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. 2005 Nov;1(5):e69.
doi: 10.1371/journal.pgen.0010069. Epub 2005 Nov 25.

Increased life span due to calorie restriction in respiratory-deficient yeast

Matt Kaeberlein  1 , Di Hu, Emily O Kerr, Mitsuhiro Tsuchiya, Eric A Westman, Nick Dang, Stanley Fields, Brian K Kennedy
Affiliations

Increased life span due to calorie restriction in respiratory-deficient yeast

Matt Kaeberlein et al. PLoS Genet. 2005 Nov.

Abstract

A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD+-dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD+ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Respiration Is Not Required for Life Span Extension by CR in BY4742
(A) BY4742 rho0 strains lack mitochondrial DNA. DAPI staining of BY4742 (i) wild-type or (ii) rho0 cells grown under standard conditions (2% glucose) and calorie-restricted (iii) wild-type or (iv) rho0 cells (CR = 0.05% glucose). (B) BY4742 rho0 strains are unable to grow on glycerol as the sole carbon source. (i) BY4742 wild-type or (ii) rho0 cells on YEP supplemented with 2% glucose or 3% glycerol. (C) CR increase life span in BY4742 rho0 cells. Replicative life span analysis for BY4742 wild-type and rho0 cells on 2% glucose and 0.05% glucose (CR). Mean life span is shown in parentheses.
Figure 2
Figure 2. Respiration Is Not Required for Life Span Extension by CR in PSY316
(A) PSY316AUT rho0 strains lack mitochondrial DNA. DAPI staining of PSY316 (i) wild-type or (ii) rho0 cells grown under standard conditions (2% glucose) and calorie-restricted (iii) wild-type or (iv) rho0 cells (CR = 0.05% glucose). (B) PSY316AUT rho0 strains are unable to grow on glycerol as the sole carbon source. (i) PSY316AUT wild-type, (ii) cyt1Δ rho0, (iii) cyt1Δ, or (iv) rho0 cells on YEP supplemented with 2% glucose or 3% glycerol. (C) CR increases life span in PSY316AUT rho0 cells. Replicative life span analysis for PSY316AUT wild-type and rho0 cells on 2% glucose and 0.05% glucose (CR). Mean life span is shown in parentheses. (D) CR increases the life span of cyt1Δ cells. Replicative life span analysis for PSY316AUT wild-type and cyt1Δ cells on 2% glucose and 0.05% glucose (CR). Mean life span is shown in parentheses. (E) CR increases the life span of cyt1Δ rho0 cells. Replicative life span analysis for PSY316AUT wild-type and cyt1Δ rho0 cells on 2% glucose and cyt1Δ rho0 cells on 0.05% glucose (CR). Mean life span is shown in parentheses.
Figure 3
Figure 3. Effect of Glucose Concentration on Life Span and Sir2 Activity in Respiratory-Deficient Mutants
(A) Mean replicative life span is significantly increased in PSY316 AUT cyt1Δ and rho0 cells as the glucose concentration is decreased to either 0.5% or 0.05%, relative to life span on 2% glucose. *p < 0.05, **p < 0.01. (B) Sir2 activity is not increased by CR but is responsive to increased expression of Sir2 or to addition of exogenous nicotinamide. Transcriptional silencing of the telomeric URA3 marker in PSY316AUT (WT) was monitored by the survival of cells plated onto medium containing 5-FOA. (C) Sir2 activity is not altered in respiratory deficient cyt1Δ cells and is unaffected by CR. Transcriptional silencing of the telomeric URA3 marker in PSY316AUT (WT) was monitored by the survival of cells plated onto medium containing 5-FOA.
Figure 4
Figure 4. Effect of Nicotinamide on Life Span Extension by CR
(A) Nicotinamide shortens the life span of wild-type cells. Replicative life span analysis for BY4742 wild-type and sir2Δ cells on 2% glucose containing or lacking 5 mM nicotinamide (nic). Mean life span is shown in parentheses. (B) Nicotinamide partially prevents Sir2-independent life span extension by CR. Replicative life span analysis for BY4742 wild type on 2% glucose, along with sir2Δ fob1Δ double mutant cells on 2% glucose or 0.05% glucose (CR) containing or lacking 5 mM nicotinamide (nic). Mean life span is shown in parentheses.
Figure 5
Figure 5. Genetic Pathways Determining Replicative Life Span in Yeast
Sir2 and CR act in parallel pathways to slow aging. Both pathways are affected by nicotinamide levels.
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