Fish Physiology and Biochemistry

, Volume 40, Issue 2, pp 595–605 | Cite as

In vitro exposure to copper influences lipid metabolism in hepatocytes from grass carp (Ctenopharyngodon idellus)

  • Qing-Ling Zhu
  • Zhi Luo
  • Mei-Qin Zhuo
  • Xiao-Ying Tan
  • Lin-Dan Sun
  • Jia-Lang Zheng
  • Qi-Liang Chen


In the present study, three different copper (Cu) concentrations (control, 10 and 100 μM, respectively) and three incubation times (24, 48 and 96 h) were chosen to assess in vitro effect of Cu on lipid metabolism in hepatocytes of grass carp Ctenopharyngodon idellus. Increased glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carnitine palmitoyltransferase I activities were observed in hepatocytes with increasing Cu concentration and exposure duration. Cu decreased mRNA levels of several lipogenic and lipolytic genes at 24 h. However, at 48 h, Cu down-regulated the process of lipogenesis but up-regulated that of lipolysis. The Cu-driven up-regulation of lipolytic genes was maintained after 96 h and accompanied by a decreased intracellular triglyceride accumulation, while no effect on lipogenic genes was shown. Thus, 96-h Cu exposure induced lipid depletion, possibly due to the up-regulation of lipolysis. Although in this process, lipogenesis might be up-regulated, it was not enough to compensate lipid consumption. Our study represents the first approach to concentration- and time-dependent in vitro effects of Cu on lipid metabolism of fish hepatocytes and provides new insights into Cu toxicity in fish at both enzymatic and molecular levels.


Hepatocytes Ctenopharyngodon idellus In vitro Lipid metabolism Cu 



This work was funded by the Fundamental Research Funds for the Central Universities (Grant No. 2013PY073) and partly by National Natural Science Foundation of China (Grant No. 31001101).

Conflict of interest

The authors declare that there is no conflict of interest.


