Fish Physiology and Biochemistry

, Volume 39, Issue 4, pp 1043–1055 | Cite as

Tissue expression of PPAR-alpha isoforms in Scophthalmus maximus and transcriptional response of target genes in the heart after exposure to WY-14643

  • R. UrbatzkaEmail author
  • S. Galante-Oliveira
  • E. Rocha
  • L. F. C. Castro
  • I. Cunha


Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and carbohydrate metabolism and can be activated either by natural ligands as fatty acids or by synthetic ligands including several environmental chemicals. In this study, two PPARα isoforms (α1 and α2) were analyzed in turbot (Scophthalmus maximus) for a different tissue distribution. PPARα1 was ubiquitously expressed, while the PPARα2 was predominantly expressed in the heart. Following this result, turbot juveniles were exposed by injection to a synthetic selective PPARα agonist, WY-14643, for 14 days. Suppression subtractive hybridization (SSH) was performed with pools of heart samples of control and exposed fish to get insights into PPARα-regulated genes in the heart of juvenile turbot. Four genes were positively identified in the forward-subtracted and 12 genes in the reverse-subtracted cDNA SSH library, corresponding to the down-regulated and up-regulated genes in response to the WY-14643 treatment, respectively. The confirmation of these results in individual samples of juvenile turbot exposed to WY-14643 revealed a statistically significant mRNA induction of two cardiac muscle proteins (myosin light chain 2 and tropomyosin 4), which were shown to be involved in heart contraction and heartbeat regulation in other teleost species. Herewith, we showed for the first time that PPARα2 is predominantly expressed in the heart and that a PPARα agonist can induce the mRNA expression of cardiac muscle proteins in teleosts.


Scophthalmus maximus PPARα WY-14643 Peroxisome proliferators Myosin light chain 2 Tropomyosin 4-1 



This work was financially supported via the project PTDC/MAR/68885/2006, funded by the Portuguese Foundation for Science and Technology (FCT) and by the “Programa Operacional Ciência e Inovação 2010” (POCI 2010), co-financed by the FEDER European Community fund. We would like to thank Professor António Afonso for having kindly provided the experimental fish.

Conflict of interest

The authors declare that they do not have any conflict of interest.


