Ruthenium (II)-derived organometallic compounds induce cytostatic and cytotoxic effects on mammalian cancer cell lines through p53-dependent and p53-independent mechanisms.

NAD+ and Vitamin B3: From Metabolism to Therapies . Anthony A. Sauve . Department of Pharmacology, Weill Medical College of Cornell University, New York, New York . Address correspondence to: Anthony A. Sauve, Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. E-mail: aas2004@med.cornell.edu Abstract The role of NAD+ metabolism in health and disease is of increased interest as the use of niacin (nicotinic acid) has emerged as a major therapy for treatment of hyperlipidemias and with the recognition that nicotinamide can protect tissues and NAD+ metabolism in a variety of disease states, including ischemia/reperfusion. In addition, a growing body of evidence supports the view that NAD+ metabolism regulates important biological effects, including lifespan. NAD+ exerts potent effects through the poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases, and the recently characterized sirtuin enzymes. These enzymes catalyze protein modifications, such as ADP-ribosylation and deacetylation, leading to changes in protein function. These enzymes regulate apoptosis, DNA repair, stress resistance, metabolism, and endocrine signaling, suggesting that these enzymes and/or NAD+ metabolism could be targeted for therapeutic benefit. This review considers current knowledge of NAD+ metabolism in humans and microbes, including new insights into mechanisms that regulate NAD+ biosynthetic pathways, current use of nicotinamide and nicotinic acid as pharmacological agents, and opportunities for drug design that are directed at modulation of NAD+ biosynthesis for treatment of human disorders and infections. Vitamin B3 (nicotinamide and nicotinic acid) is essential to all living cells. Vitamin B3 is biosynthetically converted to nicotinamide adenine dinucleotide (Fig. 1, NAD+), a versatile acceptor of hydride equivalents to form the reduced dinucleotide, NADH. The phosphorylated forms of the nicotinamide dinucleotides (NADP/NADPH) perform similar chemical functions within cells, although these are generally used in biosynthetic pathways and in cell protection mechanisms against reactive oxygen species. NAD(P)H provides reducing equivalents for cellular biochemistry and energy metabolism. Within eukaryotic cells, energy metabolism is largely mediated by electron transport chains found within the mitochondrion, and NADH plays a vital role in furnishing reducing equivalents to fuel oxidative phosphorylation. Thus, cellular energy metabolism is substantially mediated by vitamin B3-derived cofactors, and a large fraction of anabolic and catabolic pathways incorporates these cofactors within them. Nicotinamide dinucleotides also react as electrophiles to transfer the ADP-ribose (ADPR) moiety to a nucleophile. ADPR transfer to small nucleophiles forms ADPR (nucleophile/water), cyclic-ADPR (nucleophile/N1 adenine), and nicotinic acid adenine dinucleotide-phosphate (derived from NADP+, nucleophile/nicotinic acid). These compounds have been shown to regulate processes, such as channel opening and calcium release (Pollak et al., 2007). Furthermore, ADPR transfer modifies protein nucleophilic side chains (Hassa et al., 2006). The ADPR-transfer enzymes fall into three distinct families, the mono-ADP-ribosyltransferases (Hassa et al., 2006), the poly(ADPribosyl) polymerases (PARPs) (Virag and Szabo, 2002; Hassa et al., 2006), and the sirtuins (Sauve et al., 2006). The sirtuins transiently ADP-ribosylate acetyllysines of proteins causing protein deacetylation, releasing an acetyl transfer product, acetyl-ADPribose (Sauve et al., 2006). However, some sirtuins are not deacetylases and appear to catalyze protein ADPR transfer in the ordinary sense. Collectively, these enzymes regulate numerous signaling pathways and respond to changes in NAD+ metabolism. They exert profound influences on apoptosis, metabolism, proliferation, DNA repair, senescence, endocrine signaling, and lifespan (Guarente, 2006). In addition to being nutrients, nicotinamide and nicotinic acid are clinically applied pharmacological agents. Nicotinic acid is administered in large doses to lower serum lipids and cholesterol (Schachter, 2005). Nicotinamide has recently been used for prevention of type 1 diabetes (Gale et al., 2004) and is being evaluated for prevention of neurotoxicity and for treatment of ischemia. High-dose nicotinic acid and nicotinamide enter metabolism and increase NAD+ pools but also bind to proteins in cells to elicit their effects. For example, nicotinic acid has a cognate receptor, which is implicated in some of its antilipid effects (Soudijn et al., 2007). Nicotinamide inhibits PARP, leading to decreased NAD+ turnover, to provide beneficial effects in degenerative states where PARP activity is overactivated (Virag and Szabo, 2002). The multiplicity of functions attributed to nicotinamide, nicotinic acid, and the dinucleotides, as well as the linkage of powerful signaling components to NAD+ metabolism via the ADPR-transferases, has provided a surge of interest in the therapeutic possibilities inherent to targeting NAD+ metabolism for therapy (see Table 1). NAD+ metabolism has been a topic of several recent reviews (Magni et al., 2004a; Yang and Sauve, 2006; Yang et al., 2006; Belenky et al., 2007; Revollo et al., 2007). Herein, we survey knowledge of NAD+ metabolism in humans and microbes. We examine the properties of nicotinamide and nicotinic acid as nutrients and as pharmacologic agents. We consider other precursors of NAD+ distinct from nicotinamide and nicotinic