhancer and
Yang Y, Mohammed FS, Zhang N and Sauve AA
Interest in pharmacological agents capable of increasing cellular NAD concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD concentrations by 2.5-10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD increases at equivalent concentrations. NRH also provides substantial NAD increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD consumption, suggesting that it acts as a biochemical precursor to NAD Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD and a potent pharmacologic agent for NAD concentration enhancement in cells and tissues.
Chemoenzymatic Preparation of 4'-Thioribose NAD
Zhang XN, Dai Z, Cheng Q and Zhang Y
This chemoenzymatic procedure describes a strategy for the preparation of 4'-thioribose nicotinamide adenine dinucleotide (S-NAD ), including chemical synthesis of nicotinamide 4'-riboside (S-NR), recombinant expression and purification of two NAD biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S-NAD . The first basic protocol describes the procedures for introduction of nicotinamide onto 4'-thioribose and subsequent deprotection to generate S-NR as the key intermediate for enzymatically synthesizing S-NAD . In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S-NR to S-NAD . The third basic protocol presents the enzymatic approach for the generation of S-NAD from S-NR precursor. © 2019 by John Wiley & Sons, Inc.
Underpowered or negative? A crucial distinction
Dollerup OL, Treebak JT and Jessen N
Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1
Harlan BA, Pehar M, Killoy KM and Vargas MR
Sirtuins (SIRTs) are NAD-dependent deacylases that play a key role in transcription, DNA repair, metabolism, and oxidative stress resistance. Increasing NAD availability regulates endogenous SIRT activity, leading to increased resistance to oxidative stress and decreased mitochondrial reactive oxygen production in multiple cell types and disease models. This protection, at least in part, depends on the activation of antioxidant mitochondrial proteins. We now show that increasing total NAD content in astrocytes leads to the activation of the transcription factor nuclear factor, erythroid-derived 2, like 2 (Nfe2l2 or Nrf2) and up-regulation of the antioxidant proteins heme oxygenase 1 (HO-1) and sulfiredoxin 1 (SRXN1). Nrf2 activation also occurs as a result of SIRT6 overexpression. Mutations in Cu-Zn superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS). Astrocytes isolated from mutant human SOD1-overexpressing mice induce motor neuron death in coculture. Treatment with nicotinamide mononucleotide or nicotinamide riboside increases total NAD content in ALS astrocytes and abrogates their toxicity toward cocultured motor neurons. The observed neuroprotection depends on SIRT6 expression in astrocytes. Moreover, overexpression of SIRT6 in astrocytes by itself abrogates the neurotoxic phenotype of ALS astrocytes. Our results identify SIRT6 as a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.-Harlan, B. A., Pehar, M., Killoy, K. M., Vargas, M. R. Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1.
NAD supplementation rejuvenates aged gut adult stem cells
Igarashi M, Miura M, Williams E, Jaksch F, Kadowaki T, Yamauchi T and Guarente L
The tissue decline due to aging is associated with the deterioration of adult stem cell function. Here we show the number and proliferative activity of intestinal stem cells (ISCs) but not Paneth cells decline during aging, as does ISC function assessed ex vivo. Levels of SIRT1 and activity of mTORC1 also decline with aging. The treatment with the NAD(+) precursor nicotinamide riboside (NR) rejuvenates ISCs from aged mice and reverses an impaired ability to repair gut damage. The effect of NR is blocked by the mTORC1 inhibitor rapamycin or the SIRT1 inhibitor EX527. These findings demonstrate that small molecules affecting the NAD/SIRT1/mTORC1 axis may guide a translational path for maintenance of the intestine during aging.
Nicotinamide riboside protects against liver fibrosis induced by CCl via regulating the acetylation of Smads signaling pathway
Jiang R, Zhou Y, Wang S, Pang N, Huang Y, Ye M, Wan T, Qiu Y, Pei L, Jiang X, Huang Y, Yang H, Ling W, Li X, Zhang Z and Yang L
Increasing nicotinamide adenine dinucleotide (NAD) by Nicotinamide riboside (NR) provides protective benefits in multiple disorders. However, the role of NR on liver fibrosis is unclear. We performed in vivo and in vitro experiments to test the hepatic protective effects of NR against liver fibrosis and the underlying mechanisms.
PGC-1α, Sirtuins and PARPs in Huntington's Disease and Other Neurodegenerative Conditions: NAD+ to Rule Them All
Lloret A and Beal MF
In this review, we summarize the available published information on the neuroprotective effects of increasing nicotinamide adenine dinucleotide (NAD) levels in Huntington's disease models. We discuss the rationale of potential therapeutic benefit of administering nicotinamide riboside (NR), a safe and effective NAD precursor. We discuss the agonistic effect on the Sirtuin1-PGC-1α-PPAR pathway as well as Sirtuin 3, which converge in improving mitochondrial function, decreasing ROS production and ameliorating bioenergetics deficits. Also, we discuss the potential synergistic effect of increasing NAD+ combined with PARPs inhibitors, as a clinical therapeutic option not only in HD, but other neurodegenerative conditions.
Implications of altered NAD metabolism in metabolic disorders
Okabe K, Yaku K, Tobe K and Nakagawa T
Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that participates in various energy metabolism pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Besides, it is a required cofactor for post-translational modifications such as ADP-ribosylation and deacetylation by poly (ADP-ribose) polymerases (PARPs) and sirtuins, respectively. Thus, NAD regulates energy metabolism, DNA damage repair, gene expression, and stress response through these enzymes. Numerous studies have shown that NAD levels decrease with aging and under disturbed nutrient conditions, such as obesity. Additionally, a decline in NAD levels is closely related to the development of various metabolic disorders, including diabetes and fatty liver disease. In addition, many studies have revealed that administration of NAD precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), efficiently increase NAD levels in various tissues and prevent such metabolic diseases. These NAD precursors are contained in natural foods, such as cow milk, vegetables, and meats. Therefore, altered NAD metabolism can be a practical target for nutritional intervention. Recently, several human clinical trials using NAD precursors have been conducted to investigate the safety, pharmacokinetics, and efficacy against metabolic disorders such as glucose intolerance. In this review, we summarize current knowledge on the implications of NAD metabolism in metabolic diseases and discuss the outcomes of recent human clinical trials.
Drugs targeting SIRT1, a new generation of therapeutics for osteoporosis and other bone related disorders?
Zainabadi K
With an aging population and limited treatment options, osteoporosis currently represents a significant public health challenge. Recent animal studies indicate that longevity-associated SIRT1 may serve as an attractive pharmacological target for the treatment of osteoporosis and other bone related disorders. Pre-clinical studies demonstrate that mice treated with SIRT1 agonists show protection against age-related, post-menopausal, and disuse models of osteoporosis. Conversely, SIRT1 knockout models display low bone mass phenotypes associated with increased bone resorption and decreased bone formation. This review summarizes recent animal and human experimental data showing that pharmacological activation of SIRT1 may act in a manner that current treatments do not, namely by treating the imbalance in bone remodeling that is the root cause of osteoporosis and other bone disorders.
Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload
Smyrnias I, Gray SP, Okonko DO, Sawyer G, Zoccarato A, Catibog N, López B, González A, Ravassa S, Díez J and Shah AM
The mitochondrial unfolded protein response (UPR) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPR in the heart is unclear.
Transcriptional Response of White Adipose Tissue to Withdrawal of Vitamin B3
Shi W, Hegeman MA, Doncheva A, van der Stelt I, Bekkenkamp-Grovenstein M, van Schothorst EM, Brenner C, de Boer VCJ and Keijer J
Distinct markers for mild vitamin B3 deficiency are lacking. To identify these, the molecular responses of white adipose tissue (WAT) to vitamin B3 withdrawal are examined.
NR Supplementation During Lactation: Benefiting Mother and Child
Yang D and Wan Y
Abnormal nicotinamide adenine dinucleotide (NAD) metabolism causes a wide spectrum of diseases. A recent study (Cell Rep. 2019;26:969-983) shows that postpartum NAD homeostasis is depressed. By restoring NAD homeostasis, maternal nicotinamide riboside (NR) supplementation during lactation enhances postpartum weight loss, as well as juvenile development and adult neurogenesis in the offspring.
First quantification of nicotinamide riboside with B vitamers and coenzymes secreted in human milk by liquid chromatography-tandem-mass spectrometry
Redeuil K, Vulcano J, Prencipe FP, Bénet S, Campos-Giménez E and Meschiari M
The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance
Vannini N, Campos V, Girotra M, Trachsel V, Rojas-Sutterlin S, Tratwal J, Ragusa S, Stefanidis E, Ryu D, Rainer PY, Nikitin G, Giger S, Li TY, Semilietof A, Oggier A, Yersin Y, Tauzin L, Pirinen E, Cheng WC, Ratajczak J, Canto C, Ehrbar M, Sizzano F, Petrova TV, Vanhecke D, Zhang L, Romero P, Nahimana A, Cherix S, Duchosal MA, Ho PC, Deplancke B, Coukos G, Auwerx J, Lutolf MP and Naveiras O
It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.
Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer's disease model mice
Xie X, Gao Y, Zeng M, Wang Y, Wei TF, Lu YB and Zhang WP
Nicotinamide adenine dinucleotide (NAD) supplementation to repair the disabled mitochondria is a promising strategy for the treatment of Alzheimer's disease (AD) and other dementia. Nicotinamide ribose (NR) is a safe NAD precursor with high oral bioavailability, and has beneficial effects on aging. Here, we applied NR supplied food (2.5 g/kg food) to APP/PS1 transgenic AD model mice and aged mice for 3 months. Cognitive function, locomotor activity and anxiety level were assessed by standard behavioral tests. The change of body weight, the activation of microglia and astrocytes, the accumulation of Aβ and the level of serum nicotinamide phosphoribosyltransferase (NAMPT) were determined for the evaluation of pathological processes. We found that NR supplementation improved the short-term spatial memory of aged mice, and the contextual fear memory of AD mice. Moreover, NR supplementation inhibited the activation of astrocytes and the elevation of serum NAMPT of aged mice. For AD model mice, NR supplementation inhibited the accumulation of Aβ and the migration of astrocyte to Aβ. In addition, NR supplementation inhibit the body weight gain of aged and APP/PS1 mice. Thus, NR has selective benefits for both AD and aged mice, and the oral uptake of NR can be used to prevent the progression of dementia.
Acute nicotinamide riboside supplementation improves redox homeostasis and exercise performance in old individuals: a double-blind cross-over study
Dolopikou CF, Kourtzidis IA, Margaritelis NV, Vrabas IS, Koidou I, Kyparos A, Theodorou AA, Paschalis V and Nikolaidis MG
Older individuals suffer from low NADH levels. We have previously shown that nicotinamide riboside [NR; a NAD(P)(H) precursor] administration impaired exercise performance in young rats. It has been suggested that supplementation of redox agents exerts ergogenic effect only in deficient individuals. We hypothesized that old individuals would more likely benefit from NR supplementation. We investigated the effect of acute NR supplementation on redox homeostasis and physical performance in young and old individuals.
Pterostilbene raises low density lipoprotein cholesterol in people
Brenner C and Boileau AC
Nicotinamide riboside regulates inflammation and mitochondrial markers in AML12 hepatocytes
Lee HJ and Yang SJ
The NAD precursor nicotinamide riboside (NR) is a type of vitamin B found in cow's milk and yeast-containing food products such as beer. Recent studies suggested that NR prevents hearing loss, high-fat diet-induced obesity, Alzheimer's disease, and mitochondrial myopathy. The objective of this study was to investigate the effects of NR on inflammation and mitochondrial biogenesis in AML12 mouse hepatocytes.
Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes
Braidy N, Berg J, Clement J, Khorshidi F, Poljak A, Jayasena T, Grant R and Sachdev P
Nicotinamide adenine dinucleotide (NAD) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD, and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD levels. Recent studies have shown that enhancing NAD levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the NAD synthesis pathway in mammalian cells. NAD can also be produced by the NAD salvage pathway. In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD synthesis their location in the NAD anabolic pathway. Increased synthesis of NAD promotes protective cell responses, further demonstrating that NAD is a regulatory molecule associated with several biochemical pathways. In the next few years, the refinement of personalized therapy for the use of NAD precursors and improved detection methodologies allowing the administration of specific NAD precursors in the context of patients' NAD levels will lead to a better understanding of the therapeutic role of NAD precursors in human diseases.
Slc12a8 is a nicotinamide mononucleotide transporter
Grozio A, Mills KF, Yoshino J, Bruzzone S, Sociali G, Tokizane K, Lei HC, Cunningham R, Sasaki Y, Migaud ME and Imai SI
Nicotinamide mononucleotide (NMN) is a biosynthetic precursor of NAD known to promote cellular NAD production and counteract age-associated pathologies associated with a decline in tissue NAD levels. How NMN is taken up into cells has not been entirely clear. Here we show that the gene encodes a specific NMN transporter. We find that is highly expressed and regulated by NAD in the murine small intestine. knockdown abrogates the uptake of NMN and . We further show that Slc12a8 specifically transports NMN, but not nicotinamide riboside, and that NMN transport depends on the presence of sodium ion. deficiency significantly decreases NAD levels in the jejunum and ileum, which is associated with reduced NMN uptake as traced by doubly labeled isotopic NMN. Finally, we observe that expression is upregulated in the aged murine ileum, which contributes to the maintenance of ileal NAD levels. Our work identifies the first NMN transporter and demonstrates that Slc12a8 has a critical role in regulating intestinal NAD metabolism.
