NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis
Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that is present in all living cells. NAD+ acts as an important cofactor and substrate for a multitude of biological processes including energy production, DNA repair, gene expression, calcium-dependent secondary messenger signalling and immunoregulatory roles. The de novo synthesis of NAD+ is primarily dependent on the kynurenine pathway (KP), although NAD+ can also be recycled from nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NAD+ levels have been reported to decline during ageing and age-related diseases. Recent studies have shown that raising intracellular NAD+ levels represents a promising therapeutic strategy for age-associated degenerative diseases in general and to extend lifespan in small animal models. A systematic review of the literature available on Medline, Embase and Pubmed was undertaken to evaluate the potential health and/or longevity benefits due to increasing NAD+ levels. A total of 1545 articles were identified and 147 articles (113 preclinical and 34 clinical) met criteria for inclusion. Most studies indicated that the NAD+ precursors NAM, NR, nicotinamide mononucleotide (NMN), and to a lesser extent NAD+ and NADH had a favourable outcome on several age-related disorders associated with the accumulation of chronic oxidative stress, inflammation and impaired mitochondrial function. While these compounds presented with a limited acute toxicity profile, evidence is still quite limited and long-term human clinical trials are still nascent in the current literature. Potential risks in raising NAD+ levels in various clinical disorders using NAD+ precursors include the accumulation of putative toxic metabolites, tumorigenesis and promotion of cellular senescence. Therefore, NAD+ metabolism represents a promising target and further studies are needed to recapitulate the preclinical benefits in human clinical trials.
Nicotinamide riboside kinase-2 alleviates ischemia-induced heart failure through P38 signaling
Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin binding protein, predominantly expresses in skeletal muscle with a trace amount expressed in healthy cardiac tissue. NRK-2 expression dramatically increases in mouse and human ischemic heart however, the specific role of NRK-2 in the pathophysiology of ischemic cardiac diseases is unknown. We employed NRK2 knockout (KO) mice to identify the role of NRK-2 in ischemia-induced cardiac remodeling and dysfunction. Following myocardial infarction (MI), or sham surgeries, serial echocardiography was performed in the KO and littermate control mice. Cardiac contractile function rapidly declined and left ventricular interior dimension (LVID) was significantly increased in the ischemic KO vs. control mice at 2 weeks post-MI. An increase in mortality was observed in the KO vs. control group. The KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric analysis. Consistently, histological assessment revealed an extensive and thin scar and dilated LV chamber accompanied with elevated fibrosis in the KOs post-MI. Mechanistically, we observed that loss of NRK-2 enhanced p38α activation following ischemic injury. Consistently, ex vivo studies demonstrated that the gain of NRK-2 function suppresses the p38α as well as fibroblast activation (α-SMA expression) upon TGF-β stimulation, and limits cardiomyocytes death upon hypoxia/re‑oxygenation. Collectively our findings show, for the first time, that NRK-2 plays a critical role in heart failure progression following ischemic injury. NRK-2 deficiency promotes post-MI scar expansion, rapid LV chamber dilatation, cardiac dysfunction and fibrosis possibly due to increased p38α activation.
Targeting NAD in translational research to relieve diseases and conditions of metabolic stress and ageing
Nicotinamide adenine dinucleotide (NAD) plays a fundamental role in life and health through the regulation of energy biogenesis, redox homeostasis, cell metabolism, and the arbitration of cell survival via linkages to apoptosis and autophagic pathways. The importance of NAD in ageing and healthy longevity has been revealed from laboratory animal studies and early-stage clinical testing. While basic researchers and clinicians have investigated the molecular mechanisms and translation potential of NAD, there are still major gaps in applying laboratory science to design the most effective trials. This mini-review was based on the programme and discussions of the 3rd NO-Age Symposium held at the Akershus University Hospital, Norway on the 28th October 2019. This symposium brought together leading basic researchers on NAD and clinicians who are leading or are going to perform NAD augmentation-related clinical studies. This meeting covered talks about NAD synthetic pathways, subcellular homeostasis of NAD, the benefits of NAD augmentation from maternal milk to offspring, current clinical trials of the NAD precursor nicotinamide riboside (NR) on Ataxia-Telangiectasia (A-T), Parkinson's disease (PD), post-sepsis fatigue, as well as other potential NR-based clinical trials. Importantly, a consensus is emerging with respect to the design of clinical trials in order to measure meaningful parameters and ensure safety.
Nicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin-resistant men
This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase.
Nicotinamide Pathway-Dependent Sirt1 Activation Restores Calcium Homeostasis to Achieve Neuroprotection in Spinocerebellar Ataxia Type 7
Sirtuin 1 (Sirt1) is a NAD-dependent deacetylase capable of countering age-related neurodegeneration, but the basis of Sirt1 neuroprotection remains elusive. Spinocerebellar ataxia type 7 (SCA7) is an inherited CAG-polyglutamine repeat disorder. Transcriptome analysis of SCA7 mice revealed downregulation of calcium flux genes accompanied by abnormal calcium-dependent cerebellar membrane excitability. Transcription-factor binding-site analysis of downregulated genes yielded Sirt1 target sites, and we observed reduced Sirt1 activity in the SCA7 mouse cerebellum with NAD depletion. SCA7 patients displayed increased poly(ADP-ribose) in cerebellar neurons, supporting poly(ADP-ribose) polymerase-1 upregulation. We crossed Sirt1-overexpressing mice with SCA7 mice and noted rescue of neurodegeneration and calcium flux defects. NAD repletion via nicotinamide riboside ameliorated disease phenotypes in SCA7 mice and patient stem cell-derived neurons. Sirt1 thus achieves neuroprotection by promoting calcium regulation, and NAD dysregulation underlies Sirt1 dysfunction in SCA7, indicating that cerebellar ataxias exhibit altered calcium homeostasis because of metabolic dysregulation, suggesting shared therapy targets.
Nicotinamide riboside supplementation to improve skeletal muscle mitochondrial health and whole-body glucose homeostasis: does it actually work in humans?
Induction of the nicotinamide riboside kinase NAD salvage pathway in a model of sarcoplasmic reticulum dysfunction
Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation.
Silencing of PARP2 Blocks Autophagic Degradation
Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.
DNA Methylation Changes are Associated with the Programming of White Adipose Tissue Browning Features by Resveratrol and Nicotinamide Riboside Neonatal Supplementations in Mice
Neonatal supplementation with resveratrol (RSV) or nicotinamide riboside (NR) programs in male mice brown adipocyte-like features in white adipose tissue (WAT browning) together with improved metabolism in adulthood. We tested the involvement in this programming of long-term epigenetic changes in two browning-related genes that are overexpressed in WAT of supplemented mice, and . Suckling mice received orally the vehicle, RSV or NR from postnatal days 2-to-20. After weaning (d21) onto a chow diet, male mice were habituated to a normal-fat diet (NFD) starting d75, and split on d90 into continuation on the NFD or switching to a high-fat diet (HFD) until euthanization on d164. CpG methylation by bisulfite-sequencing was analyzed on inguinal WAT. Both treatments modified methylation marks in and and the HFD-dependent dynamics of these marks in the adult WAT, with distinct and common effects. The treatments also affected gene expression of DNA methylases in WAT of young animals (euthanized at d35 in independent experiments). Studies in 3T3-L1 adipocytes indicated the direct effects of RSV and NR on the DNA methylation machinery and favoring browning features. The results support epigenetic effects being involved in WAT programming by neonatal RSV or NR supplementation in male mice.
Long noncoding RNA integrates a DNA-PK-mediated DNA damage response and vascular senescence
Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 () is highly expressed in the vascular endothelium and decreases during lesion progression. knockdown accelerated atherosclerotic lesion formation by 2.4-fold in mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by knockdown. expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
Nicotinamide riboside alleviates alcohol-induced depression-like behaviours in C57BL/6J mice by altering the intestinal microbiota associated with microglial activation and BDNF expression
The gut microbiota play an important role in many central nervous system diseases through the gut microbiota-brain axis. Recent studies suggest that nicotinamide riboside (NR) has neuroprotective properties. However, it is unknown whether NR can prevent or protect against alcohol-induced depression. Furthermore, it is unclear whether its therapeutic action involves changes in the composition of the gut microbiome. Here, we investigated the effects of NR in the mouse model of alcohol-induced depression. Treatment with NR improved the alcohol-induced depressive behaviour in mice. In addition, NR decreased the number of activated microglia in the hippocampus, and it reduced the levels of pro-inflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10 and TGF-β) cytokines in the brain of mice with alcohol-induced depression. Furthermore, NR significantly upregulated BDNF and diminished the inhibition of the AKT/GSK3β/β-catenin signalling pathway in the hippocampus of these mice. 16S rRNA sequencing revealed that, compared with control and NR-treated mice, the gut microbiome richness and composition were significantly altered in the depressed mice. Spearman's correlation analysis showed that differential gut bacterial genera correlated with the levels of inflammation-related cytokines and BDNF in the brain. After faecal microbiota transplantation, cognitive behaviours, microglial activity, levels of cytokines and BDNF, and activation state of the AKT/GSK3β/β-catenin signalling pathway (which is downstream of the BDNF receptor, TrkB) in recipient mice were similar to those in donor mice. Collectively, our findings show that NR dietary supplementation protects against alcohol-induced depression-like behaviours, possibly by altering the composition of the gut microbiota.
