Nicotinic Acid Mononucleotide Research Articles

The Effectiveness of Four Nicotinamide Adenine Dinucleotide (NAD+) Precursors in Alleviating the High-Glucose-Induced Damage to Hepatocytes in Megalobrama amblycephala: Evidence in NAD+ Homeostasis, Sirt1/3 Activation, Redox Defense, Inflammatory Response, Apoptosis, and Glucose Metabolism.

The administration of NAD+ precursors is a potential approach to protect against liver damage and metabolic dysfunction. However, the effectiveness of different NAD+ precursors in alleviating metabolic disorders is still poorly elucidated. The current study was performed to compare the effectiveness of four different NAD+ precursors, including nicotinic acid (NA), niacinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) in alleviating high-glucose-induced injury to hepatocytes in a fish model, Megalobrama amblycephala. An in vitro high-glucose model was successfully established to mimic hyperglycemia-induced damage to the liver, which was evidenced by the reduced cell viability, the increased transaminase activity, and the depletion of cellular NAD+ concentration. The NAD+ precursors all improved cell viability, with the maximal effect observed in NR, which also had the most potent NAD+ boosting capacity and a significant Sirt1/3 activation effect. Meanwhile, NR presented distinct and superior effects in terms of anti-oxidative stress, inflammation inhibition, and anti-apoptosis compared with NA, NAM, and NMN. Furthermore, NR could effectively benefit glucose metabolism by activating glucose transportation, glycolysis, glycogen synthesis and the pentose phosphate pathway, as well as inhibiting gluconeogenesis. Moreover, an oral gavage test confirmed that NR presented the most potent effect in increasing hepatic NAD+ content and the NAD+/NADH ratio among four NAD+ precursors. Together, the present study results demonstrated that NR is most effective in attenuating the high-glucose-induced injury to hepatocytes in fish compared to other NAD+ precursors.

Biotechnological production of reduced and oxidized NAD+ precursors

Dysregulation of nicotinamide adenine dinucleotide (NAD+) homeostasis by increased activity of NAD+ consumers or reduced NAD+ biosynthesis plays an important role in the onset of prevalent, often age-related, diseases, such as diabetes, neuropathies or nephropathies.To counteract such dysregulation, NAD+ replenishment strategies can be used. Among these, administration of vitamin B3 derivatives (NAD+ precursors) has garnered attention in recent years.However, the high market price of these compounds and their limited availability, pose important limitations to their use in nutritional or biomedical applications. To overcome these limitations, we have designed an enzymatic method for the synthesis and purification of (1) the oxidized NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), (2) their reduced forms NMNH and NRH, and (3) their deaminated forms nicotinic acid mononucleotide (NaMN) and nicotinic acid riboside (NaR). Starting from NAD+ or NADH as substrates, we use a combination of three highly overexpressed soluble recombinant enzymes; (a) a NAD+ pyrophosphatase, (b) an NMN deamidase, and (c) a 5′-nucleotidase, to produce these six precursors. Finally, we validate the activity of the enzymatically produced molecules as NAD+ enhancers in cell culture.

Bifunctional NadC Homologue PyrZ Catalyzes Nicotinic Acid Formation in Pyridomycin Biosynthesis

Pyridomycin is a potent antimycobacterial natural product by specifically inhibiting InhA, a clinically validated antituberculosis drug discovery target. Pyridyl moieties of pyridomycin play an essential role in inhibiting InhA by occupying the reduced form of the nicotinamide adenine dinucleotide (NADH) cofactor binding site. Herein, we biochemically characterize PyrZ that is a multifunctional NadC homologue and catalyzes the successive formation, dephosphorylation, and ribose hydrolysis of nicotinic acid mononucleotide (NAMN) to generate nicotinic acid (NA), a biosynthetic precursor for the pyridyl moiety of pyridomycin. Crystal structures of PyrZ in complex with substrate quinolinic acid (QA) and the final product NA revealed a specific salt bridge formed between K184 and the C3-carboxyl group of QA. This interaction positions QA for accepting the phosphoribosyl group to generate NAMN, retains NAMN within the active site, and mediates its translocation to nucleophile D296 for dephosphorylation. Combining kinetic and thermodynamic analysis with site-directed mutagenesis, the catalytic mechanism of PyrZ dephosphorylation was proposed. Our study discovered an alternative and concise NA biosynthetic pathway involving a unique multifunctional enzyme.

