Published Research on NR 2018-03-12T11:37:25-08:00

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

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
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.
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.
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.
Role of endothelial NAD+ deficiency in age-related vascular dysfunction
Csicsar A, Tarantini S, Yabluchanskiy A, Balasubramanian P, Kiss T, Farkas E, Baur JA and Ungvari ZI
Age-related alterations in endothelium and the resulting vascular dysfunction critically contribute to a range of pathological conditions associated with old age. To rationally develop therapies that improve vascular health and thereby increase health span and lifespan in older adults, it will be essential to understand the cellular and molecular mechanisms contributing to vascular aging. Pre-clinical studies in model organisms demonstrate that NAD availability decreases with age in multiple tissues and that supplemental NAD precursors can ameliorate many age-related cellular impairments. Here we provide a comprehensive overview of NAD dependent pathways (including the NAD utilizing sirtuins and poly (ADP-ribose) polymerase enzymes) and the potential consequences of endothelial NAD deficiency in vascular aging. The multifaceted vasoprotective effects of treatments that reverse the age-related decline in cellular NAD levels are discussed. The preventive and therapeutic potential of NAD intermediates as effective, clinically relevant interventions in older adults at risk for ischemic heart disease, vascular cognitive impairment and other common geriatric conditions and diseases that involve vascular pathologies (e.g. sarcopenia, frailty) is critically discussed. We propose that NAD precursors (e.g., nicotinamide riboside, nicotinamide mononucleotide, niacin) should be considered as a critical component of combination therapies to slow the vascular aging process and increase cardiovascular health span.
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.
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.
Pterostilbene raises low density lipoprotein cholesterol in people
Brenner C and Boileau AC
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy
Diguet N, Trammell SAJ, Tannous C, Deloux R, Piquereau J, Mougenot N, Gouge A, Gressette M, Manoury B, Blanc J, Breton M, Decaux JF, Lavery GG, Baczkó I, Zoll J, Garnier A, Li Z, Brenner C and Mericskay M
Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD in the failing heart.
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.
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.
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.
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 de novo NAD synthesis pathway in mammalian cells. NAD can also be produced by the NAD salvage pathway. Recent Advances: 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.
Correction to: Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy
Raising NAD in Heart Failure: Time to Translate?
Walker MA and Tian R
ALSUntangled 42: Elysium health's "basis"
Bedlack R and
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.
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.
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).
Perturbations of NAD salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle
Agerholm M, Dall M, Jensen BAH, Prats C, Madsen S, Basse AL, Graae AS, Risis S, Goldenbaum J, Quistorff B, Larsen S, Vienberg SG and Treebak JT
Nicotinamide adenine dinucleotide (NAD) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT in maintaining NAD levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express Cre recombinase in tibialis anterior muscle of floxed Nampt mice. In sh Nampt KD C2C12 myoblasts, Nampt and NAD levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55%, and 2-deoxyglucose uptake increased by 25% in sh Nampt KD cells. Treatment with the NAD precursor nicotinamide riboside restored NAD levels in sh Nampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh Nampt KD cells, respectively. Expression of Cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NAD levels by 38% and 43%, respectively. Glucose uptake increased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxia-inducible factor (HIF)-1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known sirtuin 6 (SIRT6) target, were increased in shNampt KD cells. Thus, we propose that the shift toward glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NAD levels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.
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.
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.
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.
NAD Intermediates: The Biology and Therapeutic Potential of NMN and NR
Yoshino J, Baur JA and Imai SI
Research on the biology of NAD has been gaining momentum, providing many critical insights into the pathogenesis of age-associated functional decline and diseases. In particular, two key NAD intermediates, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), have been extensively studied over the past several years. Supplementing these NAD intermediates has shown preventive and therapeutic effects, ameliorating age-associated pathophysiologies and disease conditions. Although the pharmacokinetics and metabolic fates of NMN and NR are still under intensive investigation, these NAD intermediates can exhibit distinct behavior, and their fates appear to depend on the tissue distribution and expression levels of NAD biosynthetic enzymes, nucleotidases, and presumptive transporters for each. A comprehensive concept that connects NAD metabolism to the control of aging and longevity in mammals has been proposed, and the stage is now set to test whether these exciting preclinical results can be translated to improve human health.
