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Calorie restriction hysteretically primes aging Saccharomyces cerevisiae toward more effective oxidative metabolism.

Tahara EB, Cunha FM, Basso TO, Della Bianca BE, Gombert AK, Kowaltowski AJ - PLoS ONE (2013)

Bottom Line: Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR.Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol.Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.

ABSTRACT
Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

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Extracellular pH changes during chronological lifespan.Extracellular pH (Panels A and B) was determined as described in Materials and Methods in the culture media of WT and ρ0 (Panels A and B, respectively) S. cerevisiae over the course of chronological aging. In Panel C, the chronological lifespan of WT cells cultured under control and CR conditions was followed over time. Hepes (100 mM) was added to maintain the pH at 7.5 where indicated. Values are expressed (means±SEM) indicate the number of colonies formed from 100 cells plated onto solid YPD media.
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pone-0056388-g003: Extracellular pH changes during chronological lifespan.Extracellular pH (Panels A and B) was determined as described in Materials and Methods in the culture media of WT and ρ0 (Panels A and B, respectively) S. cerevisiae over the course of chronological aging. In Panel C, the chronological lifespan of WT cells cultured under control and CR conditions was followed over time. Hepes (100 mM) was added to maintain the pH at 7.5 where indicated. Values are expressed (means±SEM) indicate the number of colonies formed from 100 cells plated onto solid YPD media.

Mentions: The parental strain of S. cerevisiae used in this study was BY4741 (MATa, his3Δ1, leu2Δ0; met15Δ0, ura3Δ0), except in Fig. 3C, in which the RJD1144 strain was used. ρ0 mutants were obtained through the identification, isolation and characterization of spontaneous respiratory incompetent colonies, as described elsewhere [13]. Briefly, after growth of WT cells in liquid YPD for 20 h, cells were plated onto solid YPD and this plate was replicated onto YPEG, a respiratory-selective medium. Respiratory incompetent colonies were then identified and isolated. The ρ0 phenotype of selected colonies was confirmed by mating them with S. cerevisiae mit- strains containing point mutations in mitochondrial genes. After diploid selection based on heterozygous auxotrophy complementations, no reversion of respiratory incompetence was observed. We then selected one isolated colony and further characterized it by following its growth curve (which did not exhibit pos-diauxic biomass formation) and by monitoring the exhaustion of aerobic metabolites from culture media.


Calorie restriction hysteretically primes aging Saccharomyces cerevisiae toward more effective oxidative metabolism.

Tahara EB, Cunha FM, Basso TO, Della Bianca BE, Gombert AK, Kowaltowski AJ - PLoS ONE (2013)

Extracellular pH changes during chronological lifespan.Extracellular pH (Panels A and B) was determined as described in Materials and Methods in the culture media of WT and ρ0 (Panels A and B, respectively) S. cerevisiae over the course of chronological aging. In Panel C, the chronological lifespan of WT cells cultured under control and CR conditions was followed over time. Hepes (100 mM) was added to maintain the pH at 7.5 where indicated. Values are expressed (means±SEM) indicate the number of colonies formed from 100 cells plated onto solid YPD media.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569431&req=5

pone-0056388-g003: Extracellular pH changes during chronological lifespan.Extracellular pH (Panels A and B) was determined as described in Materials and Methods in the culture media of WT and ρ0 (Panels A and B, respectively) S. cerevisiae over the course of chronological aging. In Panel C, the chronological lifespan of WT cells cultured under control and CR conditions was followed over time. Hepes (100 mM) was added to maintain the pH at 7.5 where indicated. Values are expressed (means±SEM) indicate the number of colonies formed from 100 cells plated onto solid YPD media.
Mentions: The parental strain of S. cerevisiae used in this study was BY4741 (MATa, his3Δ1, leu2Δ0; met15Δ0, ura3Δ0), except in Fig. 3C, in which the RJD1144 strain was used. ρ0 mutants were obtained through the identification, isolation and characterization of spontaneous respiratory incompetent colonies, as described elsewhere [13]. Briefly, after growth of WT cells in liquid YPD for 20 h, cells were plated onto solid YPD and this plate was replicated onto YPEG, a respiratory-selective medium. Respiratory incompetent colonies were then identified and isolated. The ρ0 phenotype of selected colonies was confirmed by mating them with S. cerevisiae mit- strains containing point mutations in mitochondrial genes. After diploid selection based on heterozygous auxotrophy complementations, no reversion of respiratory incompetence was observed. We then selected one isolated colony and further characterized it by following its growth curve (which did not exhibit pos-diauxic biomass formation) and by monitoring the exhaustion of aerobic metabolites from culture media.

Bottom Line: Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR.Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol.Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.

ABSTRACT
Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

Show MeSH
Related in: MedlinePlus