NAD+ metabolism and its roles in cellular processes during ageing

NAD + is highly compartmentalized in the cytoplasm, mitochondria and nucleus, which represent its main subcellular pools ( Supplementary Box 1 ). These pools are regulated independently of each other, and consistent with this, the enzymes involved in the biosynthesis or degradation of NAD + are highly compartmentalized as well 2 – 4 . NAD + is a critical metabolite and coenzyme for multiple metabolic pathways and cellular processes. First, NAD + reduction is required to maintain energy balance and the redox state of a cell. NAD + is also continually turned over by three classes of NAD + -consuming enzymes: the NAD + glycohydrolases, also referred to as NADases (CD38, CD157 and SARM1), the protein deacylase family of sirtuins and PARPs, which have various important cellular functions. These utilize NAD + as a substrate or cofactor and generate nicotinamide (NAM) as a by-product ( FIG. 1 ). Therefore, NAD + mediates multiple major biological processes and is always in high demand ( Supplementary Boxes 2 and 3 ). To sustain NAD + levels, NAM can be recycled back to NAD + via the NAM salvage pathway (for details see BOX 1 ). Additionally, some cells, mostly in the liver, can synthesize NAD + de novo from multiple dietary sources. Thus, NAD + is constantly synthesized, catabolized and recycled in the cell to maintain stable intracellular NAD + levels ( FIG. 1 ). However, during ageing, this balance between catabolic and anabolic processes can shift, and NAD + degradation can outpace the ability of cells to make NAD + de novo or their ability to effectively recycle or salvage NAM. Furthermore, excess NAM may be catabolized via alternative metabolic pathways (for details see BOX 2 ), effectively diverting it away from the NAM salvage pathway and further impacting NAD + levels. Besides their common role as NAD + -consuming enzymes, NAD + glycohydrolases, sirtuins and PARPs have distinct roles in ageing and age-related diseases. While enhancing the activation of sirtuins has emerged as a way to increase lifespan and healthspan, aberrant activation of PARPs and NAD + glycohydrolases, such as CD38, may exert the opposite effect and exacerbate ageing phenotypes (see later). NAD + biosynthetic pathways NAD + can be made de novo from l -tryptophan via the kynurenine pathway or from vitamin precursors, such as nicotinic acid (NA), via the Preiss–Handler pathway ( FIG. 1a ). In addition to NAD + , the kynurenine pathway also utilizes l -tryptophan to produce kynurenic acid, serotonin and picolinic acid, among other bioactive molecules. Notably, the relative contribution of the de novo synthesis pathway to NAD + levels is still not well understood. Outside the liver, most cells do not express the full array of enzymes necessary to convert tryptophan to NAD + by the kynurenine pathway. Most tryptophan is metabolized to NAM in the liver, where it is released into the serum, taken up by peripheral cells and converted to NAD + by the NAM salvage pathway 5 . Additionally, under some circumstances, immune cells, such as macrophages, also make NAD + from tryptophan (discussed later) 6 . Thus, besides the liver, the de novo biosynthetic pathway seems to be a more indirect mechanism contributing to system-wide NAD + levels, with most NAD + coming from the NAM salvage pathway ( BOX 1 ).