Importance of NAD+ Anabolism in Metabolic, Cardiovascular and Neurodegenerative Disorders.

The role of nicotinamide adenine dinucleotide (NAD+) in ageing has emerged as a critical factor in understanding links to a wide range of chronic diseases. Depletion of NAD+, a central redox cofactor and substrate of numerous metabolic enzymes, has been detected in many major age-related diseases. However, the mechanisms behind age-associated NAD+ decline remains poorly understood. Despite limited conclusive evidence, supplements aimed at increasing NAD+ levels are becoming increasingly popular. This review provides renewed insights regarding the clinical utility and benefits of NAD+ precursors, namely nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), in attenuating NAD+ decline and phenotypic characterization of age-related disorders, including metabolic, cardiovascular and neurodegenerative diseases. While it is anticipated that NAD+ precursors can play beneficial protective roles in several conditions, they vary in their ability to promote NAD+ anabolism with differing adverse effects. Careful evaluation of the role of NAD+, whether friend or foe in ageing, should be considered.

Validation of a liquid chromatography coupled to tandem mass spectrometry method for simultaneous quantification of tryptophan and 10 key metabolites of the kynurenine pathway in plasma and urine: Application to a cohort of acute kidney injury patients

A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tryptophan (Trp) and ten metabolites of kynurenine pathway, including kynurenine (Kyn), 3-hydroxy-kynurenine (3-HK), kynurenic acid (KA), xanthurenic acid (XA), 3-Hydroxy-anthranilic acid (3-HANA), quinolinic acid (QA), nicotinic acid mononucleotide (NaMN), picolinic acid (Pic), nicotinamide (NAM) and nicotinic acid (NA) in both plasma and urine. This LC-MS/MS method was used to predict the occurrence of acute kidney injury (AKI) in a cohort of patients with cardiac surgery under cardiopulmonary bypass (CPB). Urinary concentrations of Pic, as well as Pic to Trp and Pic to 3-HANA ratios were highly predictive of an AKI episode the week after CPB, indicating that Pic could be a predictive biomarker of AKI. Thus, monitoring the kynurenine pathway activity with this LC–MS/MS method is a clinically relevant tool to identify new biomarkers of kidney injury.

Oral Administration of Nicotinamide Mononucleotide Is Safe and Efficiently Increases Blood Nicotinamide Adenine Dinucleotide Levels in Healthy Subjects.

Nicotinamide mononucleotide (NNM) is an orally bioavailable NAD+ precursor that has demonstrated beneficial effects against aging and aging-associated diseases in animal models. NMN is ultimately converted to NAD+, a redox cofactor that mediates many metabolic enzymes. NAD+ also serves as the substrate for poly(ADP-ribose) polymerase (PARP) and sirtuins, and regulates various biological processes, such as metabolism, DNA repair, gene expression, and stress responses. Previous mouse models showed that NMN administration can increase NAD+ in various organs and ameliorate aging-related diseases, such as obesity, diabetes, heart failure, stroke, kidney failure, and Alzheimer’s disease through NAD+-mediated pathways. However, evidence of its effect on humans is still scarce. In this study, we conducted a placebo-controlled, randomized, double blind, parallel-group trial to investigate the safety of orally administered NMN and its efficacy to increase NAD+ levels in thirty healthy subjects. Healthy volunteers received 250 mg/day of NMN (n = 15) or placebo (n = 15) for 12 weeks, and physiological and laboratory tests were performed during this period. In addition, NAD+ and its related metabolites in whole blood were examined. Oral supplementation of NMN for 12 weeks caused no abnormalities in physiological and laboratory tests, and no obvious adverse effects were observed. NAD+ levels in whole blood were significantly increased after NMN administration. We also observed the significant rise in nicotinic acid mononucleotide (NAMN) levels, but not in NMN. We also found that the increased amount of NAD+ was strongly correlated with pulse rate before the administration of NMN. These results suggest that oral administration of NMN is a safe and practical strategy to boost NAD+ levels in humans.Clinical Trial Registration: JRCT [https://jrct.niph.go.jp/], identifier: [jRCTs041200034].

