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. 2009 Apr;8(2):113-27.
doi: 10.1111/j.1474-9726.2009.00459.x. Epub 2009 Feb 23.

Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans

Eric L Greer  1 , Anne Brunet
Affiliations

Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans

Eric L Greer et al. Aging Cell. 2009 Apr.

Abstract

Dietary restriction (DR) has the remarkable ability to extend lifespan and healthspan. A variety of DR regimens have been described in species ranging from yeast to mammals. However, whether different DR regimens extend lifespan via universal, distinct, or overlapping pathways is still an open question. Here we examine the genetic pathways that mediate longevity by different DR regimens in Caenorhabditis elegans. We have previously shown that the low-energy sensing AMP-activated protein kinase AMPK/aak-2 and the Forkhead transcription factor FoxO/daf-16 are necessary for longevity induced by a DR regimen that we developed (sDR). Here we find that AMPK and FoxO are necessary for longevity induced by another DR regimen, but are dispensable for the lifespan extension induced by two different DR methods. Intriguingly, AMPK is also necessary for the lifespan extension elicited by resveratrol, a natural polyphenol that mimics some aspects of DR. Conversely, we test if genes previously reported to mediate longevity by a variety of DR methods are necessary for sDR-induced longevity. Although clk-1, a gene involved in ubiquinone biosynthesis, is also required for sDR-induced lifespan extension, we find that four other genes (sir-2.1, FoxA/pha-4, skn-1, and hsf-1) are all dispensable for longevity induced by sDR. Consistent with the observation that different DR methods extend lifespan by mostly independent genetic mechanisms, we find that the effects on lifespan of two different DR regimens are additive. Understanding the genetic network by which different DR regimens extend lifespan has important implications for harnessing the full benefits of DR on lifespan and healthspan.