The human PARP protein family is composed of 17 proteins characterized by poly(ADP-ribosyl) polymerase or mono(ADP-ribosyl) polymerase activity 24 . Briefly, the PARP-mediated cleavage of NAD + produces NAM and ADP-ribose as by-products, in which ADP-ribose is added as single or covalently linked polymers to PARP itself and other acceptor proteins, in a process called ‘poly(ADP-ribosyl)ation’ (PARylation) ( FIG. 2b ). Among all the PARPs, only PARP1, PARP2 and PARP3 localize to the nucleus ( FIG. 1b ) in response to early DNA damage and have a key role in DNA damage repair 25 – 27 ( Supplementary Box 2 ). PARP1 is the best-characterized member of the group, and it alone is responsible for about 90% of total PARP activity at least in response to DNA damage 28 , 29 . On activation, PARP1 PARylates itself along with histones and other proteins that act as a scaffold to recruit and activate other DNA repair enzymes and proteins to the lesion site to initiate DNA repair 30 . Owing to high PARP1 activity, DNA damage is associated with massive NAD + consumption. PARP1, in its role as an NAD + responsive signalling molecule, has been widely associated with the ageing process. However, PARP1 is one of the major NAD + consumers not only in cells with acute DNA damage but also under normal and other pathophysiological conditions 5 , supporting a key role of PARP1 in regulating NAD + homeostasis. For example, in mice fed a high-fat diet, those that were treated with PARP inhibitors or lacking PARP1 and PARP2 had increased NAD + levels, increased SIRT1 activity and improved mitochondrial function, and were protected from insulin resistance 31 , 32 . The strong correlation among PARP1 activation, decline in NAD + levels and inhibition of SIRT1 activity is observed in patients with xeroderma pigmentosum group A, as well as progeroid diseases, ataxia telangiectasia and Cockayne syndrome. Notably, treatment of mice with Cockayne syndrome with PARP1 inhibitors or NAD + supplements promoted lifespan extension and ameliorated the severe phenotypes caused by PARP1 hyperactivation, providing strong evidence that the negative consequences downstream of PARP1 activation are mediated by dysregulation of NAD + homeostasis in response to extensive DNA damage and genotoxic stress 33 . Of note, the ability of PARP1 to antagonize SIRT1 activity is most likely because these enzymes are localized to the same cellular compartment — in this case, the nucleus — and compete for the same NAD + pool. However, since PARP1 has lower K m and higher V max with regard to NAD + than SIRT1, PARP1 likely outcompetes SIRT1 for NAD + as a result of its greater binding affinity and faster kinetics 34 . PARP2 is structurally related to PARP1. They share a similar catalytic domain required for several cellular processes, including DNA repair and transcriptional regulation, and account for about 10% of PARP activity 35 . PARP2 activity may also influence NAD + bioavailability 25 , 26 . Like Parp1 -knockout mice, Parp2 -knockout mice show enhanced SIRT1 activity and improved metabolic function, and are protected from high-fat diet-induced obesity 36 . PARP3 is important in DNA repair as well 27 , suggesting a large overlap and potential redundancy in PARP1, PARP2 and PARP3. The functions of the other PARPs (PARP4–PARP17) in NAD + homeostasis and global metabolism in cells or organs have not been fully determined, but they are thought to be less important in modulating intracellular NAD + levels. Overall, targeting PARPs, and in particular PARP1, is a promising therapeutic strategy in the ageing field. However, more studies are needed to fully understand the contribution of PARPs to the age-related decline in NAD + levels.

SARM1 was only very recently assigned to the NAD + glycohydrolase and cyclase family along with CD38 and CD157. The enzymatic activity of SARM1 ( FIG. 2c ) relies on the Toll/interleukin receptor (TIR) domain that was previously not known to possess catalytic activity and generally to be involved in protein–protein interaction 59 . Whether SARM1 regulates NAD + under physiological conditions and to what extent is still unknown. However, SARM1-mediated NAD + degradation plays a key role in axonal degeneration after axonal injury 60 (see the section Neurodegeneration ). SARM1 is primarily expressed in neurons and promotes neuronal morphogenesis and inflammation 61 , 62 ; however, SARM1 is also expressed by immune cells such as macrophages and T lymphocytes, and regulates their functions 63 – 65 . SARM1 was originally discovered as a negative regulator of the innate immune response via direct interaction with TRIF (TIR domain-containing adapter protein inducing interferon-β), downstream of Toll-like receptor signalling 63 . However, the immunoregulatory role of SARM1 remains largely uncharacterized. Although SARM1 was originally reported to be required for chemokine CCL5 production in macrophages 66 , recent evidence revealed that SARM1 is not expressed in macrophages and that the observed chemokine phenotype was due to a background effect of the Sarm1 -knockout mouse strain 67 . Despite its controversial role in immune cells, SARM1 plays an undisputed key role in axonal degeneration and is emerging as a therapeutic target to prevent or ameliorate neurodegenerative diseases and traumatic brain injuries (as discussed in the section Neurodegeneration ).

