Marine Biotechnology

, Volume 14, Issue 5, pp 643–654 | Cite as

Dietary Lysine Imbalance Affects Muscle Proteome in Zebrafish (Danio rerio): A Comparative 2D-DIGE Study

  • Mahaut de Vareilles
  • Luis E. C. Conceição
  • Pedro Gómez-Requeni
  • Katerina Kousoulaki
  • Nadège Richard
  • Pedro M. Rodrigues
  • Kari E. Fladmark
  • Ivar Rønnestad
Original Article


Lysine (Lys) is an indispensable amino acid (AA) and generally the first limiting AA in vegetable protein sources in fish feeds. Inadequate dietary Lys availability may limit protein synthesis, accretion and growth of fish. This experiment aimed to further elucidate the role of Lys imbalance on growth by examining the myotomal muscle proteome of juvenile zebrafish (Danio rerio). Quadruplicate groups of 8 fish were fed either a low-Lys [Lys(−), 1.34 g kg−1], medium/control (Lys, 2.47 g kg−1) or high-Lys [Lys(+), 4.63 g kg−1] diet. Fish growth was monitored from 33 to 49 days post-fertilization (dpf) and trunk myotomal muscle proteome of Lys(−) and Lys(+) treatments were screened by 2D-DIGE and MALDI ToF tandem mass spectrometry. Growth rate was negatively affected by diet Lys(−). Out of 527 ± 11 (mean ± S.E.M.) protein spots detected (∼10–150 kDa and 4–7 pI value), 30 were over-expressed and 22 under-expressed in Lys(−) fish (|fold-change| >1.2, p value <0.05). Higher myosin light chains abundance and other myofibrillar proteins in Lys(−) fish pointed to increased sarcomeric degradation, indicating a higher protein turnover for supplying basal energy-saving metabolism rather than growth and muscle protein accretion. The Lys deficiency also possibly induced a higher feeding activity, reflected in the over-expression of beta enolase and mitochondrial ATP synthase. Contrarily, in the faster growing fish [Lys(+)], over-expression of apolipoprotein A-I, F-actin capping protein and Pdlim7 point to increased energy storage as fat and enhanced muscle growth, particularly by mosaic hyperplasia. Thus using an exploratory approach, this study pinpoints interesting candidates for further elucidating the role of dietary Lys on growth of juvenile fish.


Lysine Growth Skeletal muscle Myosin chain isoforms Pdlim7 Zebrafish 



This research was funded by the Research Council of Norway (165203/S40) to IR. PGR was a recipient of a research contract from the University of Bergen. Support to the Proteomics lab was provided by the Meltzer Foundation, University of Bergen (KEF). LC and MdV participated with support of project HYDRAA-PTDC/MAR/71685/2006 (Fundação para a Ciência e Tecnologia (FCT), Portugal, with the support of FEDER). MdV was also supported by a grant SFRH/BD/40698/2007 (FCT, Portugal). Authors are especially grateful to Heikki Savolainen for excellent rearing of the fish and Ann Kristin Frøyset, Shailesh Narawane, Tomé S. Silva and Odete Cordeiro for useful technical assistance.


