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Molecular and Cellular Biochemistry

, Volume 367, Issue 1–2, pp 165–173 | Cite as

Different effects of omega-3 fatty acids on the cell cycle in C2C12 myoblast proliferation

  • Yunqian Peng
  • Yu Zheng
  • Yunsheng Zhang
  • Jie Zhao
  • Fei Chang
  • Tianyu Lu
  • Ran Zhang
  • Qiuyan Li
  • Xiaoxiang Hu
  • Ning Li
Article

Abstract

Polyunsaturated fatty acids (PUFAs) are important molecules for human health. We investigated the effects of three major omega-3 PUFAs on C2C12 myoblast proliferation. Both docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids decreased cell growth, whereas linolenic (ALA) acid did not, compared with the control. Cell cycle analysis showed that G1 phase duration was increased markedly and S-phase duration was decreased by DHA and EPA. In contrast, there was no change in the G1 or S-phase duration when the cells were treated with linolenic acid. To determine how DHA and EPA affected the cell cycle, cyclins and MAPK proteins were investigated. Western blotting and real-time quantitative PCR showed that DHA and EPA decreased cyclin E and CDK2 levels at both the protein and mRNA level. Also, MAPK phosphorylation levels were decreased by treatment with DHA and EPA. Our results indicated that different kinds of n-3 PUFA differentially affected myoblast cell proliferation. DHA and EPA decreased skeletal muscle cell proliferation through a mechanism involving MAPK-ERK.

Keywords

Omega-3 fatty acids Cell cycle C2C12 myoblast Cyclin E MAPK 

Notes

Acknowledgments

The study was supported by the earmarked fund for Modern Agro-industry Technology Research Systems of China (CARAS-37), and National Transgenic Breeding Project of China (2011ZX08009-003-006).

Supplementary material

11010_2012_1329_MOESM1_ESM.tif (720 kb)
Fig. A1 Effect of fatty acids on the proliferation of C2C12 cells. DHA and EPA (10 μM) were added to cells. Cell proliferation curves are shown for 120 h of culture. The viability of cells was determined using the MTT assay. Data represent the means ± SEM of at least five separate experiments, each performed in six wells. (▲) DHA 22:6n-3; (■) EPA 20:5n-3; (◆) ALA 18:3n-3; (×) Control (TIFF 719 kb)
11010_2012_1329_MOESM2_ESM.tif (271 kb)
Fig. A2 Cell cycle of DHA-arrested cells changed back to normal after changed the growth medium. (A) cell cycle of DHA-arrested cells; (B) cell cycle of DHA-arrested cells with normal growth medium for 24 h. Results were from at least three repeated experiment. Values are means ± SEM. *, *** P < 0.05 and 0.001, respectively (TIFF 271 kb)
11010_2012_1329_MOESM3_ESM.tif (586 kb)
Fig. A3 Effects of Omega-3 fatty acids on C2C12 myoblasts cell viability. Cells were treated with different concentrations (50 μM and 100 μM) of fatty acids for 72 h. Viability was quantitated by Trypan blue exclusion method. The number of viable cells is expressed as a percentage. Black column were treated with 50 μM related fatty acids, white column were treated with 100 μM related fatty acids, Control group were treated with equivalent volume of the ethanol. Results are mean ± SEM of at least five separate experiments. There were no significant differences between control and fatty acids-treated groups (TIFF 585 kb)