During ageing, NAD + levels decline, and many enzymes associated with NAD + degradation and biosynthesis are altered. The relationship between NAD + and the 10 hallmarks of ageing was extensively reviewed 68 (see also Supplementary Table 1 ). Additionally, this decline in NAD + levels during ageing has been linked to the development and progression of ageing-related diseases, including atherosclerosis, arthritis, hypertension, cognitive decline, diabetes and cancer 3 . In this section, we focus on the major cellular processes that influence or are influenced by ageing, such as metabolic dysfunction, DNA repair failure and genomic instability, inflammageing, cellular senescence and neurodegeneration, and discuss their regulation by NAD + levels. All these processes and the age-related disorders associated with them are likely to benefit from NAD + repletion. Restoring NAD + levels with dietary precursors and targeting NAD + degradation enzymes with small-molecule inhibitors has emerged as a potential therapeutic strategy to restore NAD + levels, thereby providing opportunities for alleviating age-related decline and diseases ( FIG. 4 ) (see also the next section). Metabolic dysfunction Obesity is a growing epidemic worldwide 69 . Individuals with obesity are more likely to develop metabolic disease characterized by increased adiposity, insulin resistance, high blood glucose levels, high blood pressure and dyslipidaemia. As a result, these individuals are at higher risk of developing type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease, atherosclerosis, stroke and cancer 70 . Accordingly, obesity accelerates and exacerbates ageing, and has been associated with shortened lifespan 71 . Over the past two decades growing evidence has accumulated that targeting NAD + metabolism may provide therapeutic benefits and help treat metabolic disease and ageing 3 . NAD + was first discovered by regulating the metabolic rates of yeast extracts, and thus the links between NAD + and metabolism have been known for almost a century. We now know that NAD + lies at the heart of metabolism and regulates metabolic flux of multiple metabolic pathways (for details see BOX 1 and Supplementary Box 2 ). Accordingly, NAD + homeostasis is required for the proper function of various metabolic tissues, including fat, muscle, intestines, kidneys and liver (reviewed in 72 ). However, another key discovery was made more recently in Saccharomyces cerevisiae , showing that the longevity protein Sir2 (the yeast sirtuin) exhibited its lifespan-promoting effects in an NAD + -dependent manner 12 , 73 , 74 . This breakthrough finding suggested that Sir2 activity may be linked to metabolic status. In support of this hypothesis, it has now been demonstrated in mammalian systems that lifespan-extending metabolic manipulations, such as exercise 75 – 77 , caloric restriction 78 , time-restricted feeding and a ketogenic diet 79 , as well as healthy circadian rhythms 1 , 11 (including regular sleep patterns 80 ), work in part by increasing NAD + levels, which leads to the activation of sirtuins. Altering or disrupting metabolic status, for example as a consequence of a high-fat diet 81 , 82 , postpartum weight loss 83 and disruption of the circadian rhythm 1 , 83 , can lead to lower NAD + levels, and thereby reduced activity of sirtuins as well as other NAD + -dependent cellular processes (see Supplementary Box 2 for details). Conversely, increasing cellular NAD + levels has recently been shown to reduce reductive stress and drive the directionailty of metabolic reactions 84 . Additionally, higher NAD + levels can promote the deacetylase and deacylase activity of both nuclear SIRT1 and mitochondrial SIRT3, which regulate mitochondrial function and protect from high-fat diet-induced metabolic disease 42 , 81 , 85 – 88 . Taken together, these key studies have provided strong evidence and the rationale that targeting NAD + degradation pathways or boosting NAD + levels can influence metabolic processes and can be protective against