Quality evaluation of NMN produced via different synthesis methods: a multi-technique analysis using NMR, IR, GC-TCD, and HPLC-UV

Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the NAD-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure of the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet known is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.

Nicotinamide (NM) phosphoribosyltransferase (NMPRTase) catalyzes the reaction of NM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form NM mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the (1)H and (31)P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0-2.2 A were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and the leaving group for the requisite inversion of configuration at the C1' carbon of the ribose ring. In the active site near the C1' atom of the bound PRPP or NMN, there was neither negatively charged group nor waterproof environment necessary to support the feasibility of a ribo-oxocarbocation intermediate inherent in the S(N)1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.

Heart failure occurs at twice the rate in diabetic patients compared to normal subjects. NAD redox imbalance or depletion has emerged as a hallmark of diabetes and heart disease. We showed that NAD redox imbalance or depletion promotes heart failure progression, but its role in diabetic cardiomyopathy has not been established. We used two mouse models with altered NAD metabolism. 1. Cardiac-specific NDUFS4-KO (cKO) mice which have mitochondrial complex I deficiency, decreased NAD/NADH ratio, but normal baseline cardiac function and energetics. 2. Cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (NAMPT) in mice which elevates cardiac NAD levels. Diabetic stress induced by streptozotocin (STZ) promoted systolic (fractional shortening) and diastolic (E’/A’, e/E’) dysfunction, which were exacerbated in cKO mice. Overexpression of NAMPT to reverse NAD redox imbalance ameliorated cardiac dysfunction in both diabetic control and diabetic cKO mice. We measured expression levels of 58 genes related to NAD metabolism in these diabetic hearts. Transcript levels of genes for NAD consumption (sirtuins, PARPs and hydrolases) and NAD synthesis pathways (including NMRK1) did not change, except Nicotinamide Riboside Kinase 2 (NMRK2). NMRK2 transcript was upregulated in diabetic cKO hearts, associated with a decline in NAD levels. Expression of NAMPT to elevate NAD levels downregulated NMRK2 expression in both treated and untreated mice. The data indicate that upregulation of NMRK2 transcript is negatively correlated with cardiac function and NAD levels. NMRK2 is an enzyme involved in the Preiss-Handler and Salvage pathways of NAD synthesis by phosphorylating the NAD precursor Nicotinamide Riboside (NR) into Nicotinamide Mononucleotide (NMN). NAD metabolite analysis in the heart tissue showed a trended increase in the substrate of NMRK2, NR, and a trended decrease in the products, NMN or NAMN in diabetic cKO hearts with a decline in NAD levels. Our results suggest that NAD redox imbalance drives the progression of diabetic cardiomyopathy, and NMRK2 upregulation may regulate disease progression and NAD synthesis through mechanisms to be determined.

NAD is an essential coenzyme involved in numerous metabolic pathways and it has been demonstrated that a number of signalling pathways bring about its consumption. Different pathways leading to the formation of NAD are present in cells. Nicotinamide phosphoribosyl transferase (NAMPT), which forms nicotinamide mononucleotide (NMN) from nicotinamide (NM) and PRPP, plays a crucial role in cells to re-use nicotinamide released by NAD-metabolizing enzymes. Moreover, NAMPT has also been described as a cytokine released by immune cells and adipocytes, however the role of NAMPT in the extracellular space is still unclear. The link between NAMPT and cancer is rapidly strengthening. NAMPT has been shown to be involved in angiogenesis and to be up-regulated in a number of solid tumours. Moreover, an important role in tumorigenesis has been postulated for a number of NAD-utilizing enzymes and inhibitors of NAMPT, named FK866 and CHS 828 have entered clinical trails for cancer treatment. In particular, FK866 has entered phase II trial for metastatic melanoma. The aim of our work was to determine the role of NAMPT in melanoma progression and the possibility to use NAMPT inhibitors as anti-cancer agents in melanoma. We investigated the expression of NAMPT in normal nevi, dysplastic nevi and melanoma human samples. Surprisingly, in all melanoma samples and in dysplastic nevi NAMPT is over-expressed, suggesting a possible contribution of this enzyme in melanoma progression. To confirm this data, we investigated the expression of NAMPT in six different melanoma cell lines. All melanoma cells show high levels of NAMPT expression compared to melanocytes. To test if the inhibition of NAMPT was able to decrease melanoma cells viability, we capitalize the action of FK866. However, only one to six melanoma cell line is sensitive to FK866 treatment. To understand why melanoma cells are insensitive to NAMPT inhibition, we investigate MDR expression and the possibility that NAMPT is mutated. Unfortunately, verapamil was not able to increase the sensitivity to FK866 and NAMPT is not mutated in all melanoma cell lines tested, moreover the treatment with FK866 is able to decrease the intracellular NAD level, suggesting that this agent is able to enter cells but not to induce cell death. As NAMPT was described also as a cytokine, we speculate the possibility that melanoma cells are able to release NAMPT in the extracellular space. Indeed, in starvation condition NAMPT is released by melanoma cells in a time-dependent manner. In conclusion, we have demonstrated that NAMPT is over-expressed in human melanoma samples and in melanoma cells, however NAMPT inhibition is not able to affect melanoma cells growth and viability, suggesting that NAMPT up-regulation does not correlate with a pharmacological response. Moreover, NAMPT is released by melanoma cells as a cytokine, we can speculate that NAMPT has a role in the angiogenic process of melanoma progression Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5146. doi:1538-7445.AM2012-5146