The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion
Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase life-span of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested.
Nicotinamide riboside rescues angiotensin II-induced cerebral small vessel disease in mice
Hypertension is a leading cause of cerebral small vessel disease (CSVD). Currently, treatments for CSVD are limited. Nicotinamide riboside (NR) can protect against vascular injury and cognitive impairment in neurodegenerative diseases. In this study, the protective effects of NR against angiotensin - (Ang -)-induced CSVD were evaluated.
Evaluation of the NAD biosynthetic pathway in ALS patients and effect of modulating NAD levels in hSOD1-linked ALS mouse models
Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons. Astrocytes from diverse ALS models induce motor neuron death in co-culture. Enhancing NAD availability, or increasing the expression of the NAD-dependent deacylases SIRT3 and SIRT6, abrogates their neurotoxicity in cell culture models. To determine the effect of increasing NAD availability in ALS mouse models we used two strategies, ablation of a NAD-consuming enzyme (CD38) and supplementation with a bioavailable NAD precursor (nicotinamide riboside, NR). Deletion of CD38 had no effect in the survival of two hSOD1-linked ALS mouse models. On the other hand, NR-supplementation delayed motor neuron degeneration, decreased markers of neuroinflammation in the spinal cord, appeared to modify muscle metabolism and modestly increased the survival of hSOD1 mice. In addition, we found altered expression of enzymes involved in NAD synthesis (NAMPT and NMNAT2) and decreased SIRT6 expression in the spinal cord of ALS patients, suggesting deficits of this neuroprotective pathway in the human pathology. Our data denotes the therapeutic potential of increasing NAD levels in ALS. Moreover, the results indicate that the approach used to enhance NAD levels critically defines the biological outcome in ALS models, suggesting that boosting NAD levels with the use of bioavailable precursors would be the preferred therapeutic strategy for ALS.
Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus
The core of β-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. β-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.
Small-molecule nicotinamide for ex vivo expansion of umbilical cord blood
Umbilical cord blood transplant is an alternative graft source for patients lacking a human leukocyte antigen-matched donor; however, delayed engraftment times have historically resulted in transplant-related morbidity and mortality from complications such as infections and ineffective hematopoiesis. Recent advances in ex vivo expansion techniques have successfully augmented the initial cell dose delivered from an umbilical cord blood graft, leading to improved immune reconstitution, durable hematopoiesis, decreased transplant-related morbidity and mortality, and better outcomes. Herein we review the data for existing and developing ex vivo expansion techniques, with a focus on the preclinical and clinical data for nicotinamide-mediated cord blood expansion across both malignant and benign hematologic indications.
NAD repletion inhibits the endothelial-to-mesenchymal transition induced by TGF-β in endothelial cells through improving mitochondrial unfolded protein response
Endothelial-to-mesenchymal transition (EndMT) plays an important role in the progression of cardiac fibrosis but its mechanism and treatment need to be further understood. Herein, we have found that mitochondrial unfolded protein response (mtUPR) played a critical role in transforming growth factor beta 1 (TGF-β1)-induced EndMT in endothelial cells (ECs). MtUPR was repressed in endothelial cells after exposure to TGF-β1. NAD + precursor nicotinamide riboside (NR) could attenuate TGF-β1-induced EndMT and improve the levels of mtUPR. Significantly, prohibitin proteins (PHB and PHB2) was also regulated by nicotinamide riboside. Moreover, we found that inhibition of prohibitin proteins could prevent the protective effect of nicotinamide riboside on mtUPR and TGF-β1-induced EndMT. Overexpression of prohibitin proteins could alleviate mitochondrial function and TGF-β1-induced EndMT through improving mtUPR. In vivo, The EndMT of ECs induced by Transverse aortic constriction (TAC) in mouse was inhibited by NR. In conclusion, our results indicate that nicotinamide riboside improved the expression of prohibitin proteins to ameliorate EndMT via promotion of mtUPR. Nicotinamide riboside is a potential therapeutic target for cardiac fibrosis.
In silico pharmacophore modeling and simulation studies for searching potent antileishmanials targeted against Leishmania donovani nicotinamidase
Nicotinamidase is a key enzyme for the salvage pathway catalyzing the first step for the conversion of nicotinamide (NAm) to nicotinic acid (NA) required for the synthesis of Nicotinamide Adenine Dinucleotide (NAD) in the subsequent steps. Leishmania protozoan parasites are NAD auxotrophs and need precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD for their survival. Interestingly, absence of this enzyme in higher eukaryotes and its absolute requirement in the developmental cycle of Leishmania has led nicotinamidase an attractive drug target towards leishmaniasis. Hence, we report some potential inhibitors for nicotinamidase screened based on 3-D pharmacophore model consisting of "ML", "Hyd|Aro", "Acc" and "Excl vol" features. Subsequently, dynamics simulation studies validate the proposed pharmacophore model suggesting its reliability for future studies. Furthermore, these essential site-specific features will help in enhancing the inhibition of nicotinamidase activity. Results of our study suggest that blocking of active site of nicotinamidase by the identified lead inhibitor will have major impact on the infectious processes and the survival of the parasite. Furthermore, due to the structural homology in the enzyme among L. donovani, L. infantum, L. major, we anticipate that our study would help to design more potent drug candidates against leshmaniasis for these three species.
A reduced form of nicotinamide riboside defines a new path for NAD biosynthesis and acts as an orally bioavailable NAD precursor
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.
Genetic evidence for vitamin B3 acquisition from rice cells
Following penetration, the devastating rice blast fungus , like some other important eukaryotic phytopathogens, grows in intimate contact with living plant cells before causing disease. Cell-to-cell growth during this biotrophic growth stage must involve nutrient acquisition, but experimental evidence for the internalization and metabolism of host-derived compounds is exceedingly sparse. This striking gap in our knowledge of the infection process undermines accurate conceptualization of the plant-fungal interaction. Here, through our general interest in metabolism and with a specific focus on the signalling and redox cofactor nicotinamide adenine dinucleotide (NAD), we deleted the gene encoding quinolinate phosphoribosyltransferase, catalyst of the last step in NAD biosynthesis from tryptophan. We show how is essential for axenic growth on minimal media lacking nicotinic acid (NA, an importable NAD precursor). However, Δ mutant strains were fully pathogenic, indicating NAD biosynthesis is dispensable for lesion expansion following invasive hyphal growth in leaf tissue. Because overcoming the loss of NAD biosynthesis can only occur if importable NAD precursors (which solely comprise the NA, nicotinamide and nicotinamide riboside forms of vitamin B3) are accessible, we unexpectedly but unequivocally demonstrate that vitamin B3 can be acquired from the host and assimilated into metabolism during growth in rice cells. Our results furnish a rare, experimentally determined example of host nutrient acquisition by a fungal plant pathogen and are significant in expanding our knowledge of events at the plant-fungus metabolic interface.
Functions of aryl hydrocarbon receptor (AHR) and CD38 in NAD metabolism and nonalcoholic steatohepatitis (NASH)
Aryl hydrocarbon receptor (AHR), identified in studies of dioxin toxicity, has been characterized as ligand-activated transcription factor involved in diverse functions including microbial defense, cell proliferation, immunity and NAD metabolism. AHR targets of the latter function are PARPs/ARTs and CD38 that are regulating glucose and lipid metabolism via NAD-dependent sirtuins. Deregulation of these pathways may facilitate obesity and age-dependent pathologies. The present commentary is focused on AHR and CD38 signaling in liver. CD38 is functioning as ectoNADase and Ca mobilizing enzyme in endoplasmic reticulum and endolysosomal membranes. Deregulation of TCDD-activated AHR and CD38 may facilitate hepatic steatosis and inflammation. However, these proteins are also involved in protection against inflammation and CD38-mediated age-related decreased NAD levels that may be responsible for neurodegeneration. Further knowledge about the complexity of these pathways is needed to avoid pathologies. Therapeutic modulation of AHR and CD38 remains a challenging task.
Subcellular Characterization of Nicotinamide Adenine Dinucleotide Biosynthesis in Metastatic Melanoma by Using Organelle-Specific Biosensors
Nicotinamide adenine dinucleotide (NAD) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD levels in live cells and to address the issue of the compartmentalization of NAD biosynthesis, we exploited a recently described genetically encoded NAD biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of A375 melanoma cells, a model of metastatic melanoma (MM). FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD-producing enzyme in MM cells, was used to monitor NAD depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Throughout this work, we validated the use of genetically encoded NAD biosensors to characterize subcellular distribution of NAD production routes in MM. The chance of real-time monitoring of NAD fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD pool manipulation in cancer and metabolic diseases.
