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

Journal name:
Nature
Volume:
473,
Pages:
226–229
Date published:
DOI:
doi:10.1038/nature10007
Received
Accepted
Published online

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.

At a glance

Figures

  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).

References

  1. Mair, W. & Dillin, A. Aging and survival: the genetics of life span extension by dietary restriction. Annu. Rev. Biochem. 77, 727754 (2008)
  2. Bishop, N. A. & Guarente, L. Genetic links between diet and lifespan: shared mechanisms from yeast to humans. Nature Rev. Genet. 8, 835844 (2007)
  3. Honjoh, S., Yamamoto, T., Uno, M. & Nishida, E. Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans. Nature 457, 726730 (2009)
  4. Bishop, N. A. & Guarente, L. Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature 447, 545549 (2007)
  5. Carrano, A. C., Liu, Z., Dillin, A. & Hunter, T. A conserved ubiquitination pathway determines longevity in response to diet restriction. Nature 460, 396399 (2009)
  6. Kapahi, P. et al. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab. 11, 453465 (2010)
  7. Panowski, S. H., Wolff, S., Aguilaniu, H., Durieux, J. & Dillin, A. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature 447, 550555 (2007)
  8. Di Marzo, V. & Matias, I. Endocannabinoid control of food intake and energy balance. Nature Neurosci. 8, 585589 (2005)
  9. McPartland, J. M. & Glass, M. Functional mapping of cannabinoid receptor homologs in mammals, other vertebrates, and invertebrates. Gene 312, 297303 (2003)
  10. Hardison, S., Weintraub, S. T. & Giuffrida, A. Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations. Prostaglandins Other Lipid Mediat. 81, 106112 (2006)
  11. Lehtonen, M., Reisner, K., Auriola, S., Wong, G. & Callaway, J. C. Mass-spectrometric identification of anandamide and 2-arachidonoylglycerol in nematodes. Chem. Biodivers. 5, 24312441 (2008)
  12. Di Marzo, V., Bifulco, M. & De Petrocellis, L. The endocannabinoid system and its therapeutic exploitation. Nature Rev. Drug Discov. 3, 771784 (2004)
  13. McPartland, J. M., Matias, I., Di Marzo, V. & Glass, M. Evolutionary origins of the endocannabinoid system. Gene 370, 6474 (2006)
  14. Leung, D., Saghatelian, A., Simon, G. M. & Cravatt, B. F. Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry 45, 47204726 (2006)
  15. Fielenbach, N. & Antebi, A. C. elegans dauer formation and the molecular basis of plasticity. Genes Dev. 22, 21492165 (2008)
  16. Kirkham, T. C., Williams, C. M., Fezza, F. & Di Marzo, V. Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br. J. Pharmacol. 136, 550557 (2002)
  17. Izzo, A. A. et al. Basal and fasting/refeeding-regulated tissue levels of endogenous PPAR-α ligands in Zucker rats. Obesity 18, 5562 (2010)
  18. Chen, D., Thomas, E. L. & Kapahi, P. HIF-1 modulates dietary restriction-mediated lifespan extension via IRE-1 in Caenorhabditis elegans. PLoS Genet. 5, e1000486 (2009)
  19. Kaeberlein, T. L. et al. Lifespan extension in Caenorhabditis elegans by complete removal of food. Aging Cell 5, 487494 (2006)
  20. Banni, S. & Di Marzo, V. Effect of dietary fat on endocannabinoids and related mediators: consequences on energy homeostasis, inflammation and mood. Mol. Nutr. Food Res. 54, 8292 (2010)
  21. Watts, J. L. & Browse, J. Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 99, 58545859 (2002)
  22. Valenti, M. et al. The endocannabinoid system in the brain of Carassius auratus and its possible role in the control of food intake. J. Neurochem. 95, 662672 (2005)
  23. Soderstrom, K., Tian, Q., Valenti, M. & Di Marzo, V. Endocannabinoids link feeding state and auditory perception-related gene expression. J. Neurosci. 24, 1001310021 (2004)
  24. Breunig, E. et al. The endocannabinoid 2-arachidonoyl-glycerol controls odor sensitivity in larvae of Xenopus laevis. J. Neurosci. 30, 89658973 (2010)
  25. De Petrocellis, L., Melck, D., Bisogno, T., Milone, A. & Di Marzo, V. Finding of the endocannabinoid signalling system in Hydra, a very primitive organism: possible role in the feeding response. Neuroscience 92, 377387 (1999)
  26. Elphick, M. R. & Egertova, M. The phylogenetic distribution and evolutionary origins of endocannabinoid signalling. Handb. Exp. Pharmacol. 168, 283297 (2005)
  27. Elphick, M. R. BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus). Gene 399, 6571 (2007)
  28. Sulston, J., Hodgkin, J. & Wood, W. B. in The Nematode Caenorhabditis elegans 587606 (Cold Spring Harbor Laboratory, 1988)
  29. Held, J. M. et al. DAF-12-dependent rescue of dauer formation in Caenorhabditis elegans by (25S)-cholestenoic acid. Aging Cell 5, 283291 (2006)
  30. Lithgow, G. J., White, T. M., Melov, S. & Johnson, T. E. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc. Natl Acad. Sci. USA 92, 75407544 (1995)
  31. Fabian, T. J. & Johnson, T. E. Production of age-synchronous mass cultures of Caenorhabditis elegans. J. Gerontol. 49, B145B156 (1994)
  32. Hobert, O. PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques 32, 728730 (2002)
  33. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 6168 (1989)
  34. Dupuy, D. et al. Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans. Nature Biotechnol. 25, 663668 (2007)
  35. Knight, C. G., Patel, M. N., Azevedo, R. B. & Leroi, A. M. A novel mode of ecdysozoan growth in Caenorhabditis elegans. Evol. Dev. 4, 1627 (2002)
  36. Mair, W., Panowski, S. H., Shaw, R. J. & Dillin, A. Optimizing dietary restriction for genetic epistasis analysis and gene discovery in C. elegans. PLoS ONE 4, e4535 (2009)
  37. Sultana, T. & Johnson, M. E. Sample preparation and gas chromatography of primary fatty acid amides. J. Chromatogr. A 1101, 278285 (2006)
  38. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231237 (2003)
  39. Timmons, L., Tabara, H., Mello, C. C. & Fire, A. Z. Inducible systemic RNA silencing in Caenorhabditis elegans. Mol. Biol. Cell 14, 29722983 (2003)

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Author information

Affiliations

  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

Contributions

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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    This file contains Supplementary Tables 1-6 and Supplementary Figures 1- 17 with legends.

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