Background: Nicotinamide adenine dinucleotide (NAD) participates in redox reactions as an electron-transferring molecule, thereby serving as a cofactor for metabolism. In the NAD biosynthetic salvage pathway, nicotinamide phosphoribosyltransferase (Nampt) which forms nicotinamide mononucleotide (NMN) is the rate-limiting enzyme and exhibits the protective effect against ischemia/ reperfusion (I/R) injury in the hearts of mice with cardiac-specific overexpression of Nampt. In addition, administration of NMN is reported to reverse the pancreatic beta-cell dysfunction observed in Nampt heterozygous knockout mice. Methods: To elucidate the protective effect of NMN against cardiac I/R injury, NMN was administered to mice subjected to I/R injury either before ischemia or immediately before reperfusion. Area at risk (AAR) and infarct area (IA) were evaluated 24 hours after reperfusion. Results: To confirm the distribution of NMN in the heart, NAD and NADH, into which NMN is quickly converted in the heart, were measured. One hour after NMN administration (500mg/ kg body weight, i.p.), the NAD and NADH contents of the NMN group (n=3) were significantly increased, whereas the NAD/NADH ratio was unchanged compared to the vehicle group (n=3) (NAD: NMN=483±42 pmole/mg tissue, vehicle=183±46 pmole/mg tissue, p<0.01; NADH: NMN=262±25 pmole/mg tissue, vehicle=96±17 pmole/mg tissue, p<0.01; NAD/NADH ratio: NMN=1.85±0.09, vehicle=1.82±0.18, n.s.). In the mice administered NMN or vehicle 30 min before ischemia, IA/AAR in the NMN group was smaller than that in the vehicle group (AAR: NMN=26±0.5%, vehicle=27±0.4%, n.s.; IA/AAR: NMN=23±1.8%, vehicle=42±1.5%, p<0.01, n=4). On the other hand, in the mice administered NMN or vehicle immediately before reperfusion, IA/AAR in the NMN group was not different from that in the vehicle group (AAR: NMN=27±0.5%, vehicle=26±1.0%, n.s.; IA/AAR: NMN=37±1.4%, vehicle=34±2.2%, n.s., n=4). Conclusions: NMN administration significantly increased the NAD content in the heart and exhibited a protective effect against cardiac I/R injury. Increasing NAD content before ischemia, rather than after reperfusion, may minimize I/R injury in clinical settings, such as in acute coronary syndrome before coronary intervention.

Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD(+) and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD(+) levels. Nampt is the main source of NSPC NAD(+) levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD(+) biosynthesis is a mediator of age-associated functional declines in NSPCs.