acid, such as nicotinamide riboside. Finally, we explore current and potential applications of therapeutics that target NAD+ metabolism and consider how future therapies could develop. NAD+ Metabolism The biosynthetic and recycling metabolism of vitamin B3 converges on synthesis of the dinucleotides. Nicotinamide and nicotinic acid are synthesized as moieties within nucleotides or dinucleotides before they are available as free species within cells. Synthesis of NAD+ is achieved via both recycling and de novo pathways in most microbes and in human cells. Database searches as well as biochemical studies indicate that the de novo pathways of microbes and humans form the biosynthetic product, NAMN, via the decarboxylative coupling of PRPP and quinolinic acid (QA) (Kurnasov et al., 2003). De Novo Pathways in Humans Microbes and Bacteria The de novo pathway in most bacteria and plants starts from aspartate. The aspartate pathway is anaerobic, and molecular oxygen is not required (Kurnasov et al., 2003). The reaction of aspartate and dihydroxyacetone-phosphate leads to efficient synthesis of QA, catalyzed by aspartate oxidase and QA synthase (Fig. 2). Alternatively, yeast, humans, and some bacterial microbes make QA via an aerobic pathway from tryptophan (Kurnasov et al., 2003). Molecular oxygen as a substrate oxidizes tryptophan to downstream metabolites of the kynurenine pathway, 3-hydroxyanthranilate, and finally QA (Fig. 3) (Kurnasov et al., 2003). The respective de novo pathways are important sources of vitamin B3 in most bacteria and in humans. In humans, 1 of 67 mg of tryptophan eventually ends up as nicotinamide if another B3 source is not available in the diet (Fukuwatari et al., 2004). Recycling in Bacteria In addition to the de novo pathways, most organisms have recycling pathways capable of synthesizing NAD+ from B3 obtained via diet or obtained metabolically from decomposition of NAD+ or related nucleotides in cells. Within bacteria, decomposition of NAD+ can occur via ADPR transfer to form nicotinamide. Bacteria encode sirtuins, and these enzymes cleave nicotinamide from NAD+ (Blander and Guarente, 2004). Once formed, nicotinamide is converted to nicotinic acid with nicotinamidase. Hydrolysis of nicotinamide is an obligate step of recycling in most bacteria, and a nicotinic acid phosphoribosyltransferase couples nicotinic acid to PRPP to form NAMN (Fig. 2). The conversions of nicotinic acid to NAD+ are known as the Preiss-Handler pathway. NAMN adenylyltransferase catalyzes formation of nicotinic acid adenine dinucleotide (Fig. 2), and NAD+ synthetase completes synthesis of NAD+ by converting the carboxylate to the amide (Fig. 2). Most plants and eukaryotes (with the exception of mammals) catalyze resynthesis of NAD+ from nicotinamide, similar to bacteria, via obligate breakdown of nicotinamide to nicotinic acid followed by synthesis NAMN (Kurnasov et al., 2003). A second major pathway of NAD+ decomposition in bacteria involves breakage of the phosphate anhydride bond to form NMN and AMP (Foster et al., 1979). This latter reaction is catalyzed by pyrophosphatases as well as bacterial NAD+-dependent ligases (Fig. 2) (Wilkinson et al., 2001). NMN is resynthesized into NAD+ via NMN adenylate transferases or further converted to nicotinamide. Recycling in Humans In humans, the dominant pathways that decompose NAD+ are catalyzed by ADPribosyltransferases. Studies show that NAD+ has a half-life of 10 h in liver (Ijichi et al., 1966). Nicotinamide formed upon NAD+ turnover is not hydrolyzed but is coupled directly to PRPP to form NMN by nicotinamide phosphoribosyltransferase (nampt, also known as PBEF) (Fig. 3). This enzyme activity is found only in mammals and some classes of bacteria. It is distinct from the enzyme that couples nicotinic acid to PRPP (Yang et al., 2006; Revollo et al., 2007). Although an activity that converted nicotinamide to NAD+ independently of nicotinic acid was indicated for sometime, the enzyme was only recently identified (Rongvaux et al., 2002). The human genome also encodes a Preiss-Handler pathway, which converts nicotinic acid to NAD+ via NAMN and nicotinic acid adenine dinucleotide (Fig. 3). Humans use both nicotinic acid and nicotinamide recycling to synthesize NAD+ but utilize different pathways to achieve this. An enzyme in common between the pathways is the adenylation enzyme nmnat (Fig. 3). This enzyme has three isoforms in humans (nmnat1, nmnat-2, and nmnat-3). nmnat-1 is localized to nuclei as determined by immunofluorescence and was recently shown to stimulate PARP-1 (Schweiger et al., 2001; Berger et al., 2007). nmnat-2 is in Golgi, and nmnat-3 is in mitochondria (Berger et al., 2005). All isoforms exhibit dual specificity for both NAMN and NMN as a substrate (Raffaelli et al., 2002; Magni et al., 2004b; Berger et al., 2005). nmnat-1 is the most proficient catalyst as determined by catalytic velocity (Vmax) and efficiency (Vmax/Km). The distribution of NAD in cells and the locations of NAD+ synthesis have recently received new consideration. Implied from the fact that nmnat activity is required to complete all salvage and de novo pathways of NAD+ biosynthesis, mammalian cell NAD+ synthesis is compartmentalized. Indeed, there are stable NAD+ pools within distinct subcellular compartments. Evidence to support this idea is available from cell fractionation studies that confirm that mitochondria maintain relatively high NAD+ concentrations and that mitochondrial NAD+ does not readily leak across the inner mitochondrial membrane (Di Lisa and Ziegler, 2001). On the other hand, the majority of cytosolic NAD+ is probably made within the nucleus of cells