Mechanisms of Ageing and Development 131 (2010) 287–298

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Mechanisms of Ageing and Development

journal homepage: www.elsevier.com/locate/mechagedev

Review Vitamin B3, the nicotinamide adenine dinucleotides and aging

Ping Xu, Anthony A. Sauve *

Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue LC216, New York, NY, 10065, United States

ARTICLE INFO ABSTRACT

Article history: Organism aging is a process of time and maturation culminating in senescence and death. The molecular Available online 20 March 2010 details that define and determine aging have been intensely investigated. It has become appreciated that the process is partly an accumulation of random yet inevitable changes, but it can be strongly affected by Keywords: genes that alter lifespan. In this review, we consider how NAD+ metabolism plays important roles in the NAD random patterns of aging, and also in the more programmatic aspects. The derivatives of NAD+, such as NADPH reduced and oxidized forms of NAD(P)+, play important roles in maintaining and regulating cellular Vitamin B3 redox state, Ca2+ stores, DNA damage and repair, stress responses, cell cycle timing and lipid and energy Aging metabolism. NAD+ is also a substrate for signaling enzymes like the sirtuins and poly-ADP- Metabolism ribosylpolymerases, members of a broad family of protein deacetylases and ADP-ribosyltransferases that regulate fundamental cellular processes such as transcription, recombination, cell division, proliferation, genome maintenance, apoptosis, stress resistance and senescence. NAD+-dependent enzymes are increasingly appreciated to regulate the timing of changes that lead to aging phenotypes. We consider how metabolism, specifically connected with Vitamin B3 and the nicotinamide adenine dinucleotides and their derivatives, occupies a central place in the aging processes of mammals. ß 2010 Published by Elsevier Ireland Ltd.

1. Introduction

Aging is a complex process in which tissues and organs Abbreviations: AADPR, O-acetyl-ADP-ribose; ACCa, acetyl-CoA carboxylase-alpha; accumulate changes over the lifetime of an organism. Late in life AceCS1 and 2, acetyl-coenzyme A synthetase 1 and 2; ADPR, adenosine 5’- these changes are associated with a reduced level of tissue function diphosphoribose; ANT2, 3adenine nucleotide translocator isoform 2 and 3; ARTs, and a reduced capacity for tissue self-repair (Rossi et al., 2007). ADP-ribosyl trasnferases; ARCs, ADP-ribosyl cyclases; ATP5A, human mitochon- Accompanying this condition is an increased vulnerability to drial ATP synthase alpha-subunit; Bax, BCL2-associated X protein; cADPR, cyclic ADP-ribose; CPS1, carbamoyl phosphate synthetase 1; DBC1, deleted in breast disease. In humans, age-related diseases include cancer, arthritis, cancer-1; FOXO, forkhead box transcription factor; G6PDH, glucose-6-phosphate cataracts, osteoporosis, type II diabetes, hypertension, heart dehydrogenase; GDH, glutamate dehydrogenase; HSP70, 70 kilodalton heat shock diseases, Alzheimer’s disease, stroke and other neurodegenerative proteins; IDH, isocitrate dehydrogenase; IDP, NADP+-dependent isocitrate dehy- disorders, all of which cumulatively reflect the major causes of drogenases; IP3, inositol trisphosphate; Ku70, lupus Ku autoantigen protein p70; death late in human life (except infection). Molecular and cell IDO, indoleamine 2,3-dioxygenase; LPS, lipopolysaccharides; LXR, liver X receptor; MEF2D, myocyte-specific enhancer factor 2D; MEP, NADP+-dependent malic biological studies have revealed that aging in tissues is character- enzymes; Mtap, methylthioadenosine phosphorylase; NA, nicotinic acid; NaAD, ized by hallmark features such as telomere shortening (von Figura nicotinic acid adenine dinucleotide; NAADP, nicotinic acid adenine dinucleotide et al., 2009), reduced capacities for DNA repair (Gorbunova et al., phosphate; NADKs, NAD+ kinase; NAM, nicotinamide; NaMN, nicotinic acid 2007; Rossi et al., 2007), reduced stress responses (Knight, 2001; mononucleotide; Nampt, nicotinamide phosphoribosyltransferase; NAPRTase, nicotinic acid phosphoribosyltransferase; NF-kB, nuclear factor kappa-light- Naidoo, 2009), increased sensitivity to apoptosis and decreased cell chain-enhancer of activated B cells; Nmnat, nicotinamide/nicotinc acid mono- division (Rossi et al., 2007). In addition, aging causes profound nucleotide adenylyltransferase; NMN, nicotinamide mononucleotide; NR, nicoti- changes in tissue and systemic metabolism (Finley and Haigis, namide riboside; NOX, NADPH oxidase; Nrk1,2, nicotinamide riboside kinase; 2009) and endocrine signaling, such as slowed metabolic rate and PARPs, poly-ADP-ribosyl polymerases; PGC1-a, PPAR-gamma coactivator 1; PPAR- increased insulin resistance (Arai et al., 2009). g, peroxisome proliferator-activated receptor-gamma; p53, tumor protein 53; Pol I, + RNA polymerase I; Pnp1, purine-nucleoside phosphorylase 1; Qprtase, Quinolinate Nicotinamide dinucleotide (NAD ), and its derivatives such as phosphoribosyltransferase; SUV39H1, histone-lysine N-methyl-transferase; NADH, NADP(H) are small molecules that are central cellular Smad7, SMAD family number 7; TAFI68, TBP-associated factors; TH, transhydro- metabolites and are increasingly recognized to play important genase; TNF-a, tumor necrosis factor alpha; WRN, Werner syndrome ATP- roles in the aging process (Lin and Guarente, 2003; Sinclair and dependent helicase; 6GDH, 6-gluconate phosphate dehydrogenase. Guarente, 2006; Pollak et al., 2007; Yang et al., 2007). Consistently, * Corresponding author. Tel.: +1 212 746 6224. E-mail address: [email protected] (A.A. Sauve). these metabolites function in energy metabolism, DNA repair

0047-6374/$ – see front matter ß 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.mad.2010.03.006 288 P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298

Table 1 Cellular functions of NAD+-related metabolites and their relationship to human health.

Molecules Cellular functions Effects on human health References

Nicotinamide Increase NAD+ level; Lifespan extension of human cell; Hoane et al. (2006a,b); Kang et al. (2006); Lin et al. (2001) Inhibition on PARPs, sirtuins Neuroprotection from brain injury; Prevention of vascular injury, lung injury Treatment of pellagra

Nicotinic acid Reduce lipids (cholesterol, triglycerides, Treatment of cardiovascular diseases; Benavente et al. (2009); Penberthy (2009); low-density lipoproteins) Scanu and Bamba (2008) Increase HDL level; Neuroprotection; Increase NAD+ level Treatment of skin diseases, including pellagra

NAD+/NADH NAD+ metabolism; NAD+ depletion and cell death; Bedard and Krause (2007) Component in cellular antioxidation systems Associated with brain ischemia, diabetes, cancer, cardiovascular diseases

NADP/NADPH Component in cellular antioxidation systems; Associated with brain ischemia, diabetes, Bedard and Krause, 2007 cancer, cardiovascular diseases Substrate of NADPH oxidase

NAADP Ca2+ signaling Smooth muscle tension Galione (2006) cADPR Ca2+ signaling Unknown Galione (2006)

AADPR Substrate of yeast Ysa1; Unknown Liou et al. (2005); Tong et al. (2009) Promotion of SIR assembly in yeast