metabolic disease. In further support of this model, numerous studies have now shown that mouse models including Parp1 -knockout and Cd38 -knockout mice, or mice treated with PARP or CD38 inhibitors, have supraphysiological levels of NAD + . They are also protected from obesity, have increased metabolic rates and have (relatively) normal glucose metabolism during high-fat diet conditions and during ageing 42 , 89 – 91 . Additionally, mice receiving a high-fat diet have increased inflammation, which drives decreased expression of NAMPT, and reduced activity of the NAD + salvage pathway 82 ( BOX 1 ), providing a potential mechanistic explanation for why NAD + levels decline during obesity. In line with this mechanism, mice with adipocyte-specific deletion of NAMPT have lower levels of NAD + in their fat tissues, increased insulin resistance and increased metabolic dysfunction, which can be rescued with NMN supple

Like for the innate immune system, ageing is characterized by a reduced ability to build an effective adaptive immune response due to altered functions of adaptive immune cells, known as immunosenescence. Ageing leads to an imbalance or skewing of immune cell populations, including decreased levels of naive T and B cells, loss of T cell antigen receptor diversity and an increase in levels of virtual memory T cells. The regulatory role of NAD + and NAD + -consuming enzymes in T cell biology has been demonstrated; however, their contribution to the ageing of the adaptive immunity is largely uncharacterized. On the one hand, extracellular NAD + has been proposed as a danger signal 110 causing cell death in specific T cell subpopulations, such as regulatory T cells 111 . On the other hand, NAD + seems to exhibit immunomodulatory properties, such as influencing T cell polarization 112 , 113 . However, whether NAD + promotes a specific T cell phenotype and whether the manipulation of NAD + metabolism with NAD + precursors could result in similar immunomodulatory properties is still unknown. An established hallmark of adaptive immune ageing is the expansion of the memory population of highly cytotoxic CD8 + CD28 − T cells 114 , characterized by high secretion of effector molecules such as granzyme B 115 . This cell population is characterized by decreased SIRT1 and FOXO1 levels, resulting in an enhanced glycolytic capacity and granzyme B production 116 . These studies highlight the potential of metabolic reprogramming via manipulation of NAD + -associated pathways in age-related adaptive immune dysfunction 116 . The upregulation of the NAD + –SIRT1–FOXO1 axis via CD38 inhibition increases effector functions in T helper 1 and 17 hybrid cells 117 , and future studies using CD38 inhibitors or NAD + precursors may potentially show the therapeutic potential of targeting NAD + in the ageing adaptive immune system. Another immunological feature of adaptive immune ageing is the increase in the number of exhausted T cells, characterized by the expression of inhibitory receptor molecules (for example, PD1 and TIM3), decreased proliferative capacity and decreased effector functions 118 , 119 . PD1 is a component of the immune checkpoint, blockade of which is commonly used as an anticancer strategy 120 , 121 , but also has been proposed to restore the effector function of aged T cells 122 . With respect to NAD + metabolism, a recent study showed that CD38 overexpression was associated with a dysfunctional and exhausted CD8 T population in PD1 blockade-resistant cancers 123 , 124 , highlighting the potential of expanding studies on CD38 inhibition to age-related exhausted T cells. However, although extremely intriguing, this hypothesis is yet to be explored in depth, and more studies will be needed to determine the preclinical efficacy of this approach. Thus, overall, more work needs to be done to determine whether manipulating NAD + levels is effective in reversing ageing-related immunological dysfunction in the adaptive immune system and, equally important, whether such manipulations are safe.

