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Nicotinamide adenine dinucleotide (NAD + ) depletion has been postulated as a contributor to the severity of COVID-19; however, no study has prospectively characterized NAD + and its metabolites in relation to disease severity in patients with COVID-19. We measured NAD + and its metabolites in 56 hospitalized patients with COVID-19 and in two control groups without COVID-19: (1) 31 age- and sex-matched adults with comorbidities, and (2) 30 adults without comorbidities. Blood NAD + concentrations in COVID-19 group were only slightly lower than in the control groups (p < 0.05); however, plasma 1-methylnicotinamide concentrations were significantly higher in patients with COVID-19 (439.7 ng/mL, 95% CI: 234.0, 645.4 ng/mL) than in age- and sex-matched controls (44.5 ng/mL, 95% CI: 15.6, 73.4) and in healthy controls (18.1 ng/mL, 95% CI 15.4, 20.8; p < 0.001 for each comparison). Plasma nicotinamide concentrations were also higher in COVID-19 group and in controls with comorbidities than in healthy control group. Plasma concentrations of 2-methyl-2-pyridone-5-carboxamide (2-PY), but not NAD + , were significantly associated with increased risk of death (HR = 3.65; 95% CI 1.09, 12.2; p = 0.036) and escalation in level of care (HR = 2.90, 95% CI 1.01, 8.38, p = 0.049). RNAseq and RTqPCR analyses of PBMC mRNA found upregulation of multiple genes involved in NAD + synthesis as well as degradation, and dysregulation of NAD + -dependent processes including immune response, DNA repair, metabolism, apoptosis/autophagy, redox reactions, and mitochondrial function. Blood NAD + concentrations are modestly reduced in COVID-19; however, NAD + turnover is substantially increased with upregulation of genes involved in both NAD + biosynthesis and degradation, supporting the rationale for NAD+ augmentation to attenuate disease severity., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The coenzyme NAD + is consumed by signalling enzymes, including poly-ADP-ribosyltransferases (PARPs) and sirtuins. Ageing is associated with a decrease in cellular NAD + levels, but how cells cope with persistently decreased NAD + concentrations is unclear. Here, we show that subcellular NAD + pools are interconnected, with mitochondria acting as a rheostat to maintain NAD + levels upon excessive consumption. To evoke chronic, compartment-specific overconsumption of NAD + , we engineered cell lines stably expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum or peroxisomes, resulting in a decline of cellular NAD + concentrations by up to 50%. Isotope-tracer flux measurements and mathematical modelling show that the lowered NAD + concentration kinetically restricts NAD + consumption to maintain a balance with the NAD + biosynthesis rate, which remains unchanged. Chronic NAD + deficiency is well tolerated unless mitochondria are directly targeted. Mitochondria maintain NAD + by import through SLC25A51 and reversibly cleave NAD + to nicotinamide mononucleotide and ATP when NMNAT3 is present. Thus, these organelles can maintain an additional, virtual NAD + pool. Our results are consistent with a well-tolerated ageing-related NAD + decline as long as the vulnerable mitochondrial pool is not directly affected., Competing Interests: Competing interests: M.Z. is chief scientist at Blue Helix Health AS. All other authors declare no competing of interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Nicotinamide adenine dinucleotide, in its oxidized (NAD + ) and reduced (NADH) forms, is a reduction-oxidation (redox) co-factor and substrate for signalling enzymes that have essential roles in metabolism. The recognition that NAD + levels fall in response to stress and can be readily replenished through supplementation has fostered great interest in the potential benefits of increasing or restoring NAD + levels in humans to prevent or delay diseases and degenerative processes. However, much about the biology of NAD + and related molecules remains poorly understood. In this Review, we discuss the current knowledge of NAD + metabolism, including limitations of, assumptions about and unappreciated factors that might influence the success or contribute to risks of NAD + supplementation. We highlight several ongoing controversies in the field, and discuss the role of the microbiome in modulating the availability of NAD + precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), the presence of multiple cellular compartments that have distinct pools of NAD + and NADH, and non-canonical NAD + and NADH degradation pathways. We conclude that a substantial investment in understanding the fundamental biology of NAD + , its detection and its metabolites in specific cells and cellular compartments is needed to support current translational efforts to safely boost NAD + levels in humans., (© 2024. Springer Nature Limited.)

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders., (© 2024. The Author(s).)