  1. Abimorad EG, Favero GC, Castellani D, García F, Carneiro DJ (2009) Dietary supplementation of lysine and/or methionine on performance, nitrogen retention and excretion in pacu Piaractus mesopotamicus reared in cages. Aquaculture 295:266–270CrossRefGoogle Scholar
  2. Alami-Durante H, Wrutniak-Cabello C, Kaushik SJ, Médale F (2010) Skeletal muscle cellularity and expression of myogenic regulatory factors and myosin heavy chains in rainbow trout (Oncorhynchus mykiss): effects of changes in dietary plant protein sources and amino acid profiles. Comp Biochem Physiol Part A Mol Integr Physiol 156:561–568CrossRefGoogle Scholar
  3. Alami-Durante H, Cluzeaud M, Bazin D, Mazurais D, Zambonino-Infante JL (2011) Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expression of myogenic regulatory factors and myosin heavy chain in European sea bass larvae. J Nutr 141:2146–2151PubMedCrossRefGoogle Scholar
  4. ARC (1981) The nutrient requirements of pigs. Commonwealth Agricultural Bureau, SloughGoogle Scholar
  5. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  6. Boisen S, Hvelplund T, Weisbjerg MR (2000) Ideal amino acid profiles as a basis for feed protein evaluation. Livest Prod Sci 64:239–251CrossRefGoogle Scholar
  7. Bosworth CA, Chou C-W, Cole RB, Rees BB (2005) Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure. Proteomics 5:1362–1371PubMedCrossRefGoogle Scholar
  8. Camarata T, Snyder D, Schwend T, Klosowiak J, Holtrup B, Simon H-G (2010) Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signalling feedback loop during zebrafish pectoral fin development. BMC Dev Biol 10:104PubMedCrossRefGoogle Scholar
  9. Campos C, Valente LMP, Borges P, Bizuayehu T, Fernandes JMO (2010) Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup). J Exp Biol 213:200–2009PubMedCrossRefGoogle Scholar
  10. Cheng ZJ, Hardy RW, Usry JL (2003) Effects of lysine supplementation in plant protein-based diets on the performance of rainbow trout (Oncorhynchus mykiss) and apparent digestibility coefficients of nutrients. Aquaculture 215:255–265CrossRefGoogle Scholar
  11. Chu WY, Chen J, Zhou RX, Zhao FL, Meng T, Chen DX, Nong XX, Liu Z, Lu SQ, Zhang JS (2011) Characterization and ontogenic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi. J Fish Biol 78:1225–1238PubMedCrossRefGoogle Scholar
  12. Cohen SA, Michaud KE (1993) Synthesis of a fluorescent derivatising reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211:279–287PubMedCrossRefGoogle Scholar
  13. Conceição LEC, Grasdalen H, Rønnestad I (2003) Amino acid requirements of fish larvae and post-larvae: new tools and recent findings. Aquaculture 227:221–232CrossRefGoogle Scholar
  14. Corzo A, Kidd MT, Koter MD, Burgess SC (2005) Assessment of dietary amino acid scarcity on growth and blood plasma proteome status of broiler chickens. Poult Sci 84:419–425PubMedGoogle Scholar
  15. Dahm R, Geisler R (2006) Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species. Mar Biotechnol 8:329–345PubMedCrossRefGoogle Scholar
  16. Doran P, Donoghue P, O’Connell K, Gannon J, Ohlendieck K (2009) Proteomics of skeletal muscle aging. Proteomics 9:989–1003PubMedCrossRefGoogle Scholar
  17. Durick K, Gill GN, Taylor SS (1998) Shc and Enigma are both required for mitogenic signaling by Ret/ptc2. Mol Cell Biol 18:2298–2308PubMedGoogle Scholar
  18. Ennion S, Gauvry L, Butterworth P, Goldspink G (1995) Small-diameter white myotomal muscle fibres associated with growth hyperplasia in the carp (Cyprinus carpio) express a distinct myosin heavy chain gene. J Exp Biol 198:1603–1611PubMedGoogle Scholar
  19. Enyu Y-L, Shu-Chien AC (2011) Proteomics analysis of mitochondrial extract from liver of female zebrafish undergoing starvation and refeeding. Aquac Nutr. doi: 10.1111/j.1365-2095.2010.00776.x
  20. Ferrari S, Battini R, Cossu G (1990) Differentiation-dependent expression of apolipoprotein A-I in chicken myogenic cells in culture. Dev Bio 140:430–436CrossRefGoogle Scholar
  21. Figueiredo MA, Mareco EA, Silva MDP, Marins LF (2011) Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish. Transgenic Res. doi: 10.1007/s11248-011-9546-2
