Pyruvate kinase genes in grass carp Ctenopharyngodon idella: molecular characterization, expression patterns, and effects of dietary carbohydrate levels

  • J. J. Fan
  • X. H. Tang
  • J. J. Bai
  • Dong-Mei MaEmail author
  • P. Jiang


To explore features of carbohydrate metabolism and evolution of carbohydrate metabolism-associated genes in herbivorous fishes, the open reading frames (ORF) of PKL, PKMa, and PKMb genes of grass carp (Ctenopharyngodon idella) were obtained, encoding 538, 528, and 532 amino acids, respectively. Comparative genomic analysis showed that adjacent PK genes were highly conserved between fish and mammals. Gene expression profiles were quite different between the three PK genes in tissues and at developmental stages. PKL, PKMa, and PKMb had the highest expression levels in the liver, heart, and muscle, respectively. During embryogenesis, high expression levels of PKMa and PKMb were detected in unfertilized and fertilized eggs. Following a non-expression period, PKMa and PKMb exhibited high expressions again after the hatching stage. In contrast, PKL transcripts could not be detected in early developmental stages, and expression levels continued to increase from the hatching stage to 144 h post hatching. After the 8-week feeding trial with 18%, 30%, and 42% dietary carbohydrate levels, the concentrations of glucose and insulin in serum, pyruvate kinase enzymes, and gene expression levels in brain, muscle, and liver tissues all increased with the increase in carbohydrate levels in the diets. Furthermore, high carbohydrate levels (30% and 42% carbohydrate diets) had a greater effect on grass carp growth. This indicated that PKL, PKMa, and PKMb genes were not only very important in catalytic enzymes, which can be up-regulated by high carbohydrate dietary conditions, but also exhibited a complex and detailed division of labor in different tissues and developmental stages.


Pyruvate kinase Ctenopharyngodon idella qRT-PCR Diet Carbohydrate 



This work was supported by the Basic Research Business Fees of Chinese Academy of Fishery Sciences (2018SJ-YZ03); the Natural Science Foundation of Guangdong Province of China (2016A030313148); and China Agriculture Research System (CARS-46-03). We thank the Fish Seed Ltd. Company of Nanhai Bairong and the Fish Seed Ltd. Company of Sanshui Baijin for assistance with grass carp sample collection.

Funding information

Basic Research Business Fees of Chinese Academy of Fishery Sciences (2018SJ-YZ03); Natural Science Foundation of Guangdong (2016A030313148); and China Agriculture Research System (CARS-46-03).