Objectives: Cellular NAD + declines in inflammatory states associated with increased activity of the leukocyte-expressed NADase CD38. In this study, we tested the potential role of therapeutically targeting CD38 and NAD + in gout., Methods: We studied cultured mouse wild type and CD38 knockout (KO) murine bone marrow derived macrophages (BMDMs) stimulated by monosodium urate (MSU) crystals and used the air pouch gouty inflammation model., Results: MSU crystals induced CD38 in BMDMs in vitro, associated with NAD + depletion, and IL-1β and CXCL1 release, effects reversed by pharmacologic CD38 inhibitors (apigenin, 78c). Mouse air pouch inflammatory responses to MSU crystals were blunted by CD38 KO and apigenin. Pharmacologic CD38 inhibition suppressed MSU crystal-induced NLRP3 inflammasome activation and increased anti-inflammatory SIRT3-SOD2 activity in macrophages. BMDM RNA-seq analysis of differentially expressed genes (DEGs) revealed CD38 to control multiple MSU crystal-modulated inflammation pathways. Top DEGs included the circadian rhythm modulator GRP176, and the metalloreductase STEAP4 that mediates iron homeostasis, and promotes oxidative stress and NF-κB activation when it is overexpressed., Conclusions: CD38 and NAD + depletion are druggable targets controlling the MSU crystal- induced inflammation program. Targeting CD38 and NAD + are potentially novel selective molecular approaches to limit gouty arthritis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Exogenous exposures to the triose sugar dihydroxyacetone (DHA) occur from sunless tanning products and electronic cigarette aerosol. Once inhaled or absorbed, DHA enters cells, is converted to dihydroxyacetone phosphate (DHAP), and incorporated into several metabolic pathways. Cytotoxic effects of DHA vary across the cell types depending on the metabolic needs of the cells, and differences in the generation of reactive oxygen species (ROS), cell cycle arrest, and mitochondrial dysfunction have been reported. We have shown that cytotoxic doses of DHA induced metabolic imbalances in glycolysis and oxidative phosphorylation in liver and kidney cell models. Here, we examine the dose-dependent effects of DHA on the rat cardiomyocyte cell line, H9c2. Cells begin to experience cytotoxic effects at low millimolar doses, but an increase in cell survival was observed at 2 mM DHA. We confirmed that 2 mM DHA increased cell survival compared to the low cytotoxic 1 mM dose and investigated the metabolic differences between these two low DHA doses. Exposure to 1 mM DHA showed changes in the cell's fuel utilization, mitochondrial reactive oxygen species (ROS), and transient changes in the glycolysis and mitochondrial energetics, which normalized 24 h after exposure. The 2 mM dose induced robust changes in mitochondrial flux through acetyl CoA and elevated expression of fatty acid synthase. Distinct from the 1 mM dose, the 2 mM exposure increased mitochondrial ROS and NAD(P)H levels, and sustained changes in LDHA/LDHB and acetyl CoA-associated enzymes were observed. Although the cells were exposed to low cytotoxic (1 mM) and non-cytotoxic (2 mM) acute doses of DHA, significant changes in mitochondrial metabolic pathways occurred. Further, the proliferation increase at the acute 2 mM DHA dose suggests a metabolic adaption occurred with sustained consequences in survival and proliferation. With increased exogenous exposure to DHA through e-cigarette aerosol, this work suggests cell metabolic changes induced by acute or potentially chronic exposures could impact cell function and survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD + ) levels are hallmarks of skeletal muscle ageing and sarcopenia 1-3 , but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid 4 , NAD + levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD + pool and increases NAD + levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway 5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD + -dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD + -boosting strategy with therapeutic potential for age-associated muscle decline., (© 2024. The Author(s).)

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD + ) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1 H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD + , the key redox cofactor in metabolism and bioenergetics maintenance.