Maternal Nicotinamide Riboside Enhances Postpartum Weight Loss, Juvenile Offspring Development, and Neurogenesis of Adult Offspring
Ear PH, Chadda A, Gumusoglu SB, Schmidt MS, Vogeler S, Malicoat J, Kadel J, Moore MM, Migaud ME, Stevens HE and Brenner C
Conditions of metabolic stress dysregulate the NAD metabolome. By restoring NAD, nicotinamide riboside (NR) provides resistance to such conditions. We tested the hypotheses that postpartum might dysregulate maternal NAD and that increasing systemic NAD with NR might benefit mothers and offspring. In postpartum mothers, the liver NAD metabolome is depressed while blood increases circulation of NAD metabolites to enable a >20-fold increase in mammary NAD and NADP. Lactation and NR synergize in stimulating prolactin synthesis and mammary biosynthetic programs. NR supplementation of new mothers increases lactation and nursing behaviors and stimulates maternal transmission of macronutrients, micronutrients, and BDNF into milk. Pups of NR-supplemented mothers are advantaged in glycemic control, size at weaning, and synaptic pruning. Adult offspring of mothers supplemented during nursing retain advantages in physical performance, anti-anxiety, spatial memory, delayed onset of behavioral immobility, and promotion of adult hippocampal neurogenesis. Thus, postgestational maternal micronutrition confers lasting advantages to offspring.
Nicotinamide Mononucleotide: Exploration of Diverse Therapeutic Applications of a Potential Molecule
Poddar SK, Sifat AE, Haque S, Nahid NA, Chowdhury S and Mehedi I
Nicotinamide mononucleotide (NMN) is a nucleotide that is most recognized for its role as an intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis. Although the biosynthetic pathway of NMN varies between eukaryote and prokaryote, two pathways are mainly followed in case of eukaryotic human-one is through the salvage pathway using nicotinamide while the other follows phosphorylation of nicotinamide riboside. Due to the unavailability of a suitable transporter, NMN enters inside the mammalian cell in the form of nicotinamide riboside followed by its subsequent conversion to NMN and NAD+. This particular molecule has demonstrated several beneficial pharmacological activities in preclinical studies, which suggest its potential therapeutic use. Mostly mediated by its involvement in NAD+ biosynthesis, the pharmacological activities of NMN include its role in cellular biochemical functions, cardioprotection, diabetes, Alzheimer's disease, and complications associated with obesity. The recent groundbreaking discovery of anti-ageing activities of this chemical moiety has added a valuable essence in the research involving this molecule. This review focuses on the biosynthesis of NMN in mammalian and prokaryotic cells and mechanism of absorption along with the reported pharmacological activities in murine model.
Nicotinamide riboside has protective effects in a rat model of mesenteric ischaemia-reperfusion
Toropova YG, Pechnikova NA, Zelinskaya IA, Zhuravsky SG, Kornyushin OV, Gonchar AI, Ivkin DY, Leonova YV, Karev VE and Karabak IA
Acute mesenteric ischaemia is a syndrome caused by inadequate blood flow through the mesenteric vessels, resulting in ischaemia and eventual gangrene of the bowel wall. Although relatively rare, it is a potentially life-threatening condition. The maintenance of haemodynamic stability, along with adequate oxygen saturation, and the correction of any electrolyte imbalance, are of the utmost importance. However, nicotinamide adenine dinucleotide (NAD) biosynthesis modulation by precursor introduction can also be a powerful tool for preventing injury. Nicotinamide riboside is a pyridine-nucleoside form of vitamin B3 that functions as a precursor to NAD . The present study investigated nicotinamide riboside's effect on endothelium functional state, microcirculation and intestinal morphology in acute mesenteric ischaemia and reperfusion. Mesenteric ischaemia was simulated after the adaptation period (15 minutes) by occluding the superior mesenteric artery for 60 minutes, followed by a reperfusion period of 30 minutes. The functional state of intestinal microcirculation was evaluated by laser Doppler flowmetry. Endothelial functional activity was studied by using wire myography. Intestinal samples were stained with haematoxylin and eosin for histological analysis. The results revealed that nicotinamide riboside protects the intestinal wall from ischaemia-reperfusion injury, as well as improving the relaxation function of mesenteric vessels. Nicotinamide riboside's protective effect in small intestine ischaemia-reperfusion can be used to reduce ischaemia-reperfusion injury, as well as to preserve intestinal grafts until transplant.
NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy
Shabalin K, Nerinovski K, Yakimov A, Kulikova V, Svetlova M, Solovjeva L, Khodorkovskiy M, Gambaryan S, Cunningham R, Migaud ME, Ziegler M and Nikiforov A
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.
Metabolomes of mitochondrial diseases and inclusion body myositis patients: treatment targets and biomarkers
Buzkova J, Nikkanen J, Ahola S, Hakonen AH, Sevastianova K, Hovinen T, Yki-Järvinen H, Pietiläinen KH, Lönnqvist T, Velagapudi V, Carroll CJ and Suomalainen A
Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect.
Mitochondrial regulation of diabetic vascular disease: an emerging opportunity
Widlansky ME and Hill RB
Diabetes-related vascular complication rates remain unacceptably high despite guideline-based medical therapies that are significantly more effective in individuals without diabetes. This critical gap represents an opportunity for researchers and clinicians to collaborate on targeting mechanisms and pathways that specifically contribute to vascular pathology in patients with diabetes mellitus. Dysfunctional mitochondria producing excessive mitochondrial reactive oxygen species (mtROS) play a proximal cell-signaling role in the development of vascular endothelial dysfunction in the setting of diabetes. Targeting the mechanisms of production of mtROS or mtROS themselves represents an attractive method to reduce the prevalence and severity of diabetic vascular disease. This review focuses on the role of mitochondria in the development of diabetic vascular disease and current developments in methods to improve mitochondrial health to improve vascular outcomes in patients with DM.
Programming of the Beige Phenotype in White Adipose Tissue of Adult Mice by Mild Resveratrol and Nicotinamide Riboside Supplementations in Early Postnatal Life
Serrano A, Asnani-Kishnani M, Rodríguez AM, Palou A, Ribot J and Bonet ML
Resveratrol (RSV) and nicotinamide riboside (NR) are food compounds with anti-obesity actions in adult rodents. Here, the long-term effects of RSV and NR mild supplementation throughout lactation on adiposity-related parameters and the appearance of the beige phenotype in white adipose tissue (WAT) in adulthood are assessed.
NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis
Dorai T, Shah A, Summers F, Mathew R, Huang J, Hsieh TC and Wu JM
In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.
Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation
Vignier N, Chatzifrangkeskou M, Morales Rodriguez B, Mericskay M, Mougenot N, Wahbi K, Bonne G and Muchir A
Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscle and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for heart function. Biosynthesis of NAD+ from vitamin B3 (known as salvage pathways) is the primary source of NAD+. We showed here that NAD+ salvage pathway was altered in the heart of mouse and human carrying LMNA mutation, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Oral administration of nicotinamide riboside, a natural NAD+ precursor and a pyridine-nucleoside form of vitamin B3, leads to a marked improvement of the NAD+ cellular content, an increase of PARylation of cardiac proteins and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our results provide mechanistic and therapeutic insights into dilated cardiomyopathy caused by LMNA mutations.
Sirtuin 3 deficiency aggravates contrast-induced acute kidney injury
Zhang Q, Liu X, Li N, Zhang J, Yang J and Bu P
Sirtuin 3 (Sirt3) is a key regulator of energy metabolism and oxidative stress. To investigate the role of Sirt3 in contrast-induced acute kidney injury (CIAKI), we established the model both in vivo and in vitro to explore the potential mechanisms.
Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD Synthesis
Palzer L, Bader JJ, Angel F, Witzel M, Blaser S, McNeil A, Wandersee MK, Leu NA, Lengner CJ, Cho CE, Welch KD, Kirkland JB, Meyer RG and Meyer-Ficca ML
NAD is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD synthesis. In contrast, mice easily generate sufficient NAD solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD levels in aging and neurodegenerative diseases.
Complete genome sequence analysis of the Malacosoma neustria nucleopolyhedrovirus from Turkey
Gencer D, Nalcacioglu R, Demirbag Z and Demir I
The lackey moth, Malacosoma neustria (Linnaeus, 1758), a worldwide pest, causes extensive economic losses particularly on hazelnut, plum, oak, poplar, and willow trees. A baculovirus, Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2), has been isolated from the larvae collected in Turkey and appears to have a potential as a microbial control agent. In this study, we describe the complete genome sequence of ManeNPV-T2 and compare it to other sequenced baculovirus genomes. The ManeNPV-T2 genome is a circular double-stranded DNA molecule of 130,202 bp, has 38.2% G + C, and is predicted to contain 131 putative open reading frames (ORFs) each with a coding capacity of more then 50 amino acids. There are 27 ORFs with unknown function of which 6 are unique to ManeNPV-T2. Eleven homologous regions (hrs) and two bro genes (bro-a and bro-b) were identified in the genome. There are two homologues of chaB and nicotinamide riboside kinase-1 genes, separated from themselves with a few nucleotides. Additionally, ac145, thought to be per os infectivity factor (pif) gene, is also found as two homologues. All 38 core genes are found in the ManeNPV-T2 genome. The phylogenetic tree of ManeNPV-T2 in relation to 50 other baculoviruses whose genomes have been completely sequenced showed ManeNPV-T2 to be closely related to the group II NPVs. This study expands our knowledge on baculoviruses, describes the characterization ManeNPV, and ultimately contributes to the registration of this virus as a microbial pesticide.
Nicotinamide riboside induces a thermogenic response in lean mice
Crisol BM, Veiga CB, Lenhare L, Braga RR, Silva VRR, da Silva ASR, Cintra DE, Moura LP, Pauli JR and Ropelle ER
Nicotinamide Riboside (NR) is a NAD booster with wide physiological repercussion including the improvement on glucose and lipid homeostasis, increasing the life expectancy in mammals. However, the effects of NR on metabolism are only partially known. Here, we evaluated the effects of NR on the thermogenic response, highlighting the brown adipose tissue (BAT) in lean mice.
Pharmacological bypass of NAD salvage pathway protects neurons from chemotherapy-induced degeneration
Liu HW, Smith CB, Schmidt MS, Cambronne XA, Cohen MS, Migaud ME, Brenner C and Goodman RH
Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD, nicotinamide mononucleotide (NMN), rather than loss of NAD, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.
Nicotinamide riboside supplementation dysregulates redox and energy metabolism in rats: Implications for exercise performance
Kourtzidis IA, Dolopikou CF, Tsiftsis AN, Margaritelis NV, Theodorou AA, Zervos IA, Tsantarliotou MP, Veskoukis AS, Vrabas IS, Paschalis V, Kyparos A and Nikolaidis MG
What is the central question of this study? The aim was to investigate the potential metabolic and redox mechanisms that impaired exercise performance after 21 days of supplementation with 300 mg (kg body weight) of nicotinamide riboside in rats. What is the main finding and its importance? Nicotinamide riboside disturbed energy and redox metabolism and impaired exercise performance in heathy rats. Exogenously administered redox agents in heathy populations might lead to adverse effects.
The emergence of the nicotinamide riboside kinases in the regulation of NAD+ metabolism
Fletcher RS and Lavery GG
The concept of replenishing or elevating NAD+ availability to combat metabolic disease and ageing is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cells and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase pathway at both a tissue-specific and systemic level.
Rejuvenating Aged Hematopoietic Stem Cells Through Improvement of Mitochondrial Function
Moon J, Kim HR and Shin MG
Mitochondria are the powerhouses of the cell as well as the primary site of hematopoiesis, which also occurs in the cytoplasm. Hematopoietic stem cells (HSCs) are characterized by a very high turnover rate, and are thus considered to be relatively free from the age-related insults generated by mitochondria. However, HSCs are also subject to these age-related insults, including the incidence of myeloid proliferative diseases, marrow failure, hematopoietic neoplasms, and deterioration of the adaptive human immune system. Recently, NAD⁺ dietary supplements, known as niacin or vitamin B₃, including tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD⁺ precursor nicotinamide riboside, have been shown to play a role in restoring adult stem cell function through the amelioration of mitochondrial dysfunction. This insight motivated a study that focused on reversing aging-related cellular dysfunction in adult mouse muscle stem cells by supplementing their diet with nicotinamide riboside. The remedial effect of nicotinamide riboside enhanced mitochondrial function in these muscle stem cells in a SIRT1-dependent manner, affecting cellular respiration, membrane potential, and production of ATP. Accordingly, numerous studies have demonstrated that sirtuins, under nuclear/mitochondrial control, have age-specific effects in determining HSC phenotypes. Based on the evidence accumulated thus far, we propose a clinical intervention for the restoration of aged HSC function by improving mitochondrial function through NAD⁺ precursor supplementation.
The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage
Douiev L and Saada A
Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 μM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD) deficit combined with a replicative state, rather than oxidative stress and energy depletion.
A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects
Dollerup OL, Christensen B, Svart M, Schmidt MS, Sulek K, Ringgaard S, Stødkilde-Jørgensen H, Møller N, Brenner C, Treebak JT and Jessen N
Animal studies suggest a positive role for nicotinamide riboside (NR) on insulin sensitivity and hepatic steatosis in models of obesity and type 2 diabetes. NR, an NAD+ precursor, is a member of the vitamin B-3 family now available as an over-the-counter supplement. Although data from preclinical trials appear consistent, potential effects and safety need to be evaluated in human clinical trials.
Oxyresveratrol stimulates mucin production in an NAD-dependent manner in human intestinal goblet cells
Hwang D, Jo H, Ma SH and Lim YH
The intestinal mucus layer plays an important role in the management of inflammatory bowel disease. The aim of this study was to investigate the effects of oxyresveratrol (OXY), an antioxidant, on the stimulation of mucin production in human LS 174T goblet cells and the underlying mechanism thereof. OXY increased MUC2 expression at both the mRNA and protein levels. By performing two-dimensional gel electrophoresis, we found that the expression of nicotinic acid phosphoribosyltransferase1 (NaPRT1) in OXY-treated LS 174T cells was greatly increased compared with that in negative control cells. In addition, the NAD/NADH ratio was increased in proportion to OXY in LS 174T cells. The expression of NAD-synthesis enzymes, NaPRT1, nicotinamide riboside kinase1 (NRK1) and nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) was significantly increased at both the mRNA and protein levels in OXY-treated LS 174T cells. The inhibition of NaPRT1 and NRK1 did not decrease MUC2 expression after inhibiting by small interfering RNA (siRNA)-NaPRT1 and siRNA-NRK1, respectively; however, inhibition of Nmnat by an Nmnat inhibitor decreased MUC2 expression in a dose-dependent manner. In conclusion, OXY increases NAD levels, resulting in the stimulation of MUC2 expression in LS 174T cells. These findings present a novel role for NAD in stimulation of MUC2 expression.
