NAM, was first associated with diabetes when it was shown to protect against streptozotocin (STZ)-induced diabetes (Schein et al., 1967), which is accompanied by a robust reduction of NAD+ levels in pancreatic islet cells. NAM, but not NA, can recover this drop in NAD+ levels (Ho and Hashim, 1972). Later, it was demonstrated that the NAD+ reduction induced by STZ was due to increased DNA damage, stimulating PARP1 activity (Yamamoto et al., 1981).

Unlike other NAD+ precursors, NAM has the capacity to exert end product inhibition on SIRT1 deacetylase activity. However, long-term NAM treatment increases NAD+ levels via the NAD+ salvage pathway, which likely tips the balance of the NAD+/NAM ratio such that SIRT1 is activated. Despite NAM being suggested as a treatment for type 1 diabetes (Olmos et al., 2006), clinical trials failed to confirm this hypothesis (Cabrera-Rode et al., 2006, Gale et al., 2004). More recently, OLETF rats, a rodent model of obesity and type 2 diabetes, exhibited profound metabolic improvements following NAM treatment (100 mg/kg for 4 weeks). This treatment induced liver NAD+ levels, which were complimented by enhanced glucose control (Yang et al., 2014). However, some reports indicate that long-term or high doses of NAM are detrimental because they favor the development of a fatty liver, due to reductions in available methyl groups (Kang-Lee et al., 1983). For instance, NAM administration for 8 weeks (1–4 g/kg) resulted in methyl group deficiency, which is likely due to the conversion of NAM into 1-methyl-NAM (mNAM) by nicotinamide n-methyltransferase (NNMT) (Figure 3A). NNMT shunts NAM away from NAD+ using S-adenosylmethionine (SAM) as a methyl donor (Aksoy et al., 1994, Riederer et al., 2009). In line with this hypothesis, supplementation of methionine, a methyl group donor, prevented the formation of steatohepatosis caused by high doses of NAM (Kang-Lee et al., 1983).

Recently, NNMT expression was found to be negatively correlated with GLUT4, the insulin-responsive glucose transporter, in adipose tissue (Kraus et al., 2014). In adipose-specific Glut4-KO mice, Nnmt transcripts are increased, while they are reduced in adipose-specific Glut4-overexpressing mice Nnmt (Kraus et al., 2014). Similarly, Nnmt transcripts were increased in the WAT of ob/ob, db/db, and high-fat-fed mice compared to lean insulin-sensitive controls (Kraus et al., 2014). In addition, tissue specific knockdown of Nnmt in WAT and liver, using antisense oligonucleotides, protected against diet-induced obesity by increasing the expression of Sirt1 target genes and energy expenditure. Accordingly, treating adipocytes with mNAM, which acts as an end product inhibitor of NNMT (Aksoy et al., 1994), increased O2 consumption (Kraus et al., 2014). Coming from a totally different angle, a germline mouse model deficient in Maf1, a repressor of RNA polymerase III transcription of highly abundant cellular RNAs (Upadhya et al., 2002), also underscored the importance of NNMT in NAD+ homeostasis. Maf1−/− mice are resistant to obesity due to metabolic inefficiency as a consequence of futile tRNA production, which led to extreme reductions of NNMT levels, boosting NAM salvage to regenerate NAD+ (Bonhoure et al., 2015). In combination, these independent studies in widely different mouse models support that NNMT inhibition enhances NAD+-dependent SIRT1 activity and protects mice against obesity and type 2 diabetes. In a seemingly contradictory fashion, work in C. elegans has shown that NNMT and the methylation of NAM might actually be an integral part of the mechanism by which sirtuins provide healthspan and lifespan benefits (Schmeisser et al., 2013). For this, NNMT-produced mNAM would act as a substrate to the ortholog of the mammalian aldehyde oxidase (AOx1), GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal (Schmeisser et al., 2013). Taken together, however, NNMT activity seems strongly regulated in diverse metabolic contexts and has a major impact on NAD+ homeostasis. The discrepancies in the current findings might arise from the distinct models used (i.e., worms vs. mice) and the amplitude of the mitohormetic response in different metabolic scenarios.