Recent research has unveiled an expansive role of NAD+ in cellular energy generation, redox reactions, and as a substrate or cosubstrate in signaling pathways that regulate health span and aging. This review provides a critical appraisal of the clinical pharmacology and the preclinical and clinical evidence for therapeutic effects of NAD+ precursors for age-related conditions, with a particular focus on cardiometabolic disorders, and discusses gaps in current knowledge. NAD+ levels decrease throughout life; age-related decline in NAD+ bioavailability has been postulated to be a contributor to many age-related diseases. Raising NAD+ levels in model organisms by administration of NAD+ precursors improves glucose and lipid metabolism; attenuates diet-induced weight gain, diabetes, diabetic kidney disease, and hepatic steatosis; reduces endothelial dysfunction; protects heart from ischemic injury; improves left ventricular function in models of heart failure; attenuates cerebrovascular and neurodegenerative disorders; and increases health span. Early human studies show that NAD+ levels can be raised safely in blood and some tissues by oral NAD+ precursors and suggest benefit in preventing nonmelanotic skin cancer, modestly reducing blood pressure and improving lipid profile in older adults with obesity or overweight; preventing kidney injury in at-risk patients; and suppressing inflammation in Parkinson disease and SARS-CoV-2 infection. Clinical pharmacology, metabolism, and therapeutic mechanisms of NAD+ precursors remain incompletely understood. We suggest that these early findings provide the rationale for adequately powered randomized trials to evaluate the efficacy of NAD+ augmentation as a therapeutic strategy to prevent and treat metabolic disorders and age-related conditions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dietary vitamin B3 components, such as nicotinamide and nicotinic acid, are precursors to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD + ). NAD + levels are thought to decline with age and disease. While the drivers of this decline remain under intense investigation, strategies have emerged seeking to functionally maintain NAD + levels through supplementation with NAD + biosynthetic intermediates. These include marketed products, such as nicotinamide riboside (NR) and its phosphorylated form (NMN). More recent developments have shown that NRH (the reduced form of NR) and its phosphorylated form NMNH also increases NAD + levels upon administration, although they initially generate NADH (the reduced form of NAD + ). Other means to increase the combined levels of NAD + and NADH, NAD(H), include the inhibition of NAD + -consuming enzymes or activation of biosynthetic pathways. Multiple studies have shown that supplementation with an NAD(H) precursor changes the profile of NAD(H) catabolism. Yet, the pharmacological significance of NAD(H) catabolites is rarely considered although the distribution and abundance of these catabolites differ depending on the NAD(H) precursor used, the species in which the study is conducted, and the tissues used for the quantification. Significantly, some of these metabolites have emerged as biomarkers in physiological disorders and might not be innocuous. Herein, we review the known and emerging catabolites of the NAD(H) metabolome and highlight their biochemical and physiological function as well as key chemical and biochemical reactions leading to their formation. Furthermore, we emphasize the need for analytical methods that inform on the full NAD(H) metabolome since the relative abundance of NAD(H) catabolites informs how NAD(H) precursors are used, recycled, and eliminated.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility., Competing Interests: Declaration of interests J.D.R. is a consultant to Pfizer and an advisor and stock owner in Colorado Research Partners, L.E.A.F. Pharmaceuticals, Rafael Pharmaceuticals, Raze Therapeutics, Kadmon Pharmaceuticals, and Agios Pharmaceuticals. J.D.R. is co-inventor of SHIN2 and related SHMT inhibitors, which have been patented by Princeton University, and he is a co-founder of Toran Therapeutics. J.A.B. is consultant to Pfizer and Cytokinetics, is an inventor on a patent for using NAD precursors in liver injury, and has received research funding and materials from Elysium Health and Metro International Biotech, both of which have an interest in NAD precursors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Hernandez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pyridone adenine dinucleotides (ox-NADs) are redox inactive derivatives of the enzyme cofactor and substrate nicotinamide adenine dinucleotide (NAD) that have a carbonyl group at the C2, C4, or C6 positions of the nicotinamide ring. These aberrant cofactor analogs accumulate in cells under stress and are potential inhibitors of enzymes that use NAD(H). We studied the conformational landscape of ox-NADs in solution using molecular dynamics simulations. Compared to NAD + and NADH, 2-ox-NAD and 4-ox-NAD have an enhanced propensity for adopting the anti conformation of the pyridone ribose group, whereas 6-ox-NAD exhibits greater syn potential. Consequently, 2-ox-NAD and 4-ox-NAD have increased preference for folding into compact conformations, whereas 6-ox-NAD is more extended. ox-NADs have distinctive preferences for the orientation of the pyridone amide group, which are driven by intramolecular hydrogen bonding and steric interactions. These conformational preferences are compared to those of protein-bound NAD(H). Our results may help in identifying enzymes targeted by ox-NADs.

Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD + ), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD + precursors influence intracellular NAD + levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD + precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD + precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD + levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD + prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD + precursor in mammalian cells and how different NAD + precursors can interact and influence each other when co-administered.