and then redistributed to the cytosol by passive diffusion through nuclear pores (Berger et al., 2005). It has been asserted that the relative distribution of total NAD+ in cells is largely mitochondrial (Di Lisa and Ziegler, 2001; Di Lisa et al., 2001), although this premise derives mostly from data obtained on myocytes (Di Lisa et al., 2001), which are rich in mitochondria. In contrast, in hepatocytes, 30 to 40% of total cellular NAD+ is mitochondrial, whereas the majority is cytosolic (Tischler et al., 1977). On the extreme, erythrocytes have reasonably high concentrations of NAD+ but have no mitochondria at all. It is apparent that relative NAD+ contents in cellular compartments are probably cell- and tissue-specific. It is important to point out that, although whole-cell NAD+, nicotinamide, and nicotinic acid measurements can monitor NAD+ metabolism at a gross level, knowledge of the metabolite concentrations in subcellular compartments, such as the nucleus, cytoplasm, and mitochondria, is crucial to gauge how NAD+ metabolism affects sirtuin and PARP functions at different cellular loci. Technical and experimental progress in this area is needed before it will be possible to describe just how NAD+ metabolism is coupled to NAD+-dependent signaling processes. Pathways Involving Nicotinamide Riboside Recently, a recycling pathway independent of nicotinamide and nicotinic acid that forms NAD+, was found to be broadly conserved in bacteria, yeast, and mammals (Bieganowski and Brenner, 2004). The pathway leads from the metabolite nicotinamide riboside (NR), the dephosphorylated form of NMN. A highly biologically conserved nicotinamide riboside kinase is able to use NR as a substrate and can convert NR to NMN in cells (Bieganowski and Brenner, 2004). This activity allows NR to enter NAD+ metabolism via NMN and then to NAD+. Thus, NR is converted to NAD+ in only two metabolic steps (Figs. 2 and 3) (Bieganowski and Brenner, 2004). Yeast deficient in de novo or B3-recycling pathways but retaining an intact nmnat activity survive with NR as their only source of B3, indicating that yeast can efficiently salvage this nucleoside and synthesize adequate amounts of NAD+ via this pathway (Bieganowski and Brenner, 2004). In humans, two isoforms of the kinase (Nrk1 and Nrk2) have been cloned, although little is known about the biochemical properties of these enzymes. The Role of NAD in Energy Metabolism and Oxidation Processes Vitamin B3, in the form of the dinucleotides, plays a central role in energy metabolism, in oxidative phosphorylation, and in the redistribution of electron equivalents from catabolism redirected toward biosynthetic pathways. NADH, formed from glycolysis and from the trichloroacetic acid cycle, reacts at the point of Complex I, the NADH/coenzyme Q reductase of the mitochondrial electron transport chain (Pollak et al., 2007). Each mole of NADH consumed by the mitochondria can furnish energy for the formation of three moles of ATP from 3 mol of ADP (Pollak et al., 2007). NADH formation in cells is tightly regulated and typically represents approximately 10% of total cellular NADH and NAD+ content (Williamson et al., 1967). NADH is highly depleted in the cytosol, where it typically represents less than 1% of the combined total of NADH and NAD+ that is not in complexation with proteins (Williamson et al., 1967). In the mitochondria, NADH represents approximately 15% of the dinucleotide content uncomplexed to proteins (Williamson et al., 1967; Tischler et al., 1977). The uncomplexed dinucleotides represent the cellular pool able to interact with unliganded proteins in cells (Williamson et al., 1967). As such, the relatively low concentrations of NADH available in uncomplexed form, particularly in the cytosol, suggest that direct NADH interactions with enzymes such as sirtuins (such as nuclear SIRT1) may be insufficient to explain changes in activity attributed to NADH/NAD+ ratio (Guarente, 2006). The phosphorylated dinucleotide NADP+ in the reduced form plays important roles in biosynthesis. In contrast to NADH/NAD+, the uncomplexed and total (complexed and uncomplexed) NADPH/NADP ratios in cells are maintained high in the cytosolic and mitochondrial compartments (Tischler et al., 1977). This appears to stem from the importance of NADPH to biosynthesis and because NADPH provides several cell protective functions. For instance, NADPH is an important cofactor for P450 enzymes that detoxify xenobiotics (Pollak et al., 2007). In oxidative defense, NADPH acts as a terminal reductant for glutathione reductase, which maintains reduced glutathione. Enhanced formation of NADPH via up-regulation of glucose-6-phosphate dehydrogenase appears to increase reduced glutathione concentrations. Conversely, deletion of glucose-6-phosphate dehydrogenase causes increased sensitivity of cells to oxidative stress (Pollak et al., 2007). Finally, NADPH serves as a substrate for NADPH oxidase, which generates peroxides for release in oxidative burst processes of the immune system (Pollak et al., 2007). Clinical Manifestations of Niacin Deficiency In spite of the complexity and diversity of effects attributed to niacin in metabolism, significant redundancy in the NAD+-biosynthetic pathways of humans make modern vitamin B3 deficiency rare in industrialized nations (Graham, 1993). However, poor diets, alcoholism, AIDS, and other diseases can cause niacin deficiency or pellagra. Symptoms of pellagra include dermatitis, dementia, and diarrhea (Revollo et al., 2007). Niacin deficiency is also associated with an increased risk of cancer (Kirkland, 2003) and has been shown to increase toxicity caused by reactive oxygen