  1. Ahmad I, Maria VL, Oliveira M, Pacheco M, Santos MA (2008) Modulatory role of copper on β-naphthoflavone-induced DNA damage in European eel (Anguilla anguilla L.). Ecotoxicol Environ Saf 71:806–812PubMedCrossRefGoogle Scholar
  2. Albalat A, Saera-Vila A, Capilla E, Gutierrez J, Perez-Sanchez J, Navarro I (2007) Insulin regulation of lipoprotein lipase (LPL) activity and expression in Gilthead Sea bream (Sparus aurata). Comp Biochem Physiol 148B:151–159CrossRefGoogle Scholar
  3. Atli G, Canli M (2011) Essential metal (Cu, Zn) exposures alter the activity of ATPases in gill, kidney and muscle of tilapia Oreochromis niloticus. Ecotoxicology 20:1861–1869PubMedCrossRefGoogle Scholar
  4. Barroso JB, Peragon J, Garcia-Salguero L, de la Higuera M, Lupianez JA (1999) Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies. Int J Biochem Cell Biol 31:277–290CrossRefGoogle Scholar
  5. Boyd CE, Massaut L (1999) Risks associated with the use of chemicals in pond aquaculture. Aquacult Eng 20:113–132CrossRefGoogle Scholar
  6. 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 72:248–254PubMedCrossRefGoogle Scholar
  7. Carvalho CS, Fernandes MN (2008) Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus. Comp Biochem Physiol 151A:437–442CrossRefGoogle Scholar
  8. Chen DS, Chan KM (2009) Changes in the protein expression profiles of the Hepa-T1 cell line when exposed to Cu2+. Aquat Toxicol 94:163–176PubMedCrossRefGoogle Scholar
  9. Chen QL, Luo Z, Zheng JL, Li XD, Liu CX, Zhao YH, Gong Y (2011) Protective effects of calcium on copper toxicity in Pelteobagrus fulvidraco: copper accumulation, enzymatic activities, histology. Ecotoxicol Environ Saf 76:126–134PubMedCrossRefGoogle Scholar
  10. Chen QL, Luo Z, Liu X, Song YF, Liu CX, Zheng JL, Zhao YH (2013a) Effects of waterborne chronic copper exposure on hepatic lipid metabolism and metal-element composition in Synechogobius hasta. Arch Environ Contamin Toxicol 64:301–315CrossRefGoogle Scholar
  11. Chen QL, Luo Z, Pan YX, Zheng JL, Zhu QL, Sun LD, Zhuo MQ, Hu W (2013b) Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper. Aquat Toxicol 136–137:72–78PubMedGoogle Scholar
  12. Cheng HL, Ji NJ, Peng YX, Shen X, Xu JH, Dong ZG, Wu CC (2011) Molecular characterization and tissue-specific expression of the acetyl-CoA carboxylase alpha gene from Grass carp, Ctenopharyngodon idella. Gene 487:46–51PubMedCrossRefGoogle Scholar
  13. Cowey CB, Walton MJ (1989) Intermediary metabolism. In: Halver JE (ed) Fish nutrition, 1st edn. Academic Press, New York, pp 259–329Google Scholar
  14. Ellesat KS, Yazdani M, Holth TF, Hylland K (2011) Species-dependent sensitivity to contaminants: an approach using primary hepatocytes cultures with three marine fish species. Mar Environ Res 72:216–224PubMedCrossRefGoogle Scholar
  15. Eyckmans M, Benoot D, Van Raemdonck GA, Zegels G, Van Ostade XW, Witters E, Blust R, De Boeck G (2012) Comparative proteomics of copper exposure and toxicity in rainbow trout, common carp and gibel carp. Comp Biochem Physiol 7D:220–232Google Scholar
  16. Gong Y, Luo Z, Zhu QL, Zheng JL, Tan XY, Chen QL, Lin YC, Lu RH (2013) Characterization and tissue distribution of leptin, leptin receptor and leptin receptor overlapping transcript genes in yellow catfish Pelteobagrus fulvidraco. Gen Comp Endocrinol 182:1–6PubMedCrossRefGoogle Scholar
  17. Grosell M, McDonald M, Walsh P, Wood C (2004) Effects of prolonged copper exposure in the marine gulf toadfish (Opsanus beta) II: copper accumulation, drinking rate and Na+/K+-ATPase activity in osmoregulatory tissues. Aquat Toxicol 68:263–275PubMedCrossRefGoogle Scholar
  18. He S, Liang XF, Qu CM, Huang W, Shen D, Zhang WB, Mai KS (2012) Identification, organ expression and ligand-dependent expression levels of peroxisome proliferator activated receptors in grass carp (Ctenopharyngodon idella). Comp Biochem Physiol 155C:381–388Google Scholar
  19. Heath AG (1987) Water pollution and fish physiology. CRC Press, Florida 245Google Scholar
