N-acylethanolamine signalling mediates the effect of diet on lifespan in Caenorhabditis elegans

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Dietary restriction is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, nematode worms, flies and rodents1, 2. Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process3, 4, 5, including the nutrient-sensing target of rapamycin (TOR) pathway6 and the Foxa transcription factor PHA-4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance8. Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis9. N-acylethanolamines (NAEs) are lipid-derived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietary-restriction-induced lifespan extension in wild-type worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.

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  1. NAE levels in C. elegans are modulated by FAAH activity.
    Figure 1: NAE levels in C. elegans are modulated by FAAH activity.

    a, Levels of NAEs in first-day adult wild-type N2 worms measured by SID-GC-MS (mean+s.d., n = 5). AEA, arachidonoyl ethanolamide; EPEA, eicosapentaenoyl ethanolamide; LOEA, linoleoyl ethanolamide; OEA, oleoyl ethanolamide; PEA, palmitoyl ethanolamide; POEA, palmitoleoyl ethanolamide. b, EPEA levels are elevated in first-day eri-1(mg366); lin-15B(n744) adults after exposure to faah-1 dsRNA by soaking (mean+s.d., n = 2). c, EPEA levels are elevated in first-day wild-type N2 adults after 24h exposure to 10 μM URB597, a chemical inhibitor of mammalian FAAH (mean+s.d., n = 5, P<0.05, Wilcoxon signed rank test). d, Overexpression of faah-1 results in reduced EPEA levels in first-day wild-type N2 adults (mean+s.d.; N2, n = 9; rfIs22, n = 7; and rfIs23, n = 8, P<0.05 for both rfIs22 and rfIs23, Wilcoxon signed rank test).

  2. NAEs affect reproductive growth and dauer formation.
    Figure 2: NAEs affect reproductive growth and dauer formation.

    a, faah-1 overexpression results in developmental delay (mean+s.d.; N2, n = 54; rfIs23, n = 76). b, faah-1 RNAi rescues the growth delay of faah-1 overexpressors (mean+s.d.; N2, n = 59; rfIs23, n = 53). c, Levels of EPEA during development in N2 and daf-2(e1368) animals grown at 25°C (mean+s.d., n = 2). D, dauer; GA, gravid adult; L1, first larval stage; L2, second larval stage; L2d, alternative L2 stage preceding the dauer moult; L2d*, later time point in L2d; L3, third larval stage; L4, fourth larval stage; YA, young adult. d, Effect of treatment with exogenous NAEs on reproductive growth in daf-2(e1368) mutants at 24°C (mean+s.d., n = 2). e, Scheme illustrating genes and pathways involved in dauer formation in C. elegans. f, EPEA rescues dauer formation in multiple dauer constitutive mutants (all P<0.0001, chi-squared test, additional data in Supplementary Table 1).

  3. Reduced NAE levels are associated with dietary restriction and are sufficient to confer lifespan extension.
    Figure 3: Reduced NAE levels are associated with dietary restriction and are sufficient to confer lifespan extension.

    a, EPEA levels are reduced in starved L1 larvae and increase after 6h of exposure to food (mean+s.d., n = 3). b, EPEA levels are altered in response to food availability in adult wild-type N2 animals (mean+s.d., Mann–Whitney U-test: 12h fed (n = 6) versus dietary restriction (DR; n = 12), P<0.05; 24h fed (n = 7) versus dietary restriction (n = 7), P<0.001; 24h dietary restriction versus re-fed (n = 6), P<0.005; 24h fed versus re-fed, P = not significant). c, faah-1 overexpression extends lifespan in N2 wild-type animals under fed conditions (1×1010 colony-forming units (c.f.u.)ml−1 Escherichia coli, P<0.0001, log-rank test). d, Lifespan is not different between N2 and a faah-1 overexpressing line under conditions of optimal dietary restriction (1×109c.f.u.ml−1 E. coli). e, faah-1 overexpression extends lifespan in N2 wild-type animals under conditions of sub-optimal dietary restriction conditions (1×108c.f.u.ml−1 E. coli, P<0.0001, log-rank test). f, faah-1 overexpression affects lifespan in a nutrient-dependent manner (mean lifespan ±s.d., n = 3). g, faah-1 overexpression extends lifespan in a daf-16 mutant (P<0.0001, log-rank test). h, Lifespan extension resulting from faah-1 overexpression requires the Foxa transcription factor PHA-4 (N2 control versus N2 plus pha-4 RNAi, P<0.0001; rfIs22 control versus rfIs22 plus pha-4 RNAi, P<0.0001; rfIs22 control versus N2 control, P = 0.0014; log-rank test).

  4. EPEA suppresses the effects of dietary restriction on lifespan.
    Figure 4: EPEA suppresses the effects of dietary restriction on lifespan.

    a, EPEA treatment reduces lifespan in wild-type N2 animals on control RNAi bacteria (P<0.0001, log-rank test). b, EPEA treatment reduces lifespan in daf-2(e1368) mutants on control RNAi bacteria (P = 0.0005, log-rank test). c, EPEA has a minimal effect on N2 lifespan in the presence of high food concentrations (1×1011 c.f.u.ml−1 E. coli, P<0.0001, log-rank test). d, EPEA treatment completely suppresses the effect of optimal dietary restriction on wild-type N2 lifespan (1×109 c.f.u.ml−1 E. coli, P<0.0001; log-rank test). e, EPEA levels are reduced in rsks-1(ok1255) mutants, a genetic model of dietary restriction (mean+s.d., n = 4, P<0.05, Mann–Whitney U-test). f, EPEA treatment suppresses lifespan extension in rsks-1(ok1255) mutants (P<0.0001, log-rank test).


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  1. Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, California 94945, USA

    • Mark Lucanic,
    • Jason M. Held,
    • Maithili C. Vantipalli,
    • Ida M. Klang,
    • Jill B. Graham,
    • Bradford W. Gibson,
    • Gordon J. Lithgow &
    • Matthew S. Gill
  2. Karolinska Institute, Center for Biosciences at NOVUM, Department of Biosciences and Nutrition, Hälsovägen 7, S-141 83 Huddinge, Sweden

    • Ida M. Klang
  3. Present address: The Scripps Research Institute – Scripps Florida, 130 Scripps Way, 3B3, Jupiter, Florida 33458, USA.

    • Matthew S. Gill


M.L., J.M.H., B.W.G., G.J.L. and M.S.G. conceived of and planned experiments. M.L., M.C.V., I.M.K., J.B.G. and M.S.G. performed experiments. M.L. and M.S.G. wrote the manuscript.

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The authors declare no competing financial interests.

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