Directed Evolution of Membrane Transport Using Synthetic Selections
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 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.
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 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.
A Direct Prebiotic Synthesis of Nicotinamide Nucleotide
Kim HJ and Benner SA
The "RNA World" hypothesis proposes an early episode of the natural history of Earth, where RNA was used as the only genetically encoded molecule to catalyze steps in its metabolism. This, according to the hypothesis, included RNA catalysts that used RNA cofactors. However, the RNA World hypothesis places special demands on prebiotic chemistry, which must now deliver not only four ribonucleosides, but also must deliver the "functional" portion of these RNA cofactors. While some (e.g., methionine) present no particular challenges, nicotinamide ribose is special. Essential to its role in biological oxidations and reductions, its glycosidic bond that holds a positively charged heterocycle is especially unstable with respect to cleavage. Nevertheless, we are able to report here a prebiotic synthesis of phosphorylated nicotinamide ribose under conditions that also conveniently lead to the adenosine phosphate components of this and other RNA cofactors.
Deficiency of the Mitochondrial NAD Kinase Causes Stress-Induced Hepatic Steatosis in Mice
Zhang K, Kim H, Fu Z, Qiu Y, Yang Z, Wang J, Zhang D, Tong X, Yin L, Li J, Wu J, Qi NR, Houten SM and Zhang R
The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice.
Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity
Sorrentino V, Romani M, Mouchiroud L, Beck JS, Zhang H, D'Amico D, Moullan N, Potenza F, Schmid AW, Rietsch S, Counts SE and Auwerx J
Alzheimer's disease is a common and devastating disease characterized by aggregation of the amyloid-β peptide. However, we know relatively little about the underlying molecular mechanisms or how to treat patients with Alzheimer's disease. Here we provide bioinformatic and experimental evidence of a conserved mitochondrial stress response signature present in diseases involving amyloid-β proteotoxicity in human, mouse and Caenorhabditis elegans that involves the mitochondrial unfolded protein response and mitophagy pathways. Using a worm model of amyloid-β proteotoxicity, GMC101, we recapitulated mitochondrial features and confirmed that the induction of this mitochondrial stress response was essential for the maintenance of mitochondrial proteostasis and health. Notably, increasing mitochondrial proteostasis by pharmacologically and genetically targeting mitochondrial translation and mitophagy increases the fitness and lifespan of GMC101 worms and reduces amyloid aggregation in cells, worms and in transgenic mouse models of Alzheimer's disease. Our data support the relevance of enhancing mitochondrial proteostasis to delay amyloid-β proteotoxic diseases, such as Alzheimer's disease.
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, a form of vitamin B, protects against excitotoxicity-induced axonal degeneration
Vaur P, Brugg B, Mericskay M, Li Z, Schmidt MS, Vivien D, Orset C, Jacotot E, Brenner C and Duplus E
NAD depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD to NR prompted us to probe the effects of NAD and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD Moreover, the stronger effect of NR compared to NAD depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD homeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B, protects against excitotoxicity-induced axonal degeneration.
Circadian and Feeding Rhythms Orchestrate the Diurnal Liver Acetylome
Mauvoisin D, Atger F, Dayon L, Núñez Galindo A, Wang J, Martin E, Da Silva L, Montoliu I, Collino S, Martin FP, Ratajczak J, Cantó C, Kussmann M, Naef F and Gachon F
Lysine acetylation is involved in various biological processes and is considered a key reversible post-translational modification in the regulation of gene expression, enzyme activity, and subcellular localization. This post-translational modification is therefore highly relevant in the context of circadian biology, but its characterization on the proteome-wide scale and its circadian clock dependence are still poorly described. Here, we provide a comprehensive and rhythmic acetylome map of the mouse liver. Rhythmic acetylated proteins showed subcellular localization-specific phases that correlated with the related metabolites in the regulated pathways. Mitochondrial proteins were over-represented among the rhythmically acetylated proteins and were highly correlated with SIRT3-dependent deacetylation. SIRT3 activity being nicotinamide adenine dinucleotide (NAD) level-dependent, we show that NAD is orchestrated by both feeding rhythms and the circadian clock through the NAD salvage pathway but also via the nicotinamide riboside pathway. Hence, the diurnal acetylome relies on a functional circadian clock and affects important diurnal metabolic pathways in the mouse liver.