ADPR ADP-ribosylation of protein by ARTs Unknown Corda and Di Girolamo (2003); and PARPs Peralta-Leal et al. (2009)

pathways and cell protection pathways as well as cell death differentiate NAD+ and NADP+ functionally, and indeed the cell pathways (Hassa et al., 2006; Pollak et al., 2007; Yang et al., 2007). maintains a high NADPH/NADP ratio, consistent with use of These metabolites can be thought of as central mediators that NADPH as a reductant and as a cell protectant against oxidative influence a variety of cellular processes known to change in aging. stress (Pollak et al., 2007). Increasing evidence suggests that the In this review, we consider the recent biological and medical absolute amounts of NAD(H) and NADP(H) are important in efforts being made to understand how NAD(P)+ metabolism and its maintaining cellular health. central effects influence aging processes. 3. Metabolic transformations of NAD+ and NADH in cells 2. Vitamin B3, distribution of NAD+ and NADP+ and their redox partners NAD+ is metabolized in several ways in cells. Its metabolites include NADH, NADP+ and NADPH, nicotinic acid adenine Vitamin B3 is the precursor of nicotinamide dinucleotide dinucleotide phosphate (NAADP), adenosine 50-diphosphoribose (NAD+), and is commonly sold as a supplement in two major forms, (ADPR), cyclic ADP-ribose (cADPR) and O-acetyl-ADPR (AADPR) namely nicotinamide (NAM) and nicotinic acid (NA). The latter (Fig. 1). ADP-ribose is also generated from NAD+, and ADP- form of vitamin B3 is commonly known as niacin. Most raw foods ribosylation of proteins is an important post-translational also provide these substances, or provide them after digestive modification, expecially for genome stability (Hassa et al., 2006). action, although diets of corn are noteworthy in being a poor A major reaction of NAD+ in metabolism is to accept a hydride source of B3 (Goldsmith et al., 1952). These substances in their equivalent to generate NADH. NADH can be re-oxidized back to unmodified forms can regulate cellular activities through receptor NAD+ by many reactions within cells, but within the mitochondria, activation and also through inhibition of signaling enzymes. They electrons from NADH provide the chemical energy by which ATP are also precursors for the biosynthesis of derivatives of NAD+. can be generated. The effects of the NAD+/NADH ratio are complex Malnutrition, accompanied by deficiency of B3 and tryptophan and currently not well understood, but it is evident that among (which can be biosynthetically converted in humans to NAD+), can other things this ratio contributes to the control of several key cause the disease of pellagra. This B3 deficiency disease is metabolic enzymes involved in energy metabolism, including characterized by photo-induced rash, skin lesions, gastrointestinal glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogen- symptoms, and neuropsychiatric disturbances. Although it can be ase and pyruvate dehydrogenase. In the cytosol, the ratio of treated by replenishment of vitamin B3 to diet, it can be unbound NAD/NADH is thought to be close to 700 to 1; while in the debilitating if not identified (Prakash et al., 2008), highlighting mitochondrion the ratio of NAD/NADH is about 7-8 (Stubbs et al., the indispensible position of nicotinamide, nicotinic acid and NAD+ 1972). Unbound concentrations of these metabolites have been metabolites in the maintenance of human health (Table 1). difficult to determine, however estimates in COS7 cells have NAD+ (Fig. 1) is the first redox active form of Vitamin B3 that is provided a concentration for NADH in the nuclear compartment made in cells. Likely because of its centrality to energy close to 110 nM, and the unbound NAD+ concentration, 70 mM metabolism and to cell protection pathways, and because it is (Zhang et al., 2002; Fjeld et al., 2003). The absolute concentrations abundant, it is made by multiple biosynthetic pathways (Fig. 2). of these metabolites (bound and unbound) in different cells have NAD+ participates in metabolism largely as a redox cofactor in an been estimated to be near 10:1 in favor of NAD+, with variations in oxidative role, and thus NAD+ is kept in excess to NADH in cells this ratio recently reported to vary from 4:1 to 20:1 in mammalian (Williamson et al., 1967; Sauve, 2008). NAD+ is phosphorylated to C2C12 cells (Fulco et al., 2008). The absolute concentrations for NADP+, by action of the enzyme NAD+ kinase (Pollak et al., 2007). NAD+ in mammalian cells typically range from 200 to 700 mM (For The modification of NAD+ by a single phosphate allows the cell to recent measurements see Fulco et al., 2008; Yang et al., 2007). P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 289

Fig. 1. Chemical structures of NAD+, NAD+ precursors, and NAD+ derivatives. Abbreviations include: NAD+ nicotinamide adenine dinucleotide; NAM, nicotinamide; NR, nicotinamide riboside; NA, nicotinic acid; NADP, nicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; cADPR, cyclic- adenosine-diphosphate ribose; O-acetylADPR, O-acetyl-adenosine-diphosphate ribose; ADPR, adenosine-diphosphate ribose.

+ It has been argued that sirtuins can sense the NADH/NAD ratio Kd < 100 nM) which in turn stimulates binding of CtBP to in cells, presumably by differential recognition of the reduced and transcriptional repressor proteins (Zhang et al., 2002; Fjeld oxidized dinucleotides (Lin and Guarente, 2003), but other cellular et al., 2003). factors also seem to respond to levels of NADH (or NADPH) such as Cyclic ADP-ribose (cADPR) is formed in a reaction catalyzed by NPAS2:BMAL1 and Clock:BMAL1, which are DNA-binding proteins ADP-ribosyl cyclases (Fig. 1). CD38, is one such ADP-ribosyl involved in the maintenance of circadian rhythms. The binding to cyclase, and has been studied extensively (Malavasi et al., 2008). DNA by these complexes is stimulated by reduced dinucleotides CD38 uses NAD+ as a substrate and generates cADPR and ADPR (Rutter et al., 2001). Carboxyl-terminal binding protein (CtBP), a (Sauve et al., 1998). An additional enzymatic activity of CD38 is to transcriptional corepressor binds NADH with high affinity (with exchange nicotinic acid (NA) into NADP+ and the reaction product 290 P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298

Poly(ADP-ribose) polymerases (PARPs) generate polymers of ADP- ribose from multiple NAD+ molecules and attach them to proteins such as histones (Hassa et al., 2006). PARP-1 is chromatin- associated and appears to be involved in the regulation of various cellular and subcellular processes, including DNA repair, gene expression, genome stability, cell cycle, and cell death (Hassa et al., 2006; Peralta-Leal et al., 2009).

