Effects of Saturated Long-chain Fatty Acid on mRNA Expression of Genes Associated with Milk Fat and Protein Biosynthesis in Bovine Mammary Epithelial Cells

  • Qi, Lizhi (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Yan, Sumei (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Sheng, Ran (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Zhao, Yanli (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Guo, Xiaoyu (College of Animal Science, Inner Mongolia Agricultural University)
  • Received : 2013.08.13
  • Accepted : 2013.10.14
  • Published : 2014.03.01


This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.


  1. Banks, W, J. L. Clapperton, and M. E. Ferrie. 1976. Effect of feeding fat to dairy cows receiving a fat-deficient basal diet. II. Fatty acid composition of the milk fat. J. Dairy Res. 43:219-227.
  2. Bionaz, M. and J. J. Loor. 2008b. Gene networks driving bovine milk fat synthesis during the lactation cycle. BMC Genomics 9:366.
  3. Barber, M. C., R. A. Clegg, M. T. Travers, and R. G. Vernon. 1997. Lipid metabolism in the lactating mammary gland. Biochim Biophys Acta. 1347(2-3):101-126.
  4. Bernard, L., C. Leroux, and Y. Chilliard. 2008. Expression and nutritional regulation of lipogenic genes in the ruminant lactating mammary gland. Adv. Exp. Med. Biol. 606:67-108.
  5. Bionaz, M. and J. J. Loor. 2008a. ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 are the most abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation. J. Nutr. 138:1019-1024.
  6. Burgos, S. A., M. Dai, and J. P. Cant. 2010. Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway. J. Dairy Sci. 93:153-161.
  7. Briscoe, C. P., M. Tadayyon, J. L. Andrews, W. G. Benson, J. K. Chambers, M. M. Eilert, C. Ellis, N. A. Elshourbagy, A. S. Goetz, D. T. Minnick, P. R. Murdock, H. R. Sauls Jr., U. Shabon, L. D. Spinage, J. C. Strum, P. G. Szekeres, K. B. Tan, J. M. Way, D. M. Ignar, S. Wilson, and A. I. Muir. 2003. The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J. Biol. Chem. 278:11303-11311.
  8. Cant, J. P., E. J. Depeters, and R. L. Baldwin. 1991. Effect of dietary fat and postruminal casein administration on milk composition of lactating dairy cows. J. Dairy Sci. 74:211-219.
  9. Cousin, S. P., S. R. Hugl, C. E. Wrede, H Kajio, M. G. Myers, Jr., and C. J. Rhodes. 2001. Free Fatty Acid-Induced Inhibition of Glucose and Insulin-Like Growth Factor I-Induced Deoxyribonucleic Acid Synthesis in the Pancreatic b-Cell Line INS-1. Endocrinology 142:229-240.
  10. Invernizzi, G., B. J. Thering, M. A. McGuire, G. Savoini, and J. J. Loor. 2010. Sustained upregulation of stearoyl-CoA desaturase in bovine mammary tissue with contrasting changes in milk fat synthesis and lipogenic gene networks caused by lipid supplements. Funct. Integr. Genomics 10:561-575.
  11. Furth, P. A., R. E Nakles, S. Millman, E. S. Diaz-Cruz, and M. C. Cabrera. 2011. Signal transducer and activator of transcription 5 as a key signaling pathway in normal mammary gland developmental biology and breast cancer. Breast Cancer Res. 13:220.
  12. Hansen, H. O. and J. Knudsen. 1987. Effect of exogenous long-chain fatty acids on lipid biosynthesis in dispersed ruminant mammary gland epithelial cells: Esterification of long-chain exogenous fatty acids. J. Dairy Sci. 70:1344-1349.
  13. Hu, H., J. Q. Wang, F. D. Li, D. P. Bu, X. Y. Li, and L. Y. Zhou. 2010. Effects of free linolenic acid on transcription of mrnas of genes related to fatty acid metabolism in mammary epithelial cells of dairy cows. Chinese J. Anim. Nutr. 22(5):1342-1349.
  14. Jenkins, T. C. 1999. Lactation performance and fatty acid composition of milk from Holstein cows fed 0 to 5% oleamide. J. Dairy Sci. 82:1525-1531.
  15. Jenkins, T C. and M. A. Mcguire. 2006. Major advances in nutrition: Impact on milk composition. J. Dairy Sci. 89:1302-1310.
  16. Kadegowda, A. K. G., M Bionaz, L. S. Piperova, R. A. Erdman, and J. J. Loor. 2008. Lipogenic gene expression in MAC-T cells is affected differently by fatty acids and enhanced by PPAR-gamma activation. J. Dairy Sci. 91(E-Suppl.1):678.
  17. Lehner, R. and A. Kuksis. 1996. Biosynthesis of triacylglycerols. Prog. Lipid Res. 35:169-201.
  18. Kadegowda, A. K. G., M. Bionaz, L. S. piperova, R. A. Erdman, and J. J. Loor. 2009. Peroxisome proliferator-activated receptor-$\gamma$ activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents. J. Dairy Sci. 92:4276-4289.