  1. Berg JM, Tymoczko JL, Stryer L, Gatto GJ (2012) Biochemistry 7th Eds. W.H. Freeman, New York, pp 455–609Google Scholar
  2. Bergot F (1979) Carbohydrate in rainbow trout diets: effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture 18:157–167CrossRefGoogle Scholar
  3. Borrebaek B, Christophersen B (2000) Hepatic glucose phosphorylating activities in perch Perca fluviatilis after different dietary treatments. Comp Biochem Physiol B Biochem Mol Biol 125:387–393CrossRefGoogle Scholar
  4. Christoffels A, Koh EG, Chia JM, Brenner S, Aparicio S, Venkatesh B (2004) Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes. Mol Biol Evol 21:1146–1151CrossRefGoogle Scholar
  5. Dombrauckas JD, Santarsiero BD, Mesecar AD (2005) Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 44:9417–9429CrossRefGoogle Scholar
  6. Enes P, Panserat S, Kaushik S, Oliva-Teles A (2006) Effect of normal and waxy maize starch on growth, food utilization and hepatic glucose metabolism in European sea bass Dicentrarchus labrax juveniles. Biochem Physiol A Mol Integr Physiol 143:89–96CrossRefGoogle Scholar
  7. Fernández F, Miquel AG, Córdoba M, Varas M, Metón I, Caseras A, Baanante IV (2007) Effects of diets with distinct protein-to-carbohydrate ratios on nutrient digestibility, growth performance, body composition and liver intermediary enzyme activities in gilthead sea bream Sparus aurata L. fingerlings. J Exp Mar Biol Ecol 343:1–10CrossRefGoogle Scholar
  8. Gupta V, Bamezai RNK (2010) Human pyruvate kinase M2: A multifunctional protein. Protein Sci 19:2031–2044CrossRefGoogle Scholar
  9. Gupta N, Bianchi P, Fermo E, Kabral M, Warang P, Kedar P, Gupta N, Colah R (2007) Prenatal diagnosis for a novel homozygous mutation in PKLR gene in an Indian family. Prenat Diagn 27:117–118CrossRefGoogle Scholar
  10. Hanes J, von der Kammer H, Klaudiny J, Scheit KH (1994) Characterization by cDNA cloning of two new human protein kinases: evidence by sequence comparison of a new family of mammalian protein kinases. J Mol Biol 244:665–672CrossRefGoogle Scholar
  11. Hilton JW, Atkinson JL (1982) Response of rainbow trout Salmo gairdneri to increased levels of available carbohydrate in practical trout diets. Br J Nutr 47:597–607CrossRefGoogle Scholar
  12. Huang JY, Wang K, Vermehren-Schmaedick A, Adelman JP, Cohen MS (2016) PARP6 is a regulator of hippocampal dendritic morphogenesis. Sci Rep 6:18512CrossRefGoogle Scholar
  13. Li Y, Li X, Liu J, Wang H, Fan L, Li J, Sun G (2018) PKM2, a potential target for regulating cancer gene. Gene 668:48–53CrossRefGoogle Scholar
  14. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  15. Marie S, Diaz-Guerra MJ, Miquerol L, Kahn A, Iynedjian PB (1993) The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type. J Biol Chem 268:23881–23890Google Scholar
  16. Metón I, Fernández F, Baanante IV (2003) Short- and long-term effects of refeeding on key enzyme activities in glycolysis–gluconeogenesis in the liver of gilthead seabream Sparus aurata. Aquaculture 225:99–107CrossRefGoogle Scholar
  17. Noguchi T, Inoue H, Tanaka T (1986) The M1- and M2- type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem 261:13807–13812Google Scholar
  18. Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T (1987) The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem 262:14366–14371Google Scholar
  19. Panserat S, Plagnes-Juan E, Kaushik S (2001) Nutritional regulation and tissue specificity of gene expression for proteins involved in hepatic glucose metabolism in rainbow trout Oncorhynchus mykiss. J Exp Biol 204:2351–2360Google Scholar
  20. Rodgers JT, Haas W, Gygi SP, Puigserver P (2010) Cdc2-like kinase 2 is an insulin-regulated suppressor of hepatic gluconeogenesis. Cell Metab 11:23–34CrossRefGoogle Scholar
  21. Ruan G, Yang D, Wang W (2012) Ontogeny of the digestive tracts in grass carp Ctenopharyngodon idellus, yellowcheck carp Elopichthys bambusa and topmouth culter Culter alburnus. Acta Hydrobiologica Sinica 36:1164–1170 (in Chinese)CrossRefGoogle Scholar
