Dr. Weeks’ Comment: Dr Abram Hoffer, M.D. PhD. taught the importance of Niacin – vitamin B3 as the cheapest form of anti-aging nutrition. Since the benefits of Niacin have been well established (lowers cholesterol, reduces anxiety and now, in the NAD+ form, it offers powerful anti-aging benefits.
Abstract
Nicotinamide adenine dinucleotide (NAD+) homeostasis is constantly compromised due to degradation by NAD+‐dependent enzymes. NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT‐independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.
INTRODUCTION
Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are ubiquitous molecules in the body, which play crucial roles in energy metabolism, as they act as hydride‐accepting and hydride‐donating coenzymes during mitochondrial oxidative phosphorylation.1 Apart from its role as a redox cofactor, during the last decade NAD+ has arisen as the critical substrate for a number of protein families, such as the sirtuin (SIRT) family of protein deacetylases,2 poly(ADP‐ribose)polymerases,3 and ADP‐ribose cyclases.4 Through their downstream actions, these proteins participate in more than 500 enzymatic reactions and regulate almost all major biological processes in cells.5 This continuous enzymatic utilization of NAD+ is counterbalanced via de novo synthesis from dietary tryptophan, or through its salvage from precursors. In the Preiss‐Handler pathway, the NAD+ precursor nicotinic acid (NA) is converted into NAD+ in a three‐step enzymatic process led by the nicotinic acid phosphoribosyltransferase (NAPRT), the nicotinamide mononucleotide adenylyl transferases (NMNATs), and the NAD+ synthase (NADS). Another recycling pathway comprises intracellular nicotinamide (NAM) phosphoribosylation or nicotinamide riboside (NR) phosphorylation into nicotinamide mononucleotide (NMN), a process carried out by nicotinamide phosphoribosyltransferase (NAMPT) or nicotinamide riboside kinases (NRKs), respectively. NMN is then directly converted to NAD+ by the NMNATs.6 When given externally, NMN can also act as an NAD+ precursor. To achieve this, NMN first needs to be converted extracellularly to NR by the ectoenzyme 5′‐nucleotidase CD73, after which NR is transported into the cell by the equilibrative nucleoside transporters (ENTs) and metabolized to NAD+ via NRKs.7, 8 Very recently, it has also been reported that NMN can be incorporated into cells via the Slc12a8‐specific transporter, at least in mouse small intestine.9
The role of NAD+ in the activity of enzymes controlling major metabolic processes, together with reports supporting that decreased cellular NAD+ contributes to metabolic disturbances,10 have renewed the interest in strategies to increase NAD+ bioavailability to combat disease. In fact, NAD+ repletion and the subsequent activation of sirtuins leads to key biochemical and clinical improvements, such as enhanced mitochondrial biogenesis,11–14 protection against fatty acid‐induced liver disease15 and diabetes,11, 12 or reduced neurodegeneration16 in a variety of animal models. NAD+ homeostasis also plays a major role in kidney health and in the ability of the renal tubule to resist stressors.17 In fact, during ischemic renal injury, NAD+ consumption by poly (ADP‐ribose) polymerases (PARPs) is accelerated,18 leading to NAD+ decline in renal tissue. NAD+ replenishment through administration of the NAD+ precursor NMN has been proven effective in ameliorating tubular damage induced by ischemia‐reperfusion (IR) injury and the nephrotoxic drug cisplatin in aged mice.19
These results have turned attention to the use of NAD+ precursors for combatting metabolic disease in humans. NA and NAM have downsides, however. The former induces flushing triggered by NA binding to the GPR109A receptor,20 and NAM can act as a sirtuin inhibitor, which could limit its intended activation of these enzymes. Therefore, NMN and NR have arisen as attractive alternatives to NA or NAM, since they effectively raise NAD+ concentrations in mouse tissues without undesired adverse target effects.11, 12, 21, 22 For this reason, several clinical studies have been initiated with NR. Some studies23–28 with NR have shown that this compound is well tolerated up to 2 g per day during 12 weeks, while the first clinical trials with NMN are still ongoing (NCT03151239, UMIN000021309, UMIN000030609, and UMIN000025739). Yet, NMN and NR supplementation have some limitations of their own, including maximal NAD+‐enhancing effects of around 2‐fold, the need of high doses (from 200 to 1000 mg/kg per day) to achieve beneficial effects in animal models,11–13, 22, 29 and the rapid degradation in plasma to NAM, at least in the case of NR.30 Moreover, although an increase in NAD+ levels in whole blood has been detected upon NR administration in humans,24, 27 supplementation with this precursor has failed to increase NAD+ in other tissues, such as muscle biopsies, even after 1 g administration during 6 weeks.31 This inefficacy in raising NAD+ might explain why NR has no apparent effect on total energy expenditure, blood glucose or insulin sensitivity in humans.28
To overcome the limitations of the current repertoire of NAD+ enhancers, other molecules with a more pronounced effect on the NAD+ intracellular pool are desired. This has stimulated us to investigate the use of the reduced form of nicotinamide mononucleotide (NMNH) as an NAD+ enhancer. There is very scarce information about the role of this molecule in cells. In fact, only one enzymatic activity has been described to produce NMNH. This is the NADH diphosphatase activity of the human peroxisomal Nudix hydrolase hNUDT1232 and the murine mitochondrial Nudt13.33 It has been postulated that, in cells, NMNH would be converted to NADH via nicotinamide mononucleotide adenylyl transferases (NMNATs).34 However, both NMNH production by Nudix diphosphatases and its use by NMNATs for NADH synthesis have only been described in vitro using isolated proteins, and how NMNH participates in cellular NAD+ metabolism remains unknown.
