The above observations indicate that few sirtuins (i.e., SIRT1, SIRT3, and SIRT5) fulfill the key criteria to be considered NAD+ sensors. However, sirtuins are not exclusively regulated by NAD+. For example, NAM, the end product of the sirtuin reaction, acts as a potent and general sirtuin deacetylase inhibitor. NAM was in fact shown to inhibit Sir2p, the yeast SIRT1 ortholog in a non-competitive manner with NAD+, with an IC50 < 50 μM (Anderson et al., 2003, Bitterman et al., 2002, Borra et al., 2004). Thus, sirtuin activity can potentially be differentially regulated by the cellular concentrations of both NAD+ and NAM.
NADH has also been proposed to act as an inhibitor of SIRT1 through competitive binding of the NAD+ pocket (Lin et al., 2004). Yet, the inhibition by NADH only occurs in the millimolar range, considerably above physiological NADH levels (Schmidt et al., 2004, Smith et al., 2009, Zhang et al., 2002). For example, intracellular concentrations of NADH in muscle cells range from 50 to 100 μM (Cantó et al., 2012, Hong et al., 2014). Thus, based on the above findings, the intracellular NAD+/NAM ratio may be a better predictor of sirtuin activity compared to the popularly used NAD+/NADH ratio.

NAD+-Consuming Enzymes (II): Poly(ADP-ribose) Polymerases

Poly(ADP-ribose) polymerases (PARPs) have been the center of intense focus due to their active role in DNA repair, inflammation, and cell death, but they have now also been shown to influence circadian rhythm, neuronal function, endoplasmic reticulum stress, and metabolism, among other cellular pathways (reviewed in Cantó et al., 2013, Gibson and Kraus, 2012, Kraus and Hottiger, 2013). There are 17 different genes encoding PARP-related proteins (Gibson and Kraus, 2012), but most research has so far focused on PARP1 and PARP2, which account for the vast majority of PARP activity in the cell (Cantó et al., 2013). In general, PARP1 and PARP2 can be activated by DNA strand breaks endowing them with a role in the response to DNA damage (Amé et al., 1999, Benjamin and Gill, 1980, Gradwohl et al., 1990). However, PARPs can also be activated by interactions with the phosphorylated form of extra signal-regulated kinases (ERKs) to amplify ERK-mediated histone acetylation events (Cohen-Armon et al., 2007). Furthermore, PARPs are also activated by HSP70 during heat shock stress to alter nucleosome structure and by Trp tRNA synthetase (TrpRS) (Petesch and Lis, 2012, Sajish and Schimmel, 2015). Active PARP catalyzes the transfer of ADP-ribose subunits from NAD+ to protein acceptors, including different nuclear protein substrates, and even itself (a process called auto-poly-ADP-ribosylation), thus forming PAR chains (Kameshita et al., 1984). Classically, PARPs have been shown to play dual roles in the cell that can either result in the induction of cell death or DNA repair. PARP1, for instance, was shown to modify the effectiveness of the p53-mediated DNA damage response for different types of cytotoxic stress (Valenzuela et al., 2002). As a result, PARP inhibition can be an effective treatment for cancer (Bryant et al., 2005, Farmer et al., 2005, Fong et al., 2009), leading to the development of several potent PARP inhibitors as chemotherapeutic agents. From a purely metabolic angle, PARP1 activation has also been linked to a rapid reduction in the glycolytic rate. While this phenomenon has been classically linked to a reduction in NAD+ availability, recent evidence indicates that PARP1 might also directly PARylate hexokinase, leading to a reduction in hexokinase activity and the cellular glycolytic rate (Andrabi et al., 2014, Fouquerel et al., 2014). Indeed, the possible direct impact of PARP activities on metabolic enzymes will be a fascinating area of research for years to come.