  19. Kim, J. E. and J. Chen. 2004. Regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 53:2748-2756.
  20. Laplante, M. and D. M. Sabatini. 2009. mTOR signaling at a glance. J. Cell Sci. 122:3589-3594.
  21. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-\Delta{\Delta}Ct}$ method. Methods 25:402-408.
  22. McArthur, M. J., B. P. Atshaves, A. Frolov, W. D. Foxworth, A. B. Kier, and F. Schroeder. 1999. Cellular uptake and intracellular trafficking of long chain fatty acids. J. Lipid Res. 40:1371-1383.
  23. Noble, R. C., W. Steele, and J. H. Moore. 1969. The effects of dietary palmitic and stearic acids on milk fat composition in the cow. J. Dairy Res. 36:375-381.
  24. Ntambi, J. M. and M. Miyazaki. 2003. Recent insights into stearoyl-CoA desaturase-1. Curr. Opin. Lipidol. 14:255-261.
  25. Palmquist, D. L. 2006. Milk fat: Origin of fatty acids and influence of nutritional factors thereon. In: Advanced Dairy Chemistry Volume 2 Lipids (Ed. P. F. Fox and P. L. H. McSweeney). Springer, New York. pp. 43-92.
  26. Spitsberg, V. L., E. Matitashvili, and R. C. Gorewit. 1995. Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. Eur. J. Biochem. 230:872-878.
  27. Pauloin, A., S. Chat, C. Pechoux, C. Hue-Beauvais, S. Droineau, L. Galio, E. Devinoy, and E. Chanat. 2010. Oleate and linoleate stimulate degradation of $\beta$-casein in prolactin-treated HC11 mouse mammary epithelial cells. Cell Tissue Res. 340:91-102.
  28. Ramirez-Zacarias, J. L., F. Castro-Munozledo, and W. Kuri-Harcuch. 1992. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97:493-497.
  29. Soliman, G. A. 2011. The integral role of mTOR in lipid metabolism. Cell Cycle 10:861-862.
  30. Thering, B. J., D. E. Graugnard, P. Piantoni, and J. J. Loor. 2009. Adipose tissue lipogenic gene networks due to lipid feeding and milk fat depression in lactating cows. J. Dairy Sci. 92:4290-4300.
  31. Warntjes, J. L., P. H. Robinson, E. Galo, E. J. DePeters, and D. Howes. 2008. Effects of feeding supplemental palmitic acid (C16:0) on performance and milk fatty acid profile of lactating dairy cows under summer heat. Anim. Feed Sci. Technol. 140: 241-257.
  32. Weisbjerg, M. R., L. Wiking, N. B. Kristensen, and P. Lund. 2008. Effects of supplemental dietary fatty acids on milk yield and fatty acid composition in high and medium yielding cows. J. Dairy Res. 75:142-152.
  33. Wright, T. C., J. P. Cant, and B. W. McBrid. 2002. Inhibition of fatty acid synthesis in bovine mammary homogenate by palmitic acid is not a detergent effect. J. Dairy Sci. 85:642-647.
  34. Yonezawa, T., S. Yonekura, Y. Kobayashi, A. Hagino, K. Katoh, and Y. Obara. 2004. Effects of long-chain fatty acids on cytosolic triacylglycerol accumulation and lipid droplet formation in primary cultured bovine mammary epithelial cells. J. Dairy Sci. 87:2527-2534.
  35. Yonezawa, T., M. Sanosaka, S. Haga, Y. Kobayashi, K. Katoh, and Y. Obara. 2008a. Regulation of uncoupling protein 2 expression by long-chain fatty acids and hormones in bovine mammary epithelial cells. Biochem. Biophys. Res. Commun. 375:280-285.
  36. Yonezawa, T., S. Haga, Y. Kobayashi, K. Katoh, and Y. Obara. 2008b. Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells. Biochem. Biophys. Res. Commun. 367:729-735.

Cited by

  1. Rapamycin Inhibits Expression of Elongation of Very-long-chain Fatty Acids 1 and Synthesis of Docosahexaenoic Acid in Bovine Mammary Epithelial Cells vol.29, pp.11, 2016,
  2. Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol vol.47, pp.1, 2016,
  3. Regulation of peroxisome proliferator-activated receptor gamma on milk fat synthesis in dairy cow mammary epithelial cells vol.52, pp.10, 2016,
  4. Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells vol.88, pp.12, 2017,
  5. Dose- and type-dependent effects of long-chain fatty acids on adipogenesis and lipogenesis of bovine adipocytes pp.00220302, 2017,
  6. Metabolic transition of milk triacylglycerol synthesis in response to varying levels of palmitate in porcine mammary epithelial cells vol.13, pp.1, 2018,
  7. Transcriptional regulation of milk lipid synthesis by exogenous C16:0 and C18 fatty acids in bovine mammary epithelial cells vol.98, pp.2, 2018,