Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast

Abstract

We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions.

Keywords: Aging; Autophagy; CLS; CR; Calorie restriction; Leucine; Respiration; Saccharomyces cerevisiae; WT; calorie restriction; chronological life span; wild type.

Figure 1

Induction of autophagy during chronological aging. Macroautophagy was measured in WT cells containing plasmid pCuGFPAUT7(416) grown under the following conditions: A. Minimal medium containing 2% glucose. B. Water wash CR after growth on 2% glucose. C. Low glucose CR in 0.4% glucose medium. Equivalent amounts of total cell extracts from yeast harvested on the indicated days were analyzed by western blotting with anti-GFP antibody to detect a slower migrating GFP-Atg8p band and a faster migrating GFP band. Macroautophagy-dependent proteolysis of the GFP-Atg8p fusion protein yields GFP. The data shown in panels A and B were derived from the same yeast culture. On day 2, the culture used for panel A (days 0–1) was divided into two cultures of equal volume: one culture was used for the remaining samples in panel A (days 2–5); the other culture was washed with water and used for the samples in panel B (days 2–10). The percent conversion of GFP-Atg8p to GFP on each day is plotted in graphs below western blots and represents the degree of autophagic activation. See Materials and Methods for quantitation methodology. Day zero samples were collected during mid-log to late-log growth phase.

Figure 2

Autophagy is required for full extension of CLS by CR and promotes respiration proficiency during aging. A. CLS following growth in standard 2% glucose minimal medium. CLS was measured in yeast strains proficient (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in macroautophagy following growth in synthetic minimal medium containing 2% glucose (see Materials and Methods). Cell viability in CFU/mL is expressed as the log of the percentage of the number of viable cells on day 1 and is plotted as a function of time in days. B. CLS during water wash CR. Yeast strains were grown in standard 2% glucose minimal medium for 3 days, transferred to sterile water, and washed with water every 2–3 days thereafter. C. CLS during low glucose CR. Yeast strains were grown in standard minimal medium containing 0.4% glucose. Two independent experiments are shown in panels A-C. D-F. The percentage of petite colonies over the life span for experiment 2 in panels A-C is shown in panels D-F, respectively.

Figure 3

Autophagy is required for extension of life span during CR. A. CLS was measured in WT, atg1Δ, atg2Δ, atg7Δ, atg8Δ, and atg11Δ strains following growth in standard minimal medium containing 2% glucose. Cell viability in CFU/mL is expressed as the log of the percentage of the number of viable cells on day 1 and is plotted as a function of time in days. B. CLS during water wash CR following growth in 2% glucose minimal medium as described in Fig. 2. C. CLS during low glucose CR following growth in minimal medium containing 0.4% glucose. D. Combination of low glucose CR and water wash CR. Yeast strains were grown in minimal medium containing 0.4% glucose followed by water washing beginning on day 3 as described in Fig. 2. E-H. The percentages of petite colonies for panels A-D, respectively, are plotted as described in Fig. 2.

Figure 4

Autophagy is required for extension of life span during CR following growth in galactose medium. A. CLS was measured in WT, atg1Δ, atg2Δ, atg7Δ, atg8Δ, and atg11Δ strains following growth in standard minimal medium containing 2% galactose. Cell viability in CFU/mL is expressed as the log of the percentage of the number of viable cells on day 1 and is plotted as a function of time in days. B. CLS during water wash CR. Yeast strains were grown in minimal medium containing 2% galactose and transferred to sterile water on day 3, after which washing with sterile water was repeated every 2–3 days. C. CLS during low galactose CR. Yeast strains were grown in minimal medium containing 0.4% galactose. D. CLS following growth in standard 2% glucose minimal medium. E-H. The percentages of petite colonies for panels A-D, respectively, are plotted as described in Fig. 2.

Figure 5

Diminished requirement for autophagy during CR-mediated CLS extension in leucine prototrophs. WT, atg1Δ, atg7Δ, and atg11Δ strains with a restored LEU2 locus were previously described (Alvers et al 2009a). A. CLS was measured following growth in standard synthetic medium containing 2% glucose but lacking leucine. Cell viability in CFU/mL is expressed as the log of the percentage of the number of viable cells on day 1 and is plotted as a function of time in days. B. CLS during water wash CR following growth in 2% glucose minimal medium as described in Fig. 2. C. CLS during low glucose CR following growth in minimal medium containing 0.4% glucose. D. CLS of yeast strains were grown in standard 2% glucose minimal medium containing a three-fold elevated final concentration of leucine (+ 3XL). E-H. The percentages of petite colonies for panels A-D, respectively, are plotted as described in Fig. 2.

Figure 6

Supplemental leucine fully extends CLS in an atg1Δ strain. CLS was measured in WT and atg1Δ strains following growth in standard minimal glucose medium with: no addition (open symbols); three-fold elevated levels of histidine, lysine, and uracil (+ 3X HKu; gray symbols); or three-fold elevated levels of leucine (+ 3X L; filled symbols). Cell viability in CFU/mL is expressed as the log of the percent of the number of viable cells on day 1 and is plotted as a function of time in days. A. CLS following growth in 2% glucose medium. B. CLS during water wash CR. Yeast strains were grown in 2% glucose minimal medium for 3 days, transferred to sterile water, and washed with water every 2–3 days thereafter. C. CLS during low glucose CR. Yeast strains were grown in minimal medium containing 0.4% glucose. D-F. The percentages of petite colonies for panels A-C, respectively, are plotted as described in Fig. 2.

Figure 7

Autophagy is required for longevity in less acidic medium that is permissive for respiration deficiency during aging. A-C. CLS was measured in WT, atg1Δ, atg7Δ, and atg11Δ strains following growth in 2% glucose minimal medium containing 8, 12, or 16 mM dibasic potassium phosphate. D. CLS during low glucose CR. Yeast strains were grown in minimal medium containing 0.4% glucose containing 8 mM dibasic potassium phosphate. Cell viability in CFU/mL is expressed as the log of the percent of the number of viable cells on day 1 and is plotted as a function of time in days. E-H. Percentages of petite colonies for panels A-D, respectively, are plotted as described in Fig. 1. I-L. Media pH values for panels A-D, respectively.

Figure 8

Model for roles of autophagy and leucine in promoting respiration proficiency and chronological longevity during calorie restriction. CR upregulates macroautophagy which functions to remove dysfunctional mitochondria that would otherwise lead to respiration deficiency. Autophagy is specialized for turnover of large organelles such as mitochondria. Also shown is the role of ROS in promoting mitochondrial dysfunction. Autophagy also recycles amino acids, including leucine, which is also synthesized by functional mitochondria. Energy from functional mitochondria and amino acids and other building blocks are used to implement a post-mitotic metabolic program that supports cell survival and chronological longevity. To the extent that the post-mitotic metabolic program involves mitochondrial biogenesis for respiratory energy production in non-dividing cells (dashed arrow), there is a minimum threshold of mitochondrial function that is necessary to implement the post-mitotic metabolic program. In autophagy-deficient cells mitochondrial function drops below this threshold causing an increase in mitochondrial dysfunction, respiration deficiency, and depletion of energy and amino acids that impairs implementation of the post-mitotic program. The result is cellular damage and toxicity that leads to aging. Growth in glucose may exacerbate the problem of low mitochondrial function during aging because glucose repression inhibits mitochondrial biogenesis (not shown). Autophagy also functions directly to counteract damage accumulation and toxicity (not shown).