Czech J. Anim. Sci., 2022, 67(6):228-236 | DOI: 10.17221/121/2021-CJAS

L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathwayOriginal Paper

Yutong Sun#, Guohong Wang#, Xinbo Zhou, Yuchen Sun, Qingquan Ma*
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P.R. China

Milk production determines health status and optimal growth of suckling neonates. Recent studies revealed that the secretion of leucine into sow milk from the mammary gland is significantly lower than the amount taken up by the mammary gland from the blood, suggesting the potential and fundamental role in udder metabolism. However, little is known about the underlying mechanism. In this study, porcine mammary epithelial cells (PMECs) were employed to investigate the effects of leucine on protein synthesis and mammalian target of rapamycin (mTOR) signalling. Results showed that leucine supplementation significantly increased the proliferation of PMECs in a dose-dependent manner from 0 to 1 mmol/l. Secretions of four casein subunits were significantly increased by addition of leucine. The phosphorylation of proteins related to mTOR signalling, including mTOR, eukaryotic translation initiation factor 4E-binding protein, and p70 ribosomal protein S6 kinase, were accordingly upregulated by leucine supplementation. Casein secretion was not affected by leucine treatment when mTOR signalling was inhibited by rapamycin. This suggests that leucine stimulates protein synthesis through the mTOR signalling pathway. These findings facilitate the utilization of dietary leucine supplementation to improve milk production in lactating sows.

Keywords: leucine; sow; milk protein; mTOR signalling

Published: June 29, 2022  Show citation

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Sun Y, Wang G, Zhou X, Sun Y, Ma Q. L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathway. Czech J. Anim. Sci.. 2022;67(6):228-236. doi: 10.17221/121/2021-CJAS.
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References

