Full Text:   <2390>

Summary:  <1762>

CLC number: S823

On-line Access: 2015-06-08

Received: 2014-11-30

Revision Accepted: 2015-05-23

Crosschecked: 2015-05-25

Cited: 7

Clicked: 4457

Citations:  Bibtex RefMan EndNote GB/T7714


Nan Zheng


Hai-na Gao


-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.6 P.560-572


Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway

Author(s):  Hai-na Gao, Han Hu, Nan Zheng, Jia-qi Wang

Affiliation(s):  Ministry of Agriculture-Milk Risk Assessment Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; more

Corresponding email(s):   zhengnan_1980@126.com

Key Words:  Bovine mammary epithelial cells, Leucine, Histidine, Western blotting, mTOR, Casein

Share this article to: More <<< Previous Article|

Hai-na Gao, Han Hu, Nan Zheng, Jia-qi Wang. Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway[J]. Journal of Zhejiang University Science B, 2015, 16(6): 560-572.

@article{title="Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway",
author="Hai-na Gao, Han Hu, Nan Zheng, Jia-qi Wang",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway
%A Hai-na Gao
%A Han Hu
%A Nan Zheng
%A Jia-qi Wang
%J Journal of Zhejiang University SCIENCE B
%V 16
%N 6
%P 560-572
%@ 1673-1581
%D 2015
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1400337

T1 - Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway
A1 - Hai-na Gao
A1 - Han Hu
A1 - Nan Zheng
A1 - Jia-qi Wang
J0 - Journal of Zhejiang University Science B
VL - 16
IS - 6
SP - 560
EP - 572
%@ 1673-1581
Y1 - 2015
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1400337

The aim of this study is to investigate the effects of leucine (Leu) and histidine (His) on the expression of both the mammalian target of rapamycin (mTOR) signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells (CMEC-H), using a single supplement through western blotting. The Earle’s balanced salt solution (EBSS) was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR (Ser2481), Raptor (Ser792), eIF4E (Ser209), and eEF2 (Thr56) increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L (P<0.01). The P-4EBP1 (Thr37) at 10.80 mmol/L Leu, and P-RPS6 (Ser235/236) at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of αs2-casein, β-casein, κ-casein, P-mTOR (Ser2481), P-Raptor (Ser792), P-S6K1 (Thr389), P-4EBP1 (Thr37), P-eIF4E (Ser209), and P-eEF2 (Thr56) (P<0.01) in CMEC-H, whereas the αs1-casein expression was only reduced at 9.60 mmol/L His, G protein β subunit-like protein (GβL) at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the αs1-casein expression was positively correlated with P-mTOR (P<0.01), P-S6K1 (P<0.01), and P-eEF2 (P<0.01) for the addition of Leu, while the expressions of β-casein (P<0.01) and κ-casein (P<0.01) were positively correlated with P-eEF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H.




Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Appuhamy, J.A., Knapp, J.R., Becvar, O., et al., 2011a. Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows. J. Dairy Sci., 94(4):1952-1960.

[2]Appuhamy, J.A., Bell, A.L., Nayananjalie, W.A., et al., 2011b. Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices. J. Nutr., 141(6):1209-1215.

[3]Appuhamy, J.A., Knoebel, N.A., Nayananjalie, W.A., et al., 2012. Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices. J. Nutr., 142(3):484-491.

[4]Appuhamy, J., Nayananjalie, W.A., England, E.M., et al., 2014. 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., 97(1):419-429.

[5]Arriola Apelo, S.I., Bell, A.L., Estes, K., et al., 2014a. Effects of reduced dietary protein and supplemental rumen-protected essential amino acids on the nitrogen efficiency of dairy cows. J. Dairy Sci., 97(9):5688-5699.

[6]Arriola Apelo, S.I., Knapp, J.R., Hanigan, M.D., 2014b. Invited review: current representation and future trends of predicting amino acid utilization in the lactating dairy cow. J. Dairy Sci., 97(7):4000-4017.

[7]Arriola Apelo, S.I., Singer, L.M., Lin, X.Y., et al., 2014c. Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue. J. Dairy Sci. 97(2):1047-1056.

[8]Bequette, B.J., Hanigan, M.D., Calder, A.G., et al., 2000. Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production. J. Dairy Sci., 83(4):765-775.

[9]Beugnet, A., Tee, A.R., Taylor, P.M., et al., 2003. Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability. Biochem. J., 372(Pt 2):555-566.

[10]Bionaz, M., Hurley, W., Loor, J., 2012. Milk protein synthesis in the lactating mammary gland: insights from transcriptomics analyses. In: Hurley, W. (Ed.), Milk Protein. InTech, Chapter 11, p.285-324.

