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Inhibition of Silencing and Accelerated Aging by Nicotinamide, a Putative Negative Regulator of Yeast Sir2 and Human SIRT1*

  • Kevin J. Bitterman
    Footnotes
    Affiliations
    From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Rozalyn M. Anderson
    Footnotes
    Affiliations
    From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Haim Y. Cohen
    Affiliations
    From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Magda Latorre-Esteves
    Affiliations
    From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    David A. Sinclair
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    * This work was supported by NIA, National Institutes of Health Grant AG11972-01, the American Federation of Aging Research, a Glenn/American Federation of Aging Research Scholarship for Research in the Biology of Aging (to K. J. B.), and a John Taplin Postdoctoral Fellowship (to H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Both authors contributed equally to the results of this work.
Open AccessPublished:September 23, 2002DOI:https://doi.org/10.1074/jbc.M205670200
      The Saccharomyces cerevisiae Sir2 protein is an NAD+-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity. A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis. The Sir2 deacetylation reaction generates two products:O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B3. We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of asir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G1-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity. We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro. The degree of inhibition by nicotinamide (IC50< 50 μm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins. We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD+, thereby blocking NAD+ hydrolysis. We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes.
      Transcriptional silencing involves the heritable modification of chromatin at distinct sites in the genome. Silencing is referred to as long range repression as it is promoter nonspecific and often encompasses an entire genomic locus (
      • Courey A.J.
      • Jia S.
      ,
      • Moazed D.
      ). In yeast these silent regions, which are similar to the heterochromatin of higher eukaryotes, are subject to a wide variety of modifications (
      • Gasser M.
      • Cockell M.M.
      ). Among the best studied of these modifications is the reversible acetylation of histones (reviewed by Refs.
      • Eberharter A.
      • Becker P.B.
      and
      • Kuo M.H.
      • Allis C.D.
      ).
      There are two types of enzymes that affect the acetylation state of histones: histone acetyltransferases and the opposing histone deacetylases (HDACs).
      The abbreviations used are: HDAC, histone deacetylase; TSA, trichostatin A; SC, synthetic medium; 5-FOA, 5-fluoroorotic acid; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; GFP, green fluorescent protein.
      1The abbreviations used are: HDAC, histone deacetylase; TSA, trichostatin A; SC, synthetic medium; 5-FOA, 5-fluoroorotic acid; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; GFP, green fluorescent protein.
      Compared with more transcriptionally active areas of the genome, histones within silent regions of chromatin are known to be hypoacetylated, specifically on the NH2-terminal tails of core histones H3 and H4 (
      • Bernstein B.E.
      • Tong J.K.
      • Schreiber S.L.
      ). Three classes of histone deacetylases have been described and classified based on homology to yeast proteins. Proteins in class I (Rpd3-like) and class II (Hda1-like) are characterized by their sensitivity to the inhibitor trichostatin A (TSA) (
      • Fischle W.
      • Kiermer V.
      • Dequiedt F.
      • Verdin E.
      ,
      • Marks P.
      • Rifkind R.A.
      • Breslow V.M.
      • Miller R.
      • Kelly T.
      • Kelly W.K.
      ). Studies using this inhibitor have provided a wealth of information regarding the biochemistry and cellular function of these proteins (reviewed by Ref.
      • Yoshida M.
      • Furumai R.
      • Nishiyama M.
      • Komatsu Y.
      • Nishino N.
      • Horinouchi S.
      ).
      Yeast Sir2 is the founding member of Class III HDACs. Sir2-like deacetylases are not inhibited by TSA and have the unique characteristic of being NAD+-dependent (
      • Smith J.S.
      • Brachmann C.B.
      • Celic I.
      • Kenna M.A.
      • Muhammad S.
      • Starai V.J.
      • Avalos J.L.
      • Escalante-Semerena J.C.
      • Grubmeyer C.
      • Wolberger C.
      • Boeke J.D.
      ,
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Landry J.
      • Sutton A.
      • Tafrov S.T.
      • Heller R.C.
