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Antidepressants of the Serotonin-Antagonist Type Increase Body Fat and Decrease Lifespan of Adult Caenorhabditis elegans

Antidepressants of the Serotonin-Antagonist Type Increase Body Fat and Decrease Lifespan of Adult Caenorhabditis elegans

  • Kim Zarse, 
  • Michael Ristow
PLOS
x

Abstract

It was recently suggested that specific antidepressants of the serotonin-antagonist type, namely mianserin and methiothepin, may exert anti-aging properties and specifically extend lifespan of the nematode C.elegans by causing a state of perceived calorie restriction (Petrascheck M, Ye X, Buck LB: An antidepressant that extends lifespan in adult Caenorhabditis elegans; Nature, Nov 22, 2007;450(7169):553–6, PMID 18033297). Using the same model organism, we instead observe a reduction of life expectancy when employing the commonly used, standardized agar-based solid-phase assay while applying the same or lower concentrations of the same antidepressants. Consistent with a well-known side-effect of these compounds in humans, antidepressants not only reduced lifespan but also increased body fat accumulation in C. elegans reflecting the mammalian phenotype. Taken together and in conflict with previously published findings, we find that antidepressants of the serotonin-antagonist type not only promote obesity, but also decrease nematode lifespan.

Introduction

In recent years, the nematorde Caenorhabditis elegans has become a well-established model organism to identify compounds that may be capable of extending lifespan not only in invertebrates, but also mammals. Accordingly, several research groups have published nematode-based findings on such compounds [1][30], whereas for most of these it is currently unknown whether they might exert similar effects in mammals, while for others this was proposed in regards to rodent lifespan [31] or at least in regards to reduction of aging-associated physiological alterations, whereas no extension of lifespan was observed [32].

Like numerous other psychoactive compounds, the antidepressant mianserin has been shown to increase appetite [33] as well as body mass [34] in humans. Conversely, obesity has been shown to decrease life span in humans [35] as well as C. elegans [25], while in both species serotonin signalling has been implicated in body fat accumulation [36]. In conflict with this evidence, recently published findings unexpectedly suggest that mianserin, and additional antidepressants of the serotonin antagonist type might extend C.elegans lifespan [24], which would surprisingly implicate that obesity promotes longevity.

While the latter study has employed liquid media to determine C. elegans lifespan, we have employed standardized and widely accepted agar-based assays aiming to replicate these findings, and unexpectedly observe a dose-dependent reduction of C.elegans lifespan, primarily suggesting that different assays to determine nematode lifespan generate opposing results.

Results and Discussion

To replicate the findings of previously published experiments by Petrascheck and colleagues [24], we have we have applied both compounds described to be life-extending in the original paper, mianserin and methiothepin, to Bristol N2 C.elegans which in our case were maintained on solid-phase agar media, as described in Material and Methods.

We repeatedly observed significantly decreased life expectancies for the key compound mianserin when applying this substance at a final concentration as given in the original paper (50 µM, p<0.001), as well as at 5 µM (p<0.001) and 500 nM (p<0.001) (Fig. 1a). Similar results were obtained for a functionally related compound, methiothepin, at concentrations of 10 µM (p<0.001) as well as at 1 µM (p<0.005), whereas this compound showed no significant effect at a concentration of 100 nM (Fig. 1b). Methiothepin was shown to extend life span in the original study at a concentration of 10 µM [24].

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Figure 1. Antidepressants of the human serotonin antagonist type do not extend Caenorhabditis elegans lifespan.

Panel A: The antidepressant mianserin shortens C.elegans lifespan at concentrations of 50 µM (dark blue boxes; this concentration was shown extend lifespan in the original publication [ref. 24]), 5 µM (medium blue boxes), and 500 nM (light blue boxes). Untreated control nematodes are depicted by black circles. Panel B: The chemically and functionally related compound methiothepin similarly shortens C.elegans lifespan at concentrations of 10 µM (red boxes; this concentration was shown extend lifespan in the original publication [ref. 24]), 1 µM (orange boxes), and has no significant effect on lifespan at a lower concentration of 100 nM (yellow boxes). Untreated control nematodes are depicted by black circles.