  20. Hisar O, Sonmez AY, Beydemir S, Hisar SA, Yanik T, Cronin T (2009) Kinetic behaviour of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in different tissues of rainbow Trout (Oncorhynchus mykiss) exposed to non-lethal concentrations of cadmium. Acta Vet Brno 78:179–185CrossRefGoogle Scholar
  21. Horton JD, Goldstein JL, Brown MS (2002) SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1132PubMedCentralPubMedCrossRefGoogle Scholar
  22. Hsu SC, Huang C (2007) Changes in liver PPARα mRNA expression in response to two levels of high-safflower-oil diets correlate with changes in adiposity and serum leptin in rats and mice. J Nutr Biochem 18:86–96PubMedCrossRefGoogle Scholar
  23. Hu W, Zhi L, Zhuo MQ, Zhu QL, Zheng JL, Chen QL, Gong Y, Liu CX (2013) Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro. Fish Physiol Biochem 39:637–647PubMedCrossRefGoogle Scholar
  24. Kerner J, Hoppel C (2000) Fatty acid import into mitochondria. Biochim Biophys Acta 1486:1–17PubMedCrossRefGoogle Scholar
  25. Kurilenko AV, Zakhartsev MV, Chelomin VP (2002) In vitro effect of copper ions on transbilayer distribution of aminophospholipids in synaptosomal membrane of walleye pollock (Theragra chalcogramma). Aquat Toxicol 58:131–136PubMedCrossRefGoogle Scholar
  26. Lee CH, Olson P, Evans RM (2003) Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144:2201–2207PubMedCrossRefGoogle Scholar
  27. Levesque H, Moon T, Campbell P, Hontela A (2002) Seasonal variation in carbohydrate and lipid metabolism of yellow perch (Perca flavescens) chronically exposed to metals in the field. Aquat Toxicol 60:257–267PubMedCrossRefGoogle Scholar
  28. Liu X, Luo Z, Xiong B, Liu X, Zhao Y, Hu G, Lv G (2010) Effect of waterborne copper exposure on growth, hepatic enzymatic activities and histology in Synechogobius hasta. Ecotoxicol Environ Saf 73:1286–1291PubMedCrossRefGoogle Scholar
  29. Lu RH, Liang XF, Wang M, Zhou Y, Bai XL, He Y (2012) The role of leptin in lipid metabolism in fatty degenerated hepatocytes of the grass carp Ctenopharyngodon idellus. Fish Physiol Biochem 38:1759–1774PubMedCrossRefGoogle Scholar
  30. Magana MM, Osborne TF (1996) Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter. J Biol Chem 271:32689–32694PubMedCrossRefGoogle Scholar
  31. Magana M, Lin S, Dooley K, Osborne T (1997) Sterol regulation of acetyl coenzyme A carboxylase promoter requires two interdependent binding sites for sterol regulatory element binding proteins. J Lipid Res 38:1630–1638PubMedGoogle Scholar
  32. Manzl C, Ebner H, Kock G, Dallinger R, Krumschnabel G (2003) Copper, but not cadmium, is acutely toxic for trout hepatocytes: short-term effects on energetics and ion homeostasis. Toxicol Appl Pharmacol 191:235–244PubMedCrossRefGoogle Scholar
  33. Manzl C, Enrich J, Ebner H, Dallinger R, Krumschnabel G (2004) Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes. Toxicology 196:57–64PubMedCrossRefGoogle Scholar
  34. Minghetti M, Leaver MJ, Tocher DR (2011) Transcriptional control mechanisms of genes of lipid and fatty acid metabolism in the Atlantic salmon (Salmo salar) established cell line, SHK-1. Biochim Biophys Acta 1811:194–202PubMedCrossRefGoogle Scholar
  35. Morash AJ, Kajimura M, McClelland GB (2008) Intertissue regulation of carnitine palmitoyltransferase I (CPTI): mitochondrial membrane properties and gene expression in rainbow trout (Oncorhynchus mykiss). Biochim Biophys Acta 1778:1382–1389PubMedCrossRefGoogle Scholar
  36. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  37. Napal L, Marrero PF, Haro D (2005) An intronic peroxisome proliferator-activated receptor-binding sequence mediates fatty acid induction of the human carnitine palmitoyltransferase 1A. J Mol Biol 354:751–759PubMedCrossRefGoogle Scholar
  38. Nascimento AA, Araujo FG, Gomes ID, Mendes RM, Sales A (2012) Fish gills alterations as potential biomarkers of environmental quality in a eutrophized tropical river in south-eastern Brazil. Anat Histol Embryol 41:209–216PubMedCrossRefGoogle Scholar
  39. Nawaz M, Manzl C, Krumschnabel G (2005) In vitro toxicity of copper, cadmium, and chromium to isolated hepatocytes from carp, Cyprinus carpio L. Bull Environ Contamin Toxicol 75:652–661CrossRefGoogle Scholar