In the present study, we design and develop a new method for the purification of NMNH at scale, and explore the role of this molecule in NAD+ metabolism. We show that NMNH is effectively metabolized to NAD+ in mammalian cells, and confirm its NAD+ synthesis route is NRK and NAMPT‐independent. We also investigate the therapeutic potential of NMNH, showing that it can protect renal proximal tubular epithelial cells from hypoxia/reoxygenation‐induced injury, a crucial event in ischemic acute kidney injury (AKI),35 by accelerating processes involved in tubular regeneration.36 Finally, we explore the in vivo effects of NMNH administration in mice and demonstrate that this new precursor effectively raises NAD+ levels in blood and a variety of tissues, including kidney, to a greater extent than NMN when used at the same concentration.
These results corroborate that reduced NAD+ precursors can act as very potent NAD+ enhancers, and open doors for a new generation of highly efficient NAD+‐boosting molecules that could aid in overcoming the limitations of the current set of NAD+ enhancers.
NMN and NR are currently the most used and promising NAD+ modulators, as demonstrated by the many scientific reports showing the benefits of their use in mouse models for cardiometabolic disease,11, 12 fatty liver disease,15 or neurodegeneration,16 among many others. The fact that both molecules lack the undesirable side effects of NA (flushing) and NAM (sirtuin inhibition) has increased the interest in the potential use of these compounds as better therapeutic agents. However, supplementation with NR has so far proven to be inefficient in raising NAD+ levels in human tissue, which could explain why the beneficial effects observed in mouse studies are so far not reproducible in humans.31 Therefore, new molecules with a higher NAD+‐boosting potential that may overcome these limitations are appealing. This is the case for reduced NR (NRH), which is a very effective NAD+ booster.45, 49 In the present study, we identify the reduced form of another NAD+ precursor, namely reduced nicotinamide mononucleotide (NMNH), and show for the first time that it can act as a powerful NAD+ booster in vitro and in vivo.
To produce NMNH, we leveraged the high activity of the NAD+ pyrophosphatase from Escherichia coli over NADH, and developed an effective purification method that renders salt‐free NMNH, whose identity was confirmed by NMR and MS/MS. The molecule proved to be highly efficient and fast in increasing cellular NAD+, largely surpassing the effect of NMN in every cell line tested. In fact, while NMN was only able to double cellular NAD+ at its maximum concentration (1 mM), NMNH led to a 3‐fold NAD+ increase even at 50 µM. The response to NMNH was also faster, as 15 minutes were enough for NMNH to almost double basal NAD+, with maximal effects after 6 hours, which remained stable even 24 hours after supplementation.
This large increase in the NAD+ pool, which was in the range of 2.5 to 19‐fold depending on the cell line, was accompanied by an increase in NADH, although to a much lower extent (~3‐fold), suggesting a tight regulation of the NAD+/NADH ratio in favor of the oxidized form.
The increased NAD+ metabolic flux is also confirmed by the analysis of downstream NAD+ metabolites, such as NADP(H) and NAAD, which is considered a marker of increased NAD+ metabolism.23 In accordance with these results, and similar to those obtained with NRH,49 we detected a sharp increase in NMN and NR content upon NMNH supplementation, much higher than that obtained with NMN. This increase in oxidized precursors does not come from oxidation of the reduced ones, as demonstrated previously,49 but from an increased NAD+ flux, since blocking NRH conversion to NMNH (and therefore NADH) with an adenosine kinase inhibitor, was enough to prevent NMN and NR accumulation.
In vivo, NMNH also proved more effective than NMN in raising NAD+ levels in a variety of tissues when administered at the same concentration, confirming the results observed in cell lines.