      • Stebbins J.
      • Pillus L.
      • Sternglanz R.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ). Proteins of this class are found in a wide array of organisms, ranging from bacteria to humans. At least two Sir2 homologues, yeast Hst2 and human SIRT2, are localized to the cytoplasm and human SIRT1, a nuclear protein, has recently been shown to target p53 for deacetylation (
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ,
      • Brachmann C.B.
      • Sherman J.M.
      • Devine S.E.
      • Cameron E.E.
      • Pillus L.
      • Boeke J.D.
      ,
      • Tanny J.C.
      • Moazed D.
      ). These results indicate that only a subset of the Sir2 family are likely to be histone deacetylases. Although insensitive to TSA, several synthetic small molecule inhibitors of Sir2 have been isolated and have provided novel insights into the biology of these proteins (
      • Grozinger C.M.
      • Chao E.D.
      • Blackwell H.E.
      • Moazed D.
      • Schreiber S.L.
      ,
      • Bedalov A.
      • Gatbonton T.
      • Irvine W.P.
      • Gottschling D.E.
      • Simon J.A.
      ).
      The term silent informationregulator (SIR) was first coined to describe a set of nonessential genes required for repression of yeast mating-type loci (HML and HMR) (
      • Rine J.
      • Herskowitz I.
      ). Transcriptional silencing in yeast is also observed at telomeres and the ribosomal DNA (rDNA) locus (
      • Moazed D.
      ,
      • Wood J.G.
      • Sinclair D.A.
      ). The formation of silent heterochromatin at mating-type loci and the poly(TG1–3) tracts of yeast telomeres is mediated by a complex of Sir2, Sir3, and Sir4 (
      • Strahl-Bolsinger S.
      • Hecht A.
      • Luo K.
      • Grunstein M.
      ,
      • Hecht A.
      • Strahl-Bolsinger S.
      • Grunstein M.
      ). At the rDNA locus, Sir2 is part of the RENT (regulator of nucleolar silencing and telophase exit) complex, which includes Net1 and Cdc14 (
      • Ghidelli S.
      • Donze D.
      • Dhillon N.
      • Kamakaka R.T.
      ,
      • Shou W.
      • Sakamoto K.M.
      • Keener J.
      • Morimoto K.W.
      • Traverso E.E.
      • Azzam R.
      • Hoppe G.J.
      • Feldman R.M.R.
      • DeModena J.
      • Moazed D.
      • Charbonneaux H.
      • Nomura M.
      • Deshaies R.J.
      ). Of these proteins, Sir2 is the only factor that is indispensable for silencing at all three silent regions (
      • Gottschling D.E.
      • Aparicio O.M.
      • Billington B.L.
      • Zakian V.A.
      ,
      • Smith J.S.
      • Boeke J.D.
      ,
      • Bryk M.
      • Banerjee M.
      • Murphy M.
      • Knudsen K.E.
      • Garfinkel D.J.
      • Curcio M.J.
      ).
      The yeast rDNA locus (RDN1) consists of 100–200 tandemly repeated 9-kb units encoding ribosomal RNAs. A major cause of yeast aging has been shown to stem from recombination between these repeats (
      • Sinclair D.A.
      • Guarente L.
      ,
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ,
      • Park P.U.
      • Defossez P.A.
      • Guarente L.
      ), which can lead to the excision of an extrachromosomal rDNA circle. Extrachromosomal rDNA circles can accumulate to a DNA content greater than that of the entire yeast genome in old cells and are thought to kill cells by titrating essential transcription and/or replication factors (
      • Sinclair D.A.
      • Mills K.
      • Guarente L.
      ). Although Sir2 silences polymerase II-transcribed genes integrated at the rDNA, there is evidence that its primary function at this locus is to suppress rDNA recombination. Deletion of SIR2 eliminates rDNA silencing and increases the frequency that a marker gene is recombined of the rDNA by 10-fold (
      • Gottlieb S.
      • Esposito R.E.
      ).
