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Amino Acids

, Volume 50, Issue 5, pp 621–628 | Cite as

l-Arginine regulates protein turnover in porcine mammary epithelial cells to enhance milk protein synthesis

Original Article
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Abstract

Milk is an important food for mammalian neonates, but its insufficient production is a nutritional problem for humans and other animals. Recent studies indicate that dietary supplementation with l-arginine (Arg) increases milk production in mammals, including sows, rabbits, and cows. However, the underlying molecular mechanisms remain largely unknown. The present study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that Arg enhances milk protein synthesis via activation of the mechanistic target of rapamycin (mTOR) cell signaling. PMECs were cultured for 4 days in Arg-free basal medium supplemented with 10, 50, 200, or 500 μmol/L Arg. Rates of protein synthesis and degradation in cells were determined with the use of l-[ring-2,4-3H]phenylalanine. Cell medium was analyzed for β-casein and α-lactalbumin, whereas cells were used for quantifying total and phosphorylated levels of mTOR, ribosomal protein S6 kinase (p70S6K), 4E-binding protein 1 (4EBP1), ubiquitin, and proteasome. Addition of 50–500 μmol/L Arg to culture medium increased (P < 0.05) the proliferation of PMECs and the synthesis of proteins (including β-casein and α-lactalbumin), while reducing the rates of proteolysis, in a dose-dependent manner. The phosphorylated levels of mTOR, p70S6K and 4EBP1 were elevated (P < 0.05), but the abundances of ubiquitin and proteasome were lower (P < 0.05), in PMECs supplemented with 200–500 μmol/L Arg, compared with 10–50 μmol/L Arg. These results provide a biochemical basis for the use of Arg to enhance milk production by sows and have important implications for improving lactation in other mammals (including humans and cows).

Keywords

Arginine Mammary gland Milk protein mTOR signaling 

Abbreviations

Arg

Arginine

DMEM

Dulbecco’s modified eagle medium

4EBP1

4E-binding protein 1

FBS

Fetal bovine serum

mTOR

Mechanistic target of rapamycin

PMECs

Porcine mammary epithelial cells

p70S6K

Ribosomal protein S6 kinase

TTBS

Tris–Tween buffered saline

Notes

Acknowledgements

Q. Ma was a recipient of a postdoctoral fellowship from the China Scholarship Council. This work was supported Agriculture and Food Research Initiative Competitive Grants (2014-67015-21770) from the USDA National Institute of Food and Agriculture, and Texas A&M AgriLife Research (H-8200). The authors thank our research assistants for technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

This study involved the cultures of an established cell line and did not require an animal use protocol.

