Yeast life-span extension by calorie restriction is independent of NAD fluctuation

Abstract

Calorie restriction (CR) slows aging in numerous species. In the yeast Saccharomyces cerevisiae, this effect requires Sir2, a conserved NAD+-dependent deacetylase. We report that CR reduces nuclear NAD+ levels in vivo. Moreover, the activity of Sir2 and its human homologue SIRT1 are not affected by physiological alterations in the NAD+:NADH ratio. These data implicate alternate mechanisms of Sir2 regulation by CR.

Fig. 1

A yeast reporter assay that detects freely available nuclear NAD⁺. (A) The reporter strain with both the nadR-AD plasmid and NAD-HIS3 construct was spotted in serial 10-fold serial dilutions onto reporter assay media: 2% glucose (w/v) synthetic complete (SC) medium with or without histidine + 3-aminotriazole (3-AT). The extent of growth on the test media is indicative of the availability of NAD⁺. Also shown are assays of negative control strains: empty vector (no NadR-AD), mutant NAD boxes (mut) (TGTgTA and its inverted repeat). Spots were incubated for ~48 hours at 30°C. (B) The NAD⁺ reporter system detects changes in availability of nuclear NAD⁺. NAD⁺ may be generated from nicotinic acid via the NAD⁺ pathway or from tryptophan via the de novo pathway, which is catalyzed by Bna1-6 (17, 18). Deletion of BNA6 decreases NAD⁺ levels, whereas exogenously supplied acetaldehyde increases NAD⁺ by oxidizing the NADH pool (24). BNA6 was deleted in the NAD⁺ reporter by replacing the entire coding region with a KanMX cassette and spotted on reporter assay media. The wild-type reporter strain was pregrown in liquid SC medium with or without acetaldehyde (10 mM) for 2 hours and then spotted on reporter assay media containing acetaldehyde (10 mM). (C) Life-span extending manipulations do not correlate with increased nuclear NAD⁺. To assay calorie-restricted cells, the NAD⁺ reporter strain was grown for 2 hours in defined SC medium with either 2.0% glucose (standard concentration) or 0.5% glucose (CR). Cultures were washed and spotted in serial 10-fold dilutions on assay media with the same glucose concentrations. The retarded growth of the strain on restricted medium was rescued by acetaldehyde (10 mM). (D) The NAD⁺ reporter strain was spotted on assay media and grown under two low-intensity stresses known to extend replicative life-span: high osmolarity (4% glucose) (2) and heat stress (37°C) (8, 9).

Fig. 2

In vivo ¹³C NMR confirms that calorie restricted cells have lower NAD⁺. Intracellular concentrations of NAD⁺ as a function of time for cells grown in 2.0% glucose or 0.5% glucose as calculated by reference to a labeled lactate capillary standard. Additional glucose (2%) was added at time shown by arrow.

Fig. 3

Effect of NADH on yeast Sir2 and human SIRT1 activity. Error bars represent standard error of the mean. (A) Sir2 and SIRT1 deacetylation assays with 4 mM NAD⁺ and increasing concentrations of NADH. Activity values shown are normalized against the activities of the recombinant proteins in the absence of NADH. Activities were 23 and 100 pmol hour⁻¹ μg⁻¹ for Sir2 at 0.05 mM and 4 mM NAD⁺ respectively and 160 and 500 pmol hour ⁻¹ μg⁻¹ for SIRT1. (B) Sir2 and SIRT1 deacetylation assays with 0.05 mM NAD⁺ and increasing concentrations of NADH. For clarity, data is shown on two different scales, the upper panel for 0 to 1 μM NADH and (C) the lower panel for higher NADH concentrations. (D) Ribosomal DNA locus (RDN1) silencing assays with or without acetaldehyde (10 mM). Cells were pretreated for 2 to 3 hours and then spotted to assay plates in 10-fold dilutions or single spots for the ADE2 and MET15 assay, respectively. Silencing of the RDN1::ADE2 reporter results in growth retardation on plates lacking adenine, whereas silencing of the RDN1::MET15 reporter leads to a brown coloration on Pb²⁺-containing medium.