      Sir2 is a limiting component of yeast longevity. A single extra copy of the SIR2 gene suppresses recombination and extends life span by 40% (
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ,
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ,
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ), whereas deletion of SIR2 increases extrachromosomal rDNA circle formation and dramatically shortens life span (
      • Gottlieb S.
      • Esposito R.E.
      ,
      • Kennedy B.K.
      • Austriaco Jr., N.R.
      • Zhang J.
      • Guarente L.
      ). Recently, it has been shown that SIR2 is essential for the increased longevity provided by calorie restriction (
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ), a regimen that extends the life span of every organism it has been tested on. Moreover, increased dosage of the Sir2 homologuesir-2.1 has been shown to extend the life span of the nematode Caenorhabditis elegans (
      • Tissenbaum H.A.
      • Guarente L.
      ), and the nearest human homologue SIRT1 has been shown to inhibit apoptosis through deacetylation of p53 (
      • Vaziri H.
      • Dessain S.K.
      • Eaton E.N.
      • Imai S.I.
      • Frye R.A.
      • Pandita T.K.
      • Guarente L.
      • Weinberg R.A.
      ,
      • Luo J.
      • Nikolaev A.Y.
      • Imai S.
      • Chen D.
      • Su F.
      • Shiloh A.
      • Guarente L.
      • Gu W.
      ). These findings suggest that Sir2 and its homologues have a conserved role in the regulation of survival at both the cellular and organismal levels.
      Recently, a great deal of insight has been gained into the biochemistry of Sir2-like deacetylases (reviewed by Ref.
      • Moazed D.
      ). In vitro, Sir2 has specificity for lysine 16 of histone H4 and lysines 9 and 14 of histone H3 (
      • Smith J.S.
      • Brachmann C.B.
      • Celic I.
      • Kenna M.A.
      • Muhammad S.
      • Starai V.J.
      • Avalos J.L.
      • Escalante-Semerena J.C.
      • Grubmeyer C.
      • Wolberger C.
      • Boeke J.D.
      ,
      • Landry J.
      • Sutton A.
      • Tafrov S.T.
      • Heller R.C.
      • Stebbins J.
      • Pillus L.
      • Sternglanz R.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ). Although TSA-sensitive HDACs catalyze deacetylation without the need of a cofactor, Sir2 requires NAD+, perhaps allowing for regulation of Sir2 activity through changes in the availability of this co-substrate (
      • Smith J.S.
      • Brachmann C.B.
      • Celic I.
      • Kenna M.A.
      • Muhammad S.
      • Starai V.J.
      • Avalos J.L.
      • Escalante-Semerena J.C.
      • Grubmeyer C.
      • Wolberger C.
      • Boeke J.D.
      ,
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Landry J.
      • Sutton A.
      • Tafrov S.T.
      • Heller R.C.
      • Stebbins J.
      • Pillus L.
      • Sternglanz R.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ). The first step in Sir2-catalyzed deacetylation is the cleavage of the high energy glycosidic bond that joins the ADP-ribose moiety of NAD+ to nicotinamide. Upon cleavage, Sir2 then catalyzes the transfer of an acetyl group to ADP-ribose (
      • Smith J.S.
      • Brachmann C.B.
      • Celic I.
      • Kenna M.A.
      • Muhammad S.
      • Starai V.J.
      • Avalos J.L.
      • Escalante-Semerena J.C.
      • Grubmeyer C.
      • Wolberger C.
      • Boeke J.D.
      ,
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Tanny J.C.
      • Moazed D.
      ,
      • Sauve A.A.
      • Celic I.
      • Avalos J.
      • Deng H.
      • Boeke J.D.
      • Schramm V.L.
      ). The product of this transfer reaction is O-acetyl-ADP-ribose, a novel metabolite that has recently been shown to cause a delay/block in the cell cycle and oocyte maturation of embryos (
      • Borra M.T.
      • O'Neill F.J.
      • Jackson M.D.
      • Marshall B.
      • Verdin E.
      • Foltz K.R.
      • Denu J.M.
      ).
      The other product of deacetylation is nicotinamide, a form of vitamin B3 (
      • Dietrich L.S.