The potential of PARP inhibition as a treatment for metabolic complications was first suggested by the observation that Parp1-KO mice were protected from STZ-induced β cell death and dysfunction by maintaining NAD+ levels and therefore glucose tolerance (Masutani et al., 1999). Parp1-KO animals exhibit higher mitochondrial content, increased energy expenditure, and protection against metabolic disease brought on by a HFD (Bai et al., 2011b). Correspondingly, PARP inhibitors also prevent carbon-tetrachloride-induced liver mitochondrial dysfunction and fibrosis (Mukhopadhyay et al., 2014), and diet-induced obesity in mice (Pirinen et al., 2014). Long-term treatment of up to 18 weeks with the dual PARP1 and PARP2 inhibitor MRL-45696 was shown to enhance exercise capacity and muscle mitochondrial function in chow-diet-fed mice (Cerutti et al., 2014, Pirinen et al., 2014). Both in worm and mouse models, the effect of PARP inhibition on mitochondrial function was linked with the activation of the UPRmt, as reflected by the induction of HSP60 and CLPP, two UPRmt biomarkers (Mouchiroud et al., 2013, Pirinen et al., 2014) (Figure 4). In fact, PARP inhibitors increased mitochondrial translation without coordinate changes in cytosolic translation rates, thus leading to a mitonuclear protein imbalance (Pirinen et al., 2014), which in turn triggers the UPRmt to maintain optimal mitochondrial function (Houtkooper et al., 2013). This finding is in line with the recent discovery that mitochondrially located PARP1 activity may PARylate and disrupt the interaction between key mitochondrial-specific DNA base excision repair (BER) enzymes, namely EXOG and DNA polymerase gamma (Polγ), and the mitochondrial DNA (mtDNA), hindering mitochondrial biogenesis and reducing mtDNA copy numbers (Szczesny et al., 2014).

While the above observations set the stage for PARP inhibition to treat complex human metabolic diseases, it is important that inhibitors are selective for PARP1 and do not affect other members of the PARP family. For instance, although Parp2-KO mice were protected from diet-induced obesity, they were glucose intolerant due to defective pancreatic function (Bai et al., 2011a). In this sense, although diverse and highly efficient PARP inhibitors exist and are currently used in humans for anti-cancer therapy (Curtin and Szabo, 2013), none of them are selective for PARP1. Furthermore, since several of the PARPs play key roles in DNA damage repair upon genotoxic stress (Curtin and Szabo, 2013), further work must also ensure the long-term safety of selective PARP1 inhibition to treat metabolic diseases.

As described above, cADP-Ribose synthases, such as CD38, are primary NADases in mammalian tissues with a strong impact on SIRT1 activity (Aksoy et al., 2006a, Escande et al., 2010). This led to the hypothesis that CD38 inhibition (and subsequent increases in NAD+ levels) could be applied to treat metabolic disorders. In line with this, mice lacking CD38 are protected against diet-induced metabolic disease (Barbosa et al., 2007). Some natural flavonoids, such as quercetin, apigenin, luteolinidin, kuromanin, and luteolin, were found to inhibit CD38 in the low micromolar range (Escande et al., 2013, Kellenberger et al., 2011). Accordingly, quercetin and apigenin increased liver NAD+ levels and SIRT1 activity resulting in improved glucose homeostasis and fatty acid oxidation in the liver of these mice (Escande et al., 2013). However, the recent development of potent thiazoloquin(az)olinone inhibitors for CD38, which can enhance NAD+ levels in multiple tissues, may prove to be effective for the design of future therapies (Haffner et al., 2015). Yet, as discussed in NAD+-Consuming Enzymes (III): Cyclic ADP-Ribose Synthases, there remain several issues that require further work before CD38 inhibitors can be recommended to treat metabolic dysfunction. First, it is not entirely clear that CD38 is the main cADP-ribose synthase enzyme, therefore potentially compromising the efficacy of CD38 inhibitors for clinical use. Second, despite evidence indicating that it might also exist in nuclear and mitochondrial fractions (Aksoy et al., 2006a), CD38 activity is highest on the extracellular side of the plasma membrane, where NAD+ levels are generally very low (De Flora et al., 1997). Finally, the increase in NAD+ observed in Cd38-deficient mice is ∼30-fold, while most other strategies described to date lead to a ∼2-fold increase in NAD+ at best. The massive effect of CD38 on NAD+ levels could therefore be indicative for major alterations in additional NAD+-utilizing metabolic pathways.

