B Total amino acid concentrations in wild-type and pka1Δ cells at different aging time points as indicated. C Heatmap showing quantitation of individual amino acids, along with known amino acid properties at right. Averages of 3 time points with 3 independent biological repeats each are shown.

The amino acid quantities, obtained from nondividing cells, differed from those previously reported for fission yeast which have been obtained from rapidly proliferating cells 20 , At the onset of stationary phase, the total amino acid concentrations were lower in pka1Δ than in wild-type cells Figure 1B.

Accordingly, the individual amino acid concentrations were lower or similar in pka1Δ than in wild-type cells at that stage Figures 1C and 2. The concentration differences were particularly pronounced for the branched chain amino acids and aromatic amino acids phenylalanine, tryptophan, tyrosine Figures 1C and 2.

Changing amino acid concentrations during aging. Normalized concentration values of 19 amino acids in wild-type cells panels A, B and pka1Δ cells panels C, D, E during aging top to bottom. For each condition, 3 independent samples were analyzed. Boxplots were created with R boxplot and default settings.

During chronological aging, the concentration of total free amino acids declined in wild-type but less so in pka1Δ cells, even rising slightly at the last time point Figure 1B. This rise primarily reflected an increase in the most abundant amino acid, lysine, which compensated for the decline in other amino acids Figure 2.

In wild-type samples, the variation in free amino acids between experimental repeats noticeably increased during aging, in contrast to pka1Δ samples Figures 1B and 2. This result raises the possibility that increased variation of amino acid concentrations, reflecting less tight metabolic regulation, is a feature of wild-type aging cultures.

Wild-type cells showed a general decrease in amino acids during aging, apart from aspartate Figures 1C and 2. The branched chain amino acids were initially present at ~3-fold higher levels in wild-type cells before dropping to about half the level of pka1Δ cells Figures 1C and 2.

This result suggests that changes in amino acid concentrations are not driven by limitation of precursor molecules. Likewise, there was no clustering based on glucogenic amino acids, which can be converted into glucose through gluconeogenesis Figure 1C.

This result suggests that any need for gluconeogenesis under glucose depletion does not greatly affect free amino acid composition. Reassuringly, there was also no clustering based on membrane permeability Figure 1C , as this suggests that the results were not biased by amino acids leaking from nonviable cells during the aging time course.

This result suggests that similar metabolic changes occur in aging wild-type and mutant cells, but that these changes are delayed in the long-lived mutant cells.

Some amino acids, however, showed distinct patterns in wild-type and pka1Δ cells, including lysine, glutamate, glutamine, and aspartate Figures 1C and 2. Glutamine and aspartate showed particularly striking profiles during aging. Glutamine rapidly and strongly decreased during aging, more pronounced in wild-type cells Figures 1C and 2.

Aspartate, on the other hand, was the only amino acid that increased during aging in wild-type cells, and this increase was more pronounced in pka1Δ cells Figures 1C and 2.

These results pointed to glutamine and aspartate as markers for aging in S pombe and raised the possibility that these amino acids directly contribute to cellular life span. To examine the effect of glutamine on the CLS of wild-type and pka1Δ cells, we grew cultures to stationary phase in EMM2 minimal medium.

These manipulations did not affect cell numbers or total protein levels of the aging cultures Supplementary Figure 1.

In wild-type cells, glutamine supplementation significantly extended the CLS, both when added on Day 1 or 5 Figure 3A and B. Glutamine supplementation at Day 1 extended the medial CLS from 5 to 6. Although Day 5 was close to the median life span of wild-type cells, glutamine still extended the CLS even at this late stage Figure 3A and B.

These results indicate that glutamine is beneficial for viability of aging wild-type cells. In pka1Δ cells, on the other hand, glutamine supplementation at either Day 1 or 5 had no effect on life span, with the median CLS remaining ~8 days Figure 3C and D.

We conclude that glutamine addition during cellular aging promotes longevity in wild-type cells, but not in the long-lived pka1Δ cells which maintain relatively higher glutamine levels during aging Figure 2. Effects of amino acid supplementation on chronological life span CLS.