      ). High doses of nicotinamide and its acid derivative, nicotinic acid, are often used interchangeably to self-treat a number of conditions including anxiety, osteoarthritis, and psychosis. Furthermore, nicotinamide is currently in clinical trials as a therapy for cancer and type I diabetes (
      • Kaanders J.H.
      • Pop L.A.
      • Marres H.A.
      • Bruaset I.
      • van den Hoogen F.J.
      • Merkx M.A.
      • van der Kogel A.J.
      ). The long term safety of the high doses used in these treatments has been questioned (
      • Knip M.
      • Dovek I.F.
      • Moore W.P.
      • Gillmor H.A.
      • McLean A.E.
      • Bingley P.J.
      • Gale E.A.
      ), and the effects of these compounds at the molecular level are even less clear. Interestingly, nicotinamide has recently been shown to inhibit yeast Hst2 in vitro (
      • Landry J.
      • Slama J.T.
      • Sternglanz R.
      ), although the effects of this compound at the organismal level or on other yeast Sir2 family members have not been investigated.
      In most organisms, there are two pathways of NAD+biosynthesis. NAD+ may be synthesized de novofrom tryptophan or recycled in four steps from nicotinamide via the NAD+ salvage pathway (
      • Foster J.W.
      • Park Y.K.
      • Penfound T.
      • Fenger T.
      • Spector M.P.
      ) (Fig.1). In the salvage pathway, nicotinamide produced from NAD+ cleavage is converted to nicotinic acid by Pnc1, a nicotinamidase (
      • Ghislain M.
      • Talla E.
      • Francois J.M.
      ). Nicotinic acid is subsequently converted into nicotinic acid mononucleotide by a phosphoribosyltransferase encoded by NPT1. We recently demonstrated that increased dosage of NAD+ salvage pathway genes increases silencing at the rDNA locus, telomeres, and mating-type loci. We also showed that a single extra copy of the NPT1gene extends life span by 60% without increasing total steady-state NAD+ levels or NAD+/NADH ratios (
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ). This suggests that Sir2 may be regulated either by nuclear specific changes in NAD+ availability, flux through the salvage pathway, or by levels of an inhibitory molecule (
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ).
      Figure thumbnail gr1
      Figure 1The NAD+ salvage pathway.Nicotinamide generated by Sir2 is converted into nicotinic acid by Pnc1 and subsequently back into NAD+ in three steps.YNR073C and YEL070W are putative NAD+glycohydrolases. Question marks represent enzymes present in bacteria without obvious homologs in yeast. Abbreviations:NAD +, nicotinamide adenine dinucleotide;NaMN, nicotinic acid mononucleotide; NaAD, desamido-NAD+; NADP +, nicotinamide adenine dinucleotide phosphate.
      With regards to the later hypothesis, we wished to examine whether Sir2 enzymes might be negatively regulated by nicotinamide, a product of the deacetylation reaction. Here, we show that nicotinamide strongly inhibits silencing at yeast telomeres, rDNA, and mating-type loci, whereas the related nicotinic acid has no effect. Nicotinamide also increases recombination at the rDNA locus and shortens yeast life span to that of a sir2 mutant. We use this inhibitor to show that maintenance of silenced chromatin and the localization of Sir2/3/4 to telomeres require the continual activity of Sir2, even in non-dividing cells. Physiological concentrations of nicotinamide inhibit Sir2 and human SIRT1 noncompetitively in vitro, raising the possibility that nuclear nicotinamide negatively regulates Sir2 activity in vivo. Our findings also suggest that the medicinal use of nicotinamide should be given careful consideration.

      DISCUSSION

      We have shown that nicotinamide, a product of the Sir2 deacetylation reaction, is a strong inhibitor of Sir2 activity bothin vivo and in vitro. Addition of exogenous nicotinamide to yeast cells derepresses all three silent loci, increases recombination at the rDNA locus, and shortens yeast life span to that of a sir2 mutant. We have recently shown that strains carrying extra copies of NAD+ salvage pathway genes show increased silencing and are long-lived, yet they do not have increased total steady-state NAD+ or NADH levels (
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ). In addition, we and others have shown that two NAD+ salvage pathway enzymes, Npt1 and Nma2, are predominately nuclear (
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ,
      • Sandmeier J.J.