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes.

Treatments that elevate NAD+ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD+ is vital during oocyte maturation. This study aimed to examine the influence of different NAD+ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD+ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD+ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

Gram-scale biocatalytic preparation of the non-natural cofactor nicotinamide cytosine dinucleotide

A simple and fast method of preparation of non-natural nicotinamide adenine dinucleotide (NAD) analog nicotinamide cytosine dinucleotide (NCD) was developed. NCD synthetase (NcdS-1) was developed from nicotinic acid mononucleotide adenylyltransferase (NadD) with higher catalytic efficiency towards nicotinamide mononucleotide (NMN) and cytidine triphosphate (CTP). Reaction composition was optimized with inorganic pyrophosphatase (PPase) from E. coli to achieve near quantitative conversion of substrates. A Scalable purification procedure was determined to achieve a fast 4.19 g NCD preparation of 96 % purity.

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris

Synthetic biology offers several routes for CO2 conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a μmax of 0.008 h−1, which was further improved to 0.018 h−1 by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO2 for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.

Adenine Nucleotide Metabolites in Uremic Erythrocytes as Metabolic Markers of Chronic Kidney Disease in Children.

Chronic kidney disease (CKD) is associated with multifaceted pathophysiological lesions including metabolic pathways in red blood cells (RBC). The aim of the study was to determine the concentration of adenine nucleotide metabolites, i.e., nicotinamide adenine dinucleotide (NAD)-oxidized form, nicotinamide adenine dinucleotide hydrate (NADH)-reduced form, nicotinic acid mononucleotide (NAMN), β-nicotinamide mononucleotide (NMN), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid (NA) and nicotinamide (NAM) in RBC and to determine a relationship between NAD metabolites and CKD progression. Forty-eight CKD children and 33 age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III- stage III, Group IV- stage IV and Group RRT children on dialysis). To determine the above-mentioned metabolites concentrations in RBC liquid chromatography-mass spectrometry was used. Results: the only difference between the groups was shown concerning NAD in RBC, although the values did not differ significantly from controls. The lowest NAD values were found in Group II (188.6 ± 124.49 nmol/mL, the highest in group IV (324.94 ± 63.06 nmol/mL. Between Groups II and IV, as well as III and IV, the differences were statistically significant (p < 0.032, p < 0.046 respectively). Conclusions. CKD children do not have evident abnormalities of RBC metabolism with respect to adenine nucleotide metabolites. The significant differences in erythrocyte NAD concentrations between CKD stages may suggest the activation of adaptive defense mechanisms aimed at erythrocyte metabolic stabilization. It seems that the implementation of RRT has a positive impact on RBC NAD metabolism, but further research performed on a larger population is needed to confirm it.

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

Nicotinic acid supplementation at a supraphysiological dose increases the bioavailability of NAD+ precursors in mares.