Dihydroxyacetone (DHA) is a major byproduct of e-cigarette combustion and is the active ingredient in sunless tanning products. Mounting evidence points to its damaging effects on cellular functions. While developing a simple synthetic route to monomeric [ 13 C 3 ]DHA for flux metabolic studies that compared DHA and glyceraldehyde (GA) metabolism, we uncovered that solid DHA ages upon storage and differences in the relative abundance of each of its isomer occur when reconstituted in an aqueous solution. While all three of the dimeric forms of DHA ultimately resolve to the ketone and hydrated forms of monomeric DHA once in water at room temperature, these species require hours rather than minutes to reach an equilibrium favoring the monomeric species. Consequently, when used in bolus or flux experiments, the relative abundance of each isomer and its effects at the time of application is dependent on the initial DHA isomeric composition and concentration, and time of equilibration in solution before use. Here, we make recommendations for the more consistent handling of DHA as we report conditions that ensure that DHA is present in its monomeric form while in solutions, conditions used in an isotopic tracing study that specifically compared monomeric DHA and GA metabolism in cells.

This article contains a synthetic protocol for solvent-assisted mechanochemical synthesis of a nucleotide dimer. First, a dinucleoside phosphite is prepared by solvent-assisted mechanochemistry via the phosphoramidite method. Second, the dinucleoside phosphite is oxidized to form the dinucleotide under mechanochemical conditions. Finally, the dinucleotide is purified by column chromatography. Support protocols are also provided for preparing the acidic salts that can be utilized for phosphoramidite couplings and for demonstrating that the reaction occurs under mechanochemical conditions rather than as a result of solvent added for analysis. Mechanochemistry as applied to synthesis of dinucleotides is a recent development and it is anticipated that the principles in this protocol will be widely applicable to a range of nucleoside and ribonucleoside monomers. The advantages of mechanochemistry over traditional solution-phase chemistry are the simplicity of the procedure, improved hydrolytic stability, and elimination of the need to solubilize poorly soluble compounds. © 2022 Wiley Periodicals LLC. Basic Protocol: Solvent-assisted mechanochemical synthesis of a nucleotide dimer Supplementary Protocol 1: Synthesis of N-methylimidazolium triflate Supplementary Protocol 2: Synthesis of pyridinium trifluoroacetate Supplementary Protocol 3: Confirmation of the efficacy of mechanochemical conditions., (© 2022 Wiley Periodicals LLC.)

Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD + levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD + precursors can promote inflammation in macrophages. The effect of NRH in NAD + boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IκB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment., Competing Interests: EC holds a patent on the use of CD38 inhibitors for metabolic diseases that is licensed by Elysium health. EC is a consultant for TeneoBio, Calico, Mitobridge and Cytokinetics. EC is on the advisory board of Eolo Pharma (Santa Fe, Argentina). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chini, Peclat, Gomez, Zeidler, Warner, Kashyap, Mazdeh, Hayat, Migaud, Paulus, Chanan-Khan and Chini.)

NAD + is an essential coenzyme for all living cells. NAD + concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD + metabolism across tissues. In aged mice, we observed modest tissue NAD + depletion (median decrease ∼30%). Circulating NAD + precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD + pool was modestly faster such that absolute NAD + biosynthetic flux was maintained, consistent with more active NAD + -consuming enzymes. Calorie restriction partially mitigated age-associated NAD + decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD + by impairing synthesis in both young and aged mice. Thus, the decline in NAD + with normal aging is relatively subtle and occurs despite maintained NAD + production, likely due to increased consumption., Competing Interests: Declaration of interests J.D.R. and J.A.B. are consultants to Pfizer. J.D.R. is an advisor and stock owner in Colorado Research Partners, L.E.A.F. Pharmaceuticals, Rafael Pharmaceuticals, Raze Therapeutics, Kadmon Pharmaceuticals, and Agios pharmaceuticals. J.D.R. is a co-inventor of SHIN2 and related SHMT inhibitors, which have been patented by Princeton University, and is a co-founder of Toran Therapeutics. G.X., M.F.C., and G.J.T. are current or former employees of Pfizer who may hold Pfizer stock and/or stock options. J.A.B. and S.M. are inventors on a patent for using NAD(+) precursors in liver injury and have received research funding and materials from Elysium Health and Metro International Biotech, both of which have an interest in NAD(+) precursors. The remaining authors have nothing to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nicotinamide riboside (NR) is one of the orally bioavailable NAD + precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD + level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD + generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD + through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation., (© 2021. The Author(s).)