NAD Deficits in Age-Related Diseases and Cancer
Garrido A and Djouder N
The phenomenon of aging has gained widespread attention in recent times. Although significant advances have been made to better understand aging and its related pathologies including cancer, there is not yet a clear mechanism explaining why diseases and cancer are inherent parts of the aging process. Finding a unifying equation that could bridge aging and its related diseases would allow therapeutic development and solve an immense human health problem to live longer and better. In this review, we discuss NAD reduction as the central mechanism that may connect aging to its related pathologies and cancer. NAD boosters would ensure and ameliorate health quality during aging.
Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells
Fletcher RS, Ratajczak J, Doig CL, Oakey LA, Callingham R, Da Silva Xavier G, Garten A, Elhassan YS, Redpath P, Migaud ME, Philp A, Brenner C, Canto C and Lavery GG
Augmenting nicotinamide adenine dinucleotide (NAD) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.
Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet
Shi W, Hegeman MA, van Dartel DAM, Tang J, Suarez M, Swarts H, van der Hee B, Arola L and Keijer J
Metabolic flexibility is the ability to switch metabolism between carbohydrate oxidation (CHO) and fatty acid oxidation (FAO) and is a biomarker for metabolic health. The effect on metabolic health of nicotinamide riboside (NR) as an exclusive source of vitamin B3 is unknown and is examined here for a wide range of NR.
1 H NMR-based metabolomics study for identifying urinary biomarkers and perturbed metabolic pathways associated with severity of IgA nephropathy: a pilot study
Kalantari S, Nafar M, Samavat S and Parvin M
The severity of IgA nephropathy (IgAN), the most common primary glomerulonephritis, is judged on the basis of histologic and clinical features. A limited number of studies have considered molecular signature of IgAN for this issue, and no reliable biomarkers have been presented non-invasively for use in patient evaluations. This study aims to identify metabolite markers excreted in the urine and impaired pathways that are associated with a known marker of severity (proteinuria) to predict mild and severe stages of IgAN. Urine samples were analysed using nuclear magnetic resonance from biopsy-proven IgAN patients at mild and severe stages. Multivariate statistical analysis and pathway analysis were performed. The most changed metabolites were acetoacetate, hypotaurine, homocysteine, L-kynurenine and phenylalanine. Nine metabolites were positively correlated with proteinuria, including mesaconic acid, trans-cinnamic acid, fumaric acid, 5-thymidylic acid, anthranilic acid, indole, deoxyguanosine triphosphate, 13-cis-retinoic acid and nicotinamide riboside, while three metabolites were negatively correlated with proteinuria including acetoacetate, hypotaurine and hexanal. 'Phenylalanine metabolism' was the most significant pathway which was impaired in severe stage in comparison to mild stage of IgAN. This study indicates that nuclear magnetic resonance is a versatile technique that is capable of detecting metabolite biomarkers in combination with advanced multivariate statistical analysis. Copyright © 2017 John Wiley & Sons, Ltd.
The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD⁺ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice
Wang X, Zhang ZF, Zheng GH, Wang AM, Sun CH, Qin SP, Zhuang J, Lu J, Ma DF and Zheng YL
Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, exhibits beneficial effects on metabolic syndrome. Sustained inflammation plays a crucial role in the pathogenesis of metabolic syndrome. Here we explored the effects of PSPC on high-fat diet (HFD)-induced hepatic inflammation and the mechanisms underlying these effects. Mice were divided into four groups: Control group, HFD group, HFD + PSPC group, and PSPC group. PSPC was administered by daily oral gavage at doses of 700 mg/kg/day for 20 weeks. Nicotinamide riboside (NR) was used to increase NAD⁺ levels. Our results showed that PSPC effectively ameliorated obesity and liver injuries in HFD-fed mice. Moreover, PSPC notably blocked hepatic oxidative stress in HFD-treated mice. Furthermore, PSPC dramatically restored NAD⁺ level to abate endoplasmic reticulum stress (ER stress) in HFD-treated mouse livers, which was confirmed by NR treatment. Consequently, PSPC remarkably suppressed the nuclear factor-κB (NF-κB) p65 nuclear translocation and nucleotide oligomerization domain protein1/2 (NOD1/2) signaling in HFD-treated mouse livers. Thereby, PSPC markedly diminished the NLR family, pyrin domain containing 3 (NLRP3) inflammasome activation, ultimately lowering the expressions of inflammation-related genes in HFD-treated mouse livers. In summary, PSPC protected against HFD-induced hepatic inflammation by boosting NAD⁺ level to inhibit NLRP3 inflammasome activation.