species (Pollak et al., 2007). Prospects for Drugs Targeted to Inhibition of NAD+ Biosynthesis Antimicrobials Opportunities exist for development of antimicrobial agents that target NAD+ metabolism. Because most pathogenic bacteria have a unique aspartate-based pathway for NAD+ biosynthesis, it would seem logical that drugs could be targeted to this pathway. Bacterial mutations in the aspartate pathway to QA restrict growth of bacteria if the growth medium is deficient in a source of preformed nicotinamide or nicotinic acid (including free pyridines, nucleosides, nucleotides, or dinucleotides) (Foster et al., 1979). It is problematic that nicotinamide is relatively abundant in mammalian tissues (Qin et al., 2006; Yang and Sauve, 2006), and it is not certain how an inhibition of upstream steps of NAD+ biosynthesis would affect infectivity or virulence if recycling pathways in bacteria can biochemically salvage nicotinamide and nicotinic acid from the host. The effect of targeting this pathway on bacterial growth in a mammalian host is still undetermined. Targeting nicotinamide/nicotinic acid recycling for antibiotics may be effective because some human pathogens (e.g., Borrelia burgdorferei, Plasmodium falciparum) do not seem to encode a de novo NAD+-biosynthetic pathway. In these cases, salvage of host nicotinamide and nicotinic acid pools to complete NAD+ biosynthesis is probably required for parasite viability. It is undetermined whether small molecule inhibition of nicotinamide recycling reduces virulence or infectivity in microbial infections, and to date, no potent inhibitors of nicotinamidases have been reported. On the other hand, genetic studies have validated the importance of nicotinamidases for infectivity in pathogens that cause human disease. B. burgdorferei (Purser et al., 2003) and Brucella abortus (Kim et al., 2004) have been shown to be less infective and less pathogenic if their nicotinamidase genes are deleted. In Leishmania infantum, nicotinamide is able to restrict growth in vitro (Sereno et al., 2005). It is interesting that deletion of nicotinamidase causes abnormally high nicotinamide levels in yeast (Anderson et al., 2003; Gallo et al., 2004; Sauve et al., 2005). Thus, disruption of nicotinamidase in L. infantum may have an antileishmanial effect if it causes elevated intracellular nicotinamide concentrations. Because bacteria must use NAMN adenylation and NAD+ synthetase activity to complete both recycling and de novo pathways to NAD+ (with the exception of recycling NMN), it is likely that each of these two enzymes could be targeted for drug design with the prospect of antibiotic effects. These enzyme activities are essential for growth of most bacteria and have been identified as broad spectrum drug targets (Gerdes et al., 2002). With respect to the adenylating enzyme, humans require their own versions (nmnat-1, nmnat-2, and nmnat-3) in both recycling and de novo pathways. It is surprising that the sequence similarity of the human and bacterial enzymes is quite low, suggesting that small molecule inhibitors could be developed that are specific toward the bacterial forms (Gerdes et al., 2002). Finally, NAD+ synthetase activity is not required to recycle nicotinamide in humans, and its central role in recycling in microbes suggests that it may be an excellent target for antimicrobials. NAD+ synthetase inhibitors have proven antibiotic properties, killing Gram-positive bacteria (Velu et al., 2003) Anticancer Agents NAD+ metabolism plays a vital role in maintaining the genome, via PARPs and sirtuins, and proliferating cells appear to have higher demands for NAD+ biosynthesis and greater turnover of NAD+. The role of PARP as a protector of genomic stability has stimulated investigation of its inhibition as a way to make cancer cells more susceptible to genotoxicity (Virag and Szabo, 2002). Alternatively, compounds directed specifically to inhibition of human NAD metabolism have recently been developed. Specifically, an inhibitor of nampt/PBEF (FK-866) has recently been shown to have potent anticancer activity in cell culture and causes acute sensitivity to alkylating agents and increased apoptosis (Pogrebniak et al., 2006). It is currently in early clinical trials as an anticancer therapy. Regulation of NAD in a Model Microbe: Yeast Bakers' yeast has been a useful model organism for studying the link between NAD+ metabolism and the biological effects of NAD+-dependent sir2 (a yeast sirtuin). In yeast, the NAD+-recycling pathway is subject to regulation, and changes in this pathway have profound consequences for lifespan as well as gene silencing. The gene PNC1 when overexpressed increases gene silencing at the genetic loci HM, TEL, and rDNA and increases replicative lifespan (Anderson et al., 2003; Gallo et al., 2004). Conversely, pncΔ strains exhibit defective gene silencing and decreased replicative lifespan (Anderson et al., 2003; Gallo et al., 2004). PNC1 expression levels are subject to transcriptional regulation in response to stress, and PNC1 regulates nicotinamide concentrations (Anderson et al., 2003; Gallo et al., 2004; Sauve et al., 2005). Nicotinamide is a potent sirtuin inhibitor, implying that sirtuin catalytic activity is regulated by nicotinamide concentrations in yeast, which are in turn controlled by PNC1 expression levels (Anderson et al., 2003). Stimuli that increase PNC1 expression also extend lifespan (Anderson et al., 2003; Gallo et al., 2004), an effect that can be reproduced genetically by overexpression of SIR2. Thus, changes in NAD+ metabolism represent a mechanism for regulating heterochromatin formation and lifespan mediated by sirtuins. Regulation