Administration of nicotinamide riboside prevents oxidative stress and organ injury in sepsis
Hong G, Zheng D, Zhang L, Ni R, Wang G, Fan GC, Lu Z and Peng T
Sepsis-caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis.
The Regulatory Role of NAD in Human and Animal Cells
Kulikova VA, Gromyko DV and Nikiforov AA
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD+ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD+ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.
Nicotinamide riboside attenuates alcohol induced liver injuries via activation of SirT1/PGC-1α/mitochondrial biosynthesis pathway
Wang S, Wan T, Ye M, Qiu Y, Pei L, Jiang R, Pang N, Huang Y, Liang B, Ling W, Lin X, Zhang Z and Yang L
Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD) precursor which is present in foods such as milk and beer. It was reported that NR can prevent obesity, increase longevity, and promote liver regeneration. However, whether NR can prevent ethanol-induced liver injuries is not known. This study aimed to explore the effect of NR on ethanol induced liver injuries and the underlying mechanisms.
Nicotinamide adenine dinucleotide is transported into mammalian mitochondria
Davila A, Liu L, Chellappa K, Redpath P, Nakamaru-Ogiso E, Paolella LM, Zhang Z, Migaud ME, Rabinowitz JD and Baur JA
Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter.
Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem-mass spectrometry
Yaku K, Okabe K and Nakagawa T
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 decline 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 an 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 (reduced form of NAD) by performing a single sample preparation. Further, we validated 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 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.
The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease
Schöndorf DC, Ivanyuk D, Baden P, Sanchez-Martinez A, De Cicco S, Yu C, Giunta I, Schwarz LK, Di Napoli G, Panagiotakopoulou V, Nestel S, Keatinge M, Pruszak J, Bandmann O, Heimrich B, Gasser T, Whitworth AJ and Deleidi M
While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.
Overexpression of NRK1 ameliorates diet- and age-induced hepatic steatosis and insulin resistance
Fan R, Cui J, Ren F, Wang Q, Huang Y, Zhao B, Wei L, Qian X and Xiong X
NAD is a co-enzyme in redox reactions and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Dietary supplementation of NAD precursors nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we sought to identify the roles of nicotinamide riboside kinase 1 (NRK1) plays in regulating hepatic NAD biosynthesis and lipid metabolism. Using adenovirus mediated gene transduction to overexpress or knockdown NRK1 in mouse liver, we have demonstrated that NRK1 is critical for maintaining hepatic NAD levels and triglyceride content. We have further shown that the hepatic expression of Nmrk1 mRNA is significantly decreased either in mice treated with high-fat diet or in aged mice. However, adenoviral delivery of NRK1 in these diet- and age-induced mice elevates hepatic NAD levels, reduces hepatic steatosis, and improves glucose tolerance and insulin sensitivity. Our results provide important insights in targeting NRK1 for treating hepatic steatosis.
Loss of the Thioretinaco Ozonide Oxygen Adenosine Triphosphate Complex from Mitochondria Produces Mitochondrial Dysfunction and Carcinogenesis
McCully KS
The active site of oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis is proposed to consist of thioretinaco, a complex of two molecules of thioretinamide with cobalamin, oxidized to the disulfonium derivative, thioretinaco ozonide, and complexed with oxygen, nicotinamide adenine dinucleotide, inorganic phosphate and ATP. Reduction of the active site complex by electrons from mitochondrial electron transport complexes releases ATP from binding to the active site, producing nicotinamide riboside and hydroperoxide and generating a membrane potential from proton transport to the active site. Opening of the mitochondrial permeability transition pore from decreased mitochondrial melatonin leads to loss of the active site complex from mitochondrial membranes, as observed in aging and dementia. Loss of the active site complex from mitochondria also results from opening of the permeability transition pore and from decomposition of the disulfonium active site by electrophilic carcinogens, oncogenic viruses and microbes which cause depletion of adenosyl methionine because of increased biosynthesis of polyamines, and by free radical oxygen species generated by ionizing radiation, and by catecholamines. Thus the loss of thioretinaco ozonide from mitochondria produces the impaired oxidative phosphorylation, oxidative stress, calcium influx, apoptosis, aerobic glycolysis, and mitochondrial dysfunction that are observed in chemical carcinogenesis, microbial carcinogenesis, traumatic brain injury, aging and dementia.
Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside
Tran A, Yokose R and Cen Y
As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.
NAD repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation
Purhonen J, Rajendran J, Tegelberg S, Smolander OP, Pirinen E, Kallijärvi J and Fellman V
Biosynthetic precursors of NAD can replenish a decreased cellular NAD pool and, supposedly via sirtuin (SIRT) deacetylases, improve mitochondrial function. We found decreased hepatic NAD concentration and downregulated biosynthesis in Bcs1l knock-in mice with respiratory chain complex III deficiency and mitochondrial hepatopathy. Aiming at ameliorating disease progression via NAD repletion and improved mitochondrial function, we fed these mice nicotinamide riboside (NR), a NAD precursor. A targeted metabolomics verified successful administration and suggested enhanced NAD biosynthesis in the treated mice, although hepatic NAD concentration was unchanged at the end point. In contrast to our expectations, NR did not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1l mice. We linked this lack of therapeutic effect to NAD-independent activation of SIRT-1 and -3 via AMPK and cAMP signaling related to the starvation-like metabolic state of Bcs1l mice. In summary, we describe an unusual metabolic state with NAD depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative metabolism. Our study highlights that the knowledge of the underlying complex metabolic alterations is critical when designing therapies for mitochondrial dysfunction.-Purhonen, J., Rajendran, J., Tegelberg, S., Smolander, O.-P., Pirinen, E., Kallijärvi, J., Fellman, V. NAD repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation.
Raising NAD in Heart Failure: Time to Translate?
Walker MA and Tian R
Quantitative Analysis of NAD Synthesis-Breakdown Fluxes
Liu L, Su X, Quinn WJ, Hui S, Krukenberg K, Frederick DW, Redpath P, Zhan L, Chellappa K, White E, Migaud M, Mitchison TJ, Baur JA and Rabinowitz JD
The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism.
The Emergence of the Nicotinamide Riboside Kinases in the regulation of NAD+ Metabolism
Fletcher RS and Lavery G
The concept of replenishing or elevating nicotinamide adenine dinucleotide (NAD+) availability to combat metabolic disease and ageing (described extensively in recent reviews [1, 2]) is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cell and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase (NRK) pathway at both a tissue-specific and systemic level.
Correction to: Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy
Effects of senolytic drugs on human mesenchymal stromal cells
Grezella C, Fernandez-Rebollo E, Franzen J, Ventura Ferreira MS, Beier F and Wagner W
Senolytic drugs are thought to target senescent cells and might thereby rejuvenate tissues. In fact, such compounds were suggested to increase health and lifespan in various murine aging models. So far, effects of senolytic drugs have not been analysed during replicative senescence of human mesenchymal stromal cells (MSCs).