Our findings solidify the notion that three distinct but partially overlapping NAD+ recycling pathways exist. In one of these pathways, nicotinic acid (NA) is condensed to NAD+ via adenylyl transferases (NMNATs) and NAD+ synthase. In the other two pathways, oxidized precursors (NMN and NR) are converted to NAD+ through an NR kinases (NRKs) and NAM phosphoribosyltransferase (NAMPT)‐dependent route. However, reduced precursors (NMNH and NRH) act independently of these enzymatic activities, and require adenosine kinase (AK) to efficiently increase the cellular NAD+ pool. We hypothesize that this high efficiency of the reduced pathway could be due to the lack of degradation of the reduced precursors to NAM, which could confer an advantage over the oxidized precursors, since NAM is a weak NAD+ booster.30 This hypothesis is further supported by the fact that NMNH was able to sustain NAD+ levels in blood for much longer than NMN, suggesting reduced degradation to NAM. Another possibility would be an improved cellular uptake of NRH through the ENTs, or a higher efficiency of adenosine kinase or NMN adenylyl transferases for the reduced molecules. These possibilities will need to be addressed in follow‐up studies.
In light of the existence of a very efficient pathway for NAD+ recycling, which leads to unprecedented elevations in the intracellular NAD+ pool, one question that arises is whether this NAD+ elevation can lead to deleterious changes in the cellular redox state. In the available studies with NRH, and in the present study with NMNH, this was not the case, as supplementation with NRH or NMNH did not trigger cell apoptosis and instead protected them against genotoxic agents or hypoxia/reoxygenation injury.45 In accordance, our viability and metabolomics study indicated that NMNH increases metabolic activity by promoting NAD(P)H biosynthesis, without elevating reductive stress, as demonstrated by unchanged levels in α‐hydroxybutyrate upon NMNH supplementation.
The data presented in this study also corroborate the evidence that NAD+ boosters protect against different models of acute kidney injury, and place NMNH as a great alternative intervention to other NAD+ precursors to reduce tubular damage and accelerate recovery. To demonstrate this point, and with kidney being one of the most responsive tissues to NMNH in vivo, we adopted the in vitro model of hypoxia/reoxygenation injury as a model for damage and repair in TECs, which is the cell type that suffers the most from NAD+ depletion and mitochondrial dysfunction during ischemic acute kidney injury (AKI).
During reoxygenation, mitochondrial dysfunction and oxidative damage occur in TECs, supposedly as a direct consequence of ATP depletion due to hypoxia.66 A marker for aberrant mitochondrial metabolism and disturbed epithelial function is Tfam, whose expression is regulated by NAD+ levels.67 By efficiently increasing NAD+ levels, NMNH was also able to promote Tfam upregulation and enhance mitochondrial activity, as demonstrated by increased mitochondrial superoxide production. Superoxide can be produced by both forward and reversed electron flux in complex I of the ETC, which in the latter case could exacerbate oxidative damage and cell death. However, our gene expression analysis points toward NMNH‐driven protection against hypoxia/reoxygenation injury, as it enhances the expression of the genes encoding for the antioxidant enzymes PRDX1, and GPX1, crucially involved in resistance to renal ischemic damage.64
Despite the fact that we did not see upregulation of the genes involved in the pentose phosphate pathway (PPP), our metabolomics results suggest that NMNH supplementation in TECs may enhance antioxidant defense as a result of increased flux through this pathway, yielding necessary intermediates for TEC recovery, such as NADP(H) and nucleotides. In fact, many studies support the role of the PPP in the regeneration of renal tissue after AKI.36, 68–70 We further confirmed this event by showing an enrichment of TECs in the S phase of the cell cycle upon NMNH treatment, which is a sign of cell cycle entry and ongoing repair.71, 72
Such enhanced repair of TECs in the presence of NMNH is further corroborated by the sharp decrease of the tubular damage marker KIM‐1, and is in line with previous findings that NRH also protected against cisplatin‐induced acute kidney injury49 and that other NAD+‐boosting strategies promote tissue regeneration.73
In conclusion, owing to an efficient synthesis method for NMNH, we were able to identify this molecule as a new and potent NAD+ precursor in vitro and in vivo, and confirm the existence of a novel pathway for the recycling of reduced precursors to NAD+. We have also corroborated the potential therapeutic application of these potent NAD+ enhancers, which could succeed where NMN and NR have failed, namely boosting NAD+ in humans. Future research will need to elucidate the safety of long‐term administration of reduced precursors, and determine the beneficial effects of their administration in animal models of disease, especially in comparison with the classical boosters.
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