Simple SummaryNicotinamide mononucleotide (NMN) is the physiological circulating nicotinamide adenine dinucleotide (NAD) precursor thought to elevate the cellular level of NAD+ and ameliorate various age-related diseases. Here, we investigate how high-dose NMN functions in lung adenocarcinoma. We show that excess nicotinamide (NAM) is produced through the metabolism of high-dose NMN, while the overexpression of nicotinamide phosphoribosyltransferase (NAMPT) can significantly decrease intracellular NAM content and, in turn, boost cell proliferation in vitro and in vivo. Mechanistically, high-dose NMN promotes ferroptosis through NAM-mediated SIRT1–AMPK–ACC signaling. This study highlights the tumor influence of NMN at high doses in the manipulation of cancer cell metabolism, providing a new perspective on clinical therapy in patients with lung adenocarcinoma.Background: Nicotinamide mononucleotide (NMN) is the physiological circulating NAD precursor thought to elevate the cellular level of NAD+ and to ameliorate various age-related diseases. An inseparable link exists between aging and tumorigenesis, especially involving aberrant energetic metabolism and cell fate regulation in cancer cells. However, few studies have directly investigated the effects of NMN on another major ageing-related disease: tumors. Methods: We conducted a series of cell and mouse models to evaluate the anti-tumor effect of high-dose NMN. Transmission electron microscopy and a Mito-FerroGreen-labeled immunofluorescence assay (Fe2+) were utilized to demonstrate ferroptosis. The metabolites of NAM were detected via ELISA. The expression of the proteins involved in the SIRT1–AMPK–ACC signaling were detected using a Western blot assay. Results: The results showed that high-dose NMN inhibits lung adenocarcinoma growth in vitro and in vivo. Excess NAM is produced through the metabolism of high-dose NMN, whereas the overexpression of NAMPT significantly decreases intracellular NAM content, which, in turn, boosts cell proliferation. Mechanistically, high-dose NMN promotes ferroptosis through NAM-mediated SIRT1–AMPK–ACC signaling. Conclusions: This study highlights the tumor influence of NMN at high doses in the manipulation of cancer cell metabolism, providing a new perspective on clinical therapy in patients with lung adenocarcinoma.

Background: Nicotinamide adenine dinucleotide (NAD) participates in metabolic reactions as an electron-transferring molecule. In the NAD biosynthetic salvage pathway, nicotinamide phosphoribosyltransferase (Nampt) which forms nicotinamide mononucleotide (NMN) is the rate-limiting enzyme and exhibits the protective effect against ischemia/ reperfusion (I/R) injury in the hearts of mice with cardiac-specific overexpression of Nampt. In addition, administration of NMN is reported to reverse the pancreatic beta-cell dysfunction observed in Nampt heterozygous knockout mice. Methods: To elucidate the protective effect of NMN against cardiac I/R injury, NMN was administered to mice subjected to I/R injury either before ischemia or immediately before reperfusion. Area at risk (AAR) and infarct area (IA) were evaluated 24 hours after reperfusion. Results: To confirm the distribution of NMN in the heart, NAD and NADH, into which NMN is quickly converted in the heart, were measured. One hour after NMN administration (500mg/ kg body weight, i.p.), the NAD and NADH contents of the NMN group were significantly increased, whereas the NAD/NADH ratio was unchanged compared to the vehicle group(NAD: NMN=483±42 pmol/mg tissue, vehicle=183±46 pmol/mg tissue, p<0.01; NADH: NMN=262±25 pmol/mg tissue, vehicle=96±17 pmol/mg tissue, p<0.01; NAD/NADH ratio: NMN=1.85±0.09, vehicle=1.82±0.18, n.s.; n=3). In the mice administered NMN or vehicle 30 min before ischemia, IA/AAR in the NMN group was smaller than that in the vehicle group (AAR: NMN=30±1.9%, vehicle=29±1.7%, n.s.; IA/AAR: NMN=23±2.9%, vehicle=41±2.6%, p<0.01, n=6-7). On the other hand, in the mice administered NMN or vehicle immediately before reperfusion, IA/AAR in the NMN group was not different from that in the vehicle group (AAR: NMN=27±0.5%, vehicle=26±1.0%, n.s.; IA/AAR: NMN=37±1.4%, vehicle=34±2.2%, n.s., n=4). Conclusions: NMN administration significantly increased the NAD content in the heart and exhibited a protective effect against cardiac I/R injury. Increasing NAD content before ischemia, rather than after reperfusion, may minimize I/R injury in clinical settings, such as in acute coronary syndrome before coronary intervention.