Effects of Nicotinamide Riboside on Endocrine Pancreatic Function and Incretin Hormones in Nondiabetic Men With Obesity
Augmenting nicotinamide adenine dinucleotide (NAD+) metabolism through dietary provision of NAD+ precursor vitamins translates to improved glucose handling in rodent models of obesity and diabetes. Preclinical evidence suggests that the NAD+/SIRT1 axis may be implicated in modulating important gut-related aspects of glucose regulation. We sought to test whether NAD+ precursor supplementation with nicotinamide riboside (NR) affects β-cell function, α-cell function, and incretin hormone secretion as well as circulating bile acid levels in humans.
Degradation of Extracellular NAD Intermediates in Cultures of Human HEK293 Cells
Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.
High Dose of Dietary Nicotinamide Riboside Induces Glucose Intolerance and White Adipose Tissue Dysfunction in Mice Fed a Mildly Obesogenic Diet
Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD) precursor vitamin. The scarce reports on the adverse effects on metabolic health of supplementation with high-dose NR warrant substantiation. Here, we aimed to examine the physiological responses to high-dose NR supplementation in the context of a mildly obesogenic diet and to substantiate this with molecular data. An 18-week dietary intervention was conducted in male C57BL/6JRccHsd mice, in which a diet with 9000 mg NR per kg diet (high NR) was compared to a diet with NR at the recommended vitamin B3 level (control NR). Both diets were mildly obesogenic (40 en% fat). Metabolic flexibility and glucose tolerance were analyzed and immunoblotting, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were performed. Mice fed with high NR showed a reduced metabolic flexibility, a lower glucose clearance rate and aggravated systemic insulin resistance. This was consistent with molecular and morphological changes in eWAT, including sirtuin 1 (SIRT1)-mediated PPARγ (proliferator-activated receptor γ) repression, downregulated AKT/glucose transporter type 4 (GLUT4) signaling, an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers. In conclusion, high-dose NR induces the onset of WAT dysfunction, which may in part explain the deterioration of metabolic health.
PGC-1α, Sirtuins and PARPs in Huntington's Disease and Other Neurodegenerative Conditions: NAD+ to Rule Them All
In this review, we summarize the available published information on the neuroprotective effects of increasing nicotinamide adenine dinucleotide (NAD) levels in Huntington's disease models. We discuss the rationale of potential therapeutic benefit of administering nicotinamide riboside (NR), a safe and effective NAD precursor. We discuss the agonistic effect on the Sirtuin1-PGC-1α-PPAR pathway as well as Sirtuin 3, which converge in improving mitochondrial function, decreasing ROS production and ameliorating bioenergetics deficits. Also, we discuss the potential synergistic effect of increasing NAD+ combined with PARPs inhibitors, as a clinical therapeutic option not only in HD, but other neurodegenerative conditions.
Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters
Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.
Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in D2-mdx mice
Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD-dependent deacetylase, the depleted NAD in dystrophic skeletal muscle may limit quercetin efficacy; hence, supplementation with the NAD donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice; therefore, it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR, or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-mo-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed after treatment with Q, NR, and/or Lis for 7 mo. To mimic typical pharmacology of patients with Duchenne muscular dystrophy, a group was treated with prednisolone (Pred) in combination with Q, NR, and Lis. At 11 mo of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, whereas fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest that treatment with Q, NR, Lis, and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage. Despite a compelling rationale and previous evidence to the contrary in short-term investigations, quercetin, nicotinamide riboside, or Lisinopril, alone or in combination, failed to restore muscle function or decrease histological injury in dystrophic limb muscle from D2-mdx mice after long-term administration. Importantly, we also found that in the D2-mdx model, an emerging and relatively understudied model of Duchenne muscular dystrophy dystrophin deficiency caused profound muscle dysfunction and histopathology in skeletal muscle.
NAD precursor increases aerobic performance in mice
Nicotinamide riboside (NR) acts as a potent NAD precursor and improves mitochondrial oxidative capacity and mitochondrial biogenesis in several organisms. However, the effects of NR supplementation on aerobic performance remain unclear. Here, we evaluated the effects of NR supplementation on the muscle metabolism and aerobic capacity of sedentary and trained mice.
A ribose-functionalized NAD with unexpected high activity and selectivity for protein poly-ADP-ribosylation
Nicotinamide adenine dinucleotide (NAD)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD analogue with high efficiency and yields. Notably, the generated 3'-azido NAD exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD provides an important tool for studying cellular PARylation.
Endogenous nicotinamide riboside metabolism protects against diet-induced liver damage
Supplementation with the NAD precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders.
Combined Treatment with L-Carnitine and Nicotinamide Riboside Improves Hepatic Metabolism and Attenuates Obesity and Liver Steatosis
Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for β-oxidation and the TCA cycle, respectively. Ldlr -/-.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% /), nicotinamide riboside (NR; 0.3% /) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (-17%) and hepatic steatosis (-22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.
Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis
Liver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis.
Low Cellular NAD Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes
NAD is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD enhanced IL-1β release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD depletion, specifically TAK1, IKKβ, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.
Mitochondrial function in liver cells is resistant to perturbations in NAD salvage capacity
Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD levels, but the metabolic phenotype of deficient hepatocytes in mice is unknown. Here, we assessed role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific knockout (HNKO) mice, having a 50% reduction of liver NAD levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD, suggesting that the mitochondrial NAD pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [N]glutamine, HNKO hepatocytes had a higher [N]NAD enrichment than WT hepatocytes, indicating that HNKO mice compensate through NAD synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD levels and that the knockout has only minor consequences for mitochondrial function in the mouse liver.
Roads to the Fountain of Youth? Rejuvenating Intestinal Stem Cells
The intestinal stem cells (ISCs) of old mice and humans exhibit a reduced capacity for regeneration and repair. Compromised intestinal function may play a key role in systemic aging-related changes: not only in the affected gut, but also in the nervous and cardiovascular systems. For example, progression of age-related neurodegenerative diseases such as Alzheimer's and Parkinson's has been linked to increased inflammation from gut microbiota in old mammals, which, in turn, may be linked bidirectionally with reduced ISC function. Intestinal organoid formation has been used to dissect the mechanisms of decline of ISC function. Alterations of the Wnt pathway, including downregulation of Wnt ligands in ISCs and upregulation of Wnt ligand inhibitor Notum in Paneth cells, and dysregulation of mTORC1 contribute to the observed age-related decline. Short-term fasting, caloric restriction, and peroxisome proliferator-activated receptor delta agonists have been reported to increase ISC function in adult mice. Moreover, the mTOR inhibitor rapamycin, NAD+ precursor nicotinamide riboside, and ABC99, a small molecule Notum inhibitor, have all been reported to rejuvenate ISC function in old mice and thus may have promise in humans. However, there is some controversy over the key mechanisms involved in loss of function of ISCs, which likely results, in part, from differences in how the organoid assays are performed. Moreover, how the microbiome modulates the function of ISCs and vice versa remains to be elucidated.
Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures
Nicotinamide adenine dinucleotide (NAD) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.
Supplementation with Nicotinamide Riboside Reduces Brain Inflammation and Improves Cognitive Function in Diabetic Mice
The purpose of this study is to investigate whether nicotinamide riboside (NR) can improve inflammation and cognitive function in diabetic mice. ICR male mice were fed for 14 weeks with either high-fat chow diet (HF, 60% kcal fat) or standard chow diet (CON, 10% kcal fat). HF, streptozotocin, and nicotinamide were used to induce hyperglycemia. NR or vehicle was delivered via stomach gavage for six weeks. Oral glucose tolerance test, Y-maze test, and nest construction test were conducted before and after the NR treatment period. NR treatment induced down-regulation of NLRP3, ASC, and caspase-1. NR reduced IL-1 expression significantly by 50% in whole brains of hyperglycemic mice. Other inflammatory markers including TNF-α and IL-6 were also attenuated by NR. Brain expression of amyloid-β precursor protein and presenilin 1 were reduced by NR. In addition, NR induced significant reduction of amyloid-β in whole brains of diabetic mice. NR treatment restored hyperglycemia-induced increases in brain karyopyknosis to the levels of controls. Nest construction test showed that NR improved hippocampus functions. Spatial recognition memory and locomotor activity were also improved by NR supplementation. These findings suggest that NR may be useful for treating cognitive impairment by inhibiting amyloidogenesis and neuroinflammation.