Emerging potential benefits of modulating NAD metabolism in cardiovascular disease
Matasic DS, Brenner C and London B
Nicotinamide adenine dinucleotide (NAD) and related metabolites are central mediators of fuel oxidation and bioenergetics within cardiomyocytes. Additionally, NAD is required for the activity of multifunctional enzymes, including sirtuins and poly(ADP-ribose) polymerases that regulate posttranslational modifications, DNA damage responses, and Ca signaling. Recent research has indicated that NAD participates in a multitude of processes dysregulated in cardiovascular diseases. Therefore, supplementation of NAD precursors, including nicotinamide riboside that boosts or repletes the NAD metabolome, may be cardioprotective. This review examines the molecular physiology and preclinical data with respect to NAD precursors in heart failure-related cardiac remodeling, ischemic-reperfusion injury, and arrhythmias. In addition, alternative NAD-boosting strategies and potential systemic effects of NAD supplementation with implications on cardiovascular health and disease are surveyed.
Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence
Rajman L, Chwalek K and Sinclair DA
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, NAD controls hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NAD levels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NAD levels 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.
Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD in healthy middle-aged and older adults
Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, McQueen MB, Chonchol M and Seals DR
Nicotinamide adenine dinucleotide (NAD) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD precursors to augment NAD bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD metabolism in healthy middle-aged and older adults. Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.
A need for NAD+ in muscle development, homeostasis, and aging
Goody MF and Henry CA
Skeletal muscle enables posture, breathing, and locomotion. Skeletal muscle also impacts systemic processes such as metabolism, thermoregulation, and immunity. Skeletal muscle is energetically expensive and is a major consumer of glucose and fatty acids. Metabolism of fatty acids and glucose requires NAD+ function as a hydrogen/electron transfer molecule. Therefore, NAD+ plays a vital role in energy production. In addition, NAD+ also functions as a cosubstrate for post-translational modifications such as deacetylation and ADP-ribosylation. Therefore, NAD+ levels influence a myriad of cellular processes including mitochondrial biogenesis, transcription, and organization of the extracellular matrix. Clearly, NAD+ is a major player in skeletal muscle development, regeneration, aging, and disease. The vast majority of studies indicate that lower NAD+ levels are deleterious for muscle health and higher NAD+ levels augment muscle health. However, the downstream mechanisms of NAD+ function throughout different cellular compartments are not well understood. The purpose of this review is to highlight recent studies investigating NAD+ function in muscle development, homeostasis, disease, and regeneration. Emerging research areas include elucidating roles for NAD+ in muscle lysosome function and calcium mobilization, mechanisms controlling fluctuations in NAD+ levels during muscle development and regeneration, and interactions between targets of NAD+ signaling (especially mitochondria and the extracellular matrix). This knowledge should facilitate identification of more precise pharmacological and activity-based interventions to raise NAD+ levels in skeletal muscle, thereby promoting human health and function in normal and disease states.
Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Attenuated Hepatic Triglyceride Accumulation in Alcoholic Fatty Liver Disease in Mice
Huang S, Zhang B, Chen Y, Liu H, Liu Y, Li X, Bao Z, Song Z and Wang Z
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 NAD levels 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 NAD levels 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 NAD depletion and TG accumulation in alcohol-fed mice and may be a potential candidate for use in AFLD therapy.
Redox imbalance stress in diabetes mellitus: Role of the polyol pathway
Yan LJ
In diabetes mellitus, the polyol pathway is highly active and consumes approximately 30% glucose in the body. This pathway contains 2 reactions catalyzed by aldose reductase (AR) and sorbitol dehydrogenase, respectively. AR reduces glucose to sorbitol at the expense of NADPH, while sorbitol dehydrogenase converts sorbitol to fructose at the expense of NAD, leading to NADH production. Consumption of NADPH, accumulation of sorbitol, and generation of fructose and NADH have all been implicated in the pathogenesis of diabetes and its complications. In this review, the roles of this pathway in NADH/NAD redox imbalance stress and oxidative stress in diabetes are highlighted. A potential intervention using nicotinamide riboside to restore redox balance as an approach to fighting diabetes is also discussed.
Directed Evolution of Membrane Transport Using Synthetic Selections
Bali AP, Genee HJ and Sommer MOA
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.
A functional link between NAD homeostasis and N-terminal protein acetylation in
Croft T, James Theoga Raj C, Salemi M, Phinney BS and Lin SJ
Nicotinamide adenine dinucleotide (NAD) is an essential metabolite participating in cellular redox chemistry and signaling, and the complex regulation of NAD metabolism 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, and appeared 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 NAD We 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 NAD and 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 protein We next examined Tpm1 (tropomyosin), whose function is regulated by NatB-mediated acetylation, and Tpm1 overexpression () was shown to restore some NatB mutant defects. Interestingly, although largely suppressed NA/NAM release in NatB mutants, it did not restore NAD levels. We showed that decreased nicotinamide mononucleotide adenylyltransferase (Nma1/Nma2) levels probably caused the NAD defects, and was sufficient to restore NAD NatB-mediated N-terminal acetylation of Nma1 and Nma2 appears essential for maintaining NAD levels. In summary, our results support a connection between NatB-mediated protein acetylation and NAD homeostasis. Our findings may contribute to understanding the molecular basis and regulation of NAD metabolism.
NAD supplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency
Hou Y, Lautrup S, Cordonnier S, Wang Y, Croteau DL, Zavala E, Zhang Y, Moritoh K, O'Connell JF, Baptiste BA, Stevnsner TV, Mattson MP and Bohr VA
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/Polβ mouse that exacerbates major features of human AD including phosphorylated Tau (pTau) pathologies, synaptic dysfunction, neuronal death, and cognitive impairment. Here we report that 3xTgAD/Polβ mice 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/Polβ mice but had no impact on amyloid β peptide (Aβ) accumulation. NR-treated 3xTgAD/Polβ mice 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/Polβ mice. In general, the deficits between genotypes and the benefits of NR were greater in 3xTgAD/Polβ mice than in 3xTgAD mice. Our findings suggest a pivotal role for cellular NAD depletion upstream of neuroinflammation, pTau, DNA damage, synaptic dysfunction, and neuronal degeneration in AD. Interventions that bolster neuronal NAD levels therefore have therapeutic potential for AD.
Synthesis of β-Nicotinamide Riboside Using an Efficient Two-Step Methodology
Zhang N and Sauve AA
A two-step chemical method for the synthesis of β-nicotinamide riboside (NR) is described. NR has achieved wide use as an NAD precursor (vitamin B3) and can significantly increase central metabolite NAD concentrations 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.
Nicotinamide riboside exerts protective effect against aging-induced NAFLD-like hepatic dysfunction in mice
Han X, Bao X, Lou Q, Xie X, Zhang M, Zhou S, Guo H, Jiang G and Shi Q
Aging is one of the risk factors of non-alcoholic fatty liver disease (NAFLD). Yet, the mechanism underlying the aging-associated NAFLD-like syndrome is not fully understood. Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, has protective effects against aging. Here, we investigated the actions of NAD precursors nicotinamide riboside (NR) on the development of aging-induced NAFLD.