NAMPT inhibitor and metabolite protect mouse brain from cryoinjury through distinct mechanisms

Introduction: Nicotinamide phosphoribosyltransferase (Nampt) is a rate limiting enzyme that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), which is then converted to nicotinamide adenine dinucleotide (NAD + ) by nicotinamide mononucleotide adenylyltransferase (Nmnat). NAD + serves as an adenosine donor and source of high energy phosphate for the synthesis of ATP and regulates NAD + -dependent key cellular enzymes. Given its critical role in metabolism and energetic recovery, which is significantly associated with cardiac arrest outcomes, we hypothesized that Nampt deficiency could affect cardiac arrest survival in a mouse asystole arrest model. Methods: C57BL6 wild type and Nampt +/- mice underwent 8 min of KCl-induced asystole arrest and were monitored for up to 4-h following return of spontaneous circulation. NAM (100 mg/kg) or NMN (100 mg/kg) were administered during CPR in wild type and Nampt +/- mice and survival was assessed by Log Rank analysis. Levels of Nampt and NAD + in critical organs, heart and brain, were measured. A p < 0.05 was considered as statistically significant. Results: Compared to wild type mice, survival was decreased from 60% to 20% in Nampt +/- mice (N=10 each group, p < 0.05). The reduced Nampt expression was observed in Nampt +/- mice. NAD + content was decreased in the heart and brain of Nampt +/- mice at baseline and 30 min post-ROSC (n=5, p<0.05). NAM administration did not improve Nampt +/- mice survival. In contrast, NMN which bypasses Nampt for NAD + supplementation, significantly improved 4-h survival of Nampt +/- mice. Conclusions: Nampt deficiency decreases mouse cardiac arrest survival. NMN may serve as a new therapeutic strategy for improving cardiac arrest survival.

Nicotinamide mononucleotide (NMN) is a key precursor of nicotinamide adenine dinucleotide and an important source of cellular energy. It can prevent neuronal mitochondrial defects and alleviate heart fibrosis. Strategies to improve NMN production have important implications for human health. Through plasmid expression technology and CRISPR/Cas9 technology, we engineered Escherichia coli for efficient NMN production. First, we upregulated the expression of genes encoding key enzymes in the NMN synthesis pathway, enabling E. coli to directly produce NMN, and established the important role of the nicotinamide mononucleotide transporter in the transport of NMN from cells. The content of NMN was 0.24 g·L−1 at 24 h. Second, we strengthened the adenosine triphosphate (ATP) cycle, and the concentration of NMN was 0.49 g·L−1 at 24 h. Third, we increased the synthesis of the NMN precursor 5-phosphate ribose-1-phosphate and obtained an NMN content of 0.49 g·L−1 at 12 h and 1.11 g·L−1 at 24 h. Fourth, we introduced nicotinamide riboside kinase (NRK) and found that it was effective only for a period of time. The content of NMN was 0.54 g·L−1 at 12 h but only 1.05 g·L−1 at 24 h. Finally, we combined these strategies to regulate the whole metabolic flow, revealing that integrating multiple pathways promoted NMN production. During fermentation, we added 1 g·L−1 nicotinamide and 10 g·L−1 glucose, yielding an extracellular NMN concentration of 1.11 g·L−1.