The Competition between PARPs and Sirtuins for NAD+ as a Metabolic Determinant

Upon DNA damage, PARP enzymes utilize NAD+ to generate PAR polymers, yielding NAM as a reaction product. Excessive DNA damage dramatically reduces NAD+ levels (Berger, 1985), even down to 20%–30% of their normal levels (Houtkooper et al., 2010a). In fact, the enzymatic properties of PARP1 indicate that it is an avid NAD+ consumer, with NAD+ increasing up to 2-fold in Parp1-KO mouse tissues (Bai and Cantó, 2012). This, in turn, limits NAD+ availability for other nuclear enzymes such as SIRT1 (Figure 3A) (Bai et al., 2011b, Pillai et al., 2005, Qin et al., 2006, Rajamohan et al., 2009). In fact, the Km of PARP1 is in the ∼50–59 μM range, unlike like that of PARP2 (Km 130 μM), dictating that NAD+ is rarely rate-limiting for PARP1 activity (Table 1) (Amé et al., 1999, Mendoza-Alvarez and Alvarez-Gonzalez, 1993). The lower affinity for, and consumption rate of, NAD+ by PARP2 is in agreement with the lack of change in NAD+ levels when PARP2 is knocked down in cultured cells (Bai et al., 2011a). Interestingly, however, Parp2 deficiency increased SIRT1 expression as a consequence of a direct negative regulatory effect on the SIRT1 promoter (Figure 3B) (Bai et al., 2011a). This further illustrates how PARP activity leads to SIRT1 inactivation, either by limiting NAD+ levels, in the case of PARP1 (Bai et al., 2011b), or by acting as a transcriptional repressor, in the case of PARP2 (Bai et al., 2011a).
The complexity of this pathway was heightened when SIRT1 was shown to directly inhibit PARP1 via its deacetylation (Figure 3B). Specifically, increased PAR activity was observed in Sirt1-KO cells treated with H2O2 (Kolthur-Seetharam et al., 2006), while the ability of SIRT1 to deacetylate PARP1 was confirmed by immunoprecipitation experiments (Rajamohan et al., 2009). Furthermore, SIRT1 also negatively regulates PARP1 transcription (Rajamohan et al., 2009). Illustrating the opposing roles of both enzymes, PARP1 is required for the transcriptional co-activation of NF-κB (Hassa et al., 2003), while SIRT1 inhibits NF-κB activity through the deacetylation of RelA/p65 (Yeung et al., 2004). Furthermore, PARP1 and SIRT1 have opposing effects on p53 nuclear accumulation and activation following cytotoxic stress (Figure 3B) (Langley et al., 2002, Luo et al., 2001, Valenzuela et al., 2002, Vaziri et al., 2001). Since the Km of SIRT1 for NAD+ is higher than that of PARP1, NAD+ levels can become so low following cell stress or senescence that SIRT1 no longer has the activity to keep PARP1 in check. This is supported by the fact that NAD+-repletion by expression of NAMPT can protect against PARP1 overexpression in a SIRT1-mediated manner (Pillai et al., 2005). Thus, it is likely that diverse cellular fates and metabolic decisions are closely regulated by the balance of the reciprocal regulation of SIRT1 and PARP1 activities, under the guidance of NAD+ levels (Figure 3B).
Recent work has further strengthened the hypothesis that PARP1 and SIRT1 have counterbalancing roles in metabolism and aging. For instance, PARP1 activity is enhanced with aging (Braidy et al., 2011, Mouchiroud et al., 2013) and high caloric intake (Bai et al., 2011b) yet reduced upon nutrient scarcity (Bai et al., 2011b). Parp1 deletion in C57Bl/6 mice confers protection against diet-induced obesity (Bai et al., 2011b). Strikingly, Parp1-deficiency on a 129/SvImJ background has been reported to exacerbate HFD-induced obesity (Devalaraja-Narashimha and Padanilam, 2010). In addition, some studies have shown that Parp1 deficiency can limit adipocyte function and size, leading to higher hepatic lipid accumulation (Erener et al., 2012). Despite these discrepancies, pharmacological PARP inhibition has consistently rendered protection against diet-induced obesity (Lehmann et al., 2015, Pirinen et al., 2014), possibly through an upregulation of SIRT1-dependent mitochondrial biogenesis and energy expenditure via the mitochondrial unfolded protein response (UPRmt; see New Perspectives in NAD+ Therapeutics (IV): Aging) (Pirinen et al., 2014). In addition, when the PARP1 worm homolog, pme-1, was knocked down in C. elegans, worms lived longer and maintained a more youthful phenotype at late adult stages (Mouchiroud et al., 2013). This was correlated to a marked increase in NAD+ availability, Sir2.1 activity, and mitochondrial function that were linked to the activation of the UPRmt (Mouchiroud et al., 2013). Altogether, most studies certify that a reduction in PARP activity is beneficial against some aspects of metabolic disease.
Importantly, PARP inhibition might lead to higher NAD+ availability in a compartment-specific fashion. In line with the predominant localization of PARP1 to the nucleus, reductions in PARP1 activity/expression markedly increases nucleo/cytoplasmic NAD+ levels and SIRT1 activity, yet it does not alter mitochondrial NAD+ or SIRT3 activity (Bai et al., 2011b, Pirinen et al., 2014). However, this notion will need to be consolidated when further technical developments allow us to better directly measure NAD+ levels in a compartment-specific fashion, most notably in the nucleus.
To strengthen the hypothesis that PARPs can consume NAD+ to the point of impeding metabolism, the AHR target gene, TiPARP (TCDD-inducible poly(ADP-ribose) polymerase or PARP7), was shown to increase PARylation of proteins, reducing NAD+ levels and SIRT1-mediated PGC-1α deacetylation in liver tissue (Diani-Moore et al., 2010). Furthermore, tankyrase 2 (PARP5b)-KO mice also have reduced fat pad and body weights, although no connection has yet been made to improvements in tissue NAD+ levels (Chiang et al., 2006). Altogether, these results suggest that ADP-ribosylation by several PARP family members can lead to metabolic dysfunction, suggesting that PARP inhibitors may have beneficial effects in this context.