of NAD+ Metabolism in Humans NAD+-consuming reactions are tightly regulated in mammalian cells, and NAD+ depletion can occur rapidly in cells exposed to genotoxic stress. Genotoxins damage DNA and cause DNA strand breaks. These DNA breaks are sensed by a DNA repair system, which includes PARPs and a sirtuin, SIRT6. The activation of PARP, in particular, causes a rapid synthesis of poly(ADP-ribose) at the site of the strand break, and when this system is overactivated, it can significantly deplete cellular NAD+. On the other hand, NAD+-forming reactions are apparently subject to regulation as well. Cellular NAD+ concentrations are linked to organism nutritional status and physiologic state (Guarente, 2006). For example, NAD+ concentrations in liver increase 30% with fasting (Guarente, 2006). Thus, NAD+ metabolism is dynamically regulated by organism nutrient intake and genotoxic stress. Changes in NAD+ metabolism are now thought to initiate signaling events coupled to sirtuins or other NAD+-consuming enzymes, such as PARPs, via concentration changes of NAD+ and its metabolites, such as NADH and nicotinamide (Guarente, 2006). The mechanisms that regulate NAD+ biosynthesis in mammalian cells have recently come under increased investigation. Because the human genome does not encode a nicotinamidase, the regulation of NAD+ metabolism must be different from that of yeast. It is interesting that the nicotinamide-recycling enzyme, nampt/PBEF, is a likely regulator for both nicotinamide and NAD+ levels in cells. This enzyme is transcriptionally regulated in various conditions, and studies show that expression levels of nampt/PBEF are correlated to NAD+ concentrations in cultured cells (Revollo et al., 2007). The generality of nampt/PBEF as a determinant for NAD+ concentrations in tissues of the body and its role in activating signaling via sirtuins and other ADPribosyltransferases is still poorly determined to date. nampt/PBEF does up-regulate SIRT1 catalytic function in cultured cells (Revollo et al., 2007). It remains to be determined whether nampt/PBEF regulates NAD+ concentrations in liver where increased NAD+ concentrations associated with fasting stimulate SIRT1 and peroxisome proliferator-activated receptor γ coactivator 1-α-mediated gluconeogenesis (Guarente, 2006). In general, the mechanisms that alter human NAD+ metabolism probably include multiple processes, but the understandings of these mechanisms are currently very unclear and a considerable effort in this area is required before we know how NAD+ metabolism is controlled, how changes in NAD+ metabolism influence physiology, and how NAD+ metabolism might be manipulated for therapeutic benefit. Pharmacology of NAD+ Increasing Agents Nicotinamide Nicotinamide is a therapeutic agent that has been evaluated in several clinical studies. It is rapidly ingested and circulated into blood and is rapidly cleared to all tissues. It has a high hepatic extraction as well. Recommended intake is 0.3 mg kg–1 (20 mg for an adult), but recent clinical studies have examined ranges of 25 to 50 mg kg–1 per day (1.5– 3 g/day) (Knip et al., 2000). Nicotinamide at high doses has been reported to be protective of β-cell functions before the onset of type I diabetes. However, a large clinical study in Europe failed to show decreases in incidence of onset of type I diabetes with long-term nicotinamide dosing (Gale et al., 2004). High doses of nicotinamide administered orally or through injection are transiently metabolized in liver to increase NAD+. However, nicotinamide at elevated doses can cause hepatotoxicity. Nicotinamide is methylated to form 1-methylnicotinamide and downstream oxidized pyridones as metabolic end products (Knip et al., 2000). Large doses of nicotinamide cause methyl donor depletion (Knip et al., 2000). A large portion of nicotinamide administered to rats at 500 mg/kg was excreted unchanged within 12 h after injection. The remainder of nicotinamide was generally excreted as methylated or oxidized forms of the pyridine (Knip et al., 2000). At nonpharmacologic doses, nicotinamide is lost, mostly by excretion of the catabolic products, rather than as the unmetabolized vitamin. Nicotinic Acid Nicotinic acid is widely used in high doses to lower serum cholesterol, and it also lowers serum triglyceride levels (Capuzzi et al., 2000; Kamanna and Kashyap, 2000). This effect is unique to nicotinic acid and is not observed with high-dose nicotinamide. The doses required typically cause uncomfortable flushing in immediate release formulations (Capuzzi et al., 2000; Kamanna and Kashyap, 2000). Slow release formulations of nicotinic acid have been developed, which provide less discomfort from flushing but retain the desired lipid-lowering effects (Capuzzi et al., 2000). Nicotinic acid is rapidly metabolized by the liver and can be catabolized by glycine conjugation to nicotinuric acid (Capuzzi et al., 2000). Nicotinic acid increases NAD+ content in liver but is generally no more effective than nicotinamide in this respect (Jackson et al., 1995), indicating that NAD+ biosynthesis in liver is not a likely explanation for nicotinic acid correction effects in hyperlipidemia. The principle effects of nicotinic acid in lowering cholesterol have been proposed to stem from four basic causes: 1) inhibition of lipolysis in fat; 2) increased HDL levels; 3) lowering of serum lipoprotein-a; and 4) inhibition of synthesis and secretion of very low density lipoprotein in liver (Capuzzi et al., 2000). Some of nicotinic acids effects could be from a described interaction with the G protein HM74a (Capuzzi et al., 2000; Soudijn et al., 2007). This affinity was recently