Ageing is strongly associated with most neurodegenerative diseases and is accompanied by decreased cellular NAD + levels in the mammalian brain 133 . NAD + depletion is reported in several models of accelerated ageing which exhibit neurodegeneration 33 , 134 , 135 , as well as in neuro-degenerative diseases, including Alzheimer disease 136 , Parkinson disease 137 , 138 and amyotrophic lateral sclerosis (ALS) 139 . The underlying cause and mechanisms of NAD + loss in the brain during age-related neurodegenerative diseases are still largely unknown. However, multiple lines of evidence support a neuroprotective role for NAD + ( FIG. 3Ab ). First, axonal degeneration, which is a precursor to many age-related neuronal disorders 140 , 141 , is characterized by rapid NAD + depletion. During normal physiological conditions, the NAD + biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) ( FIG. 1 ; BOX 1 ) is a survival factor in axons, in which it needs to be constantly replenished via anterograde axonal transport due to its rapid turnover 142 . However, during axon degeneration, NMNAT2 axonal transport is blocked, and the axonal pool of the protein is rapidly degraded, leading to a critical depletion of NAD + in the axon 142 , 143 . Moreover, the NAD + -consuming enzyme SARM1 is activated by axonal injury and mediates axonal degeneration by promoting NAD + degradation 60 . This was originally shown in Wallerian degeneration slow ( Wld s ) mice, which are protected from axonal degeneration, showing absence of SARM1 expression 144 and higher neuronal NAD + levels, owing to the overexpression of a chimeric fusion protein of NMNAT1, which allows its redistribution from the nucleus to the axon, where it can substitute for the activity of NMNAT2 (REFS 145 , 146 ). The role of SARM1 in promoting axonal damage has been demonstrated in several other in vivo models. Sarm1 -knockout mice are protected from axonal degeneration 60 and can rescue the severe axon growth defects and perinatal death induced by lack of NMNAT2 (REFS 147 , 148 ). Recently, a transgenic mouse model overexpressing a dominant negative version of SARM1 in neurons showed a significant delay in axon degeneration and hints at the promising effect of gene therapy or small molecules targeting SARM1 for treating neuropathies 149 . Overall, Wld s mouse studies largely show the neuro protective function of the biosynthetic enzymes NMNAT1, NMNAT2 and NMNAT3 (REFS 142 , 150 – 152 ) and their protective role in several neurodegenerative disorders, including Parkinson disease 153 – 155 . However, the mechanism remains unclear. Besides the regulation of SARM1-dependent NAD + degradation discussed above, the degradation of their substrate and NAD + precursor, NMN, by NMNATs seems to protect axons from degeneration. In contrast to the neuroprotective role of NAD + , the precursor NMN has been reported to have a neurotoxic role promoting SARM1 activation and cyclic ADP-ribose generation, leading to axonal destruction 156 . However, the evidence that NMN accumulation leads to axonal degeneration needs to be validated and remains controversial. Nonetheless, this possibility raises questions about the therapeutic potential of increasing NAD + synthesis by supplementing NAD + precursors such as NMN. Additional lines of evidence supporting a neuroprotective role for NAD + also include studies using P7C3, an aminopropyl carbazole reported to be an allosteric activator of NAMPT in the NAM salvage pathway ( BOX 1 ). P7C3 was shown to be neuroprotective in mouse models of Parkinson disease 137 , Alzheimer disease 157 and ALS 158 . Furthermore, NAD + -consuming enzymes other than SARM1 have been reported to play a role in intracellular NAD + depletion during ageing-related neurodegenerative diseases. For example, CD38 expression increases in the course of Alzheimer disease progression 159 – 161 , and an Alzheimer disease mouse model lacking CD38 ( Cd38 -knockout mice), which results in elevated NAD + levels in their brains, shows a milder disease phenotype 159 . Consistent with the neuroprotective role of NAD + , Cd38 -knockout mice are also protected from neuronal cell death on ischaemic brain injury 162 . Multiple cells in the brain, including microglia, astrocytes, neurons and endothelial cells, express CD38 (REFS 108 , 160 ), and treating microglia and astrocytes with inflammatory cytokines induces CD38 expression 163 , 164 . Consistent with this finding, CD38 expression was associated with neuroinflammation with greater amounts of proinflammatory macrophages/microglia in mouse brains 160 , 165 . CD38 was also reported to affect social behaviour, as was its homologue CD157 (REFS 166 , 167 ), substantiating the functional impact of NAD + on neuronal function. Although there is no direct evidence of a causal role of CD38 in neurodegenerative diseases, as discussed above, CD38 is emerging as a key enzyme involved in inflammageing and senescence 45 , 105