https://doi.org/10.1371/journal.pone.0004062.g001

Petrascheck and colleagues have used liquid media not only for 96-well based screening assays, but also for final determinations of lifespans [24]. These liquid media are not commonly used for definite lifespan determinations, since they have been repeatedly reported to potentially cause differences in life span when compared to the well-established, standard solid-phase media; the first report in fact was published more the 30 years ago [37]. Liquid media have caused opposing results when being applied by different laboratories using apparently identical protocols [3], [9]. Moreover and according to their Methods Summary section [24], Petraschek et al. have not only based their liquid media on recipes from a publication [3] that was fundamentally put into question [9], but also from another laboratory [38] that has previously published a striking lack of correlation between lifespan results obtained with liquid- versus solid-phase media [39]. Lastly and most importantly, Petrascheck and colleagues observe a mean life expectancy of at least 23.6 days in N2 nematodes using their liquid media [24], whereas we [25] and others [40], [41] consistently observe a significantly shorter mean lifespan when using solid-phase media. This suggests that nematodes maintained in liquid media are kept in an a priori state of calorie restriction known to extend lifespan per se, i.e. in the absence of life-extending compounds [42], which has been recently shown to alter multiple pathways of energy metabolism [43] as to be expected in a priori states of calorie restriction [44][46].

Accordingly, and to test whether solid phase media as used in our C.elegans experiments reflect the situation in humans, we have tried to replicate the fact that mianserin increases human body mass [34] by applying this compound to nematodes. Indeed, both compounds significantly increased body fat after ten days of incubation at the concentrations that have been used by Petrascheck and colleagues [24] (Figs. 2a and 2b), whereas other pharmacological interventions known to extend C.elegans lifespan have been previously shown to decrease body fat content [25]. Nevertheless it should be noted that a specific genetic disruption that extends C.elegans lifespan, namely of the insulin-/IGF1-receptor signaling (daf-2) [40] have been shown to increase C.elegans body fat [47].

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Figure 2. Antidepressants of the human serotonin antagonist type increase Caenorhabditis elegans body fat content.

Panel A: The antidepressant mianserin increases C.elegans body fat content at a concentration of 50 µM (dark blue bar, right side; this concentration was shown extend lifespan in the original publication [ref. 24]) after ten days of treatment; untreated control nematodes are depicted as black bar. Panel B: The related compound methiothepin increases C.elegans body fat content at a concentration of 10 µM (red bar, right side; this concentration was shown extend lifespan in the original publication [ref. 24]) after ten days of treatment; untreated control nematodes are depicted as black bar.

https://doi.org/10.1371/journal.pone.0004062.g002

Taken together and consistent with the findings in humans in regards to obesity [33], [34], we find that antidepressants of the serotonin antagonist-type do not extend C.elegans lifespan at most commonly used and generally accepted experimental conditions.

Materials and Methods

Nematodes

The strain used in this study was Bristol N2 which was obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, USA). Nematodes were grown and maintained on NGM agar plates as described previously [25], [48], [49]. All experiments were performed at 20° Celsius. C. elegans stocks and prefertile animals were maintained on OP50 bacteria.

Compounds

Antidepressants mianserin and methiothepin were both obtained from Sigma-Aldrich (St. Louis, MO, USA). Agar plates containing experimental treatments were prepared from the same batch of NGM agar as the control plates except that the respective chemical was added to obtain the indicated final concentrations from a sterile stock solution (10 µM each).

Fat content analyses

Triglyceride content was performed as previously described [25] briefly by flash-freezing nematodes and storage at −80°C until further processing. Approximately 25 mg was weighed and ground in a nitrogen-chilled mortar together with 250 µl of frozen phosphate buffer. The frozen material was gathered in a reaction tube and kept on ice. Extracts were sonicated three times and centrifuged for 7 min at 12,000 g. Fat content was determined with a commercially available triglyceride determination kit (Sigma-Aldrich) as previously described [50] and normalized to protein content, which was determined according to the Bradford method [51].

Author Contributions

Conceived and designed the experiments: MR. Performed the experiments: KZ. Analyzed the data: KZ. Wrote the paper: MR.