Nicotinamide adenine dinucleotide emerges as a therapeutic target in aging and ischemic conditions
Nicotinamide adenine dinucleotide (NAD) has been described as central coenzyme of redox reactions and is a key regulator of stress resistance and longevity. Aging is a multifactorial and irreversible process that is characterized by a gradual diminution in physiological functions in an organism over time, leading to development of age-associated pathologies and eventually increasing the probability of death. Ischemia is the lack of nutritive blood flow that causes damage and mortality that mostly occurs in various organs during aging. During the process of aging and related ischemic conditions, NAD levels decline and lead to nuclear and mitochondrial dysfunctions, resulting in age-related pathologies. The majority of studies have shown that restoring of NAD using supplementation with intermediates such as nicotinamide mononucleotide and nicotinamide riboside can be a valuable strategy for recovery of ischemic injury and age-associated defects. This review summarizes the molecular mechanisms responsible for the reduction in NAD levels during ischemic disorders and aging, as well as a particular focus is given to the recent progress in the understanding of NAD precursor's effects on aging and ischemia.
Safety and Metabolism of Long-term Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy Overweight Adults
Nicotinamide riboside (NR) is a newly discovered nicotinamide adenine dinucleotide (NAD) precursor vitamin. A crystal form of NR chloride termed NIAGEN is generally recognized as safe (GRAS) for use in foods and the subject of two New Dietary Ingredient Notifications for use in dietary supplements. To evaluate the kinetics and dose-dependency of NR oral availability and safety in overweight, but otherwise healthy men and women, an 8-week randomized, double-blind, placebo-controlled clinical trial was conducted. Consumption of 100, 300 and 1000 mg NR dose-dependently and significantly increased whole blood NAD (i.e., 22%, 51% and 142%) and other NAD metabolites within 2 weeks. The increases were maintained throughout the remainder of the study. There were no reports of flushing and no significant differences in adverse events between the NR and placebo-treated groups or between groups at different NR doses. NR also did not elevate low density lipoprotein cholesterol or dysregulate 1-carbon metabolism. Together these data support the development of a tolerable upper intake limit for NR based on human data.
Aerobic and resistance exercise training reverses age-dependent decline in NAD salvage capacity in human skeletal muscle
Aging decreases skeletal muscle mass and strength, but aerobic and resistance exercise training maintains skeletal muscle function. NAD is a coenzyme for ATP production and a required substrate for enzymes regulating cellular homeostasis. In skeletal muscle, NAD is mainly generated by the NAD salvage pathway in which nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting. NAMPT decreases with age in human skeletal muscle, and aerobic exercise training increases NAMPT levels in young men. However, whether distinct modes of exercise training increase NAMPT levels in both young and old people is unknown. We assessed the effects of 12 weeks of aerobic and resistance exercise training on skeletal muscle abundance of NAMPT, nicotinamide riboside kinase 2 (NRK2), and nicotinamide mononucleotide adenylyltransferase (NMNAT) 1 and 3 in young (≤35 years) and older (≥55 years) individuals. NAMPT in skeletal muscle correlated negatively with age (r = 0.297, P < 0.001, n = 57), and VO peak was the best predictor of NAMPT levels. Moreover, aerobic exercise training increased NAMPT abundance 12% and 28% in young and older individuals, respectively, whereas resistance exercise training increased NAMPT abundance 25% and 30% in young and in older individuals, respectively. None of the other proteins changed with exercise training. In a separate cohort of young and old people, levels of NAMPT, NRK1, and NMNAT1/2 in abdominal subcutaneous adipose tissue were not affected by either age or 6 weeks of high-intensity interval training. Collectively, exercise training reverses the age-dependent decline in skeletal muscle NAMPT abundance, and our findings highlight the value of exercise training in ameliorating age-associated deterioration of skeletal muscle function.
The Potential Use of Metabolic Cofactors in Treatment of NAFLD
Non-alcoholic fatty liver disease (NAFLD) is caused by the imbalance between lipid deposition and lipid removal from the liver, and its global prevalence continues to increase dramatically. NAFLD encompasses a spectrum of pathological conditions including simple steatosis and non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and liver cancer. Even though there is a multi-disciplinary effort for development of a treatment strategy for NAFLD, there is not an approved effective medication available. Single or combined metabolic cofactors can be supplemented to boost the metabolic processes altered in NAFLD. Here, we review the dosage and usage of metabolic cofactors including l-carnitine, Nicotinamide riboside (NR), l-serine, and -acetyl-l-cysteine (NAC) in human clinical studies to improve the altered biological functions associated with different human diseases. We also discuss the potential use of these substances in treatment of NAFLD and other metabolic diseases including neurodegenerative and cardiovascular diseases of which pathogenesis is linked to mitochondrial dysfunction.
Targeting NAMPT as a therapeutic strategy against stroke
Stroke is the second and the leading most common cause of death in the world and China, respectively, but with few effective therapies. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) salvage synthesis in mammals, thereby influencing NAD-dependent enzymes and constituting a strong endogenous defence system against various stresses. Accumulating in-vitro and in-vivo studies have demonstrated the neuroprotective effect of NAMPT in stroke. Here, we review the direct evidence of NAMPT as a promising target against stroke from five potential therapeutic strategies, including NAMPT overexpression, recombinant NAMPT, NAMPT activators, NAMPT enzymatic product nicotinamide mononucleotide (NMN), and NMN precursors nicotinamide riboside and nicotinamide, and describe the relevant mechanisms and limitations, providing a promising choice for developing novel and effective therapeutic interventions against ischaemic and haemorrhagic stroke.
NAD metabolites interfere with proliferation and functional properties of THP-1 cells
Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.
Nicotinamide riboside promotes autolysosome clearance in preventing doxorubicin-induced cardiotoxicity
Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD), prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.
Chemoenzymatic Preparation of 4'-Thioribose NAD
This chemoenzymatic procedure describes a strategy for the preparation of 4'-thioribose nicotinamide adenine dinucleotide (S-NAD ), including chemical synthesis of nicotinamide 4'-riboside (S-NR), recombinant expression and purification of two NAD biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S-NAD . The first basic protocol describes the procedures for introduction of nicotinamide onto 4'-thioribose and subsequent deprotection to generate S-NR as the key intermediate for enzymatically synthesizing S-NAD . In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S-NR to S-NAD . The third basic protocol presents the enzymatic approach for the generation of S-NAD from S-NR precursor. © 2019 by John Wiley & Sons, Inc.
Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1
Sirtuins (SIRTs) are NAD-dependent deacylases that play a key role in transcription, DNA repair, metabolism, and oxidative stress resistance. Increasing NAD availability regulates endogenous SIRT activity, leading to increased resistance to oxidative stress and decreased mitochondrial reactive oxygen production in multiple cell types and disease models. This protection, at least in part, depends on the activation of antioxidant mitochondrial proteins. We now show that increasing total NAD content in astrocytes leads to the activation of the transcription factor nuclear factor, erythroid-derived 2, like 2 (Nfe2l2 or Nrf2) and up-regulation of the antioxidant proteins heme oxygenase 1 (HO-1) and sulfiredoxin 1 (SRXN1). Nrf2 activation also occurs as a result of SIRT6 overexpression. Mutations in Cu-Zn superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS). Astrocytes isolated from mutant human SOD1-overexpressing mice induce motor neuron death in coculture. Treatment with nicotinamide mononucleotide or nicotinamide riboside increases total NAD content in ALS astrocytes and abrogates their toxicity toward cocultured motor neurons. The observed neuroprotection depends on SIRT6 expression in astrocytes. Moreover, overexpression of SIRT6 in astrocytes by itself abrogates the neurotoxic phenotype of ALS astrocytes. Our results identify SIRT6 as a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.-Harlan, B. A., Pehar, M., Killoy, K. M., Vargas, M. R. Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1.
Dihydronicotinamide riboside is a potent NAD concentration enhancer and
Interest in pharmacological agents capable of increasing cellular NAD concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD concentrations by 2.5-10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD increases at equivalent concentrations. NRH also provides substantial NAD increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD consumption, suggesting that it acts as a biochemical precursor to NAD Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD and a potent pharmacologic agent for NAD concentration enhancement in cells and tissues.
NAD supplementation rejuvenates aged gut adult stem cells
The tissue decline due to aging is associated with the deterioration of adult stem cell function. Here we show the number and proliferative activity of intestinal stem cells (ISCs) but not Paneth cells decline during aging, as does ISC function assessed ex vivo. Levels of SIRT1 and activity of mTORC1 also decline with aging. The treatment with the NAD(+) precursor nicotinamide riboside (NR) rejuvenates ISCs from aged mice and reverses an impaired ability to repair gut damage. The effect of NR is blocked by the mTORC1 inhibitor rapamycin or the SIRT1 inhibitor EX527. These findings demonstrate that small molecules affecting the NAD/SIRT1/mTORC1 axis may guide a translational path for maintenance of the intestine during aging.
Underpowered or negative? A crucial distinction
Nicotinamide riboside protects against liver fibrosis induced by CCl via regulating the acetylation of Smads signaling pathway
Increasing nicotinamide adenine dinucleotide (NAD) by Nicotinamide riboside (NR) provides protective benefits in multiple disorders. However, the role of NR on liver fibrosis is unclear. We performed in vivo and in vitro experiments to test the hepatic protective effects of NR against liver fibrosis and the underlying mechanisms.