4. Role of NAD+ and nicotinamide in maintaining genome stability, regenerative fitness and protection from injury

+ Fig. 2. NAD+ synthesis in mammals. Four sources are used to synthesize NAD+. It has been long appreciated that NAD plays a vital role in The first source is tryptophan, which is converted into quinolinate and maintaining mammalian genome stability through the activity further catalyzed into nicotinic acid mononucleotide (NaMN) by quinolinate of PARP enzymes. Consistently, genetic PARP-1 deficiency leads phosphoribosyltransferase (Qprtase). The second source is nicotinic acid (NA), to genome instability (Hassa et al., 2006). In fact, in animals that which is converted into NaMN with the catalysis of nicotinic acid are deficient in NAD+ levels, because of Vitamin B3 deficiency, phosphoribosyltransferase (NAPRTase). The third source is nicotinamide (NAM), which is converted into nicotinamide mononucleotide (NMN) by the enzyme alkylating agents increase rate of progression to cancer, nicotinamide phosphoribosyltransferase (Nampt). The fourth source is consistent with increased rates of somatic mutation (Boyonoski nicotinamide riboside (NR), which is converted into NMN by nicotinamide et al., 2002; Spronck and Kirkland, 2002; Spronck et al., 2003). In riboside kinase Nrk1,2. NR also can be degraded to NAM by purine-nucleoside contrast, increased cellular NAD+ elicited by supplementation of phosphorylase 1 (Pnp1) and human methylthioadenosine phosphorylase (Mtap). The formed NaMN and NMN can be catalzyed by nicotinamide/nicotinc acid Vitamin B3 to diet can decrease rates of mutagenesis and mononucleotide adenlyltransferase (Nmnat1, 2, 3) into nicotinic acid adenine decrease rates of cancer formation (Boyonoski et al., 2002b). dinucleotide (NaAD), which is finally converted into NAD+ by the enzyme NAD+ Interestingly, recent data suggest that DNA repair may be synthetase. central to aging phenotypes in stem cells, which arguably are the source of most tissue renewal. For example, Rossi et al. (2007) showed that DNA repair deficiency ultimately limits the is nicotinic acid adenine dinucleotide phosphate (NAADP) (Mala- self-renewal capacities of haemopoietic stem cells. It is vasi et al., 2008). NAADP and cADPR are potent second messengers interesting that nicotinamide, an NAD+ precursor can promote that bind and activate Ca2+ channels in cells (Galione, 2006). the extension of replicative lifespan of human cells (Miura and O-acetyl-ADP-ribose (AADPR) is the direct product of the sirtuin Kameda, 2005; Kang et al., 2006). Kang et al. (2006) found that catalyzed NAD+-dependent protein deacetylation (Sauve et al., the supplementation to media with nicotinamide increased 2006). There is accumulating evidence to indicate that sirtuins normal human fibroblast cell lifespan, by 60% as measured by regulate lifespan in multiple organisms, such as yeast, flies, worms population doublings. The authors suggested that nicotinamide and possibly mammals (Kaeberlein et al., 1999; Tissenbaum and might increase NAD+ content, and noted that nicotinamide Guarente, 2001; Rogina and Helfand, 2004; Ho et al., 2009). reduced the level of reactive oxygen species (ROS) which can Although sirtuin catalyzed deacetylation appears to be largely damage DNA. Importantly, nicotinamide has potent inhibitory indispensible for these effects on lifespan, there is still little known effects on sirtuins (Sauve et al., 2006) and on PARP enzyme about the cellular function of the unique reaction product AADPR. activity (Preiss et al., 1971). This inhibitory effect is anticipated This compound is measurable in yeast cells (Lee et al., 2008). to reduce the rate of NAD+ turnover in cells, an effect that has Interestingly, a mitochondria protein Ysa1 of Saccharomyces been supported by observations that nicotinamide can reduce cerevisiae can cleave AADPR to acetyl-ribose-5-phosphate and NAD+ depletion in cells (Slominska et al., 2008). The inhibitory AMP (Tong et al., 2009). A similar reaction is observed for ADPR, a effects can also alter cell signaling pathways regulated by PARPs breakdown product of AADPR (Tong et al., 2009). Deletion of YSA1 and sirtuins. Therefore, determining the specific molecular results in a 50% increase in the level of ADPR and AADPR in yeast details of how nicotinamide generates observed biological cells. Dysa1 cells are less susceptible to cell death caused by outcomes is a complex problem as this compound acts in externally applied hydrogen peroxide or Cu2+ and have lower diversewaysincells.Itisapparentthatmoreextensive levels of endogenous reactive oxygen species ROS as measured by investigation is needed to ascertain the mechanisms and dihydroethidium staining (Tong et al., 2009). Tong et al. (2009) contexts where nicotinamide supports cell and tissue health. provided evidence that ADPR inhibits the glycolytic enzyme NAD+ is an important component in cellular responses to glyceraldehyde-3-phosphate dehydrogenase. This inhibition is genotoxicity. DNA damage is important in the accumulative sense, suggested to redirect glucose into the pentose phosphate pathway, and in the acute sense as well. In acute injuries, such as in stroke resulting in increased NADPH production in mutants, which was and myocardial infarction, which are disease states of aged people, supported by experimentally determined higher NADPH concen- one of the main causes of tissue damage originates from an trations in mutant cells. Increased NADPH levels is thought to increase in oxidative stress in the tissue undergoing vascular provide the observed antioxidant protection. In other studies, obstruction, a damage that becomes especially worsened with AADPR has also been found to stimulate the Sir2-mediated vascular reperfusion. This reperfusion injury appears to stem from construction of the SIR complexes that stabilize heterochromatin overactivation of PARP1 initiated by ROS generated genotoxicity. structure in yeast. AADPR can bind to Sir3 and can promote This process depletes NAD+, and ultimately causes energy failure oligomerization and association with Sir2/Sir4 (Liou et al., 2005). and cellular necrosis. Interestingly, high dosages of nicotinamide, AADPR has been found to contribute to the affinity of Sir2-3-4 which leads to PARP inhibition are protective. For example, binding to nucleosomes (Martino et al., 2009), thereby increasing nicotinamide reduces neuron damage from nitric oxide (NO) (Lin stability of heterochromatin. et al., 2000), anoxia (Lin et al., 2001), asphyxia (Klawitter et al., Protein ADP-ribosyltransfer NAD+ is a substrate for ADP-ribosyl 2007), hypoxia/reoxygenation (Chong et al., 2004; Shen et al., transferases (ARTs) and poly(ADP-ribose) polymerases (PARPs). 2004; Ji et al., 2008), retinal ischemia (Ji et al., 2008) and These enzymes modify proteins with ADP-ribose (ADPR) in either excitotoxicity induced by glutamate/N-methyl-D-aspartate monomeric or polymeric form (Corda and Di Girolamo, 2003). (NMDA) (Liu et al., 2009). P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 291