Although the elimination of neurons by axonal degradation plays a role in normal nervous system development, aberrant neuronal cell death is typical of insults such as trauma, and chemical toxicity or of aging and neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (for review, see Wang et al., 2012).

Axon degradation had originally been assumed to be a passive process. However, this view changed with the characterization of the naturally occurring Wallerian degeneration slow (WldS) dominant mutation (Conforti et al., 2000). Rodent carriers of this mutation displayed a dramatic reduction in axonal degeneration in both central and peripheral neurons. The WldS mutant protein is a chimeric protein composed of the complete sequence of NMNAT1 fused to the ubiquitination factor E4B at the N terminus (Conforti et al., 2000, Mack et al., 2001). Efforts from diverse labs have since confirmed that it is the NMNAT enzymatic activity that is required to delay axon degeneration (Araki et al., 2004, Conforti et al., 2009, Gilley and Coleman, 2010, Llopis et al., 2000, Sasaki et al., 2009, Yahata et al., 2009, Yan et al., 2010), probably by promoting an increase in NAD+-directed SIRT1 activity (Araki et al., 2004). Interestingly, WldS mutant mice exhibit enhanced insulin secretion from isolated islets with an improvement in glucose homeostasis, also via an NAD+-directed activation of SIRT1 (Wu et al., 2011). Another study specifically demonstrated that it is the cytosolic distribution of NMNAT proteins that is crucial for slowing Wallerian degeneration (Sasaki et al., 2009). Further work should define changes in nuclear and cytosolic NAD+ levels, as most studies measure NAD+ in whole brain lysate, the outcome of which is confounded by the high level of NAD+ found in neuronal mitochondria.

Following the injury of neurons, there is an induction of multiple transcripts for NAD+ biosynthetic enzymes, including a more than 20-fold increase in NRK2, which catalyzes the synthesis of NAD+ from NR, suggesting a compensatory response to elevate NAD+ levels (Sasaki et al., 2006). In line with this, the pretreatment of neurons with either high levels of NAD+ in cell culture, or precursors such as NMN or NR, protects against axonal degeneration following axotomy, hearing loss caused by excess manganese toxicity, or even noise-induced hearing loss in mice (Brown et al., 2014, Gerdts et al., 2015, Sasaki et al., 2006, Wang et al., 2014b). Similarly, rodent studies have demonstrated that pharmacological doses of NAM increases NAD+ biosynthesis and provides protection against ischemia (Klaidman et al., 2003, Sadanaga-Akiyoshi et al., 2003), fetal-alcohol-induced neurodegeneration (Ieraci and Herrera, 2006), and fetal ischemic brain injuries (Feng et al., 2006) by preventing NAD+ depletion. Further supporting the role of NAD+ in neuroprotection, a high-throughput screen identified an aminopropyl carbazole chemical P7C3 (Pieper et al., 2010), which was only recently discovered to be a pharmacological activator of NAMPT (Wang et al., 2014a), but had previously been shown to possess neuroprotective activity in models of traumatic brain injury (Yin et al., 2014), Parkinson’s disease (De Jesús-Cortés et al., 2012), and amyotrophic lateral sclerosis (Tesla et al., 2012). Increasing the activity of existing NAMPT using similar pharmacological approaches may therefore improve NAD+ depletion in aged animals, exhibiting reduced NAMPT and impairments in neural stem/progenitor cell self-renewal and differentiation, a treatment phenomenon already demonstrated using NMN on aging mice (Stein and Imai, 2014). In another model of neuronal degeneration, raised NAD+ levels after CR attenuated increases in AD-type β-amyloid content in a rodent model of AD (Qin et al., 2006). NAM was also able to improve β-amyloid peptide (1–42)-induced oxidative damage and therefore protect against neurodegeneration (Turunc Bayrakdar et al., 2014a, Turunc Bayrakdar et al., 2014b). Similarly, exposing neuronal cells to toxic prion proteins, to model protein misfolding in Alzheimer’s and Parkinson’s disease, induced NAD+ depletion that was improved with exogenous NAD+ or NAM (Zhou et al., 2015). Additionally, NR has been shown to improve the AD phenotype via PGC-1α-mediated β-secretase (BACE1) degradation and the induction of mitochondrial biogenesis (Gong et al., 2013).