References

  1. 1. Harrington LA, Harley CB (1988) Effect of vitamin E on lifespan and reproduction in Caenorhabditis elegans. Mech Ageing Dev 43: 71–78.
  2. 2. Adachi H, Ishii N (2000) Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans. J Gerontol A Biol Sci Med Sci 55: B280–285.
  3. 3. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, et al. (2000) Extension of life-span with superoxide dismutase/catalase mimetics. Science 289: 1567–1569.
  4. 4. Bakaev VV, Bakaeva LM, Nikitin YP, Shabalin AV (2002) Effect of 1-butylguanide hydrochloride on the longevity in the nematoda Caenorhabditis elegans. Biogerontology 3: Suppl 123–24.
  5. 5. Bakaev VV, Lyudmila MB (2002) Effect of ascorbic acid on longevity in the nematoda Caenorhabditis elegans. Biogerontology 3: suppl 112–16.
  6. 6. Cypser JR, Johnson TE (2002) Multiple stressors in Caenorhabditis elegans induce stress hormesis and extended longevity. J Gerontol A Biol Sci Med Sci 57: B109–114.
  7. 7. Wu Z, Smith JV, Paramasivam V, Butko P, Khan I, et al. (2002) Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans. Cell Mol Biol (Noisy-le-grand) 48: 725–731.
  8. 8. Strayer A, Wu Z, Christen Y, Link CD, Luo Y (2003) Expression of the small heat-shock protein Hsp16-2 in Caenorhabditis elegans is suppressed by Ginkgo biloba extract EGb 761. FASEB J 17: 2305–2307.
  9. 9. Keaney M, Gems D (2003) No increase in lifespan in Caenorhabditis elegans upon treatment with the superoxide dismutase mimetic EUK-8. Free Radic Biol Med 34: 277–282.
  10. 10. Ishii N, Senoo-Matsuda N, Miyake K, Yasuda K, Ishii T, et al. (2004) Coenzyme Q10 can prolong C. elegans lifespan by lowering oxidative stress. Mech Ageing Dev 125: 41–46.
  11. 11. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, et al. (2004) Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 430: 686–689.
  12. 12. Viswanathan M, Kim SK, Berdichevsky A, Guarente L (2005) A role for SIR-2.1 regulation of ER stress response genes in determining C. elegans life span. Dev Cell 9: 605–615.
  13. 13. Evason K, Huang C, Yamben I, Covey DF, Kornfeld K (2005) Anticonvulsant medications extend worm life-span. Science 307: 258–262.
  14. 14. Kornfeld K, Evason K (2006) Effects of anticonvulsant drugs on life span. Arch Neurol 63: 491–496.
  15. 15. Brown MK, Evans JL, Luo Y (2006) Beneficial effects of natural antioxidants EGCG and alpha-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans. Pharmacol Biochem Behav 85: 620–628.
  16. 16. Wilson MA, Shukitt-Hale B, Kalt W, Ingram DK, Joseph JA, et al. (2006) Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans. Aging Cell 5: 59–68.
  17. 17. Gruber J, Tang SY, Halliwell B (2007) Evidence for a trade-off between survival and fitness caused by resveratrol treatment of Caenorhabditis elegans. Ann N Y Acad Sci 1100: 530–542.
  18. 18. Kampkotter A, Gombitang Nkwonkam C, Zurawski RF, Timpel C, Chovolou Y, et al. (2007) Effects of the flavonoids kaempferol and fisetin on thermotolerance, oxidative stress and FoxO transcription factor DAF-16 in the model organism Caenorhabditis elegans. Arch Toxicol 81: 849–858.
  19. 19. Gerisch B, Rottiers V, Li D, Motola DL, Cummins CL, et al. (2007) A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling. Proc Natl Acad Sci U S A 104: 5014–5019.
  20. 20. Miller DL, Roth MB (2007) Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans. Proc Natl Acad Sci U S A 104: 20618–20622.
  21. 21. Bass TM, Weinkove D, Houthoofd K, Gems D, Partridge L (2007) Effects of resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans. Mech Ageing Dev 128: 546–552.
  22. 22. Broue F, Liere P, Kenyon C, Baulieu EE (2007) A steroid hormone that extends the lifespan of Caenorhabditis elegans. Aging Cell 6: 87–94.
  23. 23. Zou S, Sinclair J, Wilson MA, Carey JR, Liedo P, et al. (2007) Comparative approaches to facilitate the discovery of prolongevity interventions: effects of tocopherols on lifespan of three invertebrate species. Mech Ageing Dev 128: 222–226.
  24. 24. Petrascheck M, Ye X, Buck LB (2007) An antidepressant that extends lifespan in adult Caenorhabditis elegans. Nature 450: 553–556.
  25. 25. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, et al. (2007) Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab 6: 280–293.