In mice with focal cerebral ischemic stroke, nicotinamide at plasma lipid profiles at eight weeks treatment, as determined by 200 mg/kg (via intraperitoneal injection) could increase NAD+ 49%, 47% and 33% decreases in LDL cholesterol, non-HDL observed in contralateral and ipsilateral cortex (Liu et al., 2009), cholesterol and triglycerides respectively, and a 20% increase in and could shrink infarct volume, consistent with the beneficial role HDL cholesterol versus baseline before therapy, which was more of nicotinamide in the maintenance of NAD+ (Liu et al., 2009). Other effective than statin administered alone (McKenney et al., 2007). studies have found that nicotinamide is effective to prevent brain The mechanisms of nicotinic acid effects on lipid metabolism are damage from ischemia/reperfusion (Chang et al., 2002; Yang et al., still not clearly understood, although it appears that nicotinic acid 2002), asphyxia at neonatal stages (Bustamante et al., 2007) and works through several mechanisms. These include inhibiting the injurious effects of ethanol, which may cause neurological lipolysis in adipose tissue, inhibiting triglyceride synthesis, decreas- maldevelopment called fetal alcohol syndrome during the brain- ing apo B incorporation into LDL particles, and by increasing growth spurt in a mouse model (Ieraci and Herrera, 2006). retention of apo A-I and HDL in serum (Kamanna and Kashyap, In animal models, nicotinamide has been shown to protect 2008). In adipose tissue, fatty acids are stored and under stimulation against different neuronal injuries as well. Traumatic brain injury from hormones or neurotransmitters, released to liver, where fatty (TBI) causes motor, sensory, memory, cognitive and psychiatric acids are uptaken and can be assembled with lipoprotein. Nicotinic deficits of function and currently no effective drugs are available acid decreases this release of free fatty acids from adipose tissue due for this kind of brain damage (Hoane et al., 2006a). Hoane et al. to inhibition of lipolysis (Tunaru et al., 2003). Studies have revealed used thirty-four male Sprague-Dawley rats to identify proof of that G protein-coupled receptor HM74A and HM74, respectively are concept efficacy of nicotinamide in cortical contusion injury. The receptors of nicotinic acid. The binding affinity of nicotinic acid to administration of nicotinamide (500 mg/kg) for 15 min after injury HM74A is 80–250 nM (Wise et al., 2003). The activation of HM74A protected against neurodegeneration as measured by count of by nicotinic acid reduces the level of intracellular cAMP and is degenerating neurons in injured cortical tissue, and prevented proposed to inhibit lipolysis from adipocytes (Tunaru et al., 2003), edema versus control animals (Hoane et al., 2006a). Beneficial providing a mechanism for reducing serum lipid. effects of nicotinamide in cortical contusion injury was also The inhibition of triglyceride synthesis by nicotinic acid is observed in the reduced acute inflammatory responses and lesion another effect on lipid metabolism mediated by this pharmaco- cavity expansion (Hoane et al., 2006b) and produced behavioral logic agent. Nicotinic acid is a potent inhibitor of hepatocyte recovery (Hoane et al., 2008). Nicotinamide also has strong diacylglycerol acyltransferase-2 (DGAT2), an important enzyme in neuroprotective abilities following fluid percussion injury, another the manufacturing of triglycerides (TG) in liver (Ganji et al., 2004). kind of traumatic brain injury. Nicotinamide (500 mg/kg) DGAT2 is inhibited 50% by 100 mM nicotinic acid, partially improved behavior as measured by the bilateral tactile removal explaining one aspect of high nicotinic acid dose required for test (a test of responsiveness and coordination to removal of a therapy. Studies also support a potent effect of nicotinic acid on foreign adhesive patch) and working memory tests (Hoane et al., acceleration of the degradation of apo B which is an important 2006c) and protected against cortical tissue loss in rats at 7 days component of undesirable LDL and VLDL particles (Kamanna and after a moderate fluid percussion injury (Holland et al., 2008). Kashyap, 2008). Therefore, the inhibitory effects of nicotinic acid The role of nicotinamide in aging and aging related diseases is on DGAT2 and on apo B help explain decreased levels of VLDL and not limited to its ability to protect the neuron, but also to protect LDLs observed with nicotinic acid treatment. cells against vascular injury (Lin et al., 2001) and lung injury (Su Other actions of nicotinic acid positively influence the abundance et al., 2007). Collectively, these data imply that protection of of HDL particles. Apo A-I containing HDLs are synthesized and cellular NAD+ and/or other effects of nicotinamide mediated secreted by the liver and intestines. Nicotinic acid decreases the through signaling enzymes such as PARPs and sirtuins are expression of b chain adenosine triphosphate synthase in hepato- important for the preservation of tissue and for encouraging cytes, which is thought to be the receptor of HDL-apo A-I tissue regenerative capabilities. holoparticle. The downregulation of this receptor by nicotinic acid decreases hepatocyte uptake of HDL and is implicated in HDL-apo A-I 5. Nicotinic acid as a vascular protectant and as a tissue retention in serum and in size increase of HDL particles (Kamanna protectant and Kashyap, 2008). Serum retention of HDL-apo A-I increases reverse cholesterol transport, the process of movement of cholesterol The discovery that nicotinic acid can reduce serum cholesterol from the peripheral tissues to the liver, a process thought to improve (Altschul et al., 1955), stimulated broad clinical use of this form of cholesterol disposal and to reduce atherosclerosis (Kamanna and vitamin B3 to treat dyslipidemias, hypertriglyceridemia and Kashyap, 2008). Overall, through multiple pathways, nicotinic acid is atherosclerosis (Sauve, 2008; Scanu and Bamba, 2008). Clinical an effective treatment for lipid disorders and cardiovascular disease. studies show that nicotinic acid is atheroprotective, specifically It is worth pointing out that nicotinic acid has potential for because of its ability to lower plasma levels of cholesterol, treating diseases affecting neurons as well. The binding of nicotinic triglycerides, and very-low- and low-density lipoproteins (VLDL acid to HM74A causes release of prostaglandin PGE2, which in turn and LDL). Importantly it also increases the levels of high-density causes expression of the enzyme indoleamine 2,3-dioxygenase lipoprotein (HDL) but can reduce elevated lipoprotein (a). Low (IDO) in dendritic cells. Increased IDO activity can inhibit auto- levels of high-density lipoprotein (HDL) cholesterol are correlated immunity mediated demyelination in animal studies designed to with higher occurrence of adverse cardiovascular occlusive understand mechanisms of multiple sclerosis, a progressive events, even stroke. Consistently, nicotinic acid is useful to reduce disease in the central nervous system, suggesting that nicotinic the risk of stroke (Keener and Sanossian, 2008). The regulation of acid might be clinically effective to slow the pathology of this lipid metabolism by nicotinic acid is unique, and is not provided by disease (Penberthy, 2009). In addition, because it is a precursor of nicotinamide. In clinical trials, nicotinic acid is used in two NAD+ synthesis, nicotinic acid can significantly increase tissue formulations, immediate- and extended-release (ER). The latter NAD+ levels (Jackson et al., 1995). Due to this property, as well as its form slows the release of nicotinic acid and decreases the ability to absorb UV light, derivatives of nicotinic acid have been unpleasant side effect called flushing, typified by skin discomfort. developed for skin protection. Nicotinic acid can improve skin Nicotinic acid can also be used with other drugs to raise their NAD+ contents and can reduce sun sensitivity, possibly by reducing overall efficacy. A recent study demonstrates that nicotinic acid UV penetration and by preventing NAD+ depletion, cell death and (ER, 1000 mg) and rosuvastatin (20 mg) in combination improved reduced efficiency of DNA repair (Benavente et al., 2009). 292 P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298