  1. Batista-Pinto C, Rodrigues P, Rocha E, Lobo-da-Cunha A (2005) Identification and organ expression of peroxisome proliferator activated receptors in brown trout (Salmo trutta f. fario). Biochim Biophys Acta 1731:88–94CrossRefPubMedGoogle Scholar
  2. Berger J, Moller DE (2002) The mechanisms of action of PPARs. Annu Rev Med 53:409–435CrossRefPubMedGoogle Scholar
  3. Bertrand S, Thisse B, Tavares R, Sachs L, Chaumot A, Bardet PL, Escriva H, Duffraisse M, Marchand O, Safi R, Thisse C, Laudet V (2007) Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression. PLoS Genet 3:e188CrossRefPubMedGoogle Scholar
  4. Bility MT, Thompson JT, McKee RH, David RM, Butala JH, Vanden Heuvel JP, Peters JM (2004) Activation of mouse and human peroxisome proliferator-activated receptors (PPARs) by phthalate monoesters. Toxicol Sci 82:170–182CrossRefPubMedGoogle Scholar
  5. Chen Z, Huang W, Dahme T, Rottbauer W, Ackerman MJ, Xu X (2008) Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms. Cardiovasc Res 79:97–108CrossRefPubMedGoogle Scholar
  6. Colliar L, Sturm A, Leaver MJ (2011) Tributyltin is a potent inhibitor of piscine peroxisome proliferator-activated receptor alpha and beta. Comp Biochem Physiol C 153:168–173Google Scholar
  7. Corton JC, Anderson SP, Stauber A (2000) Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu Rev Pharmacol Toxicol 40:491–518CrossRefPubMedGoogle Scholar
  8. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030CrossRefPubMedGoogle Scholar
  9. Diatchenko L, Chenchik A, Siebert P (1998) Suppression subtractive hybridization: A method for generating subtracted cDNA libraries starting from poly (A+) or total RNA. In: Siebert P, Larrick J (eds) RT-PCR methods for gene cloning and analysis. BioTechniques Books, MA, pp 213–239Google Scholar
  10. Djouadi F, Brandt JM, Weinheimer CJ, Leone TC, Gonzalez FJ, Kelly DP (1999) The role of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in the control of cardiac lipid metabolism. Prostaglandins Leukot Essent Fatty Acids 60:339–343CrossRefPubMedGoogle Scholar
  11. Doud SK, Pan LX, Carleton S, Marmorstein S, Siddiqui MA (1995) Adaptational response in transcription factors during development of myocardial hypertrophy. J Mol Cell Cardiol 27:2359–2372CrossRefPubMedGoogle Scholar
  12. Escher P, Braissant O, Basu-Modak S, Michalik L, Wahli W, Desvergne B (2001) Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding. Endocrinology 142:4195–4202CrossRefPubMedGoogle Scholar
  13. Gonzalez FJ (2002) The peroxisome proliferator-activated receptor alpha (PPARalpha): role in hepatocarcinogenesis. Mol Cell Endocrinol 193:71–79CrossRefPubMedGoogle Scholar
  14. Gordon AM, Regnier M, Homsher E (2001) Skeletal and cardiac muscle contractile activation: tropomyosin “rocks and rolls”. News Physiol Sci 16:49–55PubMedGoogle Scholar
  15. Gruen F, Blumberg B (2009) Endocrine disrupters as obesogens. Mol Cell Endocrinol 304:19–29CrossRefGoogle Scholar
  16. Hamano T, Kobayashi K, Sakairi T, Hayashi M, Mutai M (2001) Peroxisome proliferator-activated receptor alpha (PPAR alpha) agonist, WY-14,643, increased transcription of myosin light chain-2 in cardiomyocytes. J Toxicol Sci 26:275–284CrossRefPubMedGoogle Scholar
  17. Henderson SA, Spencer M, Sen A, Kumar C, Siddiqui MA, Chien KR (1989) Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression. J Biol Chem 264:18142–18148PubMedGoogle Scholar
  18. Henikoff S, Henikoff JG, Alford WJ, Pietrokovski S (1995) Automated construction and graphical presentation of protein blocks from unaligned sequences. Gene 163:17–26CrossRefGoogle Scholar
  19. Hernandez OM, Jones M, Guzman G, Szczesna-Cordary D (2007) Myosin essential light chain in health and disease. Am J Physiol Heart Circ Physiol 292:H1643–H1654CrossRefPubMedGoogle Scholar
  20. Hihi AK, Michalik L, Wahli W (2002) PPARs: transcriptional effectors of fatty acids and their derivatives. Cell Mol Life Sci 59:790–798CrossRefPubMedGoogle Scholar
  21. Huang Q, Fang C, Wu X, Fan J, Dong S (2011) Perfluorooctane sulfonate impairs the cardiac development of a marine medaka (Oryzias melastigma). Aquat Toxicol 105:71–77CrossRefPubMedGoogle Scholar
  22. Infante C, Matsuoka MP, Asensio E, Canavate JP, Reith M, Manchado M (2008) Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol 9:28CrossRefPubMedGoogle Scholar
  23. Kondo H, Misaki R, Gelman L, Watabe S (2007) Ligand-dependent transcriptional activities of four torafugu pufferfish Takifugu rubripes peroxisome proliferator-activated receptors. Gen Comp Endocrinol 154:120–127CrossRefPubMedGoogle Scholar