  26. 26. Evason K, Collins JJ, Huang C, Hughes S, Kornfeld K (2008) Valproic acid extends Caenorhabditis elegans lifespan. Aging Cell 7: 305–317.
  27. 27. Benedetti MG, Foster AL, Vantipalli MC, White MP, Sampayo JN, et al. (2008) Compounds that confer thermal stress resistance and extended lifespan. Exp Gerontol 43: 882–891.
  28. 28. Kampkotter A, Timpel C, Zurawski RF, Ruhl S, Chovolou Y, et al. (2008) Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin. Comp Biochem Physiol B Biochem Mol Biol 149: 314–323.
  29. 29. Kim J, Takahashi M, Shimizu T, Shirasawa T, Kajita M, et al. (2008) Effects of a potent antioxidant, platinum nanoparticle, on the lifespan of Caenorhabditis elegans. Mech Ageing Dev 129: 322–331.
  30. 30. Wiegant FA, Surinova S, Ytsma E, Langelaar-Makkinje M, Wikman G, et al. (2008) Plant adaptogens increase lifespan and stress resistance in C. elegans. Biogerontology. in press.
  31. 31. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, et al. (2006) Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444: 337–342.
  32. 32. Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, et al. (2008) Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span. Cell Metab 8: 157–168.
  33. 33. Harris B, Harper M (1980) Unusual appetites in patients on mianserin. Lancet 1: 590.
  34. 34. Pinder RM, Blum A, Stulemeijer SM, Barres M, Molczadzki M, et al. (1980) A double-blind multicentre trial comparing the efficacy and side-effects of mianserin and chlorimipramine in depressed in- and outpatients. Int Pharmacopsychiatry 15: 218–227.
  35. 35. Fontaine KR, Redden DT, Wang C, Westfall AO, Allison DB (2003) Years of life lost due to obesity. JAMA 289: 187–193.
  36. 36. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, et al. (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421: 268–272.
  37. 37. Croll NA, Smith JM, Zuckerman BM (1977) The aging process of the nematode Caenorhabditis elegans in bacterial and axenic culture. Exp Aging Res 3: 175–189.
  38. 38. Johnson TE, de Castro E, Hegi de Castro S, Cypser J, Henderson S, et al. (2001) Relationship between increased longevity and stress resistance as assessed through gerontogene mutations in Caenorhabditis elegans. Exp Gerontol 36: 1609–1617.
  39. 39. Shook DR, Johnson TE (1999) Quantitative trait loci affecting survival and fertility-related traits in Caenorhabditis elegans show genotype-environment interactions, pleiotropy and epistasis. Genetics 153: 1233–1243.
  40. 40. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366: 461–464.
  41. 41. Tissenbaum HA, Guarente L (2001) Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410: 227–230.
  42. 42. Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, et al. (2002) Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans. Exp Gerontol 37: 1371–1378.
  43. 43. Castelein N, Hoogewijs D, De Vreese A, Braeckman BP, Vanfleteren JR (2008) Dietary restriction by growth in axenic medium induces discrete changes in the transcriptional output of genes involved in energy metabolism in Caenorhabditis elegans. Biotechnol J 3: 803–812.
  44. 44. Lenaerts I, Walker GA, Van Hoorebeke L, Gems D, Vanfleteren JR (2008) Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes. J Gerontol A Biol Sci Med Sci 63: 242–252.
  45. 45. Smith ED, Kaeberlein TL, Lydum BT, Sager J, Welton KL, et al. (2008) Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans. BMC Dev Biol 8: 49.
  46. 46. Sutphin GL, Kaeberlein M (2008) Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes. Exp Gerontol 43: 130–135.
  47. 47. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G (1997) daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277: 942–946.
  48. 48. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
  49. 49. Zarse K, Schulz TJ, Birringer M, Ristow M (2007) Impaired respiration is positively correlated with decreased life span in Caenorhabditis elegans models of Friedreich Ataxia. FASEB J 21: 1271–1275.
  50. 50. Ristow M, Pfister MF, Yee AJ, Schubert M, Michael L, et al. (2000) Frataxin activates mitochondrial energy conversion and oxidative phosphorylation. Proc Natl Acad Sci U S A 97: 12239–12243.
  51. 51. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.