Aging Cell (2006) 5, pp505–514 Doi: 10.1111/j.1474-9726.2006.00240.x © 2006 The Authors 505 Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006 Blackwell Publishing Ltd Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in yeast Mitsuhiro Tsuchiya, 1 Nick Dang, 1 Emily O. Kerr, 1 Di Hu, 1 Kristan K. Steffen, 1 Jonathan A. Oakes, 1 Brian K. Kennedy 1 and Matt Kaeberlein 2 1 Departments of Biochemistry and 2 Pathology, University of Washington, Seattle, WA 98195, USA Summary Two models have been proposed for how calorie restriction (CR) enhances replicative longevity in yeast: (i) suppression of rDNA recombination through activation of the sirtuin protein deacetylase Sir2 or (ii) decreased activity of the nutrient-responsive kinases Sch9 and TOR. We report here that CR increases lifespan independently of all Sir2-family proteins in yeast. Furthermore, we demonstrate that nicotinamide, an inhibitor of Sir2-mediated deacetylation, interferes with lifespan extension from CR, but does so independent of Sir2, Hst1, Hst2, and Hst4. We also find that 5 mM nicotinamide, a concentration sufficient to inhibit other sirtuins, does not phenocopy deletion of HST3. Thus, we propose that lifespan extension by CR is independent of sirtuins and that nicotinamide has sirtuin-independent effects on lifespan extension by CR. Key words: calorie restriction; nicotinamide; replicative aging; Sir2; yeast. Introduction Calorie restriction (CR) increases lifespan in multiple organisms, including yeast, worms, flies, and mammals. Despite several decades of study, however, the molecular mechanisms by which CR slows aging remain largely unknown. CR results in several physiological changes that may contribute to its effects on longevity, including decreased activity of nutrient and growth factor responsive pathways, increased resistance to a variety of stresses, altered translation and ribosome biogenesis, and increased autophagy (Weindruch & Walford, 1988; Masoro, 2005). It has also been suggested that CR increases the activity of Sir2-family protein deacetylases (sirtuins) in yeast, flies, and mammals (Guarente & Picard, 2005). Sir2 is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase (Imai et al., 2000; Landry et al., 2000; Smith et al., 2000) that promotes transcriptional silencing at the rDNA, the silent mating (HM) loci, and near telomeres (Rine & Herskowitz, 1987; Aparicio et al., 1991; Bryk et al., 1997; Smith & Boeke, 1997). In addition to Sir2, yeast have four sirtuin proteins, Hst1– 4, all of which are reported to deacetylate histone tails in vitro (Buck et al., 2004). Hst1 is the yeast sirtuin most similar in sequence to Sir2, and overexpression of HST1 can partially suppress the loss of silencing at HM loci in a Sir2 mutant (Brachmann et al., 1995). It has also been reported that over- expression of HST2 can partially compensate for the rDNA recombination defect of sir2∆ cells (Lamming et al., 2005). No direct link between Hst3 or Hst4 and Sir2 has been described. The role of Sir2 in yeast aging is thought to be limited to its ability to inhibit formation of extrachromosomal rDNA circles (ERC) (Kaeberlein et al., 1999) by repressing rDNA recombina- tion (Gottlieb & Esposito, 1989). ERCs are one factor that limits the replicative lifespan of a yeast cell, defined as the number of daughter cells produced by a given mother cell (Sinclair & Guarente, 1997). ERC formation is the result of homologous recombination between adjacent rDNA repeats, an event enhanced by the replication fork block protein, Fob1 (Defossez et al., 1999), and antagonized by Sir2 (Kaeberlein et al., 1999). Consistent with a link between ERCs and replicative lifespan, deletion of Fob1 or overexpression of Sir2 increases lifespan by 30–40%, and deletion of SIR2 shortens lifespan by about 50% (Kaeberlein et al., 1999). Calorie restriction, accomplished through a reduction in the glucose concentration of the media, also increases replicative lifespan in yeast by 20 – 40% (Lin et al., 2000, 2002; Kaeberlein et al., 2002). CR fails to increase the short lifespan of sir2∆ cells (Lin et al., 2000, 2002; Kaeberlein et al., 2002, 2004), which led to the hypothesis that CR slows aging by activating Sir2 and, thus, decreasing ERC accumulation (Lin et al., 2000). Lin et al. (2002, 2004) have proposed that CR activates Sir2 by increasing respiration, which results in decreased levels of NADH, an inhibitor of Sir2. Anderson et al. (2003) have proposed an alternative model whereby CR results in decreased levels of nicotinamide, a product of the Sir2 deacetylation reaction and an inhibitor of Sir2 activity. More recently, we have challenged these models by reporting that CR increases lifespan in a Sir2-independent manner and that CR fails to result in detectable activation of Sir2 in vivo (Kaeberlein et al., 2004, 2005a,c). Paradoxically, addition of the Sir2-inhibitor nicotinamide to the media partially blocks lifespan extension from CR in sir2∆ fob1∆ cells (Kaeberlein et al., 2005a; Lamming et al., 2005). Because Sir2 is absent from these cells, this suggested the possibility that lifespan extension from CR Correspondence Brian K. Kennedy, Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Tel.: 206 543 4849; fax: 206 543 3644; e-mail: bkenn@u.washington.edu Matt Kaeberlein, Department of Pathology, University of Washington, Seattle, WA 98195, USA. Tel.: 206 543 4849; fax: 206 543 3644; e-mail: kaeber@u.washington.edu Accepted for publication 29 August 2006