  1. Appuhamy JA, Knoebel NA, Nayananjalie WA, Escobar J, Hanigan MD. Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices. J Nutr. 2012 Mar; 142(3):484-91. Go to original source... Go to PubMed...
  2. Appuhamy JA, Nayananjalie WA, England EM, Gerrard DE, Akers RM, Hanigan MD. Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells. J Dairy Sci. 2014 Jan 1;97(1):419-29. Go to original source... Go to PubMed...
  3. Arriola Apelo SI, Singer LM, Lin XY, McGilliard ML, StPierre NR, Hanigan MD. Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue. J Dairy Sci. 2014 Feb;97(2):1047-56. Go to original source... Go to PubMed...
  4. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: A new generation of mTOR inhibitors. Nat Rev Drug Discov. 2011 Oct 31;10(11):868-80. Go to original source... Go to PubMed...
  5. Dahanayaka S, Rezaei R, Porter WW, Johnson GA, Burghardt RC, Bazer FW, Hou YQ, Wu ZL, Wu G. Technical note: Isolation and characterization of porcine mammary epithelial cells. J Anim Sci. 2015 Nov 1;93(11):5186-93. Go to original source... Go to PubMed...
  6. Dunshea FR, Bauman DE, Nugent EA, Kerton DJ, King RH, McCauley I. Hyperinsulinaemia, supplemental protein and branched-chain amino acids when combined can increase milk protein yield in lactating sows. Br J Nutr. 2005 Mar;93(3):325-32. Go to original source... Go to PubMed...
  7. Gao HN, Hu H, Zheng N, Wang JQ. Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway. J Zhejiang Univ Sci B. 2015 Jun;16(6):560-72. Go to original source... Go to PubMed...
  8. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004 Aug 15;18(16):1926-45. Go to original source... Go to PubMed...
  9. Kim J, Song G, Wu G, Gao H, Johnson GA, Bazer FW. Arginine, leucine, and glutamine stimulate proliferation of porcine trophectoderm cells through the MTORRPS6K-RPS6-EIF4EBP1 signal transduction pathway. Biol Reprod. 2013 May 2;88(5): 9 p. Go to original source... Go to PubMed...
  10. Lei J, Feng D, Zhang Y, Zhao FQ, Wu Z, San Gabriel A, Fujishima Y, Uneyama H, Wu G. Nutritional and regulatory role of branched-chain amino acids in lactation. Front Biosci. 2012a Jun 1;17(725):2725-39. Go to original source... Go to PubMed...
  11. Lei J, Feng D, Zhang Y, Dahanayaka S, Li X, Yao K, Wang J, Wu Z, Dai Z, Wu G. Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells. Amino Acids. 2012b Nov;43(5):2179-89. Go to original source... Go to PubMed...
  12. Lipton JO, Sahin M. The neurology of mTOR. Neuron. 2014 Oct 22;84(2):275-91. Go to original source... Go to PubMed...
  13. Ma Q, Hu S, Bannai M, Wu G. l-Arginine regulates protein turnover in porcine mammary epithelial cells to enhance milk protein synthesis. Amino Acids. 2018 May;50(5):621-8. Go to original source... Go to PubMed...
  14. Ma Q, Zhou X, Hu L, Chen J, Zhu J, Shan A. Leucine and isoleucine have similar effects on reducing lipid accumulation, improving insulin sensitivity and increasing the browning of WAT in high-fat diet-induced obese mice. Food Funct. 2020a Mar 26;11(3):2279-90. Go to original source... Go to PubMed...
  15. Ma Q, Zhou X, Sun Y, Hu L, Zhu J, Shao C, Meng Q, Shan A. Threonine, but not lysine and methionine, reduces fat accumulation by regulating lipid metabolism in obese mice. J Agric Food Chem. 2020b Apr 29;68(17):4876-83. Go to original source... Go to PubMed...
  16. Makovicky P, Rimarova K, Makovicky P, Nagy M. Genetic parameters for external udder traits of different dairy ewes. Indian J Anim Sci. 2015 Jan;85(1):89-90. Go to original source...
  17. Makovicky P, Makovicky P, Margetin M, Nagy M. Estimation of genetic parameters for chosen udder traits of different dairy ewes. Indian J Anim Sci. 2019 Jun;89(5):572-4. Go to original source...
  18. Manjarin R, Bequette BJ, Wu G, Trottier NL. Linking our understanding of mammary gland metabolism to amino acid nutrition. Amino Acids. 2014 Nov;46(11):2447-62. Go to original source... Go to PubMed...
  19. Mercier JC, Gaye P. Early events in secretion of main milk proteins: Occurrence of precursors. J Dairy Sci. 1982 Feb;65(2):299-316. Go to original source... Go to PubMed...
  20. Moser SA, Tokach MD, Dritz SS, Goodband RD, Nelssen JL, Loughmiller JA. The effects of branched-chain amino acids on sow and litter performance. J Anim Sci. 2000 Mar 1;78(3):658-67. Go to original source... Go to PubMed...
  21. Nielsen TT, Trottier NL, Stein HH, Bellaver C, Easter RA. The effect of litter size and day of lactation on amino acid uptake by the porcine mammary glands. J Anim Sci. 2002 Sep 1;80(9):2402-11. Go to original source...
  22. Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994 Oct 27;371(6500):762-7. Go to original source... Go to PubMed...
  23. Prizant RL, Barash I. Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells. J Cell Biochem. 2008 Nov 1;105 (4):1038-47. Go to original source... Go to PubMed...
  24. Pullen N, Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett. 1997 Jun 23;410(1):78-82. Go to original source... Go to PubMed...
  25. Rafiee Tari N, Arranz E, Corredig M. Effect of protein composition of a model dairy matrix containing various levels of beta-casein on the structure and anti-inflammatory activity of in vitro digestates. Food Funct. 2019 Apr; 10(4):1870-9. Go to original source... Go to PubMed...
  26. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017 Apr 6;169(2):361-71. Go to original source... Go to PubMed...
  27. Shimomura Y, Kitaura Y. Physiological and pathological roles of branched-chain amino acids in the regulation of protein and energy metabolism and neurological functions. Pharmacol Res. 2018 Jul 1;133:215-7. Go to original source... Go to PubMed...
  28. Son SM, Park SJ, Lee H, Siddiqi F, Lee JE, Menzies FM, Rubinsztein DC. Leucine signals to mTORC1 via its metabolite acetyl-coenzyme A. Cell Metab. 2019 Jan 8; 29(1):192-201. Go to original source... Go to PubMed...
  29. Suryawan A, Orellana RA, Fiorotto ML, Davis TA. Triennial growth symposium: Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs. J Anim Sci. 2011 Jul;89(7):2004-16. Go to original source... Go to PubMed...
  30. Trottier NL, Shipley CF, Easter RA. Plasma amino acid uptake by the mammary gland of the lactating sow. J Anim Sci. 1997 May 1;75(5):1266-78. Go to original source... Go to PubMed...
  31. Wang L, Lin Y, Bian Y, Liu L, Shao L, Lin L, Qu B, Zhao F, Gao X, Li Q. Leucyl-tRNA synthetase regulates lactation and cell proliferation via mTOR signaling in dairy cow mammary epithelial cells. Int J Mol Sci. 2014 Apr;15(4):5952-69. Go to original source... Go to PubMed...
  32. Zhang S, Zeng X, Ren M, Mao X, Qiao S. Novel metabolic and physiological functions of branched chain amino acids: A review. J Anim Sci Biotechnol. 2017 Jan 23;8(1): 12 p. Go to original source... Go to PubMed...
  33. Zheng YM, He XY, Zhang Y. Characteristics and EGFP expression of goat mammary gland epithelial cells. Reprod Domest Anim. 2010 Dec;45(6):e323-31. Go to original source... Go to PubMed...
  34. Zhou LM, Xu JY, Rao CP, Han S, Wan Z, Qin LQ. Effect of whey supplementation on circulating C-reactive protein: A meta-analysis of randomized controlled trials. Nutrients. 2015 Feb;7(2):1131-43. Go to original source... Go to PubMed...

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