References

  1. 1.
    De Caterina R (2011) n-3 Fatty acids in cardiovascular disease. N Engl J Med 364:2439–2450. doi: 10.1056/NEJMra1008153 PubMedCrossRefGoogle Scholar
  2. 2.
    Osterud B, Elvevoll EO (2011) Dietary omega-3 fatty acids and risk of type 2 diabetes: Lack of antioxidants? Am J Clin Nutr 94:617–8; author reply 618–9. doi: 10.3945/ajcn.111.017855 Google Scholar
  3. 3.
    Wardhana, Surachmanto ES, Datau EA (2011) The role of omega-3 fatty acids contained in olive oil on chronic inflammation. Acta Med Indones 43:138–143PubMedGoogle Scholar
  4. 4.
    Bazan NG, Molina MF, Gordon WC (2011) Docosahexaenoic acid signalolipidomics in nutrition: significance in aging, neuroinflammation, macular degeneration, Alzheimer’s, and other neurodegenerative diseases. Annu Rev Nutr 31:321–351. doi: 10.1146/annurev.nutr.012809.104635 PubMedCrossRefGoogle Scholar
  5. 5.
    van der Meij BS, van Bokhorst-de van der Schueren MA, Langius JA, Brouwer IA, van Leeuwen PA (2011) n-3 PUFAs in cancer, surgery, and critical care: a systematic review on clinical effects, incorporation, and washout of oral or enteral compared with parenteral supplementation. Am J Clin Nutr 94:1248–1265. doi: 10.3945/ajcn.110.007377 PubMedCrossRefGoogle Scholar
  6. 6.
    Davidson MH, Kling D, Maki KC (2011) Novel developments in omega-3 fatty acid-based strategies. Curr Opin Lipidol 22:437–444. doi: 10.1097/MOL.0b013e32834bd642 PubMedCrossRefGoogle Scholar
  7. 7.
    Erdman JW Jr (2000) AHA science advisory: soy protein and cardiovascular disease: a statement for healthcare professionals from the Nutrition Committee of the AHA. Circulation 102:2555–2559PubMedCrossRefGoogle Scholar
  8. 8.
    Gebauer SK, Psota TL, Harris WS, Kris-Etherton PM (2006) n-3 Fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits. Am J Clin Nutr 83:1526S–1535SPubMedGoogle Scholar
  9. 9.
    Eastwood L, Kish PR, Beaulieu AD, Leterme P (2009) Nutritional value of flaxseed meal for swine and its effects on the fatty acid profile of the carcass. J Anim Sci 87:3607–3619PubMedCrossRefGoogle Scholar
  10. 10.
    Ueda K, Ferlay A, Chabrot J, Loor JJ, Chilliard Y, Doreau M (2003) Effect of linseed oil supplementation on ruminal digestion in dairy cows fed diets with different forage: concentrate ratios. J Dairy Sci 86:3999–4007PubMedCrossRefGoogle Scholar
  11. 11.
    Wood JD, Enser M, Fisher AV, Nute GR, Sheard PR, Richardson RI, Hughes SI, Whittington FM (2008) Fat deposition, fatty acid composition and meat quality: a review. Meat Sci 78:343–358PubMedCrossRefGoogle Scholar
  12. 12.
    Wood JD, Enser M, Fisher AV, Nute GR, Richardson RI, Sheard PR (1999) Manipulating meat quality and composition. Proc Nutr Soc 58:363–370PubMedCrossRefGoogle Scholar
  13. 13.
    Wu G, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, Heinz E, Qiu X (2005) Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat Biotechnol 23:1013–1017. doi: 10.1038/nbt1107 PubMedCrossRefGoogle Scholar
  14. 14.
    Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier JA, Stobart AK, Lazarus CM (2004) Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotechnol 22:739–745. doi: 10.1038/nbt972 PubMedCrossRefGoogle Scholar
  15. 15.
    Lai L, Kang JX, Li R, Wang J, Witt WT, Yong HY, Hao Y, Wax DM, Murphy CN, Rieke A, Samuel M, Linville ML, Korte SW, Evans RW, Starzl TE, Prather RS, Dai Y (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24: 435–6Google Scholar