NAD+-Consuming Enzymes (III): Cyclic ADP-Ribose Synthases

Cyclic ADP-ribose (cADPR), a secondary messenger implicated in Ca2+ signaling, cell cycle control and insulin signaling (Malavasi et al., 2008), is produced from NAD+ by cADPR synthases. The family of cADP-ribose synthases, including CD38 and its homolog CD157, were initially described as plasma membrane antigens on thymocytes and T lymphocytes. However, these ectoenzymes have also been found in non-lymphoid tissues, including muscle, liver, and brain (Aksoy et al., 2006b, Quarona et al., 2013). In addition, recent topological studies have described the enzymatic activity of this transmembrane protein as both extra- and intra-cellular (Jackson and Bell, 1990, Lee, 2012, Zhao et al., 2012).
Mice deficient in Cd38 show significantly elevated levels of NAD+ (10- to 30-fold) in tissues such as liver, muscle, brain, and heart, with corresponding SIRT1 activation, confirming the role of CD38 as a major NAD+ consumer (Figure 3A) (Aksoy et al., 2006b, Barbosa et al., 2007). Conversely, cells overexpressing CD38 showed reductions in NAD+ levels and in the expression of proteins related to energy metabolism and antioxidant defense, as measured by quantitative proteomic analysis (Hu et al., 2014). Similar to Parp1-deficient mice, Cd38-KO animals were protected from diet-induced obesity, liver steatosis, and glucose intolerance due to enhanced energy expenditure (Barbosa et al., 2007). In fact, the influence of Cd38 defiency on metabolism is so dramatic that, despite having lower physical activity compared to WT animals, they still expend more total energy. One potential issue is that CD38-independent cADPR synthase and NAD+-glycohydrolase activity remained present in the developing brain of Cd38-KO mice (Ceni et al., 2003). Similarly, studies in heart (Kannt et al., 2012, Xie et al., 2005), skeletal muscle (Bacher et al., 2004), and kidney (Nam et al., 2006) also demonstrated that cADPR synthesis occurs independently of CD38 and CD157, suggesting the existence of other cADPR synthase family member(s). In further support of the existence of additional cADPR synthases, a small-molecule compound screen discovered two potent inhibitors, SAN2589 and SAN4825, that do not inhibit CD38 yet blunt cardiac cADPR synthase activity (Kannt et al., 2012). Despite observations that CD38 inhibition appears to enhance NAD+ levels, further work should clarify its cellular location and specific roles in various tissues to make it a viable therapeutic target.