  40. Nilsson-Ehle P, Garfinkel AS, Schotz MC (1980) Lipolytic enzymes and plasma lipoprotein metabolism. Ann Rev Biochem 49:667–693PubMedCrossRefGoogle Scholar
  41. Reddy JK, Hashimoto T (2001) Peroxisomal β-oxidation and peroxisome proliferator-activated receptor α: an adaptive metabolic system. Ann Rev Nutr 21:193–230CrossRefGoogle Scholar
  42. Sanchez W, Palluel O, Meunier L, Coquery M, Porcher JM, Ait-Aissa S (2005) Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels. Environ Toxicol Pharmacol 19:177–183PubMedCrossRefGoogle Scholar
  43. Schlenk D, Benson WH (2001) Target organ toxicity in marine and freshwater teleosts. CRC, OrgansCrossRefGoogle Scholar
  44. Seth R, Yang S, Choi S, Sabean M, Roberts E (2004) In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2. Toxicol In Vitro 18:501–509PubMedCrossRefGoogle Scholar
  45. Shaw BJ, Al-Bairuty G, Handy RD (2012) Effects of waterborne copper nanoparticles and copper sulphate on rainbow trout, (Oncorhynchus mykiss): physiology and accumulation. Aquat Toxicol 116–117:90–101PubMedCrossRefGoogle Scholar
  46. Song S, Zhang Y, Ma K, Jackson-Hayes L, Lavrentyev EN, Cook GA, Elam MB, Park EA (2004) Peroxisomal proliferator activated receptor gamma coactivator (PGC-1α) stimulates carnitine palmitoyltransferase I (CPT-Iα) through the first intron. Biochim Biophys Acta 1679:164–173PubMedCrossRefGoogle Scholar
  47. Spiegelman BM, Flier JS (2001) Obesity and the regulation of energy balance. Cell 104:531–543PubMedCrossRefGoogle Scholar
  48. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Rad Biol Med 18:321–336PubMedCrossRefGoogle Scholar
  49. Tan F, Wang M, Wang W, Lu Y (2008) Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro. Toxicol In Vitro 22:164–170PubMedCrossRefGoogle Scholar
  50. Teles M, Pacheco M, Santos MA (2005) Physiological and genetic responses of European eel (Anguilla anguilla L.) to short-term chromium or copper exposure-influence of preexposure to a PAH-like compound. Environ Toxicol 20:92–99PubMedCrossRefGoogle Scholar
  51. Thibaut R, Schnell S, Porte C (2009) Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines. Cell Biol Toxicol 25:611–622PubMedCrossRefGoogle Scholar
  52. Wan X, Ma T, Wu W, Wang Z (2004) EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbons. Ecotoxicol Environ Saf 58:84–89PubMedCrossRefGoogle Scholar
  53. Yessoufou A, Ategbo JM, Attakpa E, Hichami A, Moutairou K, Dramane KL, Khan NA (2009) Peroxisome proliferator-activated receptor-α modulates insulin gene transcription factors and inflammation in adipose tissues in mice. Mol Cell Biochem 323:101–111PubMedCrossRefGoogle Scholar
  54. Yokoyama C, Wang X, Briggs M, Admon A, Wu J, Hua X, Goldstein J, Brown M (1993) SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75:187–197PubMedCrossRefGoogle Scholar
  55. Yuan H, Shyy JYJ, Martins-Green M (2009) Second-hand smoke stimulates lipid accumulation in the liver by modulating AMPK and SREBP-1. J Hepatol 51:535–547PubMedCentralPubMedCrossRefGoogle Scholar
  56. Zheng JL, Luo Z, Zhu QL, Chen QL, Gong Y (2013a) Molecular characterization, tissue distribution and kinetic analysis of carnitine palmitoyltransferase I in juvenile yellow catfish Pelteobagrus fulvidraco. Genomics 101:195–203PubMedCrossRefGoogle Scholar
  57. Zheng JL, Luo Z, Liu CX, Chen QL, Tan XY, Zhu QL, Gong Y (2013b) Differential effects of acute and chronic zinc (Zn) exposure on hepatic lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco. Aquat Toxicol 132–133:173–181PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Qing-Ling Zhu
    • 1
    • 2
  • Zhi Luo
    • 1
    • 2
  • Mei-Qin Zhuo
    • 1
    • 2
  • Xiao-Ying Tan
    • 1
    • 2
  • Lin-Dan Sun
    • 1
    • 2
  • Jia-Lang Zheng
    • 1
    • 2
  • Qi-Liang Chen
    • 1
    • 2
  1. 1.Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture of P.R.C., Fishery CollegeHuazhong Agricultural UniversityWuhanChina
  2. 2.Freshwater Aquaculture Collaborative Innovative Centre of Hubei ProvinceWuhanChina

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