  16. 16.
    Kao BT, Lewis KA, DePeters EJ, Van Eenennaam AL (2006) Endogenous production and elevated levels of long-chain n-3 fatty acids in the milk of transgenic mice. J Dairy Sci 89:3195–3201. doi: 10.3168/jds.S0022-0302(06)72594-2 PubMedCrossRefGoogle Scholar
  17. 17.
    Sampath H, Ntambi JM (2005) Polyunsaturated fatty acid regulation of genes of lipid metabolism. Annu Rev Nutr 25:317–340. doi: 10.1146/annurev.nutr.25.051804.101917 PubMedCrossRefGoogle Scholar
  18. 18.
    Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W, Li P, Lu WJ, Watkins SM, Olefsky JM (2010) GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell 142:687–698. doi: 10.1016/j.cell.2010.07.041 PubMedCrossRefGoogle Scholar
  19. 19.
    Holy EW, Forestier M, Richter EK, Akhmedov A, Leiber F, Camici GG, Mocharla P, Luscher TF, Beer JH, Tanner FC (2011) Dietary alpha-linolenic acid inhibits arterial thrombus formation, tissue factor expression, and platelet activation. Arterioscler Thromb Vasc Biol 31:1772–1780. doi: 10.1161/ATVBAHA.111.226118 PubMedCrossRefGoogle Scholar
  20. 20.
    Lee JH, O’Keefe JH, Lavie CJ, Marchioli R, Harris WS (2008) Omega-3 fatty acids for cardioprotection. Mayo Clin Proc 83:324–332PubMedCrossRefGoogle Scholar
  21. 21.
    Arsenault D, Julien C and Calon F (2011) Chronic dietary intake of alpha-linolenic acid does not replicate the effects of DHA on passive properties of entorhinal cortex neurons. Br J Nutr: 1–13. doi: 10.1017/S0007114511004089
  22. 22.
    Zhu H, Fan C, Xu F, Tian C, Zhang F, Qi K (2010) Dietary fish oil n-3 polyunsaturated fatty acids and alpha-linolenic acid differently affect brain accretion of docosahexaenoic acid and expression of desaturases and sterol regulatory element-binding protein 1 in mice. J Nutr Biochem 21:954–960. doi: 10.1016/j.jnutbio.2009.07.011 PubMedCrossRefGoogle Scholar
  23. 23.
    Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727PubMedCrossRefGoogle Scholar
  24. 24.
    Ffrench M, Fiere D, Bryon PA, Cordier G, Viala JJ (1984) Simultaneous determination of cellular DNA and proteins by cytofluorimetry. Application to acute leukemia in adults. Pathol Biol (Paris) 32:139–143Google Scholar
  25. 25.
    Berneman ZN, Vermeulen K, Van Bockstaele DR (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 36:131–149PubMedCrossRefGoogle Scholar
  26. 26.
    Sherr CJ (1994) G1 phase progression: cycling on cue. Cell 79:551–555. doi: 0092-8674(94)90540-1 PubMedCrossRefGoogle Scholar
  27. 27.
    Dulic V, Lees E, Reed SI (1992) Association of human cyclin E with a periodic G1-S phase protein kinase. Science 257:1958–1961PubMedCrossRefGoogle Scholar
  28. 28.
    Zhang W, Liu HT (2002) MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 12:9–18. doi: 10.1038/sj.cr.7290105 PubMedCrossRefGoogle Scholar
  29. 29.
    Sugano M, Hirahara F (2000) Polyunsaturated fatty acids in the food chain in Japan. Am J Clin Nutr 71:189S–196SPubMedGoogle Scholar
  30. 30.
    Sanders TA (2000) Polyunsaturated fatty acids in the food chain in Europe. Am J Clin Nutr 71:176S–178SPubMedGoogle Scholar
  31. 31.
    Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, Zhao G, Etherton TD (2000) Polyunsaturated fatty acids in the food chain in the United States. Am J Clin Nutr 71:179S–188SPubMedGoogle Scholar
  32. 32.
