Nicotinamide adenine dinucleotide (NAD) stimulation of DNA synthesis by human bone marrow cells.

Addition of 20 to 200 μg/ml of Nicotinamide Adenine Dinucleotide (NAD) to short term cultures of normal human bone marrow cells increases DNA synthesis only if human serum is present. Although NAD derivatives; reduced (NADH) and phosphorylated (NADP and NADPH) are equally effective, the components of NAD, Nicotinamide (NA) and Adenine (Ad) have no effect or reduce DNA synthesis at high concentrations. When malignant cells are tested; acute myeloblastic leukemia cell division is unaffected or reduced by NAD, NA or Ad.     

NAD+ metabolism in health and disease

Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.     

Anticancer agent CHS-828 inhibits cellular synthesis of NAD

Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting NAD.     

Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans

NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.     

Effects of hyperthermia and nicotinamide on DNA repair synthesis, ADP-ribosyl transferase activity, NAD+ and ATP pools, and cytotoxicity in gamma-irradiated human mononuclear leukocytes

Effects of hyperthermia and nicotinamide on ADP-ribosyl transferase activity (ADPRT), unscheduled DNA synthesis (UDS), NAD+- and ATP-pools and cytotoxicity were investigated in gamma-irradiated human mononuclear leukocytes. A significant decrease in radiation-induced UDS after heat treatment for 45 min was found. Nicotinamide increased the UDS levels in irradiated cells, but no effect of hyperthermia on these increased UDS values was observed. In the presence of 2 mM nicotinamide radiation-induced ADPRT activity was reduced to about 50 per cent. However, hyperthermia for 45 min was found to have no effect on the enzyme activity for temperatures below 46 degrees C. Nicotinamide increased the NAD+ pool in unirradiated cells. Damaging the cells with gamma-radiation leads to a severe depletion of the NAD+ pool. The NAD+ pool is restored, however, if the cells repair for 5 h at 37 degrees C. When radiation-damaged cells were treated with hyperthermia, exogenously supplied nicotinamide could not be converted to NAD+ in sufficient amounts to prevent NAD+ depletion. These data indicate that the radiosensitizing effect of heat and nicotinamide could both be explained by effects on the enzyme ADPRT, i.e. nicotinamide by directly blocking the enzyme and hyperthermia by limiting the co-substrate (NAD+).     

微生物NAD合成酶及其抑制剂的研究进展

NAD(P)生物代谢在能量代谢,维持氧化还原稳态以及调节细胞寿命等许多细胞进程中有重要作用。因此,NAD生物合成途径的关键酶的抑制剂就成为备受关注的候选新药,如NAD合成酶抑制剂。本文对微生物中的NAD合成酶的催化活性特征,晶体结构,调控因子以及基于晶体结构的抑制剂设计方面进行了综述,以期为基于NAD的治疗领域打开新的思路。     

Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide

Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.     

Control of the steady-state concentrations of the nicotinamide nucleotides in rat liver

1. The effects of injecting nicotinamide, 5-methylnicotinamide, ethionine, nicotinamide+5-methylnicotinamide and nicotinamide+ethionine on concentrations in rat liver of NAD, NADP and ATP were investigated up to 5hr. after injection. 2. Nicotinamide induced three- to four-fold increases in hepatic NAD concentration even in the presence of 5-methylnicotinamide or ethionine, whereas 5-methylnicotinamide or ethionine alone did not cause marked changes in hepatic NAD concentration. 3. Nicotinamide alone also induced a twofold increase in hepatic NADP concentration. However, in the presence of 5-methylnicotinamide+nicotinamide, the NADP concentration decreased by 25% after 5hr., and in the presence of nicotinamide+ethionine by 30% in the same time. In the presence of 5-methylnicotinamide or ethionine alone hepatic NADP concentrations fell by 50% after 5hr. 4. 5-Methylnicotinamide inhibited the microsomal NAD(+) glycohydrolase (EC 3.2.2.6) by 60% at a concentration of 1mm and the NADP(+) glycohydrolase by 40% at the same concentration. 5. The rat liver NAD(+) kinase (EC 2.7.1.23) was found to have V(max.) 4.83mumoles/g. wet wt./hr. and K(m) (NAD(+)) 5.8mm. This enzyme was also inhibited by 5-methylnicotinamide in a ;mixed' fashion. 6. The results are discussed with respect to the control of NAD synthesis. It is suggested that in vivo the NAD(P)(+) glycohydrolases are effectively inactive and that the increased NAD concentrations induced by nicotinamide are due to increased substrate concentration available to both the nicotinamide and nicotinic acid pathways of NAD formation.     

Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1

NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.     

Phosphate-responsive signaling pathway is a novel component of NAD+ metabolism in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.     

Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.     

Interaction of nad with rape alcohol dehydrogenase

Rape alcohol dehydrogenase is competitively inhibited with respect to NAD by nicotinamide, as well as by compounds containing adenine (adenine, adenosine, AMP, ADP, ATP). Adenine and adenosine are bound more firmly to the enzyme than nicotinamide. The two types of compound, as component parts of the NAD coenzyme, are bound to different sites on the enzyme. Adenine and adenosine compete for the adenine nucleotide bonding site, but they do not compete for the o-phenanthroline bonding site. Nicotinamide competes with o-phenanthroline for the binding site at which the metal is apparently present.     

Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis.

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.     

Nmnat2 protects cardiomyocytes from hypertrophy via activation of SIRT6

The discovery of sirtuins (SIRT), a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, has indicated that intracellular NAD level is crucial for the hypertrophic response of cardiomyocytes. Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a central enzyme in NAD biosynthesis. Here we revealed that Nmnat2 protein expression and enzyme activity were down-regulated during cardiac hypertrophy. In neonatal rat cardiomyocytes, overexpression of Nmnat2 but not its catalytically inactive mutant blocked angiotensin II (Ang II)-induced cardiac hypertrophy, which was dependent on activation of SIRT6 through maintaining the intracellular NAD level. Our results suggested that modulation of Nmnat2 activity may be beneficial in cardiac hypertrophy.     

The content of pyridine dinucleotides in the mouse embryo before implantation

Nicotinamide adenine dinucleotide (NAD) content was found to decrease in mouse embryos during cleavage and to increase again at the blastocyst stage. The first enzyme involved in biosynthesis of NAD from nicotinamide is nicotinamide mononucleotide (NMN)-pyrophosphorylase. No such enzymatic activity was found in the embryos, but NAD-glycohydrolase activity was relatively high 24–48 hours after conception. Enzyme activity decreased in the blastocyst. The results are relevant to understanding the regulation of metabolism in preimplantation embryos.     

Interrelations of NAD and adenosine transformation in the rat liver

The interrelationship of NAD and adenosine (inosine) conversions in the rat liver is investigated. The ratio of products of NAD+ conversions (ADP-ribose, inosine, hypoxanthine and ribose phosphates) are established. AMP and adenosine are not detected, which indicates an availability of different activities of the corresponding enzymes. It is shown that under conditions of the high inorganic phosphate concentration (33 mM) ribose-1-phosphate, formed in the purine nucleoside phosphorylase reaction, is accumulated due to the phosphoribomutase inhibition, but in the presence of NAD+ the utilization of ribose phosphate increases significantly. Nicotinamide inhibits the NAD+-glycohydrolase reaction in the system containing 33 mM phosphate, NAD+ and adenosine and simultaneously it lowers the utilization of ribose.     

Nicotinamide protects target cells from cell-mediated cytolysis

Nicotinamide in concentrations of 5 mM and greater protected fibroblast target cells from lysis by lymphokine-activated killer cells (LAK cells). Protection was concentration dependent and was exerted at the level of the target cell. Nicotinamide did not interfere with effector-target cell conjugate formation or with the calcium dependent triggering step of the lytic process. Target cell lysis in cultures without nicotinamide was accompanied by fragmentation of target cell DNA. The DNA of target cells cultured with LAK cells in the presence of nicotinamide remained intact. 3-Aminobenzamide which, like nicotinamide, inhibits poly(ADP-ribose) synthetase but is not a precursor of NAD, was an effective inhibitor of target cell lysis while nicotinic acid, an alternative precursor of NAD in cells, was not. The data point to a central role for poly(ADP-ribose) synthetase in the events leading up to DNA fragmentation and the release of 51Cr from target cells damaged by lymphokine-activated killer cells.     

Nampt基因表达调控机制

烟酰胺磷酸核糖转移酶(NAMPT)是烟酰胺腺嘌呤二核苷酸(NAD)生物合成途径的关键限速酶, 也被称为内脏脂肪素(Visfatin)或前B细胞克隆增强因子(PBEF)。它通过调节机体或细胞的NAD水平以及通过其他非酶机制等途径影响代谢、炎症反应、细胞的增殖、分化和凋亡, 特别是衰老等诸多过程。文章简要综述了近年来Nampt基因的表达调控及其转录的反馈调节机制研究进展。