NAD+ deficiency has recently been linked with increased occurrences of congenital abnormalities and embryonic death in human and animal subjects. Early embryonic death is a major component of pregnancy loss in mares and very little is known regarding the requirement for NAD+ in horses. The aim of this study was to quantify NAD+ and its metabolites in the plasma and urine of mares after orally administering an acute dose of nicotinic acid and determine the absorption, metabolism and excretion of this essential precursor for NAD+ biosynthesis. Nicotinic acid (5g per os) was administered to four mares via a dosing syringe. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 22h, and urine samples were collected at 0, 3, 6 and 22h. The samples were processed and analysed by mass spectrometry. A general additive model was applied to all metabolite concentration values followed by a post-hoc multiple comparisons test. Nicotinic acid was rapidly absorbed into peripheral blood within 15min of administration and the concentrations of nicotinic acid, nicotinamide (NAM), nicotinuric acid, nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide (NaAD) increased significantly in plasma at 30min. The concentrations of NAM, nicotinic acid riboside and NaAD increased significantly in urine at 3h. The levels of NAM and NaAD remained significantly elevated in plasma at 22h, sixfold and ninefold greater, respectively, than the basal levels at 0h. While the extracellular levels of NAD+ in the samples remained undetected, the large, sustained elevation of NaAD levels in plasma indicates that the NAD+ levels were boosted within the cellular compartments. The results show that nicotinic acid supplementation increases the bioavailability of NAD+ precursors in mares, which is proposed to be beneficial during periods of peak NAD+ demand, such as during early embryo development.

Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

Of mice and men: opposing effects of nicotinamide riboside on skeletal muscle physiology at rest and during exercise.

Of mice and men: opposing effects of nicotinamide riboside on skeletal muscle physiology at rest and during exercise.

Nicotinamide riboside supplementation does not alter whole-body or skeletal muscle metabolic responses to a single bout of endurance exercise.

Acute nicotinamide riboside (NR) supplementation does not alter substrate metabolism at rest, during or in recovery from endurance exercise. NR does not alter NAD+ -sensitive signalling pathways in human skeletal muscle. NR supplementation and acute exercise influence the NAD+ metabolome. Oral supplementation of the NAD+ precursor nicotinamide riboside (NR) has been reported to alter metabolism alongside increasing sirtuin (SIRT) signalling and mitochondrial biogenesis in rodent skeletal muscle. However, whether NR supplementation can elicit a similar response in human skeletal muscle is unclear. This study assessed the effect of 7-day NR supplementation on whole-body metabolism and exercise-induced mitochondrial biogenic signalling in skeletal muscle. Eight male participants (age: 23±4years, 46.5±4.4mlkg-1 min-1 ) received 1week of NR or cellulose placebo (PLA) supplementation (1000mgday-1 ). Muscle biopsies were collected from the medial vastus lateralis prior to supplementation and pre-, immediately post- and 3h post-exercise (1h of 60% Wmax cycling) performed following the supplementation period. There was no effect of NR supplementation on substrate utilisation at rest or during exercise or on skeletal muscle mitochondrial respiration. Global acetylation, auto-PARylation of poly ADP-ribose polymerase 1 (PARP1), acetylation of Tumour protein 53 (p53)Lys382 and Manganese superoxide dismutase (MnSOD)Lys122 were also unaffected by NR supplementation or exercise. NR supplementation did not increase skeletal muscle NAD+ concentration, but it did increase the concentration of deaminated NAD+ precursors nicotinic acid riboside (NAR) and nicotinic acid mononucleotide (NAM) and methylated nicotinamide breakdown products (Me2PY and Me4PY), demonstrating the skeletal muscle bioavailability of NR supplementation. In summary, 1week of NR supplementation does not alter whole-body metabolism or skeletal muscle signal transduction pathways implicated in the mitochondrial adaptation to endurance exercise.

Caffeine and NAD+ Improve Motor Neural Integrity of Dissociated Wobbler Cells In Vitro.

