Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae

Abstract

Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals. Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism. In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref. 1). We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide. Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress. We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan. We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity. We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms.

Figure 1

Calorie restriction and heat stress extend lifespan in a PNC1-dependent manner. a, Pnc1 converts nicotinamide to nicotinic acid. b, In S. cerevisiae, NAD⁺ is synthesized de novo from tryptophan via Bna1–6 or recycled from nicotinamide. c, Average lifespan on 2.0% (w/v) glucose: wild type, 21.6 generations; pnc1Δ, 19.1; sir2Δ, 14.2. Average lifespan on 0.5% glucose: wild type, 32.7 generations; pnc1D, 18.1; sir2Δ, 14.7. d, At 30 °C, average lifespans: wild type, 19.4 generations; pnc1Δ, 18.5; sir2Δ, 12.0. At 37 °C, average lifespans: wild type, 23.4 generations; pnc1Δ, 17.5; sir2Δ, 10.6. e, Average lifespans on 2.0% glucose at 30 °C: wild type, 19.7 generations; 5×PNC1, 36.1; sir2Δ, 14.2; 5×PNC1 sir2Δ, 15.1; pnc1Δ sir2Δ, 14.4.

Figure 2

Pnc1 levels and activity are elevated in response to calorie restriction and low-intensity stress. a, Western analysis of Pnc1–GFP under conditions of glucose restriction. b, Pnc1–GFP in wild-type or cdc25-10 cells. c, Detection of Pnc1–GFP in cells subjected to mild stress as indicated (a.a., amino acid). d, Measurement of nicotinamide deamination by Pnc1. Activity (nmol ammonia min⁻¹ per mg protein) from three experiments (means ± s.d): no treatment (2% glucose), 0.9 ± 0.26; calorie restriction (CR; 0.1% glucose), 4.38 ± 0.43; heat stress (37 °C), 3.28 ± 0.32; sorbitol (1 M), 3.75 ± 0.65.

Figure 3

Pnc1–GFP is localized in the nucleus and cytoplasm, and concentrated in peroxisomes. a, Pnc1–GFP fluorescence in glucose-restricted cells (Glu 0.5% and 0.1%). b, Pnc1–GFP fluorescence under conditions of mild stress (a.a., amino acid). c, Co-localization of Pnc1–GFP (green) and RFP–PTS1 (red). Yellow indicates overlap. d, Localization of Pnc1–GFP in cells from peroxisomal mutant strains, pex6Δ, pex5Δ and pex7Δ.

Figure 4

Manipulation of nicotinamide metabolism alters silencing and lifespan. a, To monitor silencing, ADE2 was integrated at the rDNA locus. Increased growth on medium lacking adenine indicates decreased silencing. Serial dilutions of cells spotted on plates containing nicotinic acid or nicotinamide (5 mM). b, PNC1 does not have a critical role in NAD⁺ biosynthesis, even in the absence of the de novo NAD⁺ synthesis pathway. BNA6 encodes an enzyme in the NAD⁺ de novo synthesis pathway (see Fig. 1b). NPT1 encodes a phosphoribosyltransferase that converts nicotinic acid to nicotinic acid mononucleotide in the NAD⁺ salvage pathway. Spore colonies from heterozygous bna6Δ npt1Δ or bna6Δ pnc1Δ diploids. Genotypes determined by PCR with a colony/ microcolony genomic template. c, Partial rescue of silencing by additional PNC1 in the absence of the NAD⁺ salvage pathway and complete rescue in the presence of quinolinic acid (5 mM). d, Manipulation of genes involved in nicotinamide metabolism alters rDNA silencing. e, Manipulation of YLR285W affects lifespan. f, Model for the regulation of Sir2 activity and lifespan by PNC1 and nicotinamide (NAM).