shown to be quite potent (100–200 nM); nicotinic acid binding antagonizes forskolin-mediated increase of cAMP production and inhibits lipolysis in differentiated 3T3L adipocytes (Capuzzi et al., 2000). The decrease in adipose lipolysis is hypothesized to limit liver uptake of free fatty acids, which reduces synthesis of very low density lipoprotein, intermediate density lipoprotein, and low density lipoproteins (Capuzzi et al., 2000). Nicotinic acid interferes with HDL-ApoA1mediated uptake by hepatocytes, without interfering with uptake of cholesterol esters (Capuzzi et al., 2000). This inhibition of removal of HDL-ApoA1 has been proposed to increase cholesterol efflux from peripheral tissues (increased reverse cholesterol transport), mediated by an increased amount of HDL particles (Capuzzi et al., 2000). The relative importance of these mechanisms in explaining the beneficial effects of nicotinic acid, as well as the exact molecular mechanisms that explain these effects, are still under investigation. Nevertheless, it is known that nicotinic acid dose-response profiles are different for different serum lipotypes, suggesting different pharmacological mechanisms for the effects seen. Clinically, high-dose nicotinic acid leads to reduced lipidemias, reduced progression of coronary heart disease, and reduced mortality (Capuzzi et al., 2000; Kamanna and Kashyap, 2000). Effects NA and NAM on NAD+ in Tissues Nicotinamide and nicotinic acid obtained at low doses are readily absorbed and retained by the body, whereas at high doses, they are transiently absorbed and rapidly eliminated from the body, albeit with transient increases in NAD+ levels in tissues such as the liver. Two-week treatment of rats with high doses of nicotinic acid and nicotinamide (500 and 1000 mg kg–1) has been evaluated on NAD+ levels in various tissues (Jackson et al., 1995). Both blood (packed red blood cells) and liver were responsive to increased dosages of nicotinamide or nicotinic acid, leading to increases of 40 to 60% in NAD+ content for both tissues for either B3. Smaller increases in NAD+ concentrations not exceeding 15% were observed for 1000 mg kg–1 doses of nicotinamide in heart, lung, and kidneys. These findings, on the one hand, appear to confirm that nampt/PBEF activity, which is responsible for recycling nicotinamide to NAD+, is typically not rate-limited by nicotinamide concentrations in some but not all tissues. In cell culture, nampt/PBEF controls NAD+ concentrations independent of exogenous nicotinamide concentrations (Revollo et al., 2004). nampt/PBEF has a very low Km for nicotinamide (<2 µM), suggesting that it is readily saturated by endogenous nicotinamide concentrations (Revollo et al., 2004). On the other hand, the ability of nicotinamide to stimulate NAD+ synthesis in liver and blood suggests that nicotinamide is convertible to alternative forms of B3 that ultimately increase nicotinamide bioavailability and/or that nicotinamide treatment causes cellular adaptations that lead to improved NAD+ biosynthesis. Why nicotinamide is efficiently utilized in some but not all tissues for NAD+ biosynthesis is currently unexplained. Jackson et al. (1995) also showed that nicotinic acid increases NAD+ concentrations in liver and blood, similar to nicotinamide. In addition, NAD+ biosynthesis was increased in heart (50%) and kidney (100%) as well. These results show that nicotinic acid generally has a broader effect than nicotinamide for NAD+ increases in the body. These results also indicate that the Preiss-Handler pathway is typically operating below saturation in most tissues. Genome Stability A considerable body of evidence implicates NAD+ metabolism as important for the maintenance of genome stability (Kirkland, 2003). Of particular importance in this respect is the involvement of PARP-1 as a DNA damage sensor, which cooperates in the DNA damage and repair process. The importance of NAD+ and PARP is highlighted by studies that show that vitamin B3 deficiency is associated with reduced tissue NAD+ concentrations and that a reduced ability of tissues to maintain poly-ADPR concentrations at normal levels (Boyonoski et al., 2002a). In the absence of toxins, B3deficient bone marrow showed a 6.2-fold increase in micronucleus formation and a 2.8fold increase in sister chromatid exchange (Boyonoski et al., 2002a). With DNAdamaging agents, animals show reduced ability to synthesize poly-ADPR in bone marrow and were more susceptible to the formation of DNA strand breaks, as measured by comet assay (Boyonoski et al., 2002a). B3 deficiency also results in reduced latency to leukemia in animals treated with ethylnitrosourea, which is used as a model for secondary carcinogenesis arising from chemotherapies (Henning et al., 1997). Conversely, pharmacological doses of nicotinamide or nicotinic acid supplementation increase NAD+ in bone marrow and also increase poly-ADPR levels (Boyonoski et al., 2002b). This latter result provides evidence against the idea that PARP-1 is efficiently inhibited by nicotinamide concentrations at high doses, as is widely assumed, because observed poly-ADPR levels in marrow and even liver were increased by nicotinamide and nicotinic acid similarly compared to controls. B3 treatments were able to retard ethylnitrosourea-induced carcinogenesis and led to increased lifespan for animals on a normal diet (Boyonoski et al., 2002b). In vitro results indicate that PARP-1 inhibition leads to delayed DNA repair, particularly base excision repair (Hassa et al., 2006). Consistent with a role for PARP in DNA repair, PARP–/– animals exhibit hypersensitivity to alkylating agents and ionizing radiation (Hassa