Recent Advances in NAD+-Related Therapeutics

Although NA is effective to treat dyslipidemia (Altschul et al., 1955), due to its undesirable effects, niacin derivatives including acipimox and prolonged release forms, such as niaspan and enduracin, have largely replaced NA use in the clinical management of hyperlipidemia. The core of the hypothesis explaining the effectiveness of niacin rested in part on the activation of GPR109A in adipocytes, which apparently mediated the transient reduction of plasma free fatty acid (FFA) levels (Tunaru et al., 2003, Zhang et al., 2005). Yet, more recently, using both a mouse line deficient in Gpr109 and clinical trials with two GPR109 agonists, it became clear that GPR109 did not mediate niacin’s lipid efficacy, thus questioning the GPR109-mediated FFA hypothesis (Lauring et al., 2012). This, in turn, gave strength to the possibility that the effects of niacin relied on the ability of NA or NAM to elevate NAD+ levels and activate sirtuins (Cantó and Auwerx, 2012). Beyond niacin, other NAD+ precursors, such as NMN and NR, are being considered as alternatives to niacin, since they do not activate GPR109A receptors, yet still activate SIRT1 in mice (Cantó et al., 2012). Similarly, the inhibition of PARP or CD38 activities has also proven to enhance NAD+ levels and sirtuin action (Figure 3A). Further disqualifying a GPR109-mediated effect and in support an NAD+-mediated metabolic response, in human type 2 diabetes patients, acipimox increases muscle mitochondrial function, which is accompanied by a mitonuclear protein imbalance and the induction of the UPRmt (see New Perspectives in NAD+ Therapeutics (I): Metabolic Disease and New Perspectives in NAD+ Therapeutics (IV): Aging), hallmarks of SIRT1, in lieu of GPR109, activation (van de Weijer et al., 2014). In the next section, we will hence discuss the therapeutic targets, the prospective clinical indications, and the potential limitations for NAD+-boosting compounds that activate sirtuins.

New Perspectives in NAD+ Therapeutics (I): Metabolic Disease

Introducing New NAD+ Precursors: NR and NMN

NR was recently demonstrated to have a surprisingly robust effect on systemic metabolism. First, dietary supplementation with NR protected against diet-induced obesity (Cantó et al., 2012). NR treatment increased both intracellular and mitochondrial liver NAD+ levels, concomitant to an enhancement of SIRT1 as well as SIRT3 activities (Cantó et al., 2012). As a result, there was a SIRT1-dependent increase in FOXO1 deacetylation, along with elevations in SOD2 expression, a FOXO1 target gene. Furthermore, in the mitochondrial compartment, NR led to the deacetylation of the well-established SIRT3 targets SOD2 and NDUFA9. In line with the activation of SIRT1 and SIRT3 targets, mitochondrial content was higher in skeletal muscle and brown adipose tissue of NR-treated high-fat-fed animals, which increased the use of lipids as energy substrates, boosted energy expenditure, and improved insulin sensitivity (Cantó et al., 2012). In alignment, impaired glucose tolerance and glucose-stimulated insulin secretion, induced by NAD+ shortages in NAMPT-deficient heterozygous animals, could be corrected by the administration of NMN (Revollo et al., 2007). Similarly, intraperitoneally administered NMN ameliorates glucose homeostasis in age- and diet-related insulin-resistant states (Ramsey et al., 2008, Yoshino et al., 2011). Importantly, NMN reversed the loss of NAD+ levels observed in both circumstances. As with NR, NMN also safeguarded mitochondrial function in mice and improved age-related mitochondrial dysfunction (Gomes et al., 2013). Knockdown of the nuclear-localized NMNAT1 attenuated the effect of NMN, consistent with the effect of NMN being driven by increases in NAD+ levels (Gomes et al., 2013). Furthermore, as NMNAT1 is located in the nucleus, the nuclear NAD+ pool may play a more dominant role for the induction of mitochondrial-encoded OXPHOS transcripts, potentially through alterations in SIRT1-directed HIF1α destabilization, leading to c-Myc activation of the nuclear-encoded mitochondrial factor TFAM (Gomes et al., 2013). Although these findings support the use of NR or NMN as a strategy for healthy aging, their efficacy in humans still needs testing. In fact, the dosages used for NR and NMN in mice, 400–500 mg/(kg⋅day), are high and potentially suboptimal for human application. Unlike NR, the use of NMN in mice has relied on intraperitoneal delivery, which could further complicate clinical use. Thus, the dosages, routes of administration, and efficacy of NAD+ boosters need to be optimized for human use.