Stress-associated premature senescence (SAPS) has been implicated in diabetes-induced vascular dysfunction. We have shown that microRNA34a (miR-34a) is involved in promoting senescence of the retinal microvasculature. Loss of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme of NAD+ biosynthesis from nicotinamide mononucleotide (NMN), has been implicated in aging and metabolic diseases. Interestingly NAMPT is a target of miR34a, therefore, herein, we investigated the role and reciprocal relationship of miR34a and NAMPT on diabetes-induced senescence of the retinal vasculature. NAMPT expression and NAD+ content were significantly reduced in retina of streptozotocin-induced diabetic rats (STZ-rats, 8-12 weeks of hyperglycemia) and in human postmortem diabetic retinas. On the contrary, miR34a expression was found increased and positively correlated with augmented levels of senescence markers. Exposure of human retinal endothelial cells (HuREC) to glucidic stress (25mM) also decreased NAMPT and NAD+ levels while augmented the expression of miR34a and of senescence markers. Transfection of HuREC with miR-34a mimic, but not a scramble construct, in normal glucose conditions inhibited the expression of NAMPT and reduced NAD+ levels while promoting the expression of senescence markers. These effects of miR34a mimic on HuREC were halted by supplementation with NMN (0.05-1mM), but without modifying NAMPT expression. Lastly, NMN supplementation also prevented high glucose-induced loss of NAD+ and elevation of senescence markers. Our data show that high glucose/diabetes-induced SAPS in the retinal microvasculature involves miR34a-mediated blockade of NAMPT and consequent loss of NAD+. Moreover, our data suggest that NMN supplementation could be a viable therapeutic strategy to improve endothelial dysfunction in diabetic retinopathy. Disclosure M. Bartolli: None. R. Jadeja: None. M. Thounaojam: None. D. Gutsaeva: None. P. Martin: None. Funding National Institutes of Health (EY022416, EY028714)

Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo

Nicotinamide mononucleotide (NMN) is an essential precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a critical cofactor in cellular metabolism and energy regulation. With the growing interest in NMN for its antiaging and therapeutic benefits, microbial production systems, particularly Saccharomyces cerevisiae, offer a promising alternative to traditional chemical synthesis. This study explored the optimization of NMN production in S. cerevisiae BY4742 using both constitutive and inducible promoters. Yeast strains were engineered to express human nicotinamide phosphoribosyl transferase (h-NAMPT) and yeast phosphoribosyl pyrophosphate synthetase (PRS5 and PRS2) to enable the direct conversion of nicotinamide (NAM) to NMN. The genes were expressed under the control of GAL1 (inducible) and TEF1 (constitutive) promoters in the plasmids. The results demonstrated that strains with the TEF1 constitutive promoter produced higher levels of intracellular NMN and NAD+ compared with those using the GAL1 inducible promoter. Additionally, fermentation in a rich R-SD medium further enhanced NMN production, with the scTEF2g strain (overexpressing plasmid-based h-NAMPT and PRS5 genes under the TEF1 promoter) achieving 151.71 mg/L NMN, a 3-fold increase in NMN yield compared to the control strain. This is the highest intracellular NMN produced in recombinant yeast from NAM in a flask. This work highlights the importance of gene regulation through promoter selection and culture optimization in maximizing NMN yields, presenting yeast-based systems as a promising platform for NMN production from NAM.

Excessive exposure of the eye to ultraviolet B light (UVB) leads to corneal edema and opacification because of the apoptosis of the corneal endothelium. Our previous study found that nicotinamide (NIC), the precursor of nicotinamide adenine dinucleotide (NAD), could inhibit the endothelial-mesenchymal transition and accelerate healing the wound to the corneal endothelium in the rabbit. Here we hypothesize that NIC may possess the capacity to protect the cornea from UVB-induced endothelial apoptosis. Therefore, a mouse model and a cultured cell model were used to examine the effect of NAD+ precursors, including NIC, nicotinamide mononucleotide (NMN), and NAD, on the UVB-induced apoptosis of corneal endothelial cells (CECs). The results showed that UVB irradiation caused apparent corneal edema and cell apoptosis in mice, accompanied by reduced levels of NAD+ and its key biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), in the corneal endothelium. However, the subconjunctival injection of NIC, NMN, or NAD+ effectively prevented UVB-induced tissue damage and endothelial cell apoptosis in the mouse cornea. Moreover, pretreatment using NIC, NMN, and NAD+ increased the survival rate and inhibited the apoptosis of cultured human CECs irradiated by UVB. Mechanistically, pretreatment using nicotinamide (NIC) recovered the AKT activation level and decreased the BAX/BCL-2 ratio. In addition, the capacity of NIC to protect CECs was fully reversed in the presence of the AKT inhibitor LY294002. Therefore, we conclude that NAD+ precursors can effectively prevent the apoptosis of the corneal endothelium through reactivating AKT signaling; this represents a potential therapeutic approach for preventing UVB-induced corneal damage.