Implications of altered NAD metabolism in metabolic disorders
Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that participates in various energy metabolism pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Besides, it is a required cofactor for post-translational modifications such as ADP-ribosylation and deacetylation by poly (ADP-ribose) polymerases (PARPs) and sirtuins, respectively. Thus, NAD regulates energy metabolism, DNA damage repair, gene expression, and stress response through these enzymes. Numerous studies have shown that NAD levels decrease with aging and under disturbed nutrient conditions, such as obesity. Additionally, a decline in NAD levels is closely related to the development of various metabolic disorders, including diabetes and fatty liver disease. In addition, many studies have revealed that administration of NAD precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), efficiently increase NAD levels in various tissues and prevent such metabolic diseases. These NAD precursors are contained in natural foods, such as cow milk, vegetables, and meats. Therefore, altered NAD metabolism can be a practical target for nutritional intervention. Recently, several human clinical trials using NAD precursors have been conducted to investigate the safety, pharmacokinetics, and efficacy against metabolic disorders such as glucose intolerance. In this review, we summarize current knowledge on the implications of NAD metabolism in metabolic diseases and discuss the outcomes of recent human clinical trials.
Drugs targeting SIRT1, a new generation of therapeutics for osteoporosis and other bone related disorders?
With an aging population and limited treatment options, osteoporosis currently represents a significant public health challenge. Recent animal studies indicate that longevity-associated SIRT1 may serve as an attractive pharmacological target for the treatment of osteoporosis and other bone related disorders. Pre-clinical studies demonstrate that mice treated with SIRT1 agonists show protection against age-related, post-menopausal, and disuse models of osteoporosis. Conversely, SIRT1 knockout models display low bone mass phenotypes associated with increased bone resorption and decreased bone formation. This review summarizes recent animal and human experimental data showing that pharmacological activation of SIRT1 may act in a manner that current treatments do not, namely by treating the imbalance in bone remodeling that is the root cause of osteoporosis and other bone disorders.
Assessment of NADmetabolism in human cell cultures, erythrocytes, cerebrospinal fluid and primate skeletal muscle
The reduction-oxidation state of NAD/NADH is critical for cellular health with NAD and its metabolites playing critical roles in aging and pathologies. Given the inherent autooxidation of reduced dinucleotides (i.e. NADH/NADPH), and the well-established differential stability, the accurate measurement of NAD and its metabolites is technically challenging. Moreover, sample processing, normalization and measurement strategies can profoundly alter results. Here we developed a rapid and sensitive liquid chromatography mass spectrometry-based method to quantify the NAD metabolome with careful consideration of these intrinsic chemical instabilities. Utilizing this method we assess NAD metabolite stabilities and determine the presence and concentrations of NAD metabolites in clinically relevant human samples including cerebrospinal fluid, erythrocytes, and primate skeletal muscle.
Transcriptional Response of White Adipose Tissue to Withdrawal of Vitamin B3
Distinct markers for mild vitamin B3 deficiency are lacking. To identify these, the molecular responses of white adipose tissue (WAT) to vitamin B3 withdrawal are examined.
Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload
The mitochondrial unfolded protein response (UPR) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPR in the heart is unclear.
NR Supplementation During Lactation: Benefiting Mother and Child
Abnormal nicotinamide adenine dinucleotide (NAD) metabolism causes a wide spectrum of diseases. A recent study (Cell Rep. 2019;26:969-983) shows that postpartum NAD homeostasis is depressed. By restoring NAD homeostasis, maternal nicotinamide riboside (NR) supplementation during lactation enhances postpartum weight loss, as well as juvenile development and adult neurogenesis in the offspring.
The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance
It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.
First quantification of nicotinamide riboside with B vitamers and coenzymes secreted in human milk by liquid chromatography-tandem-mass spectrometry
Nicotinamide riboside regulates inflammation and mitochondrial markers in AML12 hepatocytes
The NAD precursor nicotinamide riboside (NR) is a type of vitamin B found in cow's milk and yeast-containing food products such as beer. Recent studies suggested that NR prevents hearing loss, high-fat diet-induced obesity, Alzheimer's disease, and mitochondrial myopathy. The objective of this study was to investigate the effects of NR on inflammation and mitochondrial biogenesis in AML12 mouse hepatocytes.
Pterostilbene raises low density lipoprotein cholesterol in people
Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer's disease model mice
Nicotinamide adenine dinucleotide (NAD) supplementation to repair the disabled mitochondria is a promising strategy for the treatment of Alzheimer's disease (AD) and other dementia. Nicotinamide ribose (NR) is a safe NAD precursor with high oral bioavailability, and has beneficial effects on aging. Here, we applied NR supplied food (2.5 g/kg food) to APP/PS1 transgenic AD model mice and aged mice for 3 months. Cognitive function, locomotor activity and anxiety level were assessed by standard behavioral tests. The change of body weight, the activation of microglia and astrocytes, the accumulation of Aβ and the level of serum nicotinamide phosphoribosyltransferase (NAMPT) were determined for the evaluation of pathological processes. We found that NR supplementation improved the short-term spatial memory of aged mice, and the contextual fear memory of AD mice. Moreover, NR supplementation inhibited the activation of astrocytes and the elevation of serum NAMPT of aged mice. For AD model mice, NR supplementation inhibited the accumulation of Aβ and the migration of astrocyte to Aβ. In addition, NR supplementation inhibit the body weight gain of aged and APP/PS1 mice. Thus, NR has selective benefits for both AD and aged mice, and the oral uptake of NR can be used to prevent the progression of dementia.
Acute nicotinamide riboside supplementation improves redox homeostasis and exercise performance in old individuals: a double-blind cross-over study
Older individuals suffer from low NADH levels. We have previously shown that nicotinamide riboside [NR; a NAD(P)(H) precursor] administration impaired exercise performance in young rats. It has been suggested that supplementation of redox agents exerts ergogenic effect only in deficient individuals. We hypothesized that old individuals would more likely benefit from NR supplementation. We investigated the effect of acute NR supplementation on redox homeostasis and physical performance in young and old individuals.
Slc12a8 is a nicotinamide mononucleotide transporter
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 gene encodes a specific NMN transporter. We find that is highly expressed and regulated by NAD in the murine small intestine. knockdown abrogates the uptake of NMN and . We further show that Slc12a8 specifically transports NMN, but not nicotinamide riboside, and that NMN transport depends on the presence of sodium ion. 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 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.
Nicotinamide Mononucleotide: Exploration of Diverse Therapeutic Applications of a Potential Molecule
Nicotinamide mononucleotide (NMN) is a nucleotide that is most recognized for its role as an intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis. Although the biosynthetic pathway of NMN varies between eukaryote and prokaryote, two pathways are mainly followed in case of eukaryotic human-one is through the salvage pathway using nicotinamide while the other follows phosphorylation of nicotinamide riboside. Due to the unavailability of a suitable transporter, NMN enters inside the mammalian cell in the form of nicotinamide riboside followed by its subsequent conversion to NMN and NAD+. This particular molecule has demonstrated several beneficial pharmacological activities in preclinical studies, which suggest its potential therapeutic use. Mostly mediated by its involvement in NAD+ biosynthesis, the pharmacological activities of NMN include its role in cellular biochemical functions, cardioprotection, diabetes, Alzheimer's disease, and complications associated with obesity. The recent groundbreaking discovery of anti-ageing activities of this chemical moiety has added a valuable essence in the research involving this molecule. This review focuses on the biosynthesis of NMN in mammalian and prokaryotic cells and mechanism of absorption along with the reported pharmacological activities in murine model.
Maternal Nicotinamide Riboside Enhances Postpartum Weight Loss, Juvenile Offspring Development, and Neurogenesis of Adult Offspring
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.
Mitochondrial regulation of diabetic vascular disease: an emerging opportunity
Diabetes-related vascular complication rates remain unacceptably high despite guideline-based medical therapies that are significantly more effective in individuals without diabetes. This critical gap represents an opportunity for researchers and clinicians to collaborate on targeting mechanisms and pathways that specifically contribute to vascular pathology in patients with diabetes mellitus. Dysfunctional mitochondria producing excessive mitochondrial reactive oxygen species (mtROS) play a proximal cell-signaling role in the development of vascular endothelial dysfunction in the setting of diabetes. Targeting the mechanisms of production of mtROS or mtROS themselves represents an attractive method to reduce the prevalence and severity of diabetic vascular disease. This review focuses on the role of mitochondria in the development of diabetic vascular disease and current developments in methods to improve mitochondrial health to improve vascular outcomes in patients with DM.
Metabolomes of mitochondrial diseases and inclusion body myositis patients: treatment targets and biomarkers
Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect.
NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.