6. Roles of NADP and NADPH as cell protectants NOX3, NOX4, NOX5, DUOX1 and DUOX2) are known (Bedard and Krause, 2007). ROS generated by these enzymes is involved in NADP+ is primarily generated by NAD+ kinase (NADKs), which multiple cellular functions including host defense and inflamma- transfers the gamma phosphate of ATP as the source of the tion, cellular signaling, cellular senescence, and cell growth phosphate group. In mycobacteria such as Mycobacterium tuber- (Bedard and Krause, 2007). culosis and in archaea such as Pyrococcus horikoshii, inorganic In immune responses, NOX-derived ROS kills bacteria by polyphosphate can be the phosphate donor (Sakuraba et al., 2005). generating hydrogen peroxide in concentrations that can be toxic NADKs are the sole enzymes that convert NAD+ to NADP+, and they to pathogens (El Hassani et al., 2005; Leto and Geiszt, 2006). can be seen to be centrally involved in regulating the level of NADP, Interestingly, NOX catalyzes inactivating oxidation of a methionine NADPH and perhaps even NAADP+. The reduced dinucleotide is residue of a secreted virlence peptide involved in quorum sensing produced by catalysis by glucose-6-phosphate dehydrogenase released by Staphylococcus aureus (Rothfork et al., 2004). NOX- (G6PDH) and 6-gluconate phosphate dehydrogenase (6GDH). derived ROS can also affect cell signaling within mammalian cells These metabolic steps are known as the pentose phosphate though modulation of phosphatase and kinase signaling, as well as pathway (Minich et al., 2003; Nee et al., 2009). In cytosol and altered metal ion mediated signal transduction (Cherednichenko mitochondria, the conversion from NADP+ to NADPH is also et al., 2004; Goldstein et al., 2005; Mehdi et al., 2005). In insulin catalyzed by NADP+-dependent isocitrate dehydrogenases (IDP) signal transduction, ROS enhances signaling via oxidation of and NADP+-dependent malic enzymes (MEP) (Minich et al., 2003). protein tyrosine phosphatases (Mahadev et al., 2004). Recent work In mitochondria, transhydrogenases (TH) can also catalyze the suggests that NADPH activity suppresses TGF-b (a cytokine conversion of NADH to NADPH (Fig. 3)(Minich et al., 2003). important in the stimulation of kidney fibrosis) suggesting that NADPH can regulate cellular functions via its redox function oxygen stress may positively mediate anti-fibrotic action in kidney where it serves as a reservoir of antioxidation systems in cells. It (Wolf et al., 2001). NADPH oxidase is associated with the also provides a source for reducing equivalents for multiple development of brain ischemia (Walder et al., 1997), diabetes biosynthetic pathways. NADP+, the oxidized metabolite, can be (Block et al., 2009), cancer (Kamata, 2009), and could modulate converted to NAADP by base exchange with nicotinic acid (NA) responses of pulmonary and coronary artery to hypoxia (Gupte catalyzed by mammalian ADP-ribosyl-cyclases, such as CD38. et al., 2005). Multiple functions of NADPH oxidase as well suggest NAADP is able to increase cytosolic Ca2+ concentrations by them as possible therapeutic targets. In proof of concept studies, releasing this ion from intracellular compartments (Galione, 2006). Knock-out of NOX2 in a hindlimb model of ischemia provided NADPH is a substrate for reduction of oxidized glutathione decreased oxidative stress and improved vascular outcomes in (GSSG) to reduced glutathione (GSH) by glutathione reductase endothelial progenitor cells (Haddad et al., 2009). Inhibition of (GR). GSH is a substrate for the activities of the antioxidation NADPH oxidases has become an interesting strategy for the enzymes glutathione peroxidase (GPx) and glutathione-S transfer- treatment of cardiovascular diseases (Cave, 2009). ase (GST) (Wu et al., 2004). NADPH is also a substrate for NAADP, primarily generated from NADP+, plays a putative role antioxidation systems that regenerate thioredoxin through thior- in Ca2+ signaling pathways. Ca2+ mobilization is known to be edoxin reductase (Kalinina et al., 2008). important in a number of physiological processes in aging. For Strangely, NADPH can contribute to oxidative stress as a example, during aging, skeletal and cardiac muscles are prone to substrate for NADPH oxidase. NADPH oxidase is an enzyme that reduced contractility and muscle wasting, and weakened or reacts NADPH and oxygen to generate superoxide, a precursor for abnormal intracellular Ca2+ signaling may contribute to these multiple ROS species such as peroxides and peroxynitrite. There phenotypes (Weisleder and Ma, 2008). It is known that NAADP can are multiple NOX forms in mammalian cells that have been releases Ca2+ from intracellular lumen and can act a potent second characterized, with phagocyte NADPH oxidase (NOX2 otherwise messenger distinct from IP3 and cADPR in Ca2+ signaling pathways known as gp91) the founding member and other isoforms (NOX1, (Galione, 2006). Studies in human platelets have provided evidence for distinct Ca2+ pools, with NAADP able to release a pharmacologically distinguishable reservoir (Galione, 2006; Lopez et al., 2006). NAADP is uniquely able to stimulate differentiation of PC12 cells, a neuronal precursor cell. The second messengers IP3 or cADPR, which also increase intracellular Ca2+ levels, were not effective (Brailoiu et al., 2006; Galione, 2006).