et al., 2006). Some data appear to indicate that a normal if not an augmented NAD+ level in tissues aids in DNA repair and may reduce carcinogenesis. Some hints that this may be true are found in epidemiological studies that show that PARP-1 activity levels are lower in families predisposed to cancer (Decker and Muller, 2002) and that some cancers are found to have reduced PARP activities (Decker and Muller, 2002). Another finding of interest is that PARP activity may be generally higher in long-lived people, suggesting that PARP activity levels may have an antiaging effect (Decker and Muller, 2002). It is interesting that there is growing evidence that the body naturally adapts to genotoxic, hypoxic, and caloric restriction stress by increasing NAD+ biosynthesis. These evidences suggest that physiological responses to stress may be partly cued by increased NAD+ levels. Consistent with this view, sirtuin signaling has been shown to respond to increased physiological NAD+ concentrations (Guarente, 2006). Although increased vitamin B3 intake may seem beneficial, higher dosages of nicotinamide or nicotinic acid have undesirable side effects. In addition to hepatotoxicity, nicotinamide at high doses can adversely affect thymine biosynthesis and cause an increase of DNA damage caused by depleted thymidine levels in the cell (ApSimon et al., 1995). Ischemia and Stroke NAD+ metabolism is centrally involved in damage that accompanies stroke. Stroke injury is caused by an acute blockage of arterial blood flow to the brain, which causes starvation of affected tissues for oxygen. Upon removal of the blockage, the tissue that was deprived becomes reperfused with oxygen. This oxygenation of the tissue has serious negative consequences and causes production of oxygen-reactive species, such as superoxide anion, peroxide, and hydroxyl radicals. In addition, nitric oxide produced in the brain is converted to peroxynitrite, which has potent oxidizing power. The burst of oxidative stress upon reperfusion leads to extensive tissue damage. The mechanism of loss of tissue is not strictly oxidative in nature per se but rather linked to oxidation of important cellular components such as the genetic material, DNA. The damage to DNA sets off the PARP-1 activation cascade, which if highly up-regulated can deplete most of cellular NAD+, because of hyperpolymerization of APDR in the nucleus (Hassa et al., 2006). In turn, NAD+ must be resynthesized using ATP, PRPP, and other high-energy precursors. It is believed that the demands of resynthesizing NAD+ in large quantities place serious strains on energy resources in the cell, causing the cell to die from energy depletion (Hassa et al., 2006). Evidence that this model of cell death is considerably accurate has come from many sources. The data include observations that PARP–/– mice experience significantly reduced tissue damage in cerebral ischemia, with corresponding protection of NAD+ metabolism (Hassa et al., 2006). PARP inhibitors appear to have similar effects (Virag and Szabo, 2002). Nicotinamide, which is a micromolar inhibitor of PARP, is also protective. There remain questions about the mechanisms of action of nicotinamide in this respect, because some studies show that nicotinamide may not inhibit PARP activities, as determined by ADP-ribosyl polymer measurements (Boyonoski et al., 2002b). Nicotinamide effects may also play a role in enhancing NAD+ synthesis. Nevertheless, the effects of nicotinamide are distinctly more beneficial than those of nicotinic acid in ischemia models, suggestive of its effect as a PARP-1 inhibitor (Virag and Szabo, 2002). Nicotinamide in Fetal Ischemia and Fetal Alcohol Syndrome The fetal brain is particularly sensitive to genotoxicity, alcohol, and oxidative stress. The fetal brain must undergo pattern-forming connections to other neurons, forming synapses that lead to proper information processing. It is in the early period of development and synapse formation that apoptosis becomes a susceptibility of the immature neural cells, particularly those damaged during development (Ieraci and Herrera, 2006). From a medical perspective, this issue is an important one, because alcohol abuse is considered the leading cause of mental retardation in children (Ieraci and Herrera, 2006). Few interventions are known that mitigate this damage. Recent studies have looked at nicotinamide as a potential intervention as a means to protect the fetal brain cells during this developmental stage. In fetal mice whose mothers were treated with alcohol, a single nicotinamide treatment of the mother provided protection against oxidative stress markers in the fetal brain, such as lipid peroxidation, and also prevented apoptosis (Ieraci and Herrera, 2006). When assessed for behavior, offspring whose mothers were administered nicotinamide performed better in a number of tests for anxiety, a typical side effect of fetal alcohol syndrome in mice, than their nicotinamide-untreated controls (Ieraci and Herrera, 2006). Likewise, in fetal ischemia, nicotinamide treatment has been shown to prevent neural damage versus untreated controls, suggesting that nicotinamide could represent a reasonable intervention for early neuron damage during development (Feng et al., 2006). Alzheimer's Disease and Neurodegenerative Disorders Increased interest in the involvement of NAD+ metabolism in neurodegenerative processes has hinged partly on observations that preservation of NAD+ levels protects neurons subject to either genotoxicity or trauma (Araki et al., 2004). Of recent note, Milbrandt and co-workers showed that the process of axon degeneration, which occurs when an axon is