Maintaining NAD+ levels seems to, hence, sustain basal metabolic function and health in neurons. Furthermore, based on the preliminary evidence above, NR might have a privileged position among different NAD+ precursors in the prevention of neurodegeneration, as the effect of NR may be enhanced by the increase in NRK2 during axonal damage.

The depletion of NAD+ in neurodegeneration has been generally attributed to the activation of PARP enzymes. Well-known neurodegenerative DNA repair disorders include ataxia-telangiectasia (AT), Cockayne syndrome (CS), and xeroderma pigmentosum group A (XPA), all of which demonstrate mitochondrial dysregulation due to SIRT1 inhibition and a reduction in mitophagy, the process of autophagic clearance of defective mitochondria (Fang et al., 2014). The reduction in SIRT1 activity and mitophagy in XPA-, CSB-, and ATM-deficient cells can be attributed to the aberrant activation of PARP1, as reflected by the ability of PARP inhibitor AZD2281 (olaparib) to rescue the mitochondrial defect in cells and to extend the lifespan of xpa-1 mutant worms (Fang et al., 2014). In extension of these findings, using NR or a PARP inhibitor both improved the phenotype of a mouse model of Cockayne Syndrome group B (CSB), an accelerated aging disorder featuring the disinhibition of PARP activity by CSB protein, through SIRT1-mediated improvements of metabolic, mitochondrial, and transcriptional alterations (Scheibye-Knudsen et al., 2014). Similarly, augmented PARylation in the Csa−/−/Xpa−/− (CX) mouse model of cerebellar ataxia was reduced upon NR treatment, which improved NAD+ levels, SIRT1 activity, and mitochondrial function (Fang et al., 2014). Both interventions using NAD+ precursors or PARP inhibition could hence be helpful to improve neurodegenerative phenotypes.