  22. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  23. Sato Y, Nishida M (2010) Teleost fish with specific genome duplication as unique models of vertebrate evolution. Environ Biol Fish 88:169–188CrossRefGoogle Scholar
  24. Sho O, Atsushi N, Kiichi I (2003) Molecular cloning and expression of pyruvate kinase from globefish Takifugu rubripes skeletal muscle. Comp Biochem Physiol B Biochem Mol Biol 135:397–405CrossRefGoogle Scholar
  25. Tabata M, Rodgers JT, Hall JA, Lee Y, Jedrychowski MP, Gygi SP, Puigserver P (2014) Cdc2-like kinase 2 suppresses hepatic fatty acid oxidation and ketogenesis through disruption of the PGC-1[alpha] and MED1 complex. Diabetes 63:1519–1532CrossRefGoogle Scholar
  26. Takenaka M, Noguchi T, Sadahiro S, Hirai H, Yamada K, Matsuda T, Imai E, Tanaka T (1991) Isolation and characterization of the human pyruvate kinase M gene. Eur J Biochem 198:101–106CrossRefGoogle Scholar
  27. Tani K, Yoshida MC, Satoh H, Mitamura K, Noguchi T, Tanaka T, Fujii H, Miwa S (1988) Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma. Gene 73:509–516CrossRefGoogle Scholar
  28. Tilton WM, Seaman C, Carriero D, Piomelli S (1991) Regulation of glycolysis in the erythrocyte: role of the lactate/pyruvate and NAD/NADH ratios. J Lab Clin Med 118:146–152Google Scholar
  29. Tsutsumi H, Tani K, Fujii H, Miwa S (1988) Expression of L- and M-type pyruvate kinase in human tissues. Genomics 2:86–89CrossRefGoogle Scholar
  30. Tuncel H, Tanaka S, Oka S, Nakai S, Fukutomi R, Okamoto M, Ota T, Kaneko H, Tatsuka M, Shimamoto F (2012) PARP6, a mono ADP-ribosyl transferase and a negative regulator of cell proliferation, is involved in colorectal cancer development. Int J Oncol 41:2079–2086CrossRefGoogle Scholar
  31. Varma V, Wise C, Kaput J (2010) Carbohydrate metabolic pathway genes associated with quantitative trait loci QTL for obesity and type 2 diabetes: identification by data mining. Biotechnol J 5:942–949CrossRefGoogle Scholar
  32. Vaulont S, Munnich A, Decaux JF, Kahn A (1986) Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver. J Biol Chem 261:7621–7625Google Scholar
  33. Wang Y, Lu Y, Zhang Y, Ning Z, Li Y, Zhao Q, Lu H, Huang R, Xia X, Feng Q, Liang X, Liu K, Zhang L, Lu T, Huang T, Fan D, Weng Q, Zhu C, Lu Y, Li W, Wen Z, Zhou C, Tian Q, Kang X, Shi M, Zhang W, Jang S, Du F, He S, Liao L, Li Y, Gui B, He H, Ning Z, Yang C, He L, Luo L, Yang R, Luo Q, Liu X, Li S, Huang W, Xiao L, Lin H, Han B, Zhu Z (2015) The draft genome of the grass carp Ctenopharyngodon idellus provides insights into its evolution and vegetarian adaptation. Nat Genet 47:625–631CrossRefGoogle Scholar
  34. Wilson RP (1994) Utilization of dietary carbohydrate by fish. Aquaculture 124:67–80CrossRefGoogle Scholar
  35. Wu SW, Wong SS, Yeung DC (1979) Isozymes of rat muscle pyruvate kinase. Int J Biochem 10:1013–1020CrossRefGoogle Scholar
  36. Yamada K, Noguchi T (1999) Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochem J 337:1–11CrossRefGoogle Scholar
  37. Yu LY, Bai JJ, Fan JJ, Ma DM, Quan YC, Jiang P (2015) Transcriptome analysis of the grass carp Ctenopharyngodon idella using 454 pyrosequencing methodology for gene and marker discovery. Genet Mol Res 14:19249–19263CrossRefGoogle Scholar
  38. Yuan X, Zhou Y, Liang XF, Li J, Liu L, Li B, He Y, Guo X, Liu F (2013) Molecular cloning, expression and activity of pyruvate kinase in grass carp Ctenopharyngodon idella: effects of dietary carbohydrate level. Aquaculture 410:32–40CrossRefGoogle Scholar
  39. Zanella A, Fermo E, Bianchi P, Valentini G (2005) Red cell pyruvate kinase deficiency: molecular and clinical aspects. Br J Haematol 130:11–25CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research InstituteChinese Academy of Fishery SciencesGuangzhouChina

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