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Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium.

Santos J, Leitão-Correia F, Sousa MJ, Leão C - Oncotarget (2015)

Bottom Line: New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation of ammonium concentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells.Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions.When present in the culture medium,ammonium impaired the consumption of the auxotrophy-complementing amino acids and caused in an improper cell cycle arrest of the culture.TOR1 deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation of ammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

ABSTRACT
New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation of ammonium concentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells. Here we describe that the CLS shortening under amino acid restriction can be completely reverted by removing ammonium from the culture medium. Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions. When present in the culture medium,ammonium impaired the consumption of the auxotrophy-complementing amino acids and caused in an improper cell cycle arrest of the culture.TOR1 deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation of ammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.

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Ammonium,when present in the culture medium,impaired the consumption of theauxotrophy-complementing amino acidsLeucine, lysine and histidine consumption of S. cerevisiae BY4742 cultured in SD media buffered to pH 3.4 with 2% (A and B) or 0.5% (C and D) glucose, supplemented with: high (A and C) and low (B and D) concentrations of auxotrophy-complementing amino acids (HAA and LAA, respectively), and with (w/) or without (w/o) NH4+ [0.5%, (NH4)2SO4]. Day -3 represents the day of culture inoculation and day zero represents the beginning of aging experiments. *(Peak values below detection limit ≈ 0). Values are means ± SEM (n=3).
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Figure 3: Ammonium,when present in the culture medium,impaired the consumption of theauxotrophy-complementing amino acidsLeucine, lysine and histidine consumption of S. cerevisiae BY4742 cultured in SD media buffered to pH 3.4 with 2% (A and B) or 0.5% (C and D) glucose, supplemented with: high (A and C) and low (B and D) concentrations of auxotrophy-complementing amino acids (HAA and LAA, respectively), and with (w/) or without (w/o) NH4+ [0.5%, (NH4)2SO4]. Day -3 represents the day of culture inoculation and day zero represents the beginning of aging experiments. *(Peak values below detection limit ≈ 0). Values are means ± SEM (n=3).

Mentions: To determine if the presence of NH4+ could be affecting cell viability by impairing the use of auxotrophy-complementing amino acids, we assessed the NH4+ and amino acid consumption during aging experiments in the culture medium. Regarding NH4+, there were no major differences for all the conditions tested (LAA and HAA media with 2% or 0.5% glucose at pH 3.4 and pH 6.0) and NH4+ was never completely consumed, remaining in the medium during culture aging (Fig. 2). Next, we measured by HPLC, under the same experimental conditions, the medium content of the auxotrophy-complementing amino acids in the presence and absence of NH4+. In HAA medium, either with 2% or 0.5% glucose, in the presence of NH4+ (Fig. 3A and 3C), from the three auxotrophic-complementing amino acids (leucine, lysine and histidine) only leucine was depleted from the medium after day 3 of the CLS experiment, at both pH values tested (Fig. 3A and 3C, and Fig. S2). On the contrary, in HAA medium without NH4+ either with 2% or 0.5% glucose, all three amino acids were completely depleted at both pH values, indicating that the presence of NH4+ seems to inhibit the complete consumption of these amino acids. In LAA medium pH 3.4, similar results were found, either with 2% or 0.5% glucose, in the presence or absence of NH4+ (Fig. 3B and 3D). However, in LAA medium in the presence of NH4+, poor amino acids consumption was noticed, particularly for leucine that was fully consumed for most conditions tested, except for LAA medium with NH4+ in which a fast loss of cell viability occurred (Fig. 3). At pH 6.0 similar results were obtained for the three amino acids, although a delay in their consumption was detected in comparison to pH 3.4 (Fig. S2 and Fig. 3).


Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium.

Santos J, Leitão-Correia F, Sousa MJ, Leão C - Oncotarget (2015)

Ammonium,when present in the culture medium,impaired the consumption of theauxotrophy-complementing amino acidsLeucine, lysine and histidine consumption of S. cerevisiae BY4742 cultured in SD media buffered to pH 3.4 with 2% (A and B) or 0.5% (C and D) glucose, supplemented with: high (A and C) and low (B and D) concentrations of auxotrophy-complementing amino acids (HAA and LAA, respectively), and with (w/) or without (w/o) NH4+ [0.5%, (NH4)2SO4]. Day -3 represents the day of culture inoculation and day zero represents the beginning of aging experiments. *(Peak values below detection limit ≈ 0). Values are means ± SEM (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466630&req=5

Figure 3: Ammonium,when present in the culture medium,impaired the consumption of theauxotrophy-complementing amino acidsLeucine, lysine and histidine consumption of S. cerevisiae BY4742 cultured in SD media buffered to pH 3.4 with 2% (A and B) or 0.5% (C and D) glucose, supplemented with: high (A and C) and low (B and D) concentrations of auxotrophy-complementing amino acids (HAA and LAA, respectively), and with (w/) or without (w/o) NH4+ [0.5%, (NH4)2SO4]. Day -3 represents the day of culture inoculation and day zero represents the beginning of aging experiments. *(Peak values below detection limit ≈ 0). Values are means ± SEM (n=3).
Mentions: To determine if the presence of NH4+ could be affecting cell viability by impairing the use of auxotrophy-complementing amino acids, we assessed the NH4+ and amino acid consumption during aging experiments in the culture medium. Regarding NH4+, there were no major differences for all the conditions tested (LAA and HAA media with 2% or 0.5% glucose at pH 3.4 and pH 6.0) and NH4+ was never completely consumed, remaining in the medium during culture aging (Fig. 2). Next, we measured by HPLC, under the same experimental conditions, the medium content of the auxotrophy-complementing amino acids in the presence and absence of NH4+. In HAA medium, either with 2% or 0.5% glucose, in the presence of NH4+ (Fig. 3A and 3C), from the three auxotrophic-complementing amino acids (leucine, lysine and histidine) only leucine was depleted from the medium after day 3 of the CLS experiment, at both pH values tested (Fig. 3A and 3C, and Fig. S2). On the contrary, in HAA medium without NH4+ either with 2% or 0.5% glucose, all three amino acids were completely depleted at both pH values, indicating that the presence of NH4+ seems to inhibit the complete consumption of these amino acids. In LAA medium pH 3.4, similar results were found, either with 2% or 0.5% glucose, in the presence or absence of NH4+ (Fig. 3B and 3D). However, in LAA medium in the presence of NH4+, poor amino acids consumption was noticed, particularly for leucine that was fully consumed for most conditions tested, except for LAA medium with NH4+ in which a fast loss of cell viability occurred (Fig. 3). At pH 6.0 similar results were obtained for the three amino acids, although a delay in their consumption was detected in comparison to pH 3.4 (Fig. S2 and Fig. 3).

Bottom Line: New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation of ammonium concentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells.Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions.When present in the culture medium,ammonium impaired the consumption of the auxotrophy-complementing amino acids and caused in an improper cell cycle arrest of the culture.TOR1 deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation of ammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

ABSTRACT
New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation of ammonium concentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells. Here we describe that the CLS shortening under amino acid restriction can be completely reverted by removing ammonium from the culture medium. Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions. When present in the culture medium,ammonium impaired the consumption of the auxotrophy-complementing amino acids and caused in an improper cell cycle arrest of the culture.TOR1 deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation of ammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.

Show MeSH
Related in: MedlinePlus