Genomic stress is the root of all cancers, and so maintaining genome integrity is an essential tool for the prevention of cancer. The fact that PARPs and several sirtuin enzymes are key for genomic maintenance suggests that the regulation of NAD+ could have an impact on cancer susceptibility and development (Cantó et al., 2013). As protectors of genomic stability, PARPs can potentially play a multifunctional role in various cancer-related processes, including DNA repair, recombination, cell proliferation, or cell death. In general, PARP activity protects cancer cells, especially those with high genome instability, from cellular death. Thus, PARP inhibitors are currently being clinically studied for the treatment of cancers that result from dysfunctional homologous DNA recombination repair (Curtin and Szabo, 2013). While a role for SIRT1 in cancer has been controversial, transgenic animal models demonstrate that SIRT1 protects against age-related carcinomas and sarcomas, but not lymphomas (Herranz et al., 2010). Given that PARP activity is rarely affected by physiological changes in NAD+ availability, it would be intuitive to think that most effects derived from fluctuations in NAD+ levels might crystalize into expected outcomes of modulating the activity of some sirtuins, such as SIRT1 (i.e., protection against cancer). In this sense, it has been demonstrated that niacin supplementation can decrease the development of skin cancer (Gensler et al., 1999), while NR can both reduce the incidence of cancer and have a therapeutic effect on fully formed tumors in a genetic mouse model for liver cancer (Tummala et al., 2014). Conversely, niacin deficiency can enhance cancer susceptibility, indicating that cellular NAD+ levels are inversely related to the incidence of cancer (Benavente et al., 2012, Jacobson, 1993). In another approach, some evidence suggests that the protective effects of niacin against the development of skin cancer are due to elevation in both PARPs and SIRT1 activity (Benavente et al., 2012). First, niacin can effectively restore NAD+ levels and Poly(ADP)-ribosylated proteins in keratinocytes following photodamage, indicating an increase in the activity of PARPs. Second, SIRT1, also related to DNA repair and maintaining genomic stability and nucleotide excision repair pathways (Fan and Luo, 2010, Wang et al., 2008), exhibited increased activity in this same model, as evidenced by increased protein deacetylation. However, the balance of SIRT1 and PARP activities upon genotoxic stress is further complicated by the fact that SIRT1 may reduce the expression or be a direct inhibitor of PARP1 (Kolthur-Seetharam et al., 2006, Rajamohan et al., 2009), and PARP2 is a negative regulator of SIRT1 expression (Bai et al., 2011a). This emphasizes the potential for an NAD+-dependent failsafe mechanism that can decide to elicit either repair or apoptosis depending on the severity of the cellular genotoxic insult through the manipulation and balance of SIRT1 and PARP activity levels. Furthermore, SIRT6, which, as described above, most likely does not act as an NAD+ sensor (see NAD+-Consuming Enzymes (I): Sirtuins), can activate PARP1 to stimulate highly efficient double-strand break repair, but only in response to oxidative-stress-induced DNA damage (Mao et al., 2011). In addition, SIRT3 and SIRT5 have both been shown to play roles as either tumor suppressors or oncogenes depending on the cellular and molecular context, while SIRT4 acts as a tumor repressor due to its repression of glutamine metabolism, a process that is essential during rapid cell proliferation, as is seen in cancer (reviewed in Kumar and Lombard, 2015).

In a more speculative territory, it should also be highlighted that higher NAD+ levels, either through PARP inhibition or NAD+ precursor supplementation, rewire metabolism and enhance oxidative versus glycolytic metabolism. Most cancer cells rely indisputably on glycolytic metabolism. Therefore, the metabolic remodeling by enhanced NAD+ levels could constitute a complementary mechanism to slow down cancer progression or initiate cell death. However, NAD+ depletion might also inhibit growth of several cancers. NAMPT has been found to be overexpressed in several types of tumors, and its expression is associated to tumor progression (Bi et al., 2011, Hasmann and Schemainda, 2003, Van Beijnum et al., 2002, Wang et al., 2011). Consequently, several studies showed that the NAD+ depletion triggered by the downregulation of NAMPT activity can reduce tumor cell growth and sensitize cells to chemotoxic agents (Bi et al., 2011, Hasmann and Schemainda, 2003, Wang et al., 2011, Watson et al., 2009). One must keep in mind that, in most cancer cells, PARPs are activated due to DNA damage and genome instability, leading to NAD+ depletion in cancer cells (Garten et al., 2009). As a result, the downregulation of NAMPT sensitizes cancer cells to DNA-damaging agents and apoptosis. In addition, NAD+ depletion also impairs glycolytic capacity in tumor cells (Bai and Cantó, 2012). Altogether, the above data suggest that both NAD+-boosting and depletion can impact on tumor development and progression depending on the type and the metabolic properties of the tumor.

We have only recently started to understand the key pathways involved in defining lifespan. Much of this insight came from studies on CR, the most consistent intervention that extends longevity (as reviewed in Cantó and Auwerx, 2009). Despite some evidence disputing the link between sirtuins and the longevity effects of CR, or longevity in general (Burnett et al., 2011, Jiang et al., 2000, Lamming et al., 2005), most data agree that sirtuin activation in mammals delays the onset of age-related degenerative processes (Herranz et al., 2010, Kanfi et al., 2012, Pearson et al., 2008, Satoh et al., 2013) and that defective sirtuin activity impairs some of the metabolic and physiological benefits triggered by CR (Boily et al., 2008, Hallows et al., 2011, Mercken et al., 2013, Someya et al., 2010). Of note, SIRT1 and SIRT3 have so far been the sirtuins most tightly linked to adaptations during CR. As discussed in NAD+-Consuming Enzymes (I): Sirtuins, the enzymatic characteristics of these sirtuins, but not of other sirtuins, may allow them to act as predominant NAD+ sensors, enabling them to monitor changes in nutrient availability.

