Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

SummaryCalorie restriction (CR) extends the mean and maximum lifespan of a wide variety of organisms ranging from yeast to mammals, although the molecular mechanisms of action remain unclear. For the budding yeast Saccharomyces cerevisiae reducing glucose in the growth medium extends both the replicative and chronological lifespans (CLS). The conserved NAD + -dependent histone deacetylase, Sir2p, promotes replicative longevity in S. cerevisiae by suppressing recombination within the ribosomal DNA locus and has been proposed to mediate the effects of CR on aging. In this study, we investigated the functional relationships of the yeast Sirtuins (Sir2p, Hst1p, Hst2p, Hst3p and Hst4p) with CLS and CR. SIR2 , HST2 , and HST4 were not major regulators of CLS and were not required for the lifespan extension caused by shifting the glucose concentration from 2 to 0.5% (CR). Deleting HST1 or HST3 moderately shortened CLS, but did not prevent CR from extending lifespan. CR therefore works through a Sirtuin-independent mechanism in the chronological aging system. We also show that low temperature or high osmolarity additively extends CLS when combined with CR, suggesting that these stresses and CR act through separate pathways. The CR effect on CLS was not specific to glucose. Restricting other simple sugars such as galactose or fructose also extended lifespan. Importantly, growth on nonfermentable carbon sources that force yeast to exclusively utilize respiration extended lifespan at nonrestricted concentrations and provided no additional benefit when restricted, suggesting that elevated respiration capacity is an important determinant of chronological longevity.

Genes encoding thiamine biosynthesis enzymes in microorganisms are tightly regulated such that low environmental thiamine concentrations activate transcription and high concentrations are repressive. We have determined that multiple thiamine (THI) genes in Saccharomyces cerevisiae are also regulated by the intracellular NAD ؉ concentration via the NAD ؉ -dependent histone deacetylase (HDAC) Hst1 and, to a lesser extent, Sir2. Both of these HDACs associate with a distal region of the affected THI gene promoters that does not overlap with a previously defined enhancer region bound by the thiamine-responsive Thi2/Thi3/Pdc2 transcriptional activators. The specificity of histone H3 and/or H4 deacetylation carried out by Hst1 and Sir2 at the distal promoter region depends on the THI gene being tested. Hst1/Sir2-mediated repression of the THI genes occurs at the level of basal expression, thus representing the first set of transcription factors shown to actively repress this gene class. Importantly, lowering the NAD ؉ concentration and inhibiting the Hst1/Sum1 HDAC complex elevated the intracellular thiamine concentration due to increased thiamine biosynthesis and transport, implicating NAD ؉ in the control of thiamine homeostasis.

Background: Activity of the NAD ϩ -dependent histone deacetylase Sir2 is stimulated by isonicotinamide (INAM) in vitro.

The histone deacetylase activity of Sir2p is dependent on NAD 1 and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD 1 and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD 1 concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD 1 salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD 1 concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss-Handler NAD 1 salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.