      • Celic I.
      • Boeke J.D.
      • Smith J.S.
      ). Based on these findings, we hypothesized that increased longevity is mediated by nuclear-specific increases in NAD+ availability or increased flux through the salvage pathway. The latter model implies that there may be continual cleavage of NAD+ by Sir2 family members. Consistent with this, we have shown with nicotinamide that Sir2 activity is required constitutively for the maintenance of heterochromatin. This is also consistent with the recent finding of Bedelov et al. (
      • Bedalov A.
      • Gatbonton T.
      • Irvine W.P.
      • Gottschling D.E.
      • Simon J.A.
      ) that the MATα gene at the silenced HML locus is expressed in G1 cells treated with splitomycin.
      We find that nicotinamide disrupts Sir2 localization, consistent with recent studies showing that catalytically inactive Sir2 mutants do not associate with telomeric chromatin (
      • Armstrong C.M.
      • Kaeberlein M.
      • Imai S.I.
      • Guarente L.
      ,
      • Hoppe G.J.
      • Tanny L.
      • Rudner A.D.
      • Gerber S.A.
      • Danaie S.
      • Gygi S.P.
      • Moazed D.
      ). Taking advantage of the fact that nicotinamide can rapidly inhibit Sir2, we have shown that Sir2 telomeric foci remain for up to 2 h after addition of this compound and that their eventual delocalization occurs even in nondividing cells. These findings demonstrate that continual Sir2 activity is required for its localization to telomeres, and suggest that nicotinamide interferes with the maintenance of Sir2 localization, not just its establishment during the cell cycle.
      We have shown that nicotinamide strongly inhibits the deacetylase activity of both yeast Sir2 and the human homologue, SIRT1 in vitro. The fact that nicotinamide acts noncompetitively to inhibit Sir2 enzymes, suggests that this compound does not compete with NAD+ for binding. A similar result has recently been obtained for yeast Hst2, a cytoplasmic Sir2 homologue (
      • Landry J.
      • Slama J.T.
      • Sternglanz R.
      ). Based on the reaction mechanism for Sir2 deacetylation and the crystal structure of an archeal Sir2 homologue, we propose the following model for Sir2 regulation by nicotinamide. Sir2-catalyzed deacetylation consists of two hydrolysis steps that are thought to be coupled. Cleavage of the glycosidic bond connecting nicotinamide to the ADP-ribose moiety of NAD+ is followed by cleavage of the C-N bond between an acetyl group and lysine. A recent structural analysis indicates that the NAD+ binding pocket of Sir2 enzymes contains three spatially distinct sites (A, B, and C), the later two of which are thought to be directly involved in catalysis (
      • Min J.
      • Landry J.
      • Sternglanz R.
      • Xu R.M.
      ) (Fig.7 A). In the presence of an acetyllysine, NAD+ bound to the B site can undergo a conformational change bringing the nicotinamide group in proximity to the C site, where it may be cleaved (Fig. 7 B). The ADP-ribose product of this reaction may then return to the B site where deacetylation of the acetyllysine occurs. We propose that nicotinamide binds to and blocks the internal C site, preventing the conformational change and subsequent cleavage of NAD+ (Fig.7 C).
      Figure thumbnail gr7
      Figure 7Model for noncompetitive inhibition of Sirs by nicotinamide. A, the Sir2 family of deacetylases contains an NAD+ binding pocket divided into three distinct regions. The adenine-ribose moiety of NAD+binds the A site whereas, in the absence of acetylated substrate, the nicotinamide moiety is bound to the B site. B, in the presence of an acetyllysine (K) a rotation around a phosphodiester bond of the pyrophosphate moiety would position the nicotinamide group near the C site where hydrolysis and subsequent nicotinamide release may take place. C, when present at elevated levels, free nicotinamide may bind to the C site preventing the conformational change and hydrolysis of NAD+.