Amyotrophic lateral sclerosis (ALS) is a common degenerative disease of the central nervous system concerning a progressive loss of upper and lower motor neurons. While 5%–10% of patients are diagnosed with the inherited form of the disease, the vast majority of patients suffer from the less characterized sporadic form of ALS (sALS). As the wobbler mouse and the ALS show striking similarities in view of phenotypical attributes, the mouse is rated as an animal model for the disease. Recent investigations show the importance of nicotinamide adenine dinucleotide (NAD+) and its producing enzyme nicotinic acid mononucleotide transferase 2 (Nmnat2) for neurodegeneration as well as for the preservation of health of the neuronal cells. Furthermore, it is newly determined that these molecules show significant downregulations in the spinal cord of wobbler mice in the stable phase of disease development. Here, we were able to prove a positive benefit on affected motor neurons from an additional NAD+ supply as well as an increase in the Nmnat2 level through caffeine treatment in cells in vitro. In addition, first assumptions about the importance of endogenous and exogenous factors that have an influence on the wellbeing of motor nerve cells in the model of ALS can be considered.

Metabolite analysis-aided diagnosis of papillary thyroid cancer.

Thyroid cancer is the most frequent endocrine tumor with a growing incidence worldwide. However, common diagnostic strategy for thyroid cancer classification is hardly to make a proper diagnosis in some cases. To assist classical approach, this study used metabolomics to screen and validate biomarkers from serum and urinary for papillary thyroid cancer (PTC). Overall, 124 untreated PTC, 76 untreated benign thyroid nodule (BTN), and 116 healthy control (HC) were collected in this study. Thirty-six differential metabolites were screened from non-targeted metabolomics with a discovery sample set in comparison with HC and BTN. Serum β-hydroxybutyrate (BHB), docosahexaenoic acid (DHA), 1-methyladenosine (1-MedA), pregnanediol-3-glucuronide (PdG), urinary nicotinic acid mononucleotide (NAM) and xanthosine (Xan) were validated to be significantly differential by targeted metabolomics in validation set. The logistic regression model incorporating six biomarkers had excellent discrimination from receiver-operating characteristics (ROC) analysis, with area under the receiver-operating characteristic curve (AUC) of 0.943 (95% CI 0.902 to 0.983) and 0.952 (95% CI 0.921 to 0.983) for female alone and female + male samples, respectively. The prediction accuracy and false-negative rate in the real setting of one PTC to ten suspicious nodules were 84.7 and 17.7% with the threshold at probablity of 0.5. Results of a double-blind study for PTC and BTN had true positive value of 100% and true negative value of 91.7%. To conclude, BHB, DHA, 1-MedA, PdG, NAM and Xan are suitable biomarkers for PTC, and logistic regression models with the six biomarkers can be potentially used as clinical diagnosis.

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

The Plasma NAD+ Metabolome Is Dysregulated in "Normal" Aging.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an electron carrier in cellular metabolism and plays a crucial role in the maintenance of balanced redox homeostasis. Quantification of NAD+:NADH and NADP+:NADPH ratios are pivotal to a wide variety of cellular processes, including intracellular secondary messenger signaling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase (PARP), epigenetic regulation of gene expression by NAD-dependent histone deacetylase enzymes known as sirtuins, and regulation of the oxidative pentose phosphate pathway. We quantified changes in the NAD+ metabolome in plasma samples collected from consenting healthy human subjects across a wide age range (20–87 years) using liquid chromatography coupled to tandem mass spectrometry. Our data show a significant decline in the plasma levels of NAD+, NADP+, and other important metabolites such as nicotinic acid adenine dinucleotide (NAAD) with age. However, an age-related increase in the reduced form of NAD+ and NADP+—NADH and NADPH—and nicotinamide (NAM), N-methyl-nicotinamide (MeNAM), and the products of adenosine diphosphoribosylation, including adenosine diphosphate ribose (ADPR) was also reported. Whereas, plasma levels of nicotinic acid (NA), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN) showed no statistically significant changes across age groups. Taken together, our data cumulatively suggest that age-related impairments are associated with corresponding alterations in the extracellular plasma NAD+ metabolome. Our future research will seek to elucidate the role of modulating NAD+ metabolites in the treatment and prevention of age-related diseases.

Dual control of NAD+ synthesis by purine metabolites in yeast.

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD+ biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD+. This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network.