7. NAD metabolism is regulated: Nampt/PBEF is a key NAD+ biosynthetic enzyme

Nampt (also called PBEF or Visfatin) is a dimeric type II phosphoribosyltransferase and it converts nicotinamide to nico- + + + Fig. 3. NAD metabolism in mammals. NAD in cells will be used to form NAD tinamide mononucleotide (NMN). Kim et al. (2006) and Wang et al. phosphate (NADP+)byNAD+ kinase (NADK). NADP+ is the source for the (2006) showed that mouse and rat Nampt have two active sites at formation of NADPH, which is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-gluconate phosphate dehydrogenase (6GDH), NADP+-dependent the interface of two identical monomer subunits. The sequence of isocitrate dehydrogenases (IDP) or NADP+-dependent malic enzymes (MEP). amino acids responsible for interaction with the nicotinamide ring NADPH also can be converted from NADH, a reduced form of NAD+, via catalysis (substrate) and the ribose of NMN (product) are highly conserved + of transhydrogenase (TH). NADP is the major source of nicotinic acid adenine in homologues from thirteen different organisms (Kim et al., 2006; dinucleotide phosphate (NAADP) and the formation of NAADP can be catalyzed by ADP-ribosyl cyclases (ARCs), such as CD38. Three major enzymes utilizing Wang et al., 2006). + NAD+ as the substrate are ADP-ribosyl cyclases, sirtuins, and ADP-ribosyl In the mammalian NAD biosynthetic pathway, Nampt is transferases. ADP-ribosyl cyclases (ARCs) generate cyclic ADP-ribose (cADPR) in essential to the recycling and the utilization of NAM. NAM is a the reaction. Sirtuins generate ADP-ribose (ADPR), O-acetyl-ADP-ribose (O- product of most of enzymatic reactions that consume NAD+,and acetyl-ADPR), and the deacetylated protein as the products. The third class of turnover of cellular NAD+ can be rapid. In non-stressed cells, the NAD+ utilizing enzyme is mono ADP-ribosyl transferases (ARTs) and poly-ADP- + ribosyl polymerases (PARPs), which attach mono-ADPR and poly-ADPR to the NAD halflife is as little as 4 h (Sauve unpublished results) whereas targets, such as proteins. in cells experiencing genotoxic stress, total cellular NAD+ can be P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 293 exhausted in less than 2 h (Yang et al., 2007). Consequently, the serious and often fatal acute medical condition that results from a recycling of NAM is crucial to the maintenance of NAD+ in cells. In hyperactive immune response to infection, Nampt levels have studies by the Sinclair and Sauve laboratories, it was demonstrated been determined to be very highly upregulated in neutrophils by genetic strategies that Nampt determines the amount of NAD+ in (taken from septic patients), and these cells exhibit delayed cells (Yang et al., 2007). Similarly, Imai and co-workers provided apoptosis (Jimenez et al., 1997; Taneja et al., 2004; Luk et al., 2008). evidence that Nampt catalyzed-conversion of NAM to NMN is the rate-limiting step for nicotinamide recycling, and found that Nampt 8. Other NAD biosynthetic enzymes in human aging overexpression, without nicotinamide supplementation, could increase cellular NAD+ level, indicating the amount of Nampt is Apart from Nampt, nicotinamide/nicotinic acid mononucleo- typically limiting for NAD+ synthesis. Overexpression of the Nmnat tide adenylyltransferase (Nmnat) has attracted interest because of enzyme, which adenylates NMN, was not able to change NAD+ level, a possible role in delay of axonal degeneration, a process by which consistent with adenylation being a faster metabolic step in the neurons lose axons in response to injury. Nmnat is a dual function salvage pathway (Revollo et al., 2004). Accordingly, dynamic adenylating enzyme which can convert NaMN and NMN to NaAD changes in Nampt level and activity are predicted to regulate and NAD respectively. In humans, there are three isoforms, intracellular NAD+ level and downstream cellular functions. Nmnat1, 2, 3, which recognize both NMN and NaMN with similar Nampt is induced by calorie restriction in liver (Yang et al., efficiency (Sorci et al., 2007). The isoforms have different cellular 2007), and by glucose restriction in satellite muscle cells (Fulco localizations; Nmnat1 is nuclear; Nmnat2 is Golgi localized and et al., 2008), implying that nutritional influences regulate NAD+ Nmnat3 is mitochondrial (Schweiger et al., 2001; Araki et al., 2004; level. Interestingly, calorie restriction provides anti-aging effects in Berger et al., 2005). Nmnat1 protects against programmed axonal yeasts, flies, worms and even primates. The hypothesis that NAD+ degeneration caused by injury, oxidative stress or mutations in concentration increases in mammals are a crucial component in primary cell cultures and animal models (Sasaki et al., 2006, 2009; the effect of calorie restriction on longevity is now under active Press and Milbrandt, 2008). Strikingly, NAD+and NAD+ precursors investigation by multiple laboratories. Nampt overexpression can also provide protection against axonal degeneration, suggest- increases mitochondrial NAD+ concentrations, and makes cells ing that the effect of Nmnat1 is achieved through a function more resistant to apoptosis, suggesting that Nampt and NAD+ involving NAD+ synthesis (Sasaki et al., 2006, 2009; Press and concentrations increase cell and tissue survival (Yang et al., 2007). Milbrandt, 2008; Wang and He, 2009; Yahata et al., 2009). Also of interest is the recent finding that NAD+ is subject to The possible involvement of NAD+ biosynthesis in delayed axonal circadian oscillation, a 24-h central and peripheral oscillation degeneration was appreciated by the discovery of the Wallerian involved in maintaining organismal homeostasis. Two indepen- degeneration slow (Wlds) mouse 20 years ago, in which the injured dent laboratories demonstrated that there exists a rhythmic axon maintains its structure much longer than wild type (Lunn et al., oscillation of NAD+ levels in mammalian cells, caused by the 1989). This slow degeneration phenotype was linked to a chimeric oscillation of RNA and protein levels of Nampt in a light–dark cycle gene which contained a full-length chimeric gene composed of (Nakahata et al., 2009; Ramsey et al., 2009). Nmnat1 and a partial fusion to a ubiquitin ligase Ube4b. Numerous Nampt is a very unusual enzyme since it appears to be actively studies have shown that the protection function needs Nmnat1 secreted into the plasma by certain types of cells. Imai and co- enzymatic activity as the catalytically inactive Nmnat1 and Wlds workers have suggested that extracellular Nampt (eNampt) is proteins have reduced axonal protective properties, as reported by secreted through ‘‘a non-classical secretory pathway’’ by multiple independent laboratories (Araki et al., 2004; Conforti et al., adipocytes independent of cell lysis or cell death (Revollo 2007; Jia et al., 2007; Avery et al., 2009; Conforti et al., 2009). et al., 2007). eNampt appears to be catalytically active and has However, not all studies have been supportive. In one example, similar catalytic activity to that of intracellular Nampt (iNampt) overexpression of Nmnat1 in a stably transfected Neuro2A cell line (Revollo et al., 2007). Nampt+/ mice have interesting metabolic did not delay a vincristine-induced degeneration as well as the full and endocrine phenotypes. Female heterozygotes, but not males, Wlds gene product (Watanabe et al., 2007). Further contradictory have decreased glucose tolerance that is attributed to defective data have emerged from a study in which Nmnat1 was found to have glucose-induced insulin release from pancreatic beta cells a chaperone function, which is independent of its adenylation (Revollo et al., 2007). These defects are corrected by administer- activity. Two enzymatically inactive Drosophila Nmnat proteins ing NMN (Revollo et al., 2007), and NMN has been determined to Nmnat-H30A, in which the catalytic center was mutated, and increase cellular NAD+ (Revollo et al., 2007). NMN is found as a Nmnat-WR, in which two key residues required for substrate circulating blood metabolite as well (Revollo et al., 2007). Since binding were mutated, were reported to confer neuroprotection, and eNampt can synthesize NMN with appropriate substrates, it appeared to rescue a degeneration phenotype caused by deletion of seems possible that NMN could be synthesized outside cells, in a the wildtype gene (Zhai et al., 2006). Structural superpositions have component of NAD+ metabolism that is extracellular (Revollo revealed that Nmnat1 and Nmnat3 share some structural similarity et al., 2007; Imai, 2009). CD38 is also extracellular and human with chaperones UspA and Hsp100 (Zhai et al., 2008). A chaperone CD38 can metabolize NAD+ to ADPR and nicotinamide (Sauve function is proposed to allow Nmnat1 to interact with other proteins et al., 1998). CD38 is also efficient at degrading NMN to important for neuronal protection (Zhai et al., 2006, 2008). These nicotinamide and ribose-5-phosphate (Sauve et al., 1998). results highlight the possibility of additional explanations that may Importantly, a number of blood-born immune cells such as B be involved in Wlds effects in neuroprotection, besides the proposed cells and neutrophils are enriched in CD38. The question of how metabolic effects. important NMN might be as a blood metabolite is still being Quinolinate phosphoribosyltransferase (Qprtase) is an NAD+ evaluated, but the existence of enzymes that can make it as well biosynthetic gene that is able to catalyze the conversion of as degrade it in the blood suggests an interplay of competing quinolinate to NaMN. Although a precursor to NAD+, the factors that regulate its role. accumulation of quinolinate in brain can cause excitotoxicity Nampt has been shown to have immune system functions. For and can damage neurons (Schwarcz et al., 1983, 1986; Beal et al., example, Nampt is induced by inflammatory stimuli and can be 1988). Elevated quinolinate levels are associated with Hunting- synthesized when immune cells are exposed to endotoxin/LPS, ton’s disease, epilepsy and Alzheimer’s disease (Schwarcz et al., TNF-a, IL-1b, IL-6, IL-8, G-CSF, oxidized low-density lipoprotein 1983, 1986; Beal et al., 1988). Qprtase is the only known way to (LDL) and mechanical stretch (Luk et al., 2008). In sepsis, a very metabolize quinolinate suggesting that endogenous levels of this 294 P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 enzyme could provide the dual function of reducing quinolinate substrates reported so far, many of them transcription factors that levels and increasing NAD+ levels in cells. Interestingly, increasing regulate biologically significant processes (Table 2). SirT1 is NAD+ concentrations in neuronal cells has been suggested to have located in the nucleus primarily but can also be found in cytoplasm beneficial mitigating effects in the prevention of Alzheimer’s (Michan and Sinclair, 2007). In the nucleus SirT1 can deacetylate neuropathology (Qin et al., 2006) suggesting that manipulations of histone H1 at Lys9 and 26, H3 Lys14, and H4 Lys 16 (Vaquero et al., NAD+ metabolism may provide benefits in multiple neurodegen- 2004). SIRT1 deacetylates the histone methyl-transferase erative conditions. SUV29H1 and deacetylated SUV29H1 is more effective at installing the H3K9me3 modification at chromatin. These findings suggest a 9. NAD+ utilization enzymes and programmatic human aging coordination of activities of histone deacetylation and histone methylation associated with stabilization of heterochromatin in Among the enzymes utilizing NAD+ as a substrate, the sirtuins mammalian cells (Vaquero et al., 2007). appear to regulate a wide range of cellular and physiological Besides histones, SirT1 has diverse deacetylation targets. p53, activities and they have attracted a great deal of recent and intense an important transcription factor regulating cell cycle and interest. The sirtuin deacetylase activity closely links NAD+ with a apoptosis, is a substrate of SirT1. SirT1 has been shown to variety of cellular functions that are important for regulating deacetylate multiple lysine residues of mouse p53 including development and longevity. These enzymes are activated during Lys317, Lys370, and Lys379. SirT1-dependent deacetylation of p53 calorie restriction and also have been linked to the lifespan prevents apoptosis of cells upon DNA damage or oxidative stress extending effects of calorie restriction in multiple organisms. (Vaziri et al., 2001). PGC1-a is a key transcriptional coactivator and Because these enzymes require NAD+ for their function, they have regulates glucose metabolism in liver and is important for been suggested to link NAD+ metabolic pathways with cellular, induction of mitochondrial biogenesis in skeletal muscle (Bordone tissue and adaptive phenomena important to aging. The sirtuins et al., 2006; Rodgers et al., 2008). Another important target for regulate the activities of a number of distinct pathways, including SirT1 activity is peroxisome proliferator-activated receptor-g stress resistance, mitochondrial biogenesis, adipogenesis, prolif- (PPAR-g). SIRT1 represses PPAR-g and can decrease adipogenesis eration, DNA repair, metabolism and autophagy that collectively in adipocytes. SIRT1 also increases lipolysis in white adipose tissue are thought to be involved in sustaining healthy cell types and probably by similar mechanisms (Picard et al., 2004). To date, the delaying age-related disease states. Since these processes are seen exact mechanism of repression of PPAR-g remains unclear but it to erode over time, and with age, the role of sirtuins in maintaining does require SIRT1 catalytic function (Picard et al., 2004). NF-kB these capacities suggests them to be master regulators of organism (nuclear factor kappa-light-chain-enhancer of activated B cells) is a health, that drive a program of robustness that counteracts or protein regulating immune responses. Yeung et al. (2004) delays aging in a broad multi-faceted way. demonstrated that SirT1 represses the transcriptional activities of NF-kB by deacetylating RelA/p65 subunit of NF-kB at lysine 310. 9.1. SirT1 Thus, SirT1 is a modulator of inflammatory responses. Recent studies also show that liver X receptor (LXR) protein is a Among mammalian sirtuin family members, SirT1 has been the deacetylation target of SirT1 (Li et al., 2007). LXRs are members most comprehensively studied member. It has the most cellular of the nuclear receptor superfamily that can serve as metabolic