During aging, reductions in NAD+ have been consistently observed in worms, diverse rodent tissues—including liver, pancreas, kidney, skeletal muscle, heart, and white adipose—and in human skin samples (Braidy et al., 2011, Gomes et al., 2013, Khan et al., 2014, Massudi et al., 2012, Mouchiroud et al., 2013, Yoshino et al., 2011). Several hypotheses can explain the reductions in NAD+ levels during aging. The first relies on reductions in NAMPT expression with age, which may be in part due to the dysregulation of the circadian rhythm and therefore the CLOCK/BMAL regulation of NAMPT (Nakahata et al., 2009). Another possible explanation for this lies in the higher PARP activity (due to cumulative DNA damage or alternative pathways of PARP activation, such as inflammatory or metabolic stress) observed in old worms and tissues from aged mice (Braidy et al., 2011, Mouchiroud et al., 2013). Supporting this possibility, blocking PARP activity is enough to recover NAD+ levels in aged organisms (Mouchiroud et al., 2013). The age-related reduction in NAD+, in turn, compromises mitochondrial function, which can be recovered via PARP inhibition or NAD+ precursor supplementation (Gomes et al., 2013, Mouchiroud et al., 2013). These observations are in line with the protection from metabolic dysfunction and disease in mice with genetically or pharmacologically triggered deficiencies in PARP activity (Bai et al., 2011b, Pirinen et al., 2014) or in mice treated with NR (Cantó et al., 2012) or NMN (Yoshino et al., 2011). As found in natural aging, significant reductions in skeletal muscle NAD+ occur in Deletor mice, a mouse model containing a mutation in the mitochondrial replicative helicase Twinkle, resulting in the accumulation of damage and a progressive muscle myopathy (Khan et al., 2014). Treatment of Deletor mice with NR delayed early- and late-stage disease progression by increasing mitochondrial biogenesis in skeletal muscle and brown adipose tissue while preventing mtDNA damage (Khan et al., 2014). In line with this, NR and PARP inhibition also improve the respiratory chain defect and exercise intolerance in Sco2 knockout/knockin mice, another model for mitochondrial disease (Cerutti et al., 2014). Altogether, the above data demonstrate that NAD+ supplementation maintains mitochondrial function, not only upon age-related decline but also in genetically determined mitochondrial diseases that are known to accelerate the aging process (Figure 4).