severed, can be significantly slowed when NAD+ or other NAD+ precursors are present (Araki et al., 2004). Subsequent work by other laboratories has verified that NAD+ metabolism can protect severed neurons from degeneration. It is interesting that a mouse with slowed axon degeneration has a triplicate chimeric gene consisting of a partial ubiquitin ligase gene fused to a full nmnat-1 gene (Mack et al., 2001). nmnat-1 is nuclear-localized and couples NMN or NAMN with ATP to form NAD+ or nicotinic acid adenine dinucleotide, respectively (Fig. 3). Some controversy has emerged regarding the significance of the nmnat-1 biochemical activity in slowing axon degeneration, because nmnat-1 overexpression has not been shown to increase intracellular NAD+ concentrations (Mack et al., 2001) and overexpression of nmnat-1 does not reproduce the slow degeneration phenotype in a transgenic mouse that overexpressed this enzyme (Conforti et al., 2007). Nevertheless, NAD+ appears to be protective to neural cells, and it has been reported that NAD+, NR, NMN, nicotinamide, and NA all protect neurons under different conditions in cell culture experiments. Chronic disease states such as Parkinson's and Alzheimer's, are still somewhat poorly understood. Nevertheless, recent evidence is starting to suggest that chronic neurodegenerative disorders affect NAD+ metabolism adversely and may respond favorably to interventions that target NAD+ metabolism. For example, it has been known that Parkinson's disease results in increased methylnicotinamide excretion, suggesting enhanced NAD+ breakdown. Recently, we participated in a study in which NAD metabolism was examined in transgenic mice that have a gene encoding a human amyloid precursor protein (APP). These animals develop some neuropathology of Alzheimer's disease, such as plaque formation. Upon assay of brain tissue, NAD+ levels were decreased, and nicotinamide levels were increased in animals affected severely by disease who were on normal diets compared with animals on calorie restriction diets where the neuropathology was less severe (Qin et al., 2006). NAD+ itself was implicated in mitigating disease, and exogenous NAD+ redirected how cells process amyloid precursor protein (Qin et al., 2006). It was shown that NAD+-treated cells produced less plaque-associated forms of processed APP (Aβ) through a mechanism involving upregulation of α-secretase, which cleaves APP competitively with β- and γ-secretases preventing Aβ formation (Qin et al., 2006). Increased sirtuin (SIRT1) catalytic activity was also implicated in mediating the enhanced protection from neuropathology in cell culture and in mouse brains (Qin et al., 2006). SIRT1 is transcriptionally up-regulated in neurons by calorie restriction and is activated directly by NAD+. Although the work in the area of vitamin B3 effects in neurodegenerative disorders is still very preliminary, it invites the question of how NAD+ metabolism affects long-term neurodegenerative processes and whether enhancements/modulations to NAD+ metabolism can provide therapeutically meaningful changes in long-term outcomes in these notoriously difficult to treat diseases. Conclusions The emergence of knowledge recognizing the potent role of ADPR transferases as regulators of lifespan of diverse organisms and their coupling to NAD+ metabolism has stimulated a current interest in the possibilities inherent to targeting NAD+ metabolism for therapeutic purposes. This review suggests that opportunities exist for the development of antimicrobials and anticancer drugs that inhibit the basic transformations of NAD+ metabolism. In addition, expanded use of agents such as nicotinamide and nicotinic acid in light of their beneficial characteristics in enhancing NAD+ levels in tissues deserves consideration. Agents like nicotinamide riboside, which can also enhance NAD+ concentrations, have barely been investigated for this purpose. In the broad area of neuropathology (stroke, neurodegenerative disorders, and fetal brain damage), a surge of new data has pointed to enhancement of NAD+ metabolism and attenuation of NAD+ depletion as having potentially protective effects. For new therapies to emerge, continued progress will be needed to understand the complex regulatory mechanisms that govern NAD+ metabolism in cells and tissues and how changes in NAD+ metabolism affect tissue and organism physiology in health and disease. Footnotes This work has been supported in part by National Institutes of Health Grant DK 73466-01 (to A.S.). A.S. is a consultant for Sirtris Pharmaceuticals and has financial interests related to some of the topics discussed in this review. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.120758. ABBREVIATIONS: ADPR, adenosine diphosphate ribose; APP, amyloid precursor protein; NAMN, nicotinic acid mononucleotide; nampt, nicotinamide phosphoribosyltransferase; NMN, nicotinamide mononucleotide; nmnat, nicotinamide/nicotinate mononucleotide adenylyltransferase; NR, nicotinamide riboside; PARP, poly(ADP-ribose) polymerase; PBEF, pre-B-cell colonyenhancing factor; PRPP, 5-phosphoryl-ribose-1-pyrophosphate; QA, quinolinic acid; Sir2, silencing information regulator 2; HDL, high-density lipoprotein; SIRT1, mammalian sirtuin 1; FK-866, (E)-N-[4-(1-benzoylpiperidine-4-yl)butyl]-3(pyridin-3-yl)acrylamide. Received June 14, 2007. Accepted December 27, 2007. • The American Society for Pharmacology and Experimental Therapeutics Previous Section References . ↵ Anderson RM, Bitterman KJ, Wood JG, Medvedik O, and Sinclair DA (2003) Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423: 181–185. 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