Nicotinamide riboside has protective effects in a rat model of mesenteric ischaemia-reperfusion
Acute mesenteric ischaemia is a syndrome caused by inadequate blood flow through the mesenteric vessels, resulting in ischaemia and eventual gangrene of the bowel wall. Although relatively rare, it is a potentially life-threatening condition. The maintenance of haemodynamic stability, along with adequate oxygen saturation, and the correction of any electrolyte imbalance, are of the utmost importance. However, nicotinamide adenine dinucleotide (NAD) biosynthesis modulation by precursor introduction can also be a powerful tool for preventing injury. Nicotinamide riboside is a pyridine-nucleoside form of vitamin B3 that functions as a precursor to NAD . The present study investigated nicotinamide riboside's effect on endothelium functional state, microcirculation and intestinal morphology in acute mesenteric ischaemia and reperfusion. Mesenteric ischaemia was simulated after the adaptation period (15 minutes) by occluding the superior mesenteric artery for 60 minutes, followed by a reperfusion period of 30 minutes. The functional state of intestinal microcirculation was evaluated by laser Doppler flowmetry. Endothelial functional activity was studied by using wire myography. Intestinal samples were stained with haematoxylin and eosin for histological analysis. The results revealed that nicotinamide riboside protects the intestinal wall from ischaemia-reperfusion injury, as well as improving the relaxation function of mesenteric vessels. Nicotinamide riboside's protective effect in small intestine ischaemia-reperfusion can be used to reduce ischaemia-reperfusion injury, as well as to preserve intestinal grafts until transplant.
Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation
Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscle and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for heart function. Biosynthesis of NAD+ from vitamin B3 (known as salvage pathways) is the primary source of NAD+. We showed here that NAD+ salvage pathway was altered in the heart of mouse and human carrying LMNA mutation, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Oral administration of nicotinamide riboside, a natural NAD+ precursor and a pyridine-nucleoside form of vitamin B3, leads to a marked improvement of the NAD+ cellular content, an increase of PARylation of cardiac proteins and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our results provide mechanistic and therapeutic insights into dilated cardiomyopathy caused by LMNA mutations.
Sirtuin 3 deficiency aggravates contrast-induced acute kidney injury
Sirtuin 3 (Sirt3) is a key regulator of energy metabolism and oxidative stress. To investigate the role of Sirt3 in contrast-induced acute kidney injury (CIAKI), we established the model both in vivo and in vitro to explore the potential mechanisms.
NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis
In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.
Programming of the Beige Phenotype in White Adipose Tissue of Adult Mice by Mild Resveratrol and Nicotinamide Riboside Supplementations in Early Postnatal Life
Resveratrol (RSV) and nicotinamide riboside (NR) are food compounds with anti-obesity actions in adult rodents. Here, the long-term effects of RSV and NR mild supplementation throughout lactation on adiposity-related parameters and the appearance of the beige phenotype in white adipose tissue (WAT) in adulthood are assessed.
Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD Synthesis
NAD is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD synthesis. In contrast, mice easily generate sufficient NAD solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD levels in aging and neurodegenerative diseases.
Complete genome sequence analysis of the Malacosoma neustria nucleopolyhedrovirus from Turkey
The lackey moth, Malacosoma neustria (Linnaeus, 1758), a worldwide pest, causes extensive economic losses particularly on hazelnut, plum, oak, poplar, and willow trees. A baculovirus, Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2), has been isolated from the larvae collected in Turkey and appears to have a potential as a microbial control agent. In this study, we describe the complete genome sequence of ManeNPV-T2 and compare it to other sequenced baculovirus genomes. The ManeNPV-T2 genome is a circular double-stranded DNA molecule of 130,202 bp, has 38.2% G + C, and is predicted to contain 131 putative open reading frames (ORFs) each with a coding capacity of more then 50 amino acids. There are 27 ORFs with unknown function of which 6 are unique to ManeNPV-T2. Eleven homologous regions (hrs) and two bro genes (bro-a and bro-b) were identified in the genome. There are two homologues of chaB and nicotinamide riboside kinase-1 genes, separated from themselves with a few nucleotides. Additionally, ac145, thought to be per os infectivity factor (pif) gene, is also found as two homologues. All 38 core genes are found in the ManeNPV-T2 genome. The phylogenetic tree of ManeNPV-T2 in relation to 50 other baculoviruses whose genomes have been completely sequenced showed ManeNPV-T2 to be closely related to the group II NPVs. This study expands our knowledge on baculoviruses, describes the characterization ManeNPV, and ultimately contributes to the registration of this virus as a microbial pesticide.
Nicotinamide riboside supplementation dysregulates redox and energy metabolism in rats: Implications for exercise performance
What is the central question of this study? The aim was to investigate the potential metabolic and redox mechanisms that impaired exercise performance after 21 days of supplementation with 300 mg (kg body weight) of nicotinamide riboside in rats. What is the main finding and its importance? Nicotinamide riboside disturbed energy and redox metabolism and impaired exercise performance in heathy rats. Exogenously administered redox agents in heathy populations might lead to adverse effects.
Nicotinamide riboside induces a thermogenic response in lean mice
Nicotinamide Riboside (NR) is a NAD booster with wide physiological repercussion including the improvement on glucose and lipid homeostasis, increasing the life expectancy in mammals. However, the effects of NR on metabolism are only partially known. Here, we evaluated the effects of NR on the thermogenic response, highlighting the brown adipose tissue (BAT) in lean mice.
The emergence of the nicotinamide riboside kinases in the regulation of NAD+ metabolism
The concept of replenishing or elevating NAD+ availability to combat metabolic disease and ageing is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cells and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase pathway at both a tissue-specific and systemic level.
Pharmacological bypass of NAD salvage pathway protects neurons from chemotherapy-induced degeneration
Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD, nicotinamide mononucleotide (NMN), rather than loss of NAD, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.
A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects
Animal studies suggest a positive role for nicotinamide riboside (NR) on insulin sensitivity and hepatic steatosis in models of obesity and type 2 diabetes. NR, an NAD+ precursor, is a member of the vitamin B-3 family now available as an over-the-counter supplement. Although data from preclinical trials appear consistent, potential effects and safety need to be evaluated in human clinical trials.
Oxyresveratrol stimulates mucin production in an NAD-dependent manner in human intestinal goblet cells
The intestinal mucus layer plays an important role in the management of inflammatory bowel disease. The aim of this study was to investigate the effects of oxyresveratrol (OXY), an antioxidant, on the stimulation of mucin production in human LS 174T goblet cells and the underlying mechanism thereof. OXY increased MUC2 expression at both the mRNA and protein levels. By performing two-dimensional gel electrophoresis, we found that the expression of nicotinic acid phosphoribosyltransferase1 (NaPRT1) in OXY-treated LS 174T cells was greatly increased compared with that in negative control cells. In addition, the NAD/NADH ratio was increased in proportion to OXY in LS 174T cells. The expression of NAD-synthesis enzymes, NaPRT1, nicotinamide riboside kinase1 (NRK1) and nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) was significantly increased at both the mRNA and protein levels in OXY-treated LS 174T cells. The inhibition of NaPRT1 and NRK1 did not decrease MUC2 expression after inhibiting by small interfering RNA (siRNA)-NaPRT1 and siRNA-NRK1, respectively; however, inhibition of Nmnat by an Nmnat inhibitor decreased MUC2 expression in a dose-dependent manner. In conclusion, OXY increases NAD levels, resulting in the stimulation of MUC2 expression in LS 174T cells. These findings present a novel role for NAD in stimulation of MUC2 expression.
The Regulatory Role of NAD in Human and Animal Cells
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD+ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD+ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.
Nicotinamide adenine dinucleotide is transported into mammalian mitochondria
Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter.
Loss of the Thioretinaco Ozonide Oxygen Adenosine Triphosphate Complex from Mitochondria Produces Mitochondrial Dysfunction and Carcinogenesis
The active site of oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis is proposed to consist of thioretinaco, a complex of two molecules of thioretinamide with cobalamin, oxidized to the disulfonium derivative, thioretinaco ozonide, and complexed with oxygen, nicotinamide adenine dinucleotide, inorganic phosphate and ATP. Reduction of the active site complex by electrons from mitochondrial electron transport complexes releases ATP from binding to the active site, producing nicotinamide riboside and hydroperoxide and generating a membrane potential from proton transport to the active site. Opening of the mitochondrial permeability transition pore from decreased mitochondrial melatonin leads to loss of the active site complex from mitochondrial membranes, as observed in aging and dementia. Loss of the active site complex from mitochondria also results from opening of the permeability transition pore and from decomposition of the disulfonium active site by electrophilic carcinogens, oncogenic viruses and microbes which cause depletion of adenosyl methionine because of increased biosynthesis of polyamines, and by free radical oxygen species generated by ionizing radiation, and by catecholamines. Thus the loss of thioretinaco ozonide from mitochondria produces the impaired oxidative phosphorylation, oxidative stress, calcium influx, apoptosis, aerobic glycolysis, and mitochondrial dysfunction that are observed in chemical carcinogenesis, microbial carcinogenesis, traumatic brain injury, aging and dementia.