The ability of NAD+ to induce a mitonuclear protein imbalance could provide a key link between NAD+ and mitochondrial function. Mitonuclear protein imbalance can be defined as a stoichiometric difference between nuclear and mitochondrial-encoded respiratory subunit proteins (Houtkooper et al., 2013), an effect known to promote longevity in worms (Durieux et al., 2011, Houtkooper et al., 2013). Forced expression of genes regulating UPRmt, such as Hsp60 paralogs, in Drosophila slows age-dependent mitochondrial and muscle dysfunction due to the compensatory actions of UPRmt signaling (Owusu-Ansah et al., 2013). Likewise, the activation of the UPRmt by targeting mitochondrial ribosomal protein (Mrp) translation, using a knockdown of ribosomal proteins or antibiotics that specifically inhibit mitochondrial translation, increases longevity in C. elegans (Houtkooper et al., 2013). Strikingly, even subtle changes in expression levels of the Mrp’s in the BXD mouse genetic reference population have robust effects on mouse lifespan (Houtkooper et al., 2010b, Wu et al., 2014). Similarly, both NR and PARP inhibitors increased C. elegans lifespan via an induction of the UPRmt by Sir-2.1, the worm SIRT1 ortholog (Mouchiroud et al., 2013). Part of the metabolic decline during aging is due to a PARP-directed decay in NAD+ levels, leading to reduced SIRT1, or Sir-2.1, activity and subsequent reduction in FOXO3A/Daf-16 activity and anti-oxidant defense (Mouchiroud et al., 2013) and increased HIF1α-led glycolytic reliance (Gomes et al., 2013). Furthermore, Sir-2.1-directed longevity is blunted in worms deficient in ubl-5 (Mouchiroud et al., 2013), an essential component for UPRmt-directed communication from the mitochondria to the nucleus (Durieux et al., 2011), solidifying the necessity of UPRmt induction for NAD+-induced longevity. In this sense, the decline in NAD+ observed with aging deregulates the mitonuclear protein balance and respiratory function, which accelerates the aging process. Importantly, mitochondrial dysfunction in aged mice or worms could be recovered following NMN or NR treatment (Gomes et al., 2013, Mouchiroud et al., 2013). The above results highlight how the UPRmt triggers an adaptive mitohormetic response as long as the cell is properly furnished with NAD+. However, in situations of limited NAD+ availability, SIRT1 fails to drive mitohormesis and, hence, mitochondria remain dysfunctional. As a whole, the above demonstrates a tight feedback loop between the mitonuclear protein imbalance and UPRmt on the one hand and NAD+ metabolism and SIRT1 activity on the other. In summary, this assigns a crucial role for NAD+ to synchronize the nuclear and mitochondrial genomes.

The identification of UPRmt as a main mechanism by which NAD+ levels modulate mitochondrial fitness constitutes a major leap forward in our understanding of the molecular mechanisms driving a healthy lifespan. Importantly, UPRmt is not just the principle mechanism by which NAD+ affects longevity, but is also a key mode of action for other well-studied longevity compounds, such as rapamycin and resveratrol (Houtkooper et al., 2013).

The association between metabolism, health, and lifespan have long been proposed based on similarities between metabolic dysfunction and disease (e.g., obesity, diabetes, neurodegeneration, and cancer) and the aging process. Only recently have these processes been linked so tightly by multiple proteins, including sirtuins and PARPs, all of which are tightly controlled by the regulation and subcellular balance of the metabolite NAD+. As such, we have never been so close to solving the ancient question of how we age and what we can do to slow this process while simultaneously not compromising on our quality of life. Despite these insights, several aspects of NAD+ metabolism remain obscure. On one side, the complex detection and quantification of NAD+ metabolites and fluxes has not yet allowed us to obtain a clear picture of how different NAD+ precursors are metabolized to feed cells and tissues. We also speculate that additional proteins controlling the supply or salvage of NAD+, along with proteins that are controlled by NAD+ levels, will be identified. Furthermore, the potential preventive and therapeutic use of NAD+-boosting strategies requires an assessment of the bioavailability and effectiveness of various precursor doses in human therapy. In addition, new NAD+ boosters are welcomed since the side effects of niacin generally lead to poor compliance, despite its known efficacy in a myriad of diseases. Therefore, the dosing and safety of these new NAD+ boosters (e.g., NAD+ precursors, CD38 inhibitors, and PARP inhibitors) must be thoroughly assessed to translate these exciting insights into NAD+ biology toward human relevance.

C.C. and K.J.M. both designed the outline and wrote the text. J.A. contributed to the ideas that make up this review and provided edits to all sections.

We would like to kindly thank Yoh Terada for his helpful advice during the creation of this review. K.J.M. is the recipient of a Heart and Stroke Foundation of Canada research fellowship award. C.C. is an employee of the Nestlé Institute of Health Sciences S.A. J.A. is the Nestlé Chair in Energy Metabolism. Work in the laboratory is supported by the École Polytechnique Fédérale de Lausanne, the National Institutes of Health (R01AG043930), the Swiss National Science Foundation (31003A-124713), and Systems X (51RTP0-151019).