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Figures

Fig. 1
Fig. 1
AMPK/aak-2 and FoxO/daf-16 are necessary for lifespan extension by sDR across a gradient of bacteria. (A) A serial dilution of bacteria on plates (5 × 1012 to 5 × 107 bacteria mL−1) extends WT (N2) worm lifespan but does not extend aak-2(ok524) mutant worm lifespan. Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of aak-2(ok524) mutant worms (P < 0.0001). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S1A. (B) A serial dilution of bacteria on plates extends WT (N2) worm lifespan but does not extend daf-16(mu86) mutant worm lifespan. Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of daf-16 (mu86) mutant worms (P < 0.0001). Mean, standard errors, and statistical analysis for one experiment performed in triplicate are presented in Table S1B.
Fig. 5
Fig. 5
clk-1 is necessary for sDR to extend lifespan. A serial dilution of bacteria on plates extended WT (N2) worm lifespan (28.5%, P < 0.0001) but did not extend two independent aak-2 mutant strains, aak-2(rr48) (1.0%P = 0.5787) and aak-2(ok524) (–1.4%P = 0.7804), or clk-1(e2519) mutant worm lifespans (0%P = 0.6921). Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of aak-2(ok524) mutant worms (P < 0.0001), aak-2(rr48) mutant worms (P < 0.0001), or clk-1(e2519) mutant worms (P < 0.0001). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S9.
Fig. 2
Fig. 2
AMPK and FoxO mediate longevity induced by some but not all DR methods. (A) Longevity induced by dilution of peptone (DP) is dependent on AMPK/aak-2 and FoxO/daf-16. Dilution of peptone in the plates extends WT (N2) worm lifespan (25.4%, P < 0.0001) but does not extend aak-2(ok524) mutant lifespan (–1.0%, P = 0.6371) or daf-16(mu86) mutant lifespan (5.7%, P = 0.1402). Two-way anova revealed that the lifespan extension of WT (N2) worms across a peptone gradient was significantly different from that of aak-2(ok524) mutant worms (P < 0.0001) or daf-16(mu86) mutant worms (P < 0.0001). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S2. (B) AMPK/aak-2 and FoxO/daf-16 are not completely necessary for bDR lifespan extension. The average and SEM of three independent experiments indicates that bDR increases WT (N2), aak-2(ok524), and daf-16(mu86) mutant lifespan but appears to increase WT (N2) worm lifespan to a greater extent than aak-2(ok524) or daf-16(mu86) mutant worm lifespans. For each individual experiments, see Figure S2 and Table S3. (C) As previously reported (Curtis et al., 2006), the eat-2(ad1116) mutation extends WT (N2) worm lifespan (19.8%, P < 0.0001) and aak-2(ok524) mutant lifespan (19.4%, P < 0.0001). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S4A. (D) As previously reported (Lakowski & Hekimi, 1998), the eat-2(ad1116) mutation extends WT (N2) worm lifespan (12.9%, P < 0.0001) and daf-16(mu86) mutant lifespan (32.3%P < 0.0001). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S4B.
Fig. 3
Fig. 3
Resveratrol extends lifespan in an AMPK-dependent, but FoxO-independent, manner. (A) Resveratrol (100 µm) extended WT (N2) worm lifespan (14.2%, P = 0.0005), but did not significantly extend aak-2(ok524) mutant worm lifespan (2.2%P = 0.5485). Mean, standard errors, and statistical analysis for three independent experiments performed in triplicate are presented in Table S5. (B) Resveratrol (100 µm) extended both WT (N2) worm lifespan (14.6%P < 0.0001) and daf-16(mu86) mutant worm lifespan (13.7%P < 0.0001). Res, resveratrol. Mean, standard errors, and statistical analysis for one experiment performed in triplicate are presented in Table S5.
Fig. 4
Fig. 4
sir-2.1, FoxA/pha-4, skn-1 and hsf-1 are not entirely necessary for sDR to extend lifespan. (A) A serial dilution of bacteria on plates (5 × 1012 to 5 × 107 bacteria mL−1) extends WT (N2) (26.1%, P < 0.0001) and sir-2.1(ok434) (16.6%, P < 0.0001) mutant worm lifespan but does not extend aak-2(ok524) (1.3%, P = 0.6330) mutant worm lifespan. Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of aak-2(ok524) mutant worms (P < 0.0001) but not statistically different from sir-2.1(ok434) mutant worms (P = 0.1240). Mean, standard errors, and statistical analysis for three independent experiments performed in triplicate are presented in Table S6. (B) A serial dilution of bacteria on plates extended smg-1(cc546ts) and smg-1(cc546ts); pha-4(zu225) mutant worms to a similar extend (P = 0.3724 by two-way anova). Note that this experiment was performed at 15 °C after worms reached adulthood. Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S7A. (C) A serial dilution of bacteria on plates extended WT (N2) (24.9%, P < 0.0001) worm lifespan, skn-1(zu135) (23.1%, P < 0.0001) mutant worm lifespan, and hsf-1(sy441) (33.5%, P < 0.0001) mutant worm lifespan but did not extend aak-2(rr48) (4.6%, P = 0.2566) mutant worm lifespan. Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of aak-2(ok524) mutant worms (P < 0.0001) but not statistically different from skn-1(zu135) mutant worm lifespan (P = 0.5567) or hsf-1(sy441) mutant worm lifespan (P = 0.2843). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S8 and Table S9.
Fig. 6
Fig. 6
sDR and eat-2 have an additive effect on lifespan. sDR extended WT (N2) worm lifespan (18.2%P < 0.0001) and eat-2(ad1116) mutant worm lifespan (18.0%P < 0.0001) to the same extent. sDR: 5 × 108 bacteria mL−1 and AL (ad libitum): 5 × 1011 bacteria mL−1. Mean, standard errors, and statistical analysis for four independent experiments performed in triplicate are presented in Table S4.
Fig. 7
Fig. 7
Different DR methods. (A) Table summarizing genes that have been tested for specific DR methods. Question marks indicate conflicting reports in the literature. sDR, ‘solid DR’ (Greer et al., 2007); eat-2 (Lakowski & Hekimi, 1998); bDR, liquid DR (Klass, 1977; Panowski et al., 2007); lDR, Liquid DR (Bishop & Guarente, 2007); DD, dietary deprivation (Kaeberlein et al., 2006; Lee et al., 2006); AM, axenic medium (Houthoofd et al., 2002a; Houthoofd et al., 2002b); DP, dilution of peptone in plates (Hosono et al., 1989); CDLM, chemically defined liquid media (Szewczyk et al., 2006). D, dependent; PD, partially dependent; I, independent; ND, not determined. *Not a null mutant, making results more difficult to interpret. **Experiments were performed with RNAi, making results more difficult to interpret. let-363: C. elegans TOR gene mutant (Henderson et al., 2006; Hansen et al., 2007). [1]: (Greer et al., 2007), [2]: (Curtis et al., 2006), [3]: (Lakowski & Hekimi, 1998), [4]: (Wang & Tissenbaum, 2006), [5]: (Hansen et al., 2007), [6]: (Panowski et al., 2007), [7]: (Hsu et al., 2003), [8]: (Houthoofd et al., 2003) [9]: (Henderson et al., 2006), [10]: (Hansen et al., 2005), [11]: (Bishop & Guarente, 2007), [12]: (Kaeberlein et al., 2006), [13]: (Lee et al., 2006), [14]: (Steinkraus et al., 2008), [15]: (Houthoofd et al., 2002b), [16]: (Viswanathan et al., 2005), [17]: (Wood et al., 2004), [18]: (Bass et al., 2007).(B) Different methods of DR activate distinct signaling pathways. Displayed are molecules that have been shown to play a role in mediating the longevity extension effects of DR methods. Question marks indicate conflicting reports (Henderson et al., 2006; Wang & Tissenbaum, 2006; Hansen et al., 2007). LET-363: C. elegans TOR protein.
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