Table 2 Cellular functions and targets/interaction partners of mammalian sirtuins (Note: *interaction partner).

Sirt# Telomere DNA Stress Apoptosis cell Metabolism References chromatin/ damage resistance cycle Insulin secretion/ Cholesterol Fatty acid ATP Urea cycle transcription repair gluconeogenesis metabolism oxidation synthesis

SirT1 H1K9 H1K26 Ku70 DBC1* p53 Ku70/Bax PGC1a LXRa PPARg AceCS1 Jeong et al. (2007);

H3K14 H4K16 TAFI68 FOXO4 FOXO FOXO1 PGC1a Kahyo et al. (2008); WRN NF-kB p300 ACCa* Kim et al. (2008); SUV39H1 MEF2D Law et al. (2009); p70 Li et al. (2007); Smad7 Michan et al. (2007); Vaquero et al. (2007)

SirT2 H4K16 FOXO3a Tubulin FOXO1 Jing et al. (2007); North et al. (2003); Wang et al. (2007)

SirT3 Hsp70* Ku70 AceCS2 Law et al. (2009); GDH Lombard et al. (2007); IDH Schwer et al. (2006); ATP5A* Sundaresan et al. (2008)

SirT4 Histone GDH, ANT2* Ahuja et al. (2007); ANT3* Haigis et al. (2006)

SirT5 Cytochrome c CPS1 Nakagawa et al. (2009); Schlicker et al. (2008)

SirT6 H3K9 NF-kB Michishita et al. (2008); TNF Van Gool et al. (2009)

SirT7 RNA Pol I* p53, Ford et al. (2006); UBF* Grob et al. (2009); Vakhrusheva et al. (2008) P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 295 sensors in their ability to directly bind to oxysterols, which can inflammatory diseases. As a nuclear protein, SirT7 was reported to activate their transcriptional activities. The downstream effects of interact with RNA polymerase I (Pol I) and histones (Ford et al., LXRs are in regulation of reverse cholesterol transport and they can 2006). SirT7 increases stress resistance of cardiomyocytes and also alter systemic lipid profiles, making them important possible prevents apoptosis and inflammatory cardiomyopathy in mice targets for therapeutics. The deacetylation of SirT1 activates LXRs (Vakhrusheva et al., 2008). This sirtuin may have impact on cell and SIRT1/ mice have lower HDL cholesterol and defective viability in mammals (Ford et al., 2006) and tissue integrity during responses to an LXR agonist, indicating that SIRT1 is a probable in aging (Vakhrusheva et al., 2008). vivo regulator of LXR function (Li et al., 2007). A putative modulator of SIRT1 is Deleted in Breast Cancer-1 10. Strategies for longevity by regulating NAD metabolism in (DBC1) which can interact directly with SIRT1 thereby inhibiting its cells catalytic functions as determined by studies in vitro and in vivo (Kim et al., 2008; Zhao et al., 2008). DBC1 binds directly to the catalytic Modulation of NAD+ metabolism is an approach to delay aging domain of SirT1. As DBC1 is implicated in breast, lung and colon and prevent age-related diseases. It is proven effective in treatment cancer, the interaction of DBC1 with SirT1 suggests the involvement of cardiovascular disease with nicotinic acid. However, NAD+ of SirT1 in cancer, one of the dominant diseases of aging. precursors like NAM, NA or nicotinamide riboside could also be effective for the treatment of varieties of neurodegenerative 9.2. SirT2-7 diseases, lipid disorders, and cardiovascular diseases. Nampt, a key enzyme in NAD synthesis, is currently seen as an Sirtuins SirT2 to SirT7 are also implicated in maintenance of interesting target for new therapy. FK866, a potent inhibitor of cellular functions. SirT2 is largely cytoplasmic where tubulin has Nampt, is being evaluated to treat cancer and inflammatory diseases. been suggested to be a substrate (North et al., 2003) although it can Treatment of HEPG2 carcinoma cells with FK866 (IC50 = 1–3 nM) occasionally reside in the nucleus, where it can deacetylate histone caused a delayed apoptosis associated with a slow depletion of H4 Lys16 (Michan and Sinclair, 2007). SIRT2 can associate with cellular NAD+, suggesting the potential efficacy of a therapeutic FOXO1 and deacetylate this transcription factor, thereby causing approach to cancer involving interference with NAD+ homeostasis inhibition of pre-adipocyte differentiation to adipocytes (Jing et al., (Hasmann and Schemainda, 2003). The inhibition of FK866 of Nampt 2007). SIRT2 also modulates stress resistance through FOXO3a decreased intracellularNAD and circulatingTNFa, mitigating disease (Wang et al., 2007), indicating that SirT2 has functions in lipid severityin mice duringcollagen-induced arthritis (Bussoetal.,2008). metabolism and cellular response to oxidative stress. Recently another chemical CHS-8282 was discovered to be a potent