The salvage and de novo synthesis pathways of NAD + are also potential targets for therapies boosting NAD + levels in vivo. Specifically, activators of NAMPT, the rate-limiting enzyme in the NAD + salvage pathway, and NMNATs, which contribute to the NAD + de novo and salvage pathways ( FIG. 1 ), have been suggested as possible therapeutic interventions for boosting tissue NAD + levels 213 . Indeed, the neuroprotective agent P7C3 enhances NAMPT activity and boosts NAD + levels in doxorubicin-treated human cells, suggesting it may be a functional therapeutic for ageing and age-related disease processes, such as neurodegeneration 214 . However, the real efficacy of P7C3 in increasing NAMPT activity remains controversial 215 . Recently, another small molecule, SBI-797812, was proposed as a potent NAMPT activator active in the nanomolar range. This compound increased NAMPT-mediated NMN production in vitro and, importantly, NAD + levels in cell lines and in vivo 214 , 215 . Despite the limited effect of SBI-797812 of boosting only hepatic NAD + levels in vivo, this is a promising pharmacological approach to raise intracellular NAD + levels. Moreover, pharmacological inhibition of ACMSD by TES-991 and TES-102524 boosts de novo NAD + synthesis and SIRT1 activity, ultimately enhancing mitochondrial function in the liver, kidneys and brain of mice 216 . Although these strategies are promising for therapeutic increase of NAD + levels, one of the challenges that remains is identifying with greater precision how some of these molecules, such as P7C3, work at the molecular level, determining their targets and exploring potential off-target effects. In addition to modulating NAD + levels, in recent years, attention has been given to modulating the NAD + /NADH redox balance primarily through the administration of the redox-cycling quinone β-lapachone, an exogenous co-substrate of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), which regenerates NAD + from NADH. NQO1 was shown to be increased by caloric restriction (although it was not sufficient to mediate anti-ageing effects of this dietary intervention) 217 , and enhancing NQO1 activity by administering β-lapachone prevented age-dependent decline of motor and cognitive function in aged mice by ameliorating mitochondrial dysfunction 218 , 219 . However, NQO1 is commonly overexpressed in most solid tumours in which β-lapachone administration induces imbalance of redox cycle and oxidative stresses 220 ; therefore, β-lapachone can have a variety of effects depending on the context and should be administered with caution.

Almost 90 years after its initial discovery, NAD + is emerging as a central metabolite in the ageing field, and NAD + level decline is becoming an established feature of several age-associated diseases. The NAD + field is moving rapidly and has evolved as one of the major exciting research areas in biomedical research. In the past 5 years there has been a plethora of advancements, including the development of exciting tools and technology ( Supplementary Box 4 ), to further advance our understanding of how NAD + levels influence or are influenced by complex signalling, metabolic and cellular pathways. Furthermore, with the use of stable-isotope tracing and NAD + biosensors, there have also been major advances and refinement in our understanding of how NAD + levels are regulated both at the cellular level and at the systems level. We now have greater understanding of the mechanisms that lead to decline of NAD + levels with age and the emerging role of NAD + -consuming enzymes such as CD38 and SARM1, along with PARPs in this process. Furthermore, this decline in NAD + levels is now known to affect multiple age-dependent cellular processes, including DNA repair, oxidative stress and immune cell function. Moreover, recent pre-clinical trials have demonstrated, using different animal models, that NAD + depletion is a key pathway in age-associated diseases, including neurodegenerative, metabolic and progeroid disorders. However, it is still not clear which cells and enzymes drive the decline in NAD + levels in specific disorders. Moreover, which targets or pathways can be exploited to efficiently and safely restore NAD + homeostasis is still under investigation. The good news is that different NAD + -boosting strategies have been shown to be effective in extending healthspan and lifespan (see the section Therapeutic targeting of NAD + level decline and TABLE 1 ). Thus, the use of NAD + precursors such as NMN and NR, along with small molecules that boost NAD + biosynthesis or inhibit NAD + degradation, offers an exciting therapeutic approach to treat ageing-related diseases and increase human healthspan. This is particularly important as elderly populations are rising rapidly, and ageing-related diseases are predicted to cause a great deal of societal and economic burden in the coming decades. However, the translatability of NAD + -boosting therapies to humans remains the key question to be answered. Several human clinical trials are ongoing to assess the safety and efficacy of NAD + augmentation ( TABLE 2 ) and, importantly, early-phase trials of short-term NR/NMN administration have proven it to be safe and to increase NAD + levels in healthy participants. However, despite the promising preliminary results, whether long-term supplementation with NAD + precursors has any side effects is still unknown. Moreover, many other questions need to be answered to deepen our understanding of the potential of NAD + -boosting therapies: is there any tissue/disease specificity for NMN and NR? What are the therapeutic doses of NAD + precursors necessary for different diseases? Could the combination of NAD + -boosting strategies, such as CD38 and/or PARP1 inhibitors with NAD + precursor supplementation, be something to consider? Hopefully, the upcoming results of current clinical trials will shed some light on our unsolved questions and set the basis for future directions in deciphering the role of NAD + during ageing in humans.