References

  1. Assaad H, Zhou L, Carroll RJ, Wu G (2014) Rapid publication-ready MS-word tables for one-way ANOVA. SpringerPlus 3:474CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bar-Peled L, Sabatini DM (2014) Regulation of mTORC1 by amino acids. Trends Cell Biol 24:400–406CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bauchart-Thevret C, Cui L, Wu G, Burrin DG (2010) Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide. Am J Physiol Endocinol Metab 299:E899–E909CrossRefGoogle Scholar
  4. Bazer FW, Johnson GA, Wu G (2015) Amino acids and conceptus development during the peri-implantation period of pregnancy. Adv Exp Med Biol 843:23–52CrossRefPubMedGoogle Scholar
  5. Chantranupong L, Scaria SM, Saxton RA, Gygi MP, Shen K, Wyant GA, Wang T, Harper JW, Gygi SP, Sabatini DM (2016) The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 165:153–164CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chew BP, Eisenman JR, Tanaka TS (1984) Arginine infusion stimulates prolactin, growth hormone, insulin, and subsequent lactation in pregnant dairy cows. J Dairy Sci 67:2507–2518CrossRefPubMedGoogle Scholar
  7. Chin SF, Robbins KR (1991) Relation of arginine nutrition to mammary gland development in the rat. Nutr Res 11:1317–1327CrossRefGoogle Scholar
  8. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci USA 105:17414–17419CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chotechuang N, Azzout-Marniche D, Bos C, Chaumontet C, Gaudichon C, Tomé D (2011) Down-regulation of the ubiquitin–proteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes. Amino Acids 41:457–468CrossRefPubMedGoogle Scholar
  10. Dahanayaka S, Rezaei R, Porter WW, Johnson GA, Burghardt RC, Bazer FW, Hou YQ, Wu ZL, Wu G (2015) Isolation and characterization of porcine mammary epithelial cells. J Anim Sci 93:5186–5193CrossRefPubMedGoogle Scholar
  11. Delgado R, Abad-Guamán R, De la Mata E, Menoyo D, Nicodemus N, García J, Carabaño R (2017) Effect of dietary supplementation with arginine and glutamine on the performance of rabbit does and their litters during the first three lactations. Anim Feed Sci Technol 227:84–94CrossRefGoogle Scholar
  12. Glickman MH, Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428CrossRefPubMedGoogle Scholar
  13. Hay N, Sonenberg N (2004) Upstream and downstream of mTOR. Genes Dev 18:1926–1945CrossRefPubMedGoogle Scholar
  14. Hou YQ, Yin YL, Wu G (2015) Dietary essentiality of “nutritionally nonessential amino acids” for animals and humans. Exp Biol Med 240:997–1007CrossRefGoogle Scholar
  15. Hou YQ, Yao K, Yin YL, Wu G (2016) Endogenous synthesis of amino acids limits growth, lactation and reproduction of animals. Adv Nutr 7:331–342CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jiang Q, He LQ, Hou YQ, Chen JS, Duan YH, Deng D, Wu GY, Yin YL, Yao K (2016) Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells. Amino Acids 48:2179–2188CrossRefPubMedGoogle Scholar
  17. Kim SW, Wu G (2009) Regulatory role for amino acids in mammary gland growth and milk synthesis. Amino Acids 37:89–95CrossRefPubMedGoogle Scholar
  18. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175CrossRefPubMedGoogle Scholar
  19. Kirchgessner VM, Rader G, Roth-Maier DA (1991) Influence of an oral arginine supplementation on lactation performance of sows. J Anim Physiol Anim Nutr 66:38–44CrossRefGoogle Scholar
  20. Kong X, Tan B, Yin Y, Gao H, Li X, Jaeger LA, Bazer FW, Wu G (2012) l-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells. J Nutr Biochem 23:1178–1183CrossRefPubMedGoogle Scholar
  21. Kong XF, Wang XQ, Yin YL, Li XL, Gao HJ, Bazer FW, Wu G (2014) Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells. Biol Reprod 91(5):106CrossRefPubMedGoogle Scholar
  22. Lecker SH, Goldberg AL, Mitch WE (2006) Protein degradation by the ubiquitin–proteasome pathway in normal and disease states. J Am Soc Nephrol 17:1807–1819CrossRefPubMedGoogle Scholar
  23. Lei J, Feng DY, Zhang YL, Zhao FQ, Wu ZL, San Gabriel A, Fujishima Y, Uneyama H, Wu G (2012) Nutritional and regulatory role of branched-chain amino acids in lactation. Front Biosci 17:2725–2739CrossRefGoogle Scholar
  24. Li H, Meininger CJ, Bazer FW, Wu G (2016) Intracellular sources of ornithine for polyamine synthesis in endothelial cells. Amino Acids 48:2401–2410CrossRefPubMedGoogle Scholar
  25. Ma X, Han M, Li DF, Hu S, Gilbreath KR, Bazer FW, Wu G (2017) l-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway. Amino Acids 49:957–964CrossRefPubMedGoogle Scholar