SirT3, SirT4 and SirT5 are mitochondria proteins (See Table 2 for inhibitor of Nampt with Ki of 2.6 nM, and it can efficiently deplete a list of interaction partners for these sirtuins). So far, three targets cellular NAD+ level and kill cancer cells (Olesen et al., 2008). of SirT3 have been reported, acetyl-coenzymes A synthetase 2 Nmnat, an enzyme in NAD synthesis, is another target of drug (AcCS2) (Schwer et al., 2006), Ku-70 (Sundaresan et al., 2008) and design. Overexpression of Nmnat has been shown to delay axonal glutamate dehydrogenase (GDH) (Lombard et al., 2007). Law et al. degeneration (Press and Milbrandt, 2008; Yahata et al., 2009). (2009) showed that ATP5A and HSP70 are interacting partners of Modulation of the activity of this enzyme is potentially useful in SirT3. ATP5A, is a protein subunit of the ATP synthase residing in axonal protection. It has been reported that 50 mM epigalloca- mitochondria, and HSP70 is a chaperone protein that co-complexes techin gallate (EGCG) can activate human Nmnat in vitro. It with other cellular proteins, and is implicated in both normal and doubles the NAD+ synthesis activity of Nmnat2, while the effect on abnormal cell physiology. The interaction of ATP5A and HSP70 Nmnat3 was 42% whereas Nmnat 1 was not affected (Berger et al., with SirT3 suggests that SirT3 has functions in energy metabolism 2005). In contrast to EGCG, gallotannin is a potent inhibitor of and cell resistance to stress. Deacetylation activity has not been Nmnat, with 10 mM, 55 mM and 2 mMofIC50 respectively for reported for SirT4. It was demonstrated to inhibit glutamate Nmnat1, Nmnat 2, and Nmnat 3 (Berger et al., 2005). dehydrogenase activity by ADP-ribosylation, and it is now In addition to regulating the activity of enzymes involved with considered likely that SirT4 regulates insulin secretion (Haigis NAD+ synthesis,modulatingtheactivity of sirtuin enzymes is another et al., 2006). In another study, SirT3 and SirT4 were necessary for approach to improving health and delaying aging. Resveratrol has protection of cells under conditions of genotoxic stress (Yang et al., been shown to inhibit carcinogenesis, prevent cardiovascular 2007), suggesting SirT3 and SirT4 are important for maintaining disease, protect against brain damage following cerebral ischaemia, mitochondrial integrity and cell survival. Similar with SirT4, there and enhance stress resistance to oxidative stress (Baur and Sinclair, are few targets that have been identified for SirT5. SirT5 can 2006). Treatment with resveratrol increases the lifespan of several deacetylate cytochrome c, a mitochondrial protein important for different species and resveratrol was identified as a potent activator electron transfer and for apoptosis (Schlicker et al., 2008). SirT5 of SirT1 (Howitz et al., 2003). Interestingly, Milne et al. discovered also deacetylates carbamoyl phosphate synthetase 1 (CPS1), which SRT2183, which activates SirT1 1,000-fold more potently than regulates the urea cycle (Nakagawa et al., 2009), indicating a direct resveratrol. The administration of this compound in mice as well as connection of SirT5 with metabolism. Zucker fa/fa rats increased insulin sensitivity and lowered plasma SirT6 and SirT7 are nuclear proteins (See Table 2 for a list of glucose (Milne et al., 2007), providing proof of concept that SIRT1 interaction partners of these sirtuins). SirT6 deficiency causes the activation could lead to treatments of type 2 diabetes. Although this most striking phenotype among all the sirtuin knockouts. SirT6 work has been contradicted by another laboratory (Pacholec et al., knock out cells grow slowly and show more sensitivity to genotoxic 2010), the therapeutic potential of sirtuin activation for treating the damage than wild type; SirT6 deficient mice do not survive past 4 diseases of aging is still very attractive given the observed potency of weeks and suffer a multitude of apparent abnormalities, including sirtuin-mediated biological effects. loss of subcutaneous fat and metabolic disorders such as progressive hypoglycemia (Mostoslavsky et al., 2006), indicating SirT6 may take 11. Conclusions part in diverse physiologic functions. Indeed, Michishita et al. (2008) demonstrated that SirT6 deacetylated histone H3 at lysine 9 and This review has examined a multitude of biological effects modulated telomere chromatin. Recently, SirT6 was reported to associated with NAD(P)+ metabolism and the use of agents like regulate tumor necrosis factor (TNF) protein synthesis at a post- nicotinamide and nicotinic acid in a pharmacological context. It is transcriptional step (Van Gool et al., 2009), connecting SirT6 with clear that because of the diverse influences of NAD(P)+ and associated 296 P. Xu, A.A. Sauve / Mechanisms of Ageing and Development 131 (2010) 287–298 metabolites on cells and tissues that this review cannot fully explore Cherednichenko, G., Zima, A.V., Feng, W., Schaefer, S., Blatter, L.A., Pessah, I.N., 2004. NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium- the full biological consequences of these agents on cell, tissue and induced calcium release. Circ. Res. 94, 478–486. organism aging. It is the authors’ view that the medical strategies that Chong, Z.Z., Lin, S.H., Maiese, K., 2004. The NAD+ precursor nicotinamide governs will ultimately emerge centered on manipulating NAD+ metabolism neuronal survival during oxidative stress through protein kinase B coupled to FOXO3a and mitochondrial membrane potential. J. Cereb. Blood Flow Metab. 24, are still in a relatively early period of development and that 728–743. opportunities still exist for treating disease, particularly in acute Conforti, L., Fang, G., Beirowski, B., Wang, M.S., Sorci, L., Asress, S., Adalbert, R., Silva, trauma, neurodegenerative conditions and possibly in cancer A., Bridge, K., Huang, X.P., Magni, G., Glass, J.D., Coleman, M.P., 2007. NAD(+) and prevention. The period of the present features a new appreciation axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration. Cell Death Differ. 14, 116–127. for an old metabolism, withconnections emerging that could provide Conforti, L., Wilbrey, A., Morreale, G., Janeckova, L., Beirowski, B., Adalbert, R., new approaches to treating some of the most common disorders of Mazzola, F., Di Stefano, M., Hartley, R., Babetto, E., Smith, T., Gilley, J., Billington, human experience: those associated with age. R.A., Genazzani, A.A., Ribchester, R.R., Magni, G., Coleman, M., 2009. Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice. J. Cell Biol. 184, 491–500. Acknowledgments Corda, D., Di Girolamo, M., 2003. Functional aspects of protein mono-ADP-ribosyla- tion. EMBO J. 22, 1953–1958. El Hassani, R.A., Benfares, N., Caillou, B., Talbot, M., Sabourin, J.C., Belotte, V., The author has received support from NIH grant R01 DK Morand, S., Gnidehou, S., Agnandji, D., Ohayon, R., Kaniewski, J., Noel-Hudson, 074366, NY State Spinal Cord Injury Board C023832 and AAS also M.S., Bidart, J.M., Schlumberger, M., Virion, A., Dupuy, C., 2005. Dual oxidase2 is expressed all along the digestive tract. Am. J. Physiol. Gastrointest. Liver Physiol. acknowledges support of the Ellison Medical Foundation New 288, G933–G942. Scholar Award in Aging (2007–2011). AAS is a Scientific Advisory Finley, L.W., Haigis, M.C., 2009. The coordination of nuclear and mitochondrial Board Member for Sirtris Pharmaceuticals (A GlaxoSmithKline communication during aging and calorie restriction. Age. Res. Rev. 8, 173– Company) and has financial interests related to novel precursors of 188. Fjeld, C.C., Birdsong, W.T., Goodman, R.H., 2003. Differential binding of NAD(+) and + NAD and sirtuins. 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