  24. Leaver MJ, Boukouvala E, Antonopoulou E, Diez A, Favre-Krey L, Ezaz MT, Bautista JM, Tocher DR, Krey G (2005) Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish. Endocrinology 146:3150–3162CrossRefPubMedGoogle Scholar
  25. Maglich JM, Caravella JA, Lambert MH, Willson TM, Moore JT, Ramamurthy L (2003) The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily. Nucleic Acids Res 31:4051–4058CrossRefPubMedGoogle Scholar
  26. Morano I (1999) Tuning the human heart molecular motors by myosin light chains. J Mol Med 77:544–555CrossRefPubMedGoogle Scholar
  27. Ogino Y, Katoh H, Kuraku S, Yamada G (2009) Evolutionary history and functional characterization of androgen receptor genes in jawed vertebrates. Endocrinology 150:5415–5427CrossRefPubMedGoogle Scholar
  28. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedGoogle Scholar
  29. Raingeard D, Cancio I, Cajaraville MP (2009) Cloning and expression pattern of peroxisome proliferator-activated receptors, estrogen receptor alpha and retinoid X receptor alpha in the thicklip grey mullet Chelon labrosus. Comp Biochem Physiol C 149:26–35Google Scholar
  30. Robinson-Rechavi M, Marchand O, Escriva H, Bardet PL, Zelus D, Hughes S, Laudet V (2001) Euteleost fish genomes are characterized by expansion of gene families. Genome Res 11:781–788CrossRefPubMedGoogle Scholar
  31. Rosal R, Rodea-Palomares I, Boltes K, Fernandez-Pinas F, Leganes F, Gonzalo S, Petre A (2010) Ecotoxicity assessment of lipid regulators in water and biologically treated wastewater using three aquatic organisms. Environ Sci Pollut Res Int 17:135–144CrossRefPubMedGoogle Scholar
  32. Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences. Nucleic Acids Res 26:1628–1635CrossRefPubMedGoogle Scholar
  33. Rottbauer W, Wessels G, Dahme T, Just S, Trano N, Hassel D, Burns CG, Katus HA, Fishman MC (2006) Cardiac myosin light chain-2: a novel essential component of thick-myofilament assembly and contractility of the heart. Circ Res 99:323–331CrossRefPubMedGoogle Scholar
  34. Shimada E, Kinoshita M, Murata K (2009) Expression of cardiac myosin light chain 2 during embryonic heart development in medaka fish, Oryzias latipes, and phylogenetic relationship with other myosin light chains. Dev Growth Differ 51:1–16CrossRefPubMedGoogle Scholar
  35. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum persimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedGoogle Scholar
  36. Toramoto T, Ikeda D, Ochiai Y, Minoshima S, Shimizu N, Watabe S (2004) Multiple gene organization of pufferfish Fugu rubripes tropomyosin isoforms and tissue distribution of their transcripts. Gene 331:41–51CrossRefPubMedGoogle Scholar
  37. Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP (2011) Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS One 6:e18180CrossRefPubMedGoogle Scholar
  38. Urbatzka R, Lorenz C, Lutz I, Kloas W (2010) Expression profiles of LHbeta, FSHbeta and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles. Gen Comp Endocrinol 168:239–244CrossRefPubMedGoogle Scholar
  39. Wagner KD, Wagner N (2010) Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) acts as regulator of metabolism linked to multiple cellular functions. Pharmacol Ther 125:423–435CrossRefPubMedGoogle Scholar
  40. Wall CE, Cozza S, Riquelme CA, McCombie WR, Heimiller JK, Marr TG, Leinwand LA (2011) Whole transcriptome analysis of fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptation. Physiol Genomics 43:69–76CrossRefPubMedGoogle Scholar
  41. Zhao L, Zhao X, Tian T, Lu Q, Skrbo-Larssen N, Wu D, Kuang Z, Zheng X, Han Y, Yang S, Zhang C, Meng A (2008) Heart-specific isoform of tropomyosin4 is essential for heartbeat in zebrafish embryos. Cardiovasc Res 80:200–208CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • R. Urbatzka
    • 1
    Email author
  • S. Galante-Oliveira
    • 1
    • 2
  • E. Rocha
    • 1
    • 3
  • L. F. C. Castro
    • 1
  • I. Cunha
    • 1
  1. 1.Laboratory of Cellular, Molecular and Analytical Studies (LECEMA), Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), CIMAR Associated Laboratory (CIMAR LA)University of Porto (U.Porto)PortoPortugal
  2. 2.Department of Biology and CESAMUniversity of AveiroAveiroPortugal
  3. 3.Laboratory of Histology and Embryology, Institute of Biomedical Sciences Abel Salazar (ICBAS)University of Porto (U.Porto)PortoPortugal

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