Nicotinamide riboside exerts protective effect against aging-induced NAFLD-like hepatic dysfunction in mice
Aging is one of the risk factors of non-alcoholic fatty liver disease (NAFLD). Yet, the mechanism underlying the aging-associated NAFLD-like syndrome is not fully understood. Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, has protective effects against aging. Here, we investigated the actions of NAD precursors nicotinamide riboside (NR) on the development of aging-induced NAFLD.
Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD depletion
Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD homeostasis on these pathways is unknown. We utilised skeletal muscle myotube models to induce NAD depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. NAD excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD , which was rapidly reversed with repletion of the NAD pool using NR. NAD depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD depletion remains unclear, and requires further investigation. These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD precursor usage in models of skeletal muscle physiology.
A Pilot Study Investigating Changes in the Human Plasma and Urine NAD+ Metabolome During a 6 Hour Intravenous Infusion of NAD
Accumulating evidence suggests that active maintenance of optimal levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+) is beneficial in conditions of either increased NAD+ turnover or inadequate synthesis, including Alzheimer's disease and other neurodegenerative disorders and the aging process. While studies have documented the efficacy of some NAD+ precursors such as nicotinamide riboside (NR) in raising plasma NAD+, no data are currently available on the fate of directly infused NAD+ in a human cohort. This study, therefore, documented changes in plasma and urine levels of NAD+ and its metabolites during and after a 6 h 3 μmol/min NAD+ intravenous (IV) infusion. Surprisingly, no change in plasma (NAD+) or metabolites [nicotinamide, methylnicotinamide, adenosine phosphoribose ribose (ADPR) and nicotinamide mononucleotide (NMN)] were observed until after 2 h. Increased urinary excretion of methylnicotinamide and NAD+ were detected at 6 h, however, no significant rise in urinary nicotinamide was observed. This study revealed for the first time that: (i) at an infusion rate of 3 μmol/min NAD+ is rapidly and completely removed from the plasma for at least the first 2 h; (ii) the profile of metabolites is consistent with NAD+ glycohydrolase and NAD+ pyrophosphatase activity; and (iii) urinary excretion products arising from an NAD+ infusion include NAD+ itself and methyl nicotinamide (meNAM) but not NAM.
Why Is Mom Stressed: Homeorhesis as the Potential Problem and Nicotinamide Riboside as the Potential Solution
The remodeling of female mammalian physiology to support the development of a fertilized egg into an externally breathing individual and then to provide all the nutrition to this individual while remodeling back to nearly her pregestational state is without parallel in male mammalian physiological transitions. While it is common parlance to refer to postpartum depression as a not infrequent stress in women, the postpartum physiological changes after every birth constitute profound metabolic stresses that are understudied and have important nutritional, behavioral, and neurodevelopmental implications for the maternal and neonatal health of every mammalian species. We discovered that the postpartum liver of a lactating female mouse has a depressed nicotinamide adenine dinucleotide (NAD) metabolome linked to circulation of higher levels of NAD metabolites in support of a >20-fold increase in NAD coenzymes in the mammary. Furthermore, by supporting a new mother's apparent higher demand for NAD precursors, we increased circulation of prolactin, superinduced mammary biosynthetic programs, increased her time of arched-back nursing, enhanced mammary production of brain-derived neurotrophic factor, promoted postgestational weight loss, advanced the neurobehavioral development of her offspring, and allowed them to mature as stronger and more resilient adults with advantages in hippocampal neurogenesis and body composition. These results show that a new mother's capacity for biosynthesis and functionally important nurturing is apparently limited by NAD. Here, we discuss homeorhetic flow of resources from a new mother to her offspring in the context of NAD metabolism and suggest avenues for future investigation.
Role of pseudohypoxia in the pathogenesis of type 2 diabetes
Type 2 diabetes is caused by persistent high blood glucose, which is known as diabetic hyperglycemia. This hyperglycemic situation, when not controlled, can overproduce NADH and lower nicotinamide adenine dinucleotide (NAD), thereby creating NADH/NAD redox imbalance and leading to cellular pseudohypoxia. In this review, we discussed two major enzymatic systems that are activated by diabetic hyperglycemia and are involved in creation of this pseudohypoxic condition. One system is aldose reductase in the polyol pathway, and the other is poly (ADP ribose) polymerase. While aldose reductase drives overproduction of NADH, PARP could in contrast deplete NAD. Therefore, activation of the two pathways underlies the major mechanisms of NADH/NAD redox imbalance and diabetic pseudohypoxia. Consequently, reductive stress occurs, followed by oxidative stress and eventual cell death and tissue dysfunction. Additionally, fructose formed in the polyol pathway can also cause metabolic syndrome such as hypertension and nonalcoholic fatty liver disease. Moreover, pseudohypoxia can also lower sirtuin protein contents and induce protein acetylation which can impair protein function. Finally, we discussed the possibility of using nicotinamide riboside, an NAD precursor, as a promising therapeutic agent for restoring NADH/NAD redox balance and for preventing the occurrence of diabetic pseudohypoxia.
Understanding the physicochemical properties and degradation kinetics of nicotinamide riboside, a promising vitamin Bnutritional supplement
Nicotinamide riboside (NR), a newly recognised form of vitamin B and a precursor to nicotinamide adenine dinucleotide (NAD), has been demonstrated to show therapeutic potential and the possibility of becoming a drug compound in addition to its proven role in rejuvenating ageing cells in mice. However, current literature is devoid of information relating to the physicochemical characterisation of NR and its respective impact upon formulation and final product processing. Here we report physicochemical properties of NR including pa, log , solubility, melting point, degradation mechanics, and kinetics, with a special focus on its stability under thermal and physiologically relevant conditions. A simple and rapid HPLC method confirms a base-catalysed hydrolysis degradation of NRCl to nicotinamide and sugar in simulated gastrointestinal (GI) fluids. Given the antagonising effect of nicotinamide against NR, the presented data have a profound impact on how NRCl should be handled both during formulation and storage to prevent formation and to limit accumulation of nicotinamide. The innovative combinatorial use of H NMR and Differential Scanning Calorimetry (DSC) was employed to investigate thermal events during NR melting. NRCl degrades upon melting and in solution undergoes hydrolysis in a buffer and in simulated intestinal environments. The results suggest that a proper and evidence-based formulation of NRCl is vital to enable further investigation and clinical analysis of this promising and novel nutrient. Any formulation would need to promote the stability of NRCl and protect it from hostile environments to prevent the accumulation of a potentially antagonistic degradation product. With the current work, we have filled a niche but vital gap in NR literature and the data presented may prove useful in furthering the understanding, specifically the formulation and processing of NRCl.
Vitamin B3 in Health and Disease: Toward the Second Century of Discovery
This introductory chapter briefly reviews the history, chemistry, and biochemistry of NAD (the term NAD as it is used here refers to both oxidized and reduced forms of the molecule) consuming ADP-ribose transfer enzymes as components of the involvement of vitamin B3 in health and disease.
Role of mitochondria in diabetic peripheral neuropathy: Influencing the NAD-dependent SIRT1-PGC-1α-TFAM pathway
Survival of human peripheral nervous system neurons and associated distal axons is highly dependent on energy. Diabetes invokes a maladaptation in glucose and lipid energy metabolism in adult sensory neurons, axons and Schwann cells. Mitochondrial (Mt) dysfunction has been implicated as an etiological factor in failure of energy homeostasis that results in a low intrinsic aerobic capacity within the neuron. Over time, this energy failure can lead to neuronal and axonal degeneration and results in increased oxidative injury in the neuron and axon. One of the key pathways that is impaired in diabetic peripheral neuropathy (DPN) is the energy sensing pathway comprising the nicotinamide-adenine dinucleotide (NAD)-dependent Sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α)/Mt transcription factor A (TFAM or mtTFA) signaling pathway. Knockout of PGC-1α exacerbates DPN, whereas overexpression of human TFAM is protective. LY379268, a selective metabolomic glutamate receptor 2/3 (mGluR2/3) receptor agonist, also upregulates the SIRT1/PGC-1α/TFAM signaling pathway and prevents DPN through glutamate recycling in Schwann/satellite glial (SG) cells and by improving dorsal root ganglion (DRG) neuronal Mt function. Furthermore, administration of nicotinamide riboside (NR), a precursor of NAD, prevents and reverses DPN, in part by increasing NAD levels and SIRT1 activity. In summary, we review the role of NAD, mitochondria and the SIRT1-PGC-1α-TFAM pathway both from the perspective of pathogenesis and therapy in DPN.
Erratum: Author Correction: Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study
[This corrects the article DOI: 10.1038/s41514-017-0016-9.].