  22. Gatlin DM III, Barrows FT, Brown P, Dabrowski K, Gaylord TG, Hardy RW, Herman E, Hu G, Krogdahl Å, Nelson R, Overturf K, Rust M, Sealey W, Skonberg D, Souza EJ, Stone D, Wilson R, Wurtele E (2007) Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquac Res 38:551–579CrossRefGoogle Scholar
  23. Gómez-Requeni P, Mingarro M, Kirchner S, Calduch-Giner JA, Médale F, Corraze G, Panserat S, Martin SAM, Houlihan DF, Kaushik SJ, Pérez-Sánchez J (2003) Effects of dietary amino acid profile on growth performance, key metabolic enzymes and somatotropic axis responsiveness of gilthead sea bream (Sparus aurata). Aquaculture 220:749–767CrossRefGoogle Scholar
  24. Gómez-Requeni P, Conceição LEC, Jordal A-EO, Rønnestad I (2010) A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development. Fish Physiol Biochem 36:1199–1215PubMedCrossRefGoogle Scholar
  25. Gómez-Requeni P, de Vareilles M, Kousoulaki K, Jordal A-E, Conceição LEC, Rønnestad I (2011) Whole-body proteome response to a dietary lysine imbalance in zebrafish Danio rerio. Comp Biochem Physiol Part D Genomics Proteomics 6:178–186PubMedCrossRefGoogle Scholar
  26. Hevrøy EM, Jordal A-EO, Hordvik I, Espe M, Hemre GI, Olsvik PA (2006) Myosin heavy chain mRNA expression correlates higher with muscle protein accretion than growth in Atlantic salmon, Salmo salar. Aquaculture 252:453–461CrossRefGoogle Scholar
  27. Hochstrasser DF, Sanchez JC, Appel RD (2002) Proteomics and its trends facing nature’s complexity. Proteomics 2:807–812PubMedCrossRefGoogle Scholar
  28. Hug C, Jay PY, Reddy I, McNally JG, Bridgman PC, Elson EL, Cooper JA (1995) Capping protein levels influence actin assembly and cell motility in Dictyostelium. Cell 81:591–600PubMedCrossRefGoogle Scholar
  29. Jagoe RT, Lecker SH, Gomes M, Goldberg AL (2002) Patterns of gene expression in atrophying skeletal muscles: response to food deprivation. FASEB J 16:1697–1712PubMedCrossRefGoogle Scholar
  30. Johansen KA, Sealey WM, Overturf K (2006) The effects of chronic immune stimulation on muscle growth in rainbow trout. Comp Biochem Physiol Part B Biochem Mol Biol 144:520–531CrossRefGoogle Scholar
  31. Johnston IA, Temple GK (2002) Thermal plasticity of skeletal muscle phenotype in ectothermic vertebrates and its significance for locomotory behavior. J Exp Biol 205:2305–2322PubMedGoogle Scholar
  32. Johnston IA, Lee H-T, Macqueen DJ, Paranthaman K, Kawashima C, Anwar A, Kinghorn JR, Dalmay T (2009) Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genomewide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. J Exp Biol 212:1781–1793PubMedCrossRefGoogle Scholar
  33. Johnston IA, Bower NI, Macqueen DJ (2011) Growth and the regulation of muscle myotomal mass in teleost fish. J Exp Biol 214:1617–1628PubMedCrossRefGoogle Scholar
  34. Jung C-R, Lim JH, Choi Y, Kim D-G, Kang KJ, Noh S-M, Im D-S (2010) Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice. J Clin Invest 120:4493–4506PubMedCrossRefGoogle Scholar
  35. Jury DR, Kaveti S, Duan ZH, Willard B, Kinter M, Londraville R (2008) Effects of calorie restriction on the zebrafish liver proteome. Comp Biochem Physiol Part D Genomics Proteomics 3:275–282PubMedCrossRefGoogle Scholar
  36. Kjærsgård IVH, Jessen F (2003) Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins. J Agric Food Chem 51:3985–3991PubMedCrossRefGoogle Scholar
  37. Krcmery J, Camarata T, Kulisz A, Simon H-G (2010) Nucleocytoplasmic functions of the PDZ-LIM protein family: new insights into organ development. BioEssays 32:100–108PubMedCrossRefGoogle Scholar
  38. Li P, Mai K, Trushenski J, Wu G (2009) New developments in fish amino acid nutrition: towards functional and environmentally oriented aquafeeds. Amino acids 37:43–53PubMedCrossRefGoogle Scholar
  39. López-Albors O, Ayala MD, Gil F, García-Alcázar A, Abellán E, Latorre R, Ramírez-Zarzosa G, Vázquez JM (2003) Early temperature effects on muscle growth dynamics and histochemical profile of muscle fibres of sea bass Dicentrarchus labrax L., during larval and juvenile stages. Aquaculture 220:285–406CrossRefGoogle Scholar