A CLS assays average of 3 biological repeats with 3 technical repeats each for wild-type cells with and without glutamine supplementation at Days 1 and 5 as indicated. Median CLS in control cells indicated with vertical lines and treated cells indicated with dotted vertical lines are shown.

B Areas under curve AUCs of CLS assays in A as indicated left panel: AUCs from Day 1; right panel: AUCs from Day 5. The p -values t test indicate significance of difference in CLS triggered by glutamine supplementation. C CLS assays as in A for pka1Δ cells with and without glutamine supplementation at Days 1 and 5.

D AUC of CLS assays in C, as described in B. E CLS assays as in A for wild-type cells with and without aspartate supplementation at Days 1 and 5. F AUC of CLS assays in E, as described in B. G CLS assays as in A for pka1Δ cells with and without aspartate supplementation at Days 1 and 5.

H AUC of CLS assays in G, as described in B. To examine the effect of aspartate on the CLS of wild-type and pka1Δ cells, we performed the same experiment as with glutamine, but supplementing aspartate to chronologically aging cultures. Again, these manipulations did not affect cell numbers or total protein levels of the aging cultures Supplementary Figure 1.

In wild-type cells, aspartate supplementation at either Day 1 or 5 had no effect on the CLS Figure 3E and F. In pka1Δ cells, on the other hand, aspartate led to a significant shorter CLS, both when applied on Day 1 or 5 median CLS shortened from 8 to 6.

We conclude that aspartate addition during cellular aging has no effect in wild-type cells but shortens the CLS in pka1Δ cells where aspartate naturally strongly increases during aging Figure 2.

We report intracellular amino acid concentrations in S pombe as a function of both chronological aging and genetic background. Our results show an overall decrease in amino acids during chronological aging, especially in wild-type cells. Such a decrease has also been observed in budding yeast We cannot exclude the possibility that some dead cells contributed to these amino acid measurements, although amino acids with high membrane permeability do not increase with age and thus do not preferentially leak from dead cells.

Such amino acid signatures might therefore serve as aging biomarkers for S pombe. Glutamine and aspartate show the most distinct profiles during aging, with a strong decrease of glutamine, especially in wild-type cells, and a strong increase of aspartate, especially in pka1Δ cells.

The antagonistic changes in glutamine and aspartate are probably linked. During glucose deprivation, yeast cells turn to glutamine and glutamate for energy by making aspartate via glutaminolysis Increased aspartate levels in aging pka1Δ cells could reflect that long-lived cells feature more efficient glutaminolysis, although alanine, another product of glutaminolysis, did not show the same trend.

Induced glutaminolysis in long-lived cells could explain why aspartate did not increase life span in pka1Δ cells which naturally feature high aspartate levels. Glutamine supplementation promotes longevity of wild-type but not of long-lived pka1Δ cells.

Aspartate supplementation, on the other hand, shortens the life span of pka1Δ but not of wild-type cells. These amino acids also affect life span in worms 15 : glutamine at high doses extends life span but at a lower dose shortens life span, while aspartate shortens life span.

Glutamine and aspartate both affect mitochondrial functions which might mediate their life-span effects. Glutamine, derived from the Krebs cycle metabolite alpha-ketoglutarate, is one of the amino acids recently shown to become limited when blocking respiration in fermentatively growing S pombe cells Amino acid supplementations at a later time point Day 5 have weaker effects on longevity, likely reflecting the lower viability of the cell population at that time which diminishes any beneficial.

It is actually surprising that the late supplementations, when cells are aging, still have some effect on life span. Further experiments will provide mechanistic insights into the roles of glutamine and aspartate during aging.

These findings highlight the metabolic complexity of aging and its relationship with nutrient-sensing pathways like PKA. Interestingly, decreased glutamine levels are associated with aging also in budding yeast, rats, and humans 16 , 26 , suggesting that conserved cellular processes are involved in this phenomenon.

We thank Shajahan Anver, Clara Correia-Melo, and Stephan Kamrad for critical reading and valuable comments on the manuscript. Colman RJ , Anderson RM , Johnson SC , et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. doi: Google Scholar.

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