Nicotinamide Riboside Enhances Mitochondrial Proteostasis and Adult Neurogenesis through Activation of Mitochondrial Unfolded Protein Response Signaling in the Brain of ALS SOD1 Mice
Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, the brain stem, and the motor cortex. So far, there is still a lack of effective drugs. Nicotinamide adenine dinucleotide (NAD+) takes part in redox reactions and the NAD-dependent signaling pathway. The NAD+ decline is related with many neurological diseases, leading to the accumulation of neurotoxic protein in the central nervous system. Moreover, the NAD+ supplementation is shown to promote neural stem cells/neuronal precursor cells (NSCs/NPCs) pool maintenance. Regulatory mechanisms and functions of NAD+ metabolism in ALS are still unknown. Thus, we hypothesized the aggregation of human SOD1 toxic protein and the fate of NSCs/NPCs in the ALS disease could be improved by the administration of nicotinamide riboside (NR), an NAD+ precursor. In this study, we treated SOD1 transgenic and wild-type mice by the oral administration of 20 mg/ml NR starting at 50 days of age. Effects of NR on the body weight, the motor function, the onset and the survival were assessed during the experiment. The expression of mutant hSOD1 protein, mitochondrial unfolded protein response (UPR) related protein, mitophagy markers and NAD+ metabolism related protein were detected by immunoblotting. Effects of NR on the NSCs/NPCs in neurogenic niches of brain were identified by the immunofluorescence staining. Our investigation elucidated that the NR treatment exhibited better hanging wire endurance but did not postpone the onset or extend the life span of SOD1 mice. Besides, we observed that the NR repletion promoted the clearance of mitochondrial hSOD1 neurotoxic protein. Meanwhile, the mitochondrial function pathway was disrupted in the brain of SOD1 mice. What's more, we demonstrated that the inadequate function of NAD+ salvage synthesis pathway was the primary explanation behind the decline of NAD+, and the NR treatment enhanced the proliferation and migration of NSCs/NPCs in the brain of SOD1 mice. At last, we found that levels of UPR related protein were significantly increased in the brain of SOD1 mice after the NR treatment. In summary, these findings reveal that the administration of NR activates UPR signaling, modulates mitochondrial proteostasis and improves the adult neurogenesis in the brain of SOD1 mice.
Short-term NAD supplementation prevents hearing loss in mouse models of Cockayne syndrome
Age-related hearing loss (ARHL) is one of the most common disorders affecting elderly individuals. There is an urgent need for effective preventive measures for ARHL because none are currently available. Cockayne syndrome (CS) is a premature aging disease that presents with progressive hearing loss at a young age, but is otherwise similar to ARHL. There are two human genetic complementation groups of CS, A and B. While the clinical phenotypes in patients are similar, the proteins have very diverse functions, and insight into their convergence is of great interest. Here, we use mouse models for CS ( and ) that recapitulate the hearing loss in human CS patients. We previously showed that NAD, a key metabolite with various essential functions, is reduced in CS and associated with multiple CS phenotypes. In this study, we report that NAD levels are reduced in the cochlea of mice and that short-term treatment (10 days) with the NAD precursor nicotinamide riboside (NR), prevents hearing loss, restores outer hair cell loss, and improves cochlear health in mice. Similar, but more modest effects were observed in mice. Remarkably, we observed a reduction in synaptic ribbon counts in the presynaptic zones of inner hair cells in both and mice, pointing to a converging mechanism for cochlear defects in CS. Ribbon synapses facilitate rapid and sustained synaptic transmission over long periods of time. Ribeye, a core protein of synaptic ribbons, possesses an NAD(H) binding pocket which regulates its activity. Intriguingly, NAD supplementation rescues reduced synaptic ribbon formation in both and mutant cochleae. These findings provide valuable insight into the mechanism of CS- and ARHL-associated hearing loss, and suggest a possible intervention.
Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies
4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function. The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma ( = 48) and control group ( = 100). The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099 ± 0.009 vs. 0.066 ± 0.006 µmol/L and 1.10 ± 0.08 vs. 1.97 ± 0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253 ± 17 vs. 369 ± 19) as well as plasma MNA (0.057 ± 0.004 vs. 0.069 ± 0.003 µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116 ± 0.019 vs. 0.131 ± 0.014 and 0.102 ± 0.006 vs. 0.113 ± 0.011, respectively). In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.
Syntheses and chemical properties of β-nicotinamide riboside and its analogues and derivatives
The β-anomeric form of nicotinamide riboside (NR) is a precursor for nicotinamide adenine dinucleotide (NAD), a redox cofactor playing a critical role in cell metabolism. Recently, it has been demonstrated that its chloride salt (NRCl) has beneficial effects, and now NRCl is available as a dietary supplement. Syntheses and studies of analogues and derivatives of NR are of high importance to unravel the role of NR in biochemical processes in living cells and to elaborate the next generation of NR derivatives and conjugates with the view of developing novel drug and food supplement candidates. This review provides an overview of the synthetic approaches, the chemical properties, and the structural and functional modifications which have been undertaken on the nicotinoyl riboside scaffold.
Neonatal Resveratrol and Nicotinamide Riboside Supplementations Sex-Dependently Affect Beige Transcriptional Programming of Preadipocytes in Mouse Adipose Tissue
Nutritional programming of the thermogenic and fuel oxidation capacity of white adipose tissue (WAT) through dietary interventions in early life is a potential strategy to enhance future metabolic health. We previously showed that mild neonatal supplementations with the polyphenol resveratrol (RSV) and the vitamin B3 form nicotinamide riboside (NR) have sex-dependent, long-term effects on the thermogenic/oxidative phenotype of WAT of mice in adulthood, enhancing this phenotype selectively in male animals. Here, we tested the hypothesis that these dietary interventions may impact the commitment of progenitor cells resident in the developing WAT toward brown-like (beige) adipogenesis. NMRI mice received orally from postnatal day 2-20 (P2-20) a mild dose of RSV or NR, in independent experiments; control littermates received the vehicle. Sex-separated primary cultures were established at P35 from the stromovascular fraction of inguinal WAT (iWAT) and of brown adipose tissue (BAT). Expression of genes related to thermogenesis and oxidative metabolism was assessed in the differentiated cultures, and in the iWAT depot of young (P35) animals. Neonatal RSV and NR treatments had little impact on the animals' growth during early postnatal life and the expression of thermogenesis- and oxidative metabolism-related genes in the iWAT depot of young mice. However, the expression of brown/beige adipocyte marker genes was upregulated in the iWAT primary cultures from RSV supplemented and NR supplemented male mice, and downregulated in those from supplemented female mice, as compared to cultures derived from sex-matched control littermates. RSV supplementation had similar sex-dependent effects on the expression of thermogenesis-related genes in the BAT primary cultures. A link between the sex-dependent short-term effects of neonatal RSV and NR supplementations on primary iWAT preadipocyte differentiation observed herein and their previously reported sex-dependent long-term effects on the thermogenic/oxidative capacity of adult iWAT is suggested. The results provide proof-of-concept that the fate of preadipocytes resident in WAT of young animals toward the beige adipogenesis transcriptional program can be modulated by specific food bioactives/micronutrients received in early postnatal life.
Aged Nicotinamide Riboside Kinase 2 Deficient Mice Present an Altered Response to Endurance Exercise Training
Skeletal muscle aging is marked by the development of a sarcopenic phenotype, a global decline of muscle energetic capacities, and an intolerance to exercise. Among the metabolic disorders involved in this syndrome, NAD metabolism was shown to be altered in skeletalmuscle, with an important role for the NAMPT enzyme recycling the nicotinamide precursor. An alternative pathway for NAD biosynthesis has been described for the nicotinamide riboside vitamin B3 precursor used by the NMRK kinases, including the striated muscle-specific NMRK2. With this study, our goal is to explore the ability of 16-month-old mice to perform endurance exercise and study the consequences on muscle adaptation to exercise. 10 control and 6 mice were used and randomly assigned to sedentary and treadmill endurance training groups. After 9 weeks of training, heart and skeletal muscle samples were harvested and used for gene expression analysis, NAD levels measurements and immunohistochemistry staining. Endurance training triggered a reduction in the expression of Cpt1b and AcadL genes involved in fatty acid catabolism in the heart of mice, not in control mice. NAD levels were not altered in heart or skeletal muscle, nor at baseline neither after exercise training in any group. gene encoding for the slow MHC-I was more strongly induced by exercise in mice than in controls. Moreover, -15 expression levels is higher in mice skeletal muscle at baseline compared to controls. No fiber type switch was observed in plantaris after exercise, but fast fibers diameter was reduced in aged control mice, not in mice. No fiber type switch or diameter modification was observed in soleus muscle. In this study, we demonstrated for the first time a phenotype in old mice in response to endurance exercise training. Although NMRK2 seems to be predominantly dispensable to maintain global NAD levels in heart and skeletal muscle, we demonstrated a maladaptive metabolic response to exercise in cardiac and skeletal muscle, showing that NMRK2 has a specific and restricted role in NAD signaling compared to the NAMPT pathway.