      We have shown that the potency of nicotinamide rivals that of the most effective synthetic Sir2 inhibitors identified thus far. The fact that SIRT1 is inhibited by such low concentrations of nicotinamide in vitro raises the possibility that this mode of inhibition may be physiologically relevant. Levels of nicotinamide in mammalian tissues have been reported to lie in the range of 11–400 μm (
      • Dietrich L.S.
      ,
      • Hoshino J.
      • Schluter U.
      • Kroger H.
      ,
      • Ijichi H.
      • Ichiyama A.
      • Hayaishi O.
      ,
      • Hagino Y.L.
      • Henderson J.M.
      ). More recently, levels of nicotinamide in cerebrospinal fluid were determined with high accuracy to be 54.2 μm (
      • Smythe G.A.
      • Braga O.
      • Brew B.J.
      • Grant R.S.
      • Guillemin G.J.
      • Kerr S.J.
      • Walker D.W.
      ), a value that is similar to the IC50 for nicotinamide reported here. We propose that fluctuations in cellular nicotinamide levels may directly control the activity of Sir2 proteins in vivo. These fluctuations may in turn be regulated by enzymes involved in nicotinamide metabolism, including Pnc1.
      The yeast PNC1 gene encodes a nicotinamidase that is situated in a key position to regulate NAD+-dependent deacetylases. By converting nicotinamide into nicotinic acid as part of the NAD+salvage pathway, Pnc1 may reduce levels of this inhibitor and simultaneously increase the availability of NAD+ to Sir2 (see Fig. 1). Interestingly, PNC1 is one of the most highly induced genes in response to stress and conditions that resemble calorie restriction (
      • Gasch A.P.
      • Spellman P.T.
      • Kao C.M.
      • Carmel-Harel O.
      • Eisen M.B.
      • Storz G.
      • Botstein D.
      • Brown P.O.
      ,
      • Moskvina E.
      • Schuller C.
      • Maurer C.T.
      • Mager W.H.
      • Ruis H.
      ), both of which have been shown to extend replicative life span (
      • Swiecilo A.
      • Krawiec Z.
      • Wawryn J.
      • Bartosz G.
      • Bilinski T.
      ,
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ). This raises the possibility that high levels of Pnc1 induce silencing under conditions of stress or nutrient limitation, by removing the inhibitory effects of nicotinamide and increasing NAD+ production. Our previous finding that a single extra copy of PNC1 increases Sir2-dependent silencing (
      • Anderson R.M.
      • Bitterman K.J.
      • Wood J.G.
      • Medvedik O.
      • Cohen H.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      • Sinclair D.A.
      ) adds further support to this model. It will be interesting to determine whether intracellular nicotinamide levels vary in response to such conditions.
      Nicotinamide and nicotinic acid are used at high doses (up to 10 g/day) to self-treat a wide variety of ailments (
      • Knip M.
      • Dovek I.F.
      • Moore W.P.
      • Gillmor H.A.
      • McLean A.E.
      • Bingley P.J.
      • Gale E.A.
      ). Both are considered forms of vitamin B3 and are often used interchangeably, although nicotinamide has become preferred in many cases because of an apparent lack of side effects. We have shown that these two related compounds have drastically different effects at the molecular level. In addition, nicotinamide is currently in trials as a therapy to prevent cancer recurrence and insulin-dependent (type I) diabetes (
      • Kaanders J.H.
      • Pop L.A.
      • Marres H.A.
      • Bruaset I.
      • van den Hoogen F.J.
      • Merkx M.A.
      • van der Kogel A.J.
      ). Our results clearly demonstrate that nicotinamide can inhibit Sir2 enzymes, even in noncycling cells, and raise the concern that there may be deleterious consequences of long term nicotinamide therapy in humans.

      Acknowledgments

      We thank D. Moazed, J. Tanny, J. Simon, J. Wood, and B. Forrester for reagents and advice.

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