Nicotinamide N -methyltransferase (NNMT) is an enzyme that uses S -methyladenosine (SAM) as a methyl donor to methylate the ring nitrogen of nicotinamide (NAM), converting it to N 1 -methylnicotinamide (MNAM) (see FIG. 1a ). MNAM can be further oxidized via aldehyde oxidase to produce N 1 -methyl-2-pyridone-5-carboxamide and N 1 -methyl-4-pyridone-3-carboxamide, which along with MNAM are secreted in the urine. NNMT conversion of NAM to MNAM effectively diverts NAM from being recycled back to nicotinamide adenine dinucleotide (NAD + ) by the NAM salvage pathway and affects global NAD + levels. Thus, there has been growing interest in the role of NNMT as a key regulator of NAD + levels during disease states, such as obesity, cancer and ageing 264 . For example, NNMT expression increases in visceral white adipose tissue and liver during obesity, and appears to have mostly negative consequences 265 – 268 . This includes the depletion of the methyl donor SAM and NAD + in white adipose tissue and the liver in response to a high-fat diet. As a result, increased NNMT activity leads to reduced methylation of the promoters of genes involved in fibrosis and aberrant gene expression of metabolic and inflammatory genes 266 . Thus, NNMT appears to be a critical metabolic enzyme linking NAD + metabolism to control of gene expression via its ability to regulate levels of bioactive molecules, such as consumption of NAD + and SAM, to produce MNAM. The role of NNMT in regulating NAD + suggests that NNMT is important in ageing. In support of this, the Caenorhabditis elegans homologue of NNMT, ANMT-1, regulates the production of MNAM. In worms, MNAM serves as a substrate for the aldehyde oxidase GAD-3, which produces hydrogen peroxide, which, in turn, acts as a hormesis signal to promote longevity. Worms lacking anmt-1 no longer benefit from sir-2.1 -dependent lifespan extension 191 . These data suggest that consumption of NAD + via NNMT is beneficial for lifespan extension at least in C. elegans . However, it is unclear what role NNMT activation plays in mammalian ageing, although NNMT expression and activity do appear to increase in rodents as they age. For example, our group recently saw NNMT gene expression increase in the hepatocytes of ageing mice 45 , and a similar increase was observed in hepatocytes of older mice fed a high-protein diet 269 . Additionally, treating old mice with an NNMT inhibitor activated senescent muscle stem cells and promoted muscle regeneration during ageing 270 . Thus, in contrast to C. elegans , in mammals early evidence suggests that NNMT may promote ageing-related diseases. Thus, targeting NNMT may provide a viable therapeutic avenue to treat ageing-related diseases. Another metabolic fate of NAD + is direct phosphorylation by NAD + kinase (NADK) to produce NADP(H), which has a key role as a major source of reducing power that regulates intracellular redox balance and anabolic processes, such as lipogenesis. A recent study using in vitro isotopic tracing label incorporation into NADP(H) showed that NADK accounts for 10% of NAD + consumption 5 . However, the total NADP + pool is 20 times less than the NAD + pool. Furthermore, in conditions where NAD + levels decline, such as in ageing, NADP + levels also decline. Thus, NADP + levels appear to be directly linked to NAD + levels and rise and fall in tandem, making it unlikely that NADK is a major NAD + sink. However, NADK has recently been shown to be a direct target of AKT kinase, phosphorylating the residues Ser44, Ser46 and Ser48 and leading to enhanced activation of NADK 271 . Thus, given that AKT signalling is abnormally increased during ageing 272 , aberrant activation of NADK may account for some of the decline in NAD + levels during ageing. Therefore, further studies will be needed to determine the role of NADK in regulating NAD + and NADP + pools in ageing and ageing-related diseases.