Role of pseudohypoxia in the pathogenesis of type 2 diabetes
Song J, Yang X and Yan LJ
Type 2 diabetes is caused by persistent high blood glucose, which is known as diabetic hyperglycemia. This hyperglycemic situation, when not controlled, can overproduce NADH and lower nicotinamide adenine dinucleotide (NAD), thereby creating NADH/NAD redox imbalance and leading to cellular pseudohypoxia. In this review, we discussed two major enzymatic systems that are activated by diabetic hyperglycemia and are involved in creation of this pseudohypoxic condition. One system is aldose reductase in the polyol pathway, and the other is poly (ADP ribose) polymerase. While aldose reductase drives overproduction of NADH, PARP could in contrast deplete NAD. Therefore, activation of the two pathways underlies the major mechanisms of NADH/NAD redox imbalance and diabetic pseudohypoxia. Consequently, reductive stress occurs, followed by oxidative stress and eventual cell death and tissue dysfunction. Additionally, fructose formed in the polyol pathway can also cause metabolic syndrome such as hypertension and nonalcoholic fatty liver disease. Moreover, pseudohypoxia can also lower sirtuin protein contents and induce protein acetylation which can impair protein function. Finally, we discussed the possibility of using nicotinamide riboside, an NAD precursor, as a promising therapeutic agent for restoring NADH/NAD redox balance and for preventing the occurrence of diabetic pseudohypoxia.
Why Is Mom Stressed: Homeorhesis as the Potential Problem and Nicotinamide Riboside as the Potential Solution
Brenner C
The remodeling of female mammalian physiology to support the development of a fertilized egg into an externally breathing individual and then to provide all the nutrition to this individual while remodeling back to nearly her pregestational state is without parallel in male mammalian physiological transitions. While it is common parlance to refer to postpartum depression as a not infrequent stress in women, the postpartum physiological changes after every birth constitute profound metabolic stresses that are understudied and have important nutritional, behavioral, and neurodevelopmental implications for the maternal and neonatal health of every mammalian species. We discovered that the postpartum liver of a lactating female mouse has a depressed nicotinamide adenine dinucleotide (NAD) metabolome linked to circulation of higher levels of NAD metabolites in support of a >20-fold increase in NAD coenzymes in the mammary. Furthermore, by supporting a new mother's apparent higher demand for NAD precursors, we increased circulation of prolactin, superinduced mammary biosynthetic programs, increased her time of arched-back nursing, enhanced mammary production of brain-derived neurotrophic factor, promoted postgestational weight loss, advanced the neurobehavioral development of her offspring, and allowed them to mature as stronger and more resilient adults with advantages in hippocampal neurogenesis and body composition. These results show that a new mother's capacity for biosynthesis and functionally important nurturing is apparently limited by NAD. Here, we discuss homeorhetic flow of resources from a new mother to her offspring in the context of NAD metabolism and suggest avenues for future investigation.
Regulatory Effects of NAD Metabolic Pathways on Sirtuin Activity
Zhang N and Sauve AA
NAD acts 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 NAD with acyllysine groups to remove the acyl modification from substrate proteins. This deacetylation provides a regulatory function and integrates cellular NAD metabolism 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 NAD concentrations can impact sirtuin activities has motivated openings of new areas of research, including investigations of how NAD levels are regulated at the subcellular level, and searches for more potent NAD precursors typified by nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). This review describes current results and thinking of how NAD metabolic pathways regulate sirtuin activities and how regulated NAD levels can impact cell physiology. In addition, NAD precursors are discussed, with attention to how these might be harnessed to generate novel therapeutic options to treat the diseases of aging.
Role of mitochondria in diabetic peripheral neuropathy: Influencing the NAD-dependent SIRT1-PGC-1α-TFAM pathway
Chandrasekaran K, Anjaneyulu M, Choi J, Kumar P, Salimian M, Ho CY and Russell JW
Survival of human peripheral nervous system neurons and associated distal axons is highly dependent on energy. Diabetes invokes a maladaptation in glucose and lipid energy metabolism in adult sensory neurons, axons and Schwann cells. Mitochondrial (Mt) dysfunction has been implicated as an etiological factor in failure of energy homeostasis that results in a low intrinsic aerobic capacity within the neuron. Over time, this energy failure can lead to neuronal and axonal degeneration and results in increased oxidative injury in the neuron and axon. One of the key pathways that is impaired in diabetic peripheral neuropathy (DPN) is the energy sensing pathway comprising the nicotinamide-adenine dinucleotide (NAD)-dependent Sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α)/Mt transcription factor A (TFAM or mtTFA) signaling pathway. Knockout of PGC-1α exacerbates DPN, whereas overexpression of human TFAM is protective. LY379268, a selective metabolomic glutamate receptor 2/3 (mGluR2/3) receptor agonist, also upregulates the SIRT1/PGC-1α/TFAM signaling pathway and prevents DPN through glutamate recycling in Schwann/satellite glial (SG) cells and by improving dorsal root ganglion (DRG) neuronal Mt function. Furthermore, administration of nicotinamide riboside (NR), a precursor of NAD, prevents and reverses DPN, in part by increasing NAD levels and SIRT1 activity. In summary, we review the role of NAD, mitochondria and the SIRT1-PGC-1α-TFAM pathway both from the perspective of pathogenesis and therapy in DPN.
Syntheses and chemical properties of β-nicotinamide riboside and its analogues and derivatives
Makarov MV and Migaud ME
The β-anomeric form of nicotinamide riboside (NR) is a precursor for nicotinamide adenine dinucleotide (NAD), a redox cofactor playing a critical role in cell metabolism. Recently, it has been demonstrated that its chloride salt (NRCl) has beneficial effects, and now NRCl is available as a dietary supplement. Syntheses and studies of analogues and derivatives of NR are of high importance to unravel the role of NR in biochemical processes in living cells and to elaborate the next generation of NR derivatives and conjugates with the view of developing novel drug and food supplement candidates. This review provides an overview of the synthetic approaches, the chemical properties, and the structural and functional modifications which have been undertaken on the nicotinoyl riboside scaffold.
Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies
Jablonska P, Mierzejewska P, Kutryb-Zajac B, Rzyman W, Dziadziuszko R, Polanska J, Sitkiewicz M, Smolenski RT and Slominska EM
4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function. The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma ( = 48) and control group ( = 100). The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099 ± 0.009 vs. 0.066 ± 0.006 µmol/L and 1.10 ± 0.08 vs. 1.97 ± 0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253 ± 17 vs. 369 ± 19) as well as plasma MNA (0.057 ± 0.004 vs. 0.069 ± 0.003 µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116 ± 0.019 vs. 0.131 ± 0.014 and 0.102 ± 0.006 vs. 0.113 ± 0.011, respectively). In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.
Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD depletion
Oakey LA, Fletcher RS, Elhassan YS, Cartwright DM, Doig CL, Garten A, Thakker A, Maddocks ODK, Zhang T, Tennant DA, Ludwig C and Lavery GG
Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD homeostasis on these pathways is unknown. We utilised skeletal muscle myotube models to induce NAD depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. NAD excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells results in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity is increased in response to low NAD , which is rapidly reversed with repletion of the NAD pool using NR. NAD depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reverses these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD depletion remains unclear, and requires further investigation. These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD precursor usage in models of skeletal muscle physiology.