  40. Lu J, Zheng J, Liu H, Li J, Chen H, Chen K (2010) Protein profiling of skeletal muscle of pufferfish, Takifugu rubripes. Mol Biol Rep 37:2141–2147PubMedCrossRefGoogle Scholar
  41. Moreno-Sánchez N, Rueda J, Carabaño MJ, Reverter A, McWilliam S, González C, Díaz C (2010) Skeletal muscle specific gene networks in cattle. Funct Integr Genomics 10:609–618PubMedCrossRefGoogle Scholar
  42. Moutou KA, Canario AVM, Mamuris Z, Power DM (2001) Molecular cloning and sequence of Sparus aurata skeletal myosin light chains expressed in white muscle: developmental expression and thyroid regulation. J Exp Biol 204:3009–3018PubMedGoogle Scholar
  43. Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brillant Blue G-250 and R-250. Electrophoresis 9:255–262PubMedCrossRefGoogle Scholar
  44. NRC (National Research Council) (1993) Nutrient requirements of fish. National Academy Press, Washington D.CGoogle Scholar
  45. Ohlendieck K (2011) Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques. Skeletal Muscle 1:6PubMedCrossRefGoogle Scholar
  46. Patterson SE, Mook LB, Devoto SH (2008) Growth in the larval zebrafish pectoral fin and trunk musculature. Dev Dyn 237:307–315PubMedCrossRefGoogle Scholar
  47. Rathore RM, Liaset B, Hevrøy EM, El-Mowafi A, Espe M (2010) Lysine limitation alters the storage pattern of protein, lipid and glycogen in on-growing Atlantic salmon. Aquac Res. doi: 10.1111/j.1365-2109.2010.02576.x
  48. Rehfeldt C, Te Pas MFW, Wimmers K, Brameld JM, Nissen PM, Berri C, Valente LMP, Power DM, Picard B, Stickland NC, Oksbjerg N (2010) Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact. Animal. doi: 10.1017/S1751731110002089
  49. Rowlerson A, Scapolo P, Mascarello F, Carpene E, Veggetti A (1985) Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle. J Muscle Res Cell Motil 6:601PubMedCrossRefGoogle Scholar
  50. Salem M, Kenney PB, Rexroad CE III, Yao J (2010) Proteomic signature of muscle atrophy in rainbow trout. J Proteomics 73:778–789PubMedCrossRefGoogle Scholar
  51. Silva P, Valente LMP, Galante MH, Andrade CAP, Monteiro RAF, Rocha E (2009) Dietary protein content influences both growth and size distribution of anterior and posterior muscle fibres in juveniles of Pagellus bogaraveo (Brunnich). J Musc Res Cell Motil 30:29–39CrossRefGoogle Scholar
  52. Silva P, Power DM, Valente LMP, Silva N, Monteiro RAF, Rocha E (2010) Expression of the myosin light chains 1, 2 and 3 in the muscle of blackspot seabream (Pagellus bogaraveo, Brunnich), during development. Fish Physiol Biochem 36:1125–1132PubMedCrossRefGoogle Scholar
  53. Van Raamsdonk W, Van’t Veer L, Veeken K, Heyting C, Pool C (1982) Differentiation of muscle fiber types in the teleost, Brachydanio rerio, the zebrafish. Posthatching development. Anat Embryol 164:51PubMedCrossRefGoogle Scholar
  54. Veiseth-Kent E, Grove H, Faergestad EM, Fjaera SO (2010) Changes in muscle and blood plasma proteomes of Atlantic salmon (Salmo salar) induced by crowding. Aquaculture 309:272–279CrossRefGoogle Scholar
  55. Wilson RP (2002) In: Halver JE, Hardy RW (eds) Fish nutrition, 3rd edn. Academic, San DiegoGoogle Scholar
  56. Winder SJ, Ayscough KR (2005) Actin-binding proteins. J Cell Sci 118:651–654PubMedCrossRefGoogle Scholar
  57. Wu R, Durick K, Songyang Z, Cantley LC, Taylor SS, Gill GN (1996) Specificity of LIM domain interactions with receptor tyrosine kinases. J Biol Chem 271:15934–15941PubMedCrossRefGoogle Scholar
  58. Zimmerman AM, Lowery MS (1999) Hyperplasic development and hypertrophic growth of muscle fibers in the white seabass (Atractoscion nobilis). J Exp Zool 284:299–308PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Mahaut de Vareilles
    • 1
    • 2
  • Luis E. C. Conceição
    • 1
  • Pedro Gómez-Requeni
    • 2
  • Katerina Kousoulaki
    • 3
  • Nadège Richard
    • 1
  • Pedro M. Rodrigues
    • 1
  • Kari E. Fladmark
    • 4
  • Ivar Rønnestad
    • 2
  1. 1.CIMAR/CCMARUniversidade do Algarve, Campus de GambelasFaroPortugal
  2. 2.Department of BiologyUniversity of BergenBergenNorway
  3. 3.NOFIMA ASFyllingsdalenNorway
  4. 4.Department of Molecular BiologyUniversity of BergenBergenNorway

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