  26. Mateo RD, Wu G, Moon HK, Carroll JA, Kim SW (2008) Effects of dietary arginine supplementation during gestation and lactation on the performance of lactating primiparous sows and nursing piglets. J Anim Sci 86:827–835CrossRefPubMedGoogle Scholar
  27. Mezl VA, Knox WE (1977) Metabolism of arginine in lactating rat mammary gland. Biochem J 166:105–113CrossRefPubMedPubMedCentralGoogle Scholar
  28. O’Quinn PR, Knabe DA, Wu G (2002) Arginine catabolism in lactating porcine mammary tissue. J Anim Sci 80:467–474CrossRefPubMedGoogle Scholar
  29. Pau MY, Milner JA (1982) Effect of arginine deficiency on mammary gland development in the rat. J Nutr 112:1827–1833CrossRefPubMedGoogle Scholar
  30. Rezaei R, Wu Z, Hou Y, Bazer FW, Wu G (2016) Amino acids and mammary gland development: nutritional implications for milk production and neonatal growth. J Anim Sci Biotechno 7:20CrossRefGoogle Scholar
  31. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761–771CrossRefPubMedGoogle Scholar
  32. Sun KJ, Wu ZL, Ji Y, Wu G (2016) Glycine regulates protein turnover by activating Akt/mTOR and inhibiting expression of genes involved in protein degradation in C2C12 myoblasts. J Nutr 146:2461–2467CrossRefPubMedGoogle Scholar
  33. Suryawan A, Davis TA (2011) Regulation of protein synthesis by amino acids in muscle of neonates. Front Biosci (Landmark Ed) 16:1445–1460CrossRefPubMedCentralGoogle Scholar
  34. Tan B, Yin Y, Kong X, Li P, Li X, Gao H, Li X, Huang R, Wu G (2010) l-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells. Amino Acids 38:1227–1235CrossRefPubMedGoogle Scholar
  35. Trottier NL, Shipley CF, Easter RA (1997) Plasma amino acid uptake by the mammary gland of the lactating sow. J Anim Sci 75:1266–1278CrossRefPubMedGoogle Scholar
  36. Wang WW, Wu ZL, Lin G, Hu SD, Wang B, Dai ZL, Wu G (2014) Glycine stimulates protein synthesis and inhibits oxidative stress in pig small-intestinal epithelial cells. J Nutr 144:1540–1548CrossRefPubMedGoogle Scholar
  37. Wang S, Tsun Z-Y, Wolfson RL, Shen K, Wyant GA, Plovanich ME, Yuan ED, Jones TD, Chantranupong L, Comb W, Wang T, Bar-Peled L, Zoncu R, Straub C, Kim C, Park J, Sabatini BL, Sabatini DM (2015) Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 347:188–194CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, Sabatini DM (2016) Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 351:43–48CrossRefPubMedGoogle Scholar
  39. Wu G (2009) Amino acids: metabolism, functions, and nutrition. Amino Acids 37:1–17CrossRefPubMedGoogle Scholar
  40. Wu G (2013) Amino acids: biochemistry and nutrition. CRC Press, Boca RatonCrossRefGoogle Scholar
  41. Wu G (2014) Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition. J Anim Sci Biotechnol 5:34CrossRefPubMedPubMedCentralGoogle Scholar
  42. Wu G (2018) Principles of animal nutrition. CRC Press, Boca RatonGoogle Scholar
  43. Wu G, Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17CrossRefPubMedPubMedCentralGoogle Scholar
  44. Wu GY, Thompson JR (1990) The effect of glutamine on protein turnover in chick skeletal muscle in vitro. Biochem J 265:593–598CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wu G, Flynn NE, Knabe DA (2000a) Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets. Am J Physiol 279:E395–E402Google Scholar
  46. Wu G, Flynn NE, Knabe DA, Jaeger LA (2000b) A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning. Am J Physiol 279:R554–R559Google Scholar
  47. Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE, Yin Y (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37:153–168CrossRefPubMedGoogle Scholar
  48. Wu G, Bazer FW, Dai ZL, Li DF, Wang JJ, Wu ZL (2014) Amino acid nutrition in animals: protein synthesis and beyond. Annu Rev Anim Biosci 2:387–417CrossRefPubMedGoogle Scholar
  49. Yao K, Yin YL, Chu W, Liu Z, Deng D, Li T, Huang R, Zhang J, Tan B, Wu G (2008) Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs. J Nutr 138:867–872CrossRefPubMedGoogle Scholar
  50. Zhu C, Guo C, Gao K, Wang L, Chen Z, Ma X, Jiang Z (2017) Dietary arginine supplementation in multiparous sows during lactation improves the weight gain of suckling piglets. J Integr Agric 16:648–655CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Qingquan Ma
    • 1
    • 2
  • Shengdi Hu
    • 2
  • Makoto Bannai
    • 3
  • Guoyao Wu
    • 2
  1. 1.Institute of Animal Nutrition, Northeast Agricultural UniversityHarbinChina
  2. 2.Department of Animal ScienceTexas A&M UniversityCollege StationUSA
  3. 3.AminoScience Division, Department of Business Strategy and PlanningAjinomoto Co., Inc.TokyoJapan

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