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD + precursor dihydronicotinamide riboside (NRH) rapidly increased NAD + levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Cancer.)

Assembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase β (Polβ) and XRCC1 to DNA lesion sites identifies a role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polβ's dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD + biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD + bioavailability to facilitate BER., Competing Interests: Declaration of interests R.W.S. is a scientific consultant for Canal House Biosciences, but this company was not involved in this study, nor was the consulting work related to this study. The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acute respiratory failure (ARF) requiring mechanical ventilation, a complicating factor in sepsis and other disorders, is associated with high morbidity and mortality. Despite its severity and prevalence, treatment options are limited. In light of accumulating evidence that mitochondrial abnormalities are common in ARF, here we applied broad spectrum quantitative and semiquantitative metabolomic analyses of serum from ARF patients to detect bioenergetic dysfunction and determine its association with survival. Plasma samples from surviving and non-surviving patients (N = 15/group) were taken at day 1 and day 3 after admission to the medical intensive care unit and, in survivors, at hospital discharge. Significant differences between survivors and non-survivors (ANOVA, 5% FDR) include bioenergetically relevant intermediates of redox cofactors nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), increased acyl-carnitines, bile acids, and decreased acyl-glycerophosphocholines. Many metabolites associated with poor outcomes are substrates of NAD(P)-dependent enzymatic processes, while alterations in NAD cofactors rely on bioavailability of dietary B-vitamins thiamine, riboflavin and pyridoxine. Changes in the efficiency of the nicotinamide-derived cofactors' biosynthetic pathways also associate with alterations in glutathione-dependent drug metabolism characterized by substantial differences observed in the acetaminophen metabolome. Based on these findings, a four-feature model developed with semi-quantitative and quantitative metabolomic results predicted patient outcomes with high accuracy (AUROC = 0.91). Collectively, this metabolomic endotype points to a close association between mitochondrial and bioenergetic dysfunction and mortality in human ARF, thus pointing to new pharmacologic targets to reduce mortality in this condition.

All life forms require nicotinamide adenine dinucleotide, NAD + , and its reduced form NADH. They are redox partners in hundreds of cellular enzymatic reactions. Changes in the intracellular levels of total NAD (NAD + + NADH) and the (NAD + /NADH) ratio can cause cellular dysfunction. When not present in protein complexes, NADH and its phosphorylated form NADPH degrade through intricate mechanisms. Replenishment of a declining total NAD pool can be achieved with biosynthetic precursors that include one of the reduced forms of nicotinamide riboside (NR + ), NRH. NRH, like NADH and NADPH, is prone to degradation via oxidation, hydration, and isomerization and, as such, is an excellent model compound to rationalize the nonenzymatic metabolism of NAD(P)H in a biological context. Here, we report on the stability of NRH and its propensity to isomerize and irreversibly degrade. We also report the preparation of two of its naturally occurring isomers, their chemical stability, their reactivity toward NRH-processing enzymes, and their cell-specific cytotoxicity. Furthermore, we identify a mechanism by which NRH degradation causes covalent peptide modifications, a process that could expose a novel type of NADH-protein modifications and correlate NADH accumulation with "protein aging." This work highlights the current limitations in detecting NADH's endogenous catabolites and in establishing the capacity for inducing cellular dysfunction.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD + pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca 2+ -binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

Dihydroxyacetone (DHA) is a three-carbon sugar that is the active ingredient in sunless tanning products and a by-product of electronic cigarette (e-cigarette) combustion. Increased use of sunless tanning products and e-cigarettes has elevated exposures to DHA through inhalation and absorption. Studies have confirmed that DHA is rapidly absorbed into cells and can enter into metabolic pathways following phosphorylation to dihydroxyacetone phosphate (DHAP), a product of fructose metabolism. Recent reports have suggested metabolic imbalance and cellular stress results from DHA exposures. However, the impact of elevated exposure to DHA on human health is currently under-investigated. We propose that exogenous exposures to DHA increase DHAP levels in cells and mimic fructose exposures to produce oxidative stress, mitochondrial dysfunction, and gene and protein expression changes. Here, we review cell line and animal model exposures to fructose to highlight similarities in the effects produced by exogenous exposures to DHA. Given the long-term health consequences of fructose exposure, this review emphasizes the pressing need to further examine DHA exposures from sunless tanning products and e-cigarettes., (© 2021 Wiley Periodicals LLC.)