    Khan NA, Nishimura K, Aires V, Yamashita T, Oaxaca-Castillo D, Kashiwagi K, Igarashi K (2006) Docosahexaenoic acid inhibits cancer cell growth via p27(Kip1), CDK2, ERK1/ERK2, and retinoblastoma phosphorylation. J Lipid Res 47:2306–2313PubMedCrossRefGoogle Scholar
  33. 33.
    Bordoni A, Astolfi A, Morandi L, Pession A, Danesi F, Di Nunzio M, Franzoni M, Biagi P (2007) N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure. FEBS Lett 581:923–929. doi: 10.1016/j.febslet.2007.01.070 PubMedCrossRefGoogle Scholar
  34. 34.
    Sauer LA, Dauchy RT, Blask DE, Krause JA, Davidson LK, Dauchy EM (2005) Eicosapentaenoic acid suppresses cell proliferation in MCF-7 human breast cancer xenografts in nude rats via a pertussis toxin-sensitive signal transduction pathway. J Nutr 135:2124–2129PubMedGoogle Scholar
  35. 35.
    Siddiqui RA, Jenski LJ, Harvey KA, Wiesehan JD, Stillwell W, Zaloga GP (2003) Cell-cycle arrest in Jurkat leukaemic cells: a possible role for docosahexaenoic acid. Biochem J 371:621–629PubMedCrossRefGoogle Scholar
  36. 36.
    Terano T, Tanaka T, Tamura Y, Kitagawa M, Higashi H, Saito Y, Hirai A (1999) Eicosapentaenoic acid and docosahexaenoic acid inhibit vascular smooth muscle cell proliferation by inhibiting phosphorylation of Cdk2 cyclin E complex. Biochem Biophys Res Commun 254:502–506PubMedCrossRefGoogle Scholar
  37. 37.
    Lee CY, Sit WH, Fan ST, Man K, Jor IW, Wong LL, Wan ML, Tan-Un KC, Wan JM (2010) The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 36:991–998PubMedGoogle Scholar
  38. 38.
    Jourdan ML, Barascu A, Besson P, Le Floch O, Bougnoux P (2006) CDK1-cyclin B1 mediates the inhibition of proliferation induced by omega-3 fatty acids in MDA-MB-231 breast cancer cells. Int J Biochem Cell Biol 38:196–208PubMedCrossRefGoogle Scholar
  39. 39.
    Harris WS (2005) Alpha-linolenic acid—A gift from the land? Circulation 111:2872–2874PubMedCrossRefGoogle Scholar
  40. 40.
    Abedin L, Lien EL, Vingrys AJ, Sinclair AJ (1999) The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig. Lipids 34:475–482PubMedCrossRefGoogle Scholar
  41. 41.
    Obaya AJ, Sedivy JM (2002) Regulation of cyclin-Cdk activity in mammalian cells. Cell Mol Life Sci 59:126–142PubMedCrossRefGoogle Scholar
  42. 42.
    Tarn WY, Lai MC (2011) Translational control of cyclins. Cell Div 6:5. doi: 10.1186/1747-1028-6-5 PubMedCrossRefGoogle Scholar
  43. 43.
    Wilkinson MG, Millar JB (2000) Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J 14:2147–2157. doi: 10.1096/fj.00-0102 PubMedCrossRefGoogle Scholar
  44. 44.
    Gysin S, Lee SH, Dean NM, McMahon M (2005) Pharmacologic inhibition of RAF→MEK→ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1. Cancer Res 65:4870–4880. doi: 10.1158/0008-5472.CAN-04-2848 PubMedCrossRefGoogle Scholar
  45. 45.
    Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E (2004) C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem 48:223–233PubMedGoogle Scholar
  46. 46.
    Hurley MS, Flux C, Salter AM, Brameld JM (2006) Effects of fatty acids on skeletal muscle cell differentiation in vitro. Br J Nutr 95:623–630. doi: S0007114506000833 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Yunqian Peng
    • 1
  • Yu Zheng
    • 1
  • Yunsheng Zhang
    • 1
  • Jie Zhao
    • 1
  • Fei Chang
    • 1
  • Tianyu Lu
    • 1
  • Ran Zhang
    • 1
  • Qiuyan Li
    • 1
  • Xiaoxiang Hu
    • 1
  • Ning Li
    • 1
  1. 1.State Key Laboratory of AgrobiotechnologyChina Agricultural UniversityBeijingChina

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