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. 2014 Dec;13(6):1075-85.
doi: 10.1111/acel.12273. Epub 2014 Sep 25.

Dietary Restriction Involves NAD⁺ -Dependent Mechanisms and a Shift Toward Oxidative Metabolism

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Free PMC article

Dietary Restriction Involves NAD⁺ -Dependent Mechanisms and a Shift Toward Oxidative Metabolism

Natalie Moroz et al. Aging Cell. .
Free PMC article

Abstract

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺ -dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF-16 (FOXO), SKN-1 (Nrf1/2/3), PHA-4 (FOXA), AAK-2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR-2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC-1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc-1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺ -dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺ -dependent processes may be supported by a DR-induced shift toward oxidative metabolism rather than an increase in total respiration.

Keywords: C. elegans; NAD+; aging; dietary restriction; sirtuins; stress response.

Figures

Figure 1
Figure 1
Lifespan extension from liquid-based DR and DD. (A) Description of the liquid DR method. C. elegans were allowed to develop under standard C. elegans conditions, then moved to NGM plates seeded with treated bacterial food (strain OP50) as day-one adults. These animals were moved to 12-well plates containing 2.5 mL of different concentrations of treated OP50 after 2 days of AL feeding, then moved to fresh cultures every 2–3 weeks. (B) Representative lifespans of wild-type (N2) worms that were fed different concentrations of treated bacteria (indicated as bacterial OD) and examined in parallel. Mean values and analysis of this experiment are presented in Table S12 (experiment 1C). (C) Effect of food concentration on mean lifespan. This graph describes a composite of 21 independent experiments, each of which examined a range of serially diluted treated bacteria in parallel. Lifespans were normalized to AL by dividing the mean lifespan at each food concentration by the AL value determined in parallel. DR and DD increased lifespan by 59.4% and 76%, respectively. SEM is shown. Compared to AL, a two-tailed t-test for all A600 values, < 0.001. Individual experiments are presented in Table S12.
Figure 2
Figure 2
Importance of stress response mechanisms for DR lifespan extension. (A) Lack of DAF-16 impaired DR lifespan extension. AL lifespans obtained in parallel are shown in (B). (C,D) skn-1 was required for DR to extend lifespan. Whereas WT worms experienced an average of 48.3% and 91.0% increase in lifespan upon DR and DD, respectively, these increases were only 20.9% and 13.3% in predicted null skn-1 mutants. (D) Under AL liquid conditions, skn-1 mutants’ mean lifespan was equal to that of WT, in contrast to results obtained on plates (Tullet et al., 2008). (E, F) pha-4(zu225) mutation eliminated DR longevity in the control smg-1(cc546ts) background. smg-1 lifespan was increased 33.2% by DR and 38.8% by DD, respectively. (G,H) The AMPK subunit AAK-2 is required for full DR and DD lifespan extension. Composites of all analyses are shown, with individual experiments presented in Table S13 (Fig.2A,B), Table S14 (Fig.2C,D), Table S15 (Fig.2E,F), and Table S16 (Fig.2G,H). Two-way ANOVA analysis across the bacterial gradient (compared to control): *= 0.0004, **= 0.0001. The food concentration 0.50 A600 is underlined to facilitate comparison of results.
Figure 3
Figure 3
The NAD+ salvage pathway and NAD+-dependent SIRT1/sir-2.1 regulate DR lifespan. (A, B) Reduced DR response in sir-2.1(ok434) mutants. Note that the sir-2.1 mutants live longer than WT worms under AL feeding. (C-F) Impairment of DR lifespan extension in sir-2.1;sir-2.2;sir-2.4 (C, D) and sir-2.1;sir-2.3;sir-2.4 (E, F) triple mutants. (G, H) pnc-1 is required for DR lifespan extension. Composites are shown, with data from individual experiments presented in Table S17 (Fig.3A,B), Table S18 (Fig.3C,D), Table S19 (Fig.3E,F), and Table S20 (Fig.3G,H). Two-way ANOVA analysis: **P = 0.0001.
Figure 4
Figure 4
DR increases movement and stress resistance independently of NAD+ salvage. (A,B) DR increased the rate of spontaneous movement comparably in aging WT and pnc-1(pk9605) animals. Body bends per minute were scored. Note that pnc-1 mutation did not affect the percentage increase associated with DR. (C,D) DR comparably increased thermotolerance (survival at 38 °C) in WT and pnc-1 animals. The time points indicated refer to days after hatching. Composites of all analyses are shown, with individual experimental data, mean, standard error, percent change, and statistical analysis presented in Table S21 (Fig.4A,B), and Table S22 (Fig.4C,D). *t-test vs. AL,P < 0.05; **t-test vs. AL,P < 0.001; $t-test vs. age 13, P < 0.01; @t-test vs. age 10, P < 0.065; #t-test vs. age 10, P < 0.025.
Figure 5
Figure 5
Differential effects of DR on overall and productive respiration. (A) Oxygen consumption per animal decreases with age. One-way ANOVA WT day 9 vs. day 12 vs. day 16, P < 0.0001, one-way ANOVA pnc-1 day 9 vs. day 12 vs. day 16, P < 0.0001. (B) Oxygen consumption per protein mass is elevated in older animals. One-way ANOVA WT day 9 vs. day 12 vs. day 16, P < 0.001, one-way ANOVA pnc-1 day 9 vs. day 12 vs. day 16, P < 0.0001. (C,D) DR reduces the overall respiration rate. One-way ANOVA WT AL vs. DR,P < 0.0001, two-way ANOVA WT AL/DR and age, P < 0.0003, one-way ANOVA pnc-1AL vs. DR,P < 0.032, two-way ANOVA pnc-1AL/DR and age, P < 0.0025. OCR is shown as normalized to worm number (C) and protein (D). Note that OCR values and trends are comparable in WT and pnc-1(pk9605) animals. AL data for WT and pnc-1(pk9605) that were used for normalization in (C,D) are shown in (A,B) and were obtained and analyzed in parallel to DR data. (E,F) DR increases the productive fraction of respiration, as detected by the increase in OCR seen upon administration of the mitochondrial uncoupler FCCP. Note that this trend was similar in WT and pnc-1(pk9605) animals. One-way ANOVA WT AL vs. DR,P < 0.023, two-way ANOVA WT AL vs. DR and age, P < 0.0635, one-way ANOVA pnc-1AL vs. DR,P < 0.011, two-way ANOVA pnc-1AL vs. DR and age, P < 0.0208, two-way ANOVA WT/pnc-1 and age, P < 0.5548, two-way ANOVA WT/pnc-1 and AL vs. DR,P < 0.0021. OCR is shown as normalized to worm number. Similar trends are seen in data normalized to protein content, presented in Fig. S3. In each experiment, samples were assayed in 3–5 replicates per experiment. Wells that did not respond were censored. Individual experimental data, mean, standard error, percent change, and statistical analysis are presented in (A) Table S23, S24, (B) Table S25, S26, (C) Table S27, (D) Table S28, (E) Table S23, S24, (F) Table S23, S24. *t-test vs. AL,P < 0.06; **t-test vs. AL,P < 0.01; @t-test vs. age 9, P < 0.055; #t-test vs. age 9, P < 0.01.
Figure 6
Figure 6
Importance of stress defense and metabolic mechanisms in DR. As described in the text, genetic experiments implicate the indicated growth-regulated stress-defense pathways and NAD+-associated mechanisms in DR. Other mechanisms that have been implicated in DR in C. elegans are consistent with this overall model. For example, both AMPK and PHA-4 promote autophagy (Hansen et al., ; Egan et al., 2011).

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