Targeting NAD+ in Metabolic Disease: New Insights Into an Old Molecule
Elhassan YS, Philp AA and Lavery GG
Nicotinamide adenine dinucleotide (NAD+) is an established cofactor for enzymes serving cellular metabolic reactions. More recent research identified NAD+ as a signaling molecule and substrate for sirtuins and poly-adenosine 5'-diphosphate polymerases; enzymes that regulate protein deacetylation and DNA repair, and translate changes in energy status into metabolic adaptations. Deranged NAD+ homeostasis and concurrent alterations in mitochondrial function are intrinsic in metabolic disorders, such as type 2 diabetes, nonalcoholic fatty liver, and age-related diseases. Contemporary NAD+ precursors show promise as nutraceuticals to restore target tissue NAD+ and have demonstrated the ability to improve mitochondrial function and sirtuin-dependent signaling. This review discusses the accumulating evidence for targeting NAD+ metabolism in metabolic disease, maps the different strategies for NAD+ boosting, and addresses the challenges and open questions in the field. The health potential of targeting NAD+ homeostasis will inform clinical study design to identify nutraceutical approaches for combating metabolic disease and the unwanted effects of aging.
Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease
Watson A, Nong Z, Yin H, O'Neil C, Fox S, Balint B, Guo L, Leo O, Chu MWA, Gros R and Pickering JG
The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown.
The NAD+/PARP1/SIRT1 Axis in Aging
Mendelsohn AR and Larrick JW
NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.
Nicotinamide riboside, a form of vitamin B3 and NAD+ precursor, relieves the nociceptive and aversive dimensions of paclitaxel-induced peripheral neuropathy in female rats
Hamity MV, White SR, Walder RY, Schmidt MS, Brenner C and Hammond DL
Injury to sensory afferents may contribute to the peripheral neuropathies that develop after administration of chemotherapeutic agents. Manipulations that increase levels of nicotinamide adenine dinucleotide (NAD) can protect against neuronal injury. This study examined whether nicotinamide riboside (NR), a third form of vitamin B3 and precursor of NAD, diminishes tactile hypersensitivity and place escape-avoidance behaviors in a rodent model of paclitaxel-induced peripheral neuropathy. Female Sprague-Dawley rats received 3 intravenous injections of 6.6 mg/kg paclitaxel over 5 days. Daily oral administration of 200 mg/kg NR beginning 7 days before paclitaxel treatment and continuing for another 24 days prevented the development of tactile hypersensitivity and blunted place escape-avoidance behaviors. These effects were sustained after a 2-week washout period. This dose of NR increased blood levels of NAD by 50%, did not interfere with the myelosuppressive effects of paclitaxel, and did not produce adverse locomotor effects. Treatment with 200 mg/kg NR for 3 weeks after paclitaxel reversed the well-established tactile hypersensitivity in a subset of rats and blunted escape-avoidance behaviors. Pretreatment with 100 mg/kg oral acetyl-L-carnitine (ALCAR) did not prevent paclitaxel-induced tactile hypersensitivity or blunt escape-avoidance behaviors. ALCAR by itself produced tactile hypersensitivity. These findings suggest that agents that increase NAD, a critical cofactor for mitochondrial oxidative phosphorylation systems and cellular redox systems involved with fuel utilization and energy metabolism, represent a novel therapeutic approach for relief of chemotherapy-induced peripheral neuropathies. Because NR is a vitamin B3 precursor of NAD and a nutritional supplement, clinical tests of this hypothesis may be accelerated.
Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay
Ummarino S, Mozzon M, Zamporlini F, Amici A, Mazzola F, Orsomando G, Ruggieri S and Raffaelli N
Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.
The NAD precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation
Weidele K, Beneke S and Bürkle A
NAD is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD levels may be limiting under conditions of genotoxic stress and that boosting the NAD system with nicotinic acid can improve genomic stability.
Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs
Nübel G, Sorgenfrei FA and Jäschke A
RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels. The boronyl groups inside the gel form relatively stable complexes with 1,2-cis diols, occurring naturally at the 3'-end of RNA, and also in the nicotinamide riboside of NAD-modified RNA at the 5'-end. The transient formation of diesters between the immobilized boronic acid and the diols causes lower mobility of the modified RNAs, compared to unmodified RNAs, resulting in two distinct bands for one RNA sequence. We used APB affinity gel electrophoresis to preparatively purify in vitro transcribed NAD-RNA from triphosphorylated RNA, to study the enzyme kinetics of the NAD-RNA decapping enzyme NudC, and to determine the NAD modification ratios of various cellular sRNAs. In summary, APB affinity gels can be used to study cofactor-modified RNAs with low amounts of material, and to rapidly screen for their occurrence in total RNA while avoiding complex sample treatments.
Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration
Mukherjee S, Chellappa K, Moffitt A, Ndungu J, Dellinger RW, Davis JG, Agarwal B and Baur JA
The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.
Nicotinamide riboside or IL-17A signaling blockers to prevent liver disorders
Teijeiro A and Djouder N
Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism
James EL, Lane JA, Michalek RD, Karoly ED and Parkinson EK
Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.
NAD(+) metabolism: Bioenergetics, signaling and manipulation for therapy
Yang Y and Sauve AA
We survey the historical development of scientific knowledge surrounding Vitamin B3, and describe the active metabolite forms of Vitamin B3, the pyridine dinucleotides NAD and NADP which are essential to cellular processes of energy metabolism, cell protection and biosynthesis. The study of NAD has become reinvigorated by new understandings that dynamics within NAD metabolism trigger major signaling processes coupled to effectors (sirtuins, PARPs, and CD38) that reprogram cellular metabolism using NAD as an effector substrate. Cellular adaptations include stimulation of mitochondrial biogenesis, a process fundamental to adjusting cellular and tissue physiology to reduced nutrient availability and/or increased energy demand. Several mammalian metabolic pathways converge to NAD, including tryptophan-derived de novo pathways, nicotinamide salvage pathways, nicotinic acid salvage and nucleoside salvage pathways incorporating nicotinamide riboside and nicotinic acid riboside. Key discoveries highlight a therapeutic potential for targeting NAD biosynthetic pathways for treatment of human diseases. A recent emergence of understanding that NAD homeostasis is vulnerable to aging and disease processes has stimulated testing to determine if replenishment or augmentation of cellular or tissue NAD can have ameliorative effects on aging or disease phenotypes. This experimental approach has provided several proofs of concept successes demonstrating that replenishment or augmentation of NAD concentrations can provide ameliorative or curative benefits. Thus NAD metabolic pathways can provide key biomarkers and parameters for assessing and modulating organism health.
Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding
Drew JE, Farquharson AJ, Horgan GW and Williams LM
The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D.
NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation
Ryu D, Zhang H, Ropelle ER, Sorrentino V, Mázala DA, Mouchiroud L, Marshall PL, Campbell MD, Ali AS, Knowels GM, Bellemin S, Iyer SR, Wang X, Gariani K, Sauve AA, Cantó C, Conley KE, Walter L, Lovering RM, Chin ER, Jasmin BJ, Marcinek DJ, Menzies KJ and Auwerx J
Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD) synthesis, consistent with a potential role for the essential cofactor NAD in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD biosynthesis. Replenishing NAD stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.
Overcoming ATM Deficiency by Activating the NAD/SIRT1 Axis
Guarente L
In this issue, Fang et al. (2016) show that both the DNA repair defect and mitochondrial dysfunction in ATM cells or mice are mitigated by the anti-aging compound nicotinamide riboside or a SIRT1 activator. This broad suppression by activating the NAD/SIRT1 axis may generally apply to diseases and aging maladies.
NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells
Ratajczak J, Joffraud M, Trammell SA, Ras R, Canela N, Boutant M, Kulkarni SS, Rodrigues M, Redpath P, Migaud ME, Auwerx J, Yanes O, Brenner C and Cantó C
NAD is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD synthesis from other NAD precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD synthesis, explaining the overlapping metabolic effects observed with the two compounds.
Nicotinamide riboside is uniquely and orally bioavailable in mice and humans
Trammell SA, Schmidt MS, Weidemann BJ, Redpath P, Jaksch F, Dellinger RW, Li Z, Abel ED, Migaud ME and Brenner C
Nicotinamide riboside (NR) is in wide use as an NAD precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD metabolism in humans. We report that human blood NAD can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD, is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD repletion.
The pathophysiological importance and therapeutic potential of NAD' biosynthesis and mitochondrial sirtuin SIRT3 in age-associated diseases
Yamaguchi S and Yoshino J
Nicotinamide adenine dinucleotide(NAD') is a classic coenzyme playing a critical role in cellular redox reactions. Emerging evidence demonstrates that NAD' and its key mediators, NAD+-dependent protein deacetylases (sirtuins), together regulate many important meta- bolic pathways including mitochondrial function. Thus, impaired NAD' biosynthesis is critically involved in the pathophysiology of aging and age-associated diseases. Importantly, administration of key NAD+ intermediates, such as nicotinamide mononucleotide(NMN) or nicotinamide riboside (NR), improves mitochondrial function and exerts remarkable therapeutic effects for various age-associated diseases, such as diabetes and cancer, in mice. In this review, we will summarize and discuss pathophysiological relevance and translational potential of NAD' biology and mitochondrial sirtuin(SIRT3) in age-associated diseases.
Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle
Frederick DW, Loro E, Liu L, Davila A, Chellappa K, Silverman IM, Quinn WJ, Gosai SJ, Tichy ED, Davis JG, Mourkioti F, Gregory BD, Dellinger RW, Redpath P, Migaud ME, Nakamaru-Ogiso E, Rabinowitz JD, Khurana TS and Baur JA
NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.
Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD homeostasis and contributes to longevity
Tsang F and Lin SJ
Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, , and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD precursor) homeostasis. Maintaining adequate NAD pools has been shown to play key roles in life span extension, but factors regulating NAD metabolism and homeostasis are not completely understood. Recently, NAD metabolism was also linked to the phosphate (Pi)-sensing pathway in yeast. Canonical activation requires Pi-starvation. Interestingly, NAD depletion without Pi-starvation was sufficient to induce activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with signaling components to regulate NR/NAD homeostasis. These studies suggest that NAD metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of , PKA, TOR and Sch9 pathways was reported to potentially affect NAD metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD homeostasis, and cross-talk among nutrient sensing pathways and NAD homeostasis.
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.].
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.
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.
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.
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.
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.
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.
An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers
Airhart SE, Shireman LM, Risler LJ, Anderson GD, Nagana Gowda GA, Raftery D, Tian R, Shen DD and O'Brien KD
The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels.
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
NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD precursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NAD in whole blood demonstrated that NRPT significantly increases the concentration of NAD in a dose-dependent manner. NAD levels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NAD levels was sustained throughout the entire 8-week trial. NAD levels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NAD levels sustainably.
The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats
Kourtzidis IA, Stoupas AT, Gioris IS, Veskoukis AS, Margaritelis NV, Tsantarliotou M, Taitzoglou I, Vrabas IS, Paschalis V, Kyparos A and Nikolaidis MG
Nicotinamide adenine dinucleotide (NAD(+)) and its phosphorylated form (NADP(+)) are key molecules in ubiquitous bioenergetic and cellular signaling pathways, regulating cellular metabolism and homeostasis. Thus, supplementation with NAD(+) and NADP(+) precursors emerged as a promising strategy to gain many and multifaceted health benefits. In this proof-of-concept study, we sought to investigate whether chronic nicotinamide riboside administration (an NAD(+) precursor) affects exercise performance.
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.
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