Neonatal Resveratrol and Nicotinamide Riboside Supplementations Sex-Dependently Affect Beige Transcriptional Programming of Preadipocytes in Mouse Adipose Tissue
Asnani-Kishnani M, Rodríguez AM, Serrano A, Palou A, Bonet ML and Ribot J
Nutritional programming of the thermogenic and fuel oxidation capacity of white adipose tissue (WAT) through dietary interventions in early life is a potential strategy to enhance future metabolic health. We previously showed that mild neonatal supplementations with the polyphenol resveratrol (RSV) and the vitamin B3 form nicotinamide riboside (NR) have sex-dependent, long-term effects on the thermogenic/oxidative phenotype of WAT of mice in adulthood, enhancing this phenotype selectively in male animals. Here, we tested the hypothesis that these dietary interventions may impact the commitment of progenitor cells resident in the developing WAT toward brown-like (beige) adipogenesis. NMRI mice received orally from postnatal day 2-20 (P2-20) a mild dose of RSV or NR, in independent experiments; control littermates received the vehicle. Sex-separated primary cultures were established at P35 from the stromovascular fraction of inguinal WAT (iWAT) and of brown adipose tissue (BAT). Expression of genes related to thermogenesis and oxidative metabolism was assessed in the differentiated cultures, and in the iWAT depot of young (P35) animals. Neonatal RSV and NR treatments had little impact on the animals' growth during early postnatal life and the expression of thermogenesis- and oxidative metabolism-related genes in the iWAT depot of young mice. However, the expression of brown/beige adipocyte marker genes was upregulated in the iWAT primary cultures from RSV supplemented and NR supplemented male mice, and downregulated in those from supplemented female mice, as compared to cultures derived from sex-matched control littermates. RSV supplementation had similar sex-dependent effects on the expression of thermogenesis-related genes in the BAT primary cultures. A link between the sex-dependent short-term effects of neonatal RSV and NR supplementations on primary iWAT preadipocyte differentiation observed herein and their previously reported sex-dependent long-term effects on the thermogenic/oxidative capacity of adult iWAT is suggested. The results provide proof-of-concept that the fate of preadipocytes resident in WAT of young animals toward the beige adipogenesis transcriptional program can be modulated by specific food bioactives/micronutrients received in early postnatal life.
Aged Nicotinamide Riboside Kinase 2 Deficient Mice Present an Altered Response to Endurance Exercise Training
Deloux R, Tannous C, Ferry A, Li Z and Mericskay M
Skeletal muscle aging is marked by the development of a sarcopenic phenotype, a global decline of muscle energetic capacities, and an intolerance to exercise. Among the metabolic disorders involved in this syndrome, NAD metabolism was shown to be altered in skeletalmuscle, with an important role for the NAMPT enzyme recycling the nicotinamide precursor. An alternative pathway for NAD biosynthesis has been described for the nicotinamide riboside vitamin B3 precursor used by the NMRK kinases, including the striated muscle-specific NMRK2. With this study, our goal is to explore the ability of 16-month-old mice to perform endurance exercise and study the consequences on muscle adaptation to exercise. 10 control and 6 mice were used and randomly assigned to sedentary and treadmill endurance training groups. After 9 weeks of training, heart and skeletal muscle samples were harvested and used for gene expression analysis, NAD levels measurements and immunohistochemistry staining. Endurance training triggered a reduction in the expression of Cpt1b and AcadL genes involved in fatty acid catabolism in the heart of mice, not in control mice. NAD levels were not altered in heart or skeletal muscle, nor at baseline neither after exercise training in any group. gene encoding for the slow MHC-I was more strongly induced by exercise in mice than in controls. Moreover, -15 expression levels is higher in mice skeletal muscle at baseline compared to controls. No fiber type switch was observed in plantaris after exercise, but fast fibers diameter was reduced in aged control mice, not in mice. No fiber type switch or diameter modification was observed in soleus muscle. In this study, we demonstrated for the first time a phenotype in old mice in response to endurance exercise training. Although NMRK2 seems to be predominantly dispensable to maintain global NAD levels in heart and skeletal muscle, we demonstrated a maladaptive metabolic response to exercise in cardiac and skeletal muscle, showing that NMRK2 has a specific and restricted role in NAD signaling compared to the NAMPT pathway.
Erratum: Author Correction: Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study
Dellinger RW, Santos SR, Morris M, Evans M, Alminana D, Guarente L and Marcotulli E
[This corrects the article DOI: 10.1038/s41514-017-0016-9.].
Vitamin B3 in Health and Disease: Toward the Second Century of Discovery
Jacobson MK and Jacobson EL
This introductory chapter briefly reviews the history, chemistry, and biochemistry of NAD (the term NAD as it is used here refers to both oxidized and reduced forms of the molecule) consuming ADP-ribose transfer enzymes as components of the involvement of vitamin B3 in health and disease.
Serum Metabolomics Study Based on LC-MS and Antihypertensive Effect of on Spontaneously Hypertensive Rats
Liu A, Chu YJ, Wang X, Yu R, Jiang H, Li Y, Zhou H, Gong LL, Yang WQ and Ju J
Our previous studies have shown that has an important role in lowering blood pressure, but its intervention mechanism has not been clarified completely in the metabolic level. Therefore, in this study, a combination method of HPLC-TOF/MS-based metabolomics and multivariate statistical analyses was employed to explore the mechanism and evaluate the antihypertensive effect of . Serum samples were analyzed and identified by HPLC-TOF/MS, while the acquired data was further processed by partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to discover the perturbed metabolites. A clear cluster among the different groups was obtained, and 7 significantly changed potential biomarkers were screened out. These biomarkers were mainly associated with lipid metabolism (dihydroceramide, ceramide, PC, LysoPC, and TXA2) and vitamin and amino acids metabolism (nicotinamide riboside, 5-HTP). The result indicated that could decrease the blood pressure effectively, partially by regulating the above biomarkers and metabolic pathways. Analyzing and verifying the specific biomarkers, further understanding of the therapeutic mechanism and antihypertensive effect of was acquired. Metabolomics provided a new insight into estimate of the therapeutic effect and dissection of the potential mechanisms of traditional Chinese medicine (TCM) in treating hypertension.
NAD biosynthesis, aging, and disease
Johnson S and Imai SI
Nicotinamide adenine dinucleotide (NAD ) biosynthesis and its regulation have recently been attracting markedly increasing interest. Aging is marked by a systemic decrease in NAD across multiple tissues. The dysfunction of NAD biosynthesis plays a critical role in the pathophysiologies of multiple diseases, including age-associated metabolic disorders, neurodegenerative diseases, and mental disorders. As downstream effectors, NAD -dependent enzymes, such as sirtuins, are involved in the progression of such disorders. These recent studies implicate NAD biosynthesis as a potential target for preventing and treating age-associated diseases. Indeed, new studies have demonstrated the therapeutic potential of supplementing NAD intermediates, such as nicotinamide mononucleotide and nicotinamide riboside, providing a proof of concept for the development of an effective anti-aging intervention.
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