Mitochondria require nicotinamide adenine dinucleotide (NAD + ) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD + transporters have been identified in yeast and plants 1,2 , but their existence in mammals remains controversial 3-5 . Here we demonstrate that mammalian mitochondria can take up intact NAD + , and identify SLC25A51 (also known as MCART1)-an essential 6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD + transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD + content, impairs mitochondrial respiration, and blocks the uptake of NAD + into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD + levels and restores NAD + uptake into yeast mitochondria lacking endogenous NAD + transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD + into mitochondria.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism., Competing Interests: Declaration of Interests Authors declare no competing interests., (Published by Elsevier Inc.)

Objective: A decay in intracellular NAD + levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD + precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD + precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD + biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD + precursors that can overcome these limitations., Methods: Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD + precursor in mammalian cells and mice., Results: NRH acts as a more potent and faster NAD + precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD + , thus revealing a new NRK1-independent pathway for NAD + synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury., Conclusions: Our data identify a new pathway for NAD + synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD + levels., (Copyright © 2019 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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.

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD + ) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD + derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo NAD + synthesis depleted NAD + , suppressed mitochondrial NAD + -dependent signaling and respiration, and impaired phagocytosis and resolution of inflammation. Innate immune challenge triggered upstream KP activation but paradoxically suppressed cell-autonomous NAD + synthesis by limiting the conversion of downstream quinolinate to NAD + , a profile recapitulated in aging macrophages. Increasing de novo NAD + generation in immune-challenged or aged macrophages restored oxidative phosphorylation and homeostatic immune responses. Thus, KP-derived NAD + operates as a metabolic switch to specify macrophage effector responses. Breakdown of de novo NAD + synthesis may underlie declining NAD + levels and rising innate immune dysfunction in aging and age-associated diseases.

Nicotinamide mononucleotide (NMN) is a biosynthetic precursor of NAD + known to promote cellular NAD + production and counteract age-associated pathologies associated with a decline in tissue NAD + levels. How NMN is taken up into cells has not been entirely clear. Here we show that the Slc12a8 gene encodes a specific NMN transporter. We find that Slc12a8 is highly expressed and regulated by NAD + in the murine small intestine. Slc12a8 knockdown abrogates the uptake of NMN in vitro and in vivo . We further show that Slc12a8 specifically transports NMN, but not nicotinamide riboside, and that NMN transport depends on the presence of sodium ion. Slc12a8 deficiency significantly decreases NAD + levels in the jejunum and ileum, which is associated with reduced NMN uptake as traced by doubly labeled isotopic NMN. Finally, we observe that Slc12a8 expression is upregulated in the aged murine ileum, which contributes to the maintenance of ileal NAD + levels. Our work identifies the first NMN transporter and demonstrates that Slc12a8 has a critical role in regulating intestinal NAD + metabolism.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: 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., Methods: We exploited expression profiling of muscle NAD + biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD + recycling to evaluate NMN and NR utilization., Results: Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD + . NAMPT inhibition depletes muscle NAD + availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD + in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD + . Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD + availability., Conclusions: These results identify skeletal muscle cells as requiring NAMPT to maintain NAD + availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD + availability.

NAD + availability decreases with age and in certain disease conditions. Nicotinamide mononucleotide (NMN), a key NAD + intermediate, has been shown to enhance NAD + biosynthesis and ameliorate various pathologies in mouse disease models. In this study, we conducted a 12-month-long NMN administration to regular chow-fed wild-type C57BL/6N mice during their normal aging. Orally administered NMN was quickly utilized to synthesize NAD + in tissues. Remarkably, NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies. Consistent with these phenotypes, NMN prevented age-associated gene expression changes in key metabolic organs and enhanced mitochondrial oxidative metabolism and mitonuclear protein imbalance in skeletal muscle. These effects of NMN highlight the preventive and therapeutic potential of NAD + intermediates as effective anti-aging interventions in humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD(+) depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD(+) depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD(+) depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Novel 5,5-fused thiophene lactams are potent inhibitors and acylating agents of HNE and PPE.