[11]Burgos, S.A., Cant, J.P., 2010. IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells. Domest. Anim. Endocrinol., 38(4):211-221.

[12]Cheng, S.W., Fryer, L.G., Carling, D., 2004. Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status. J. Biol. Chem., 279(16):15719-15722.

[13]Christophersen, C.T., Karlsen, J., Nielsen, M.O., et al., 2002. Eukaryotic elongation factor-2 (eEF-2) activity in bovine mammary tissue. J. Dairy Res., 69(2):205-212.

[14]Durán, R.V., Hall, M.N., 2012. Leucyl-tRNA synthetase: double duty in amino acid sensing. Cell Res., 22(8):1207-1209.

[15]Gao, H.N., Hu, H., Wang, J.Q., et al., 2015. Effects of leucine and histidine on milk protein synthesis via mammalian target of rapamycin signaling pathway in the bovine mammary epithelial cells. Chin. J. Anim. Nutr., 27(4):1124-1134 (in Chinese).

[16]Gerasimovskaya, E.V., Tucker, D.A., Stenmark, K.R., 2005. Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation. J. Appl. Physiol., 98(2):722-731.

[17]Gingras, A.C., Raught, B., Sonenberg, N., 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem., 68(1):913-963.

[18]Hanigan, M.D., Crompton, L.A., Metcalf, J.A., et al., 2001. Modelling mammary metabolism in the dairy cow to predict milk constituent yield, with emphasis on amino acid metabolism and milk protein production: model evaluation. J. Theor. Biol., 213(2):223-239.

[19]Hara, K., Maruki, Y., Long, X., et al., 2002. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell, 110(2):177-189.

[20]Harris, T.E., Lawrence, J.C.Jr., 2003. TOR signaling. Sci. STKE, 212:re15.

[21]Hristov, A.N., Price, W.J., Shafii, B., 2004. A meta-analysis examining the relationship among dietary factors, dry matter intake, and milk and milk protein yield in dairy cows. J. Dairy Sci., 87(7):2184-2196.

[22]Hu, H., Zheng, N., Dai, W.T., 2014. Immortalization of a primary bovine mammary epithelial cell line by the SV40 large T-antigen gene. The American Dairy Science Association. Kansas City, MO.

[23]Kalscheur, K.F., Baldwin VI, R.L., Glenn, B.P., et al., 2006. Milk production of dairy cows fed differing concentrations of rumen-degraded protein. J. Dairy Sci., 89(1):249-259.

[24]Kaul, G., Pattan, G., Rafeequi, T., 2011. Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation. Cell Biochem. Funct., 29(3):227-234.

[25]Kim, C.H., Choung, J.J., Chamberlain, D.G., 2001. Estimates of the efficiency of transfer of L-histidine from blood to milk when it is the first-limiting amino acid for secretion of milk protein in the dairy cow. J. Sci. Food Agric., 81(12):1150-1155.

[26]Kim, D.H., Sarbassov, D.D., Ali, S.M., 2002. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell, 110(2):163-175.

[27]Kim, D.H., Sarbassov, D.D., Ali, S.M., et al., 2003. GβL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell, 11(4):895-904.

[28]Kim, E., 2009. Mechanisms of amino acid sensing in mTOR signaling pathway. Nutr. Res. Pract., 3(1):64-71.

[29]Kim, S.G., Buel, G.R., Blenis, J., 2013. Nutrient regulation of the mTOR complex 1 signaling pathway. Mol. Cells, 35(6):463-473.

[30]Kimball, S.R., 2002. Regulation of global and specific mRNA translation by amino acids. J. Nutr., 132(5):883-886.

[31]Korhonen, M., Vanhatalo, A., Huhtanen, P., 2002. Evaluation of isoleucine, leucine, and valine as a second-limiting amino acid for milk production in dairy cows fed grass silage diet. J. Dairy Sci., 85(6):1533-1545

[32]Laplante, M., Sabatini, D.M., 2012. mTOR signaling in growth control and disease. Cell, 149(2):274-293.

[33]Li, X., Alafuzoff, I., Soininen, H., et al., 2005. Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer’s disease brain. FEBS J., 272(16):4211-4220.

[34]Merrick, W.C., 1992. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev., 56(2):291-315.

[35]Moshel, Y., Rhoads, R.E., Barash, I., 2006. Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells. J. Cell. Biochem., 98(3):685-700.

[36]Peterson, R.T., Beal, P.A., Comb, M.J., et al., 2000. FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J. Biol. Chem., 275(10):7416-7423.

[37]Prizant, R.L., Barash, I., 2008. Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells. J. Cell. Biochem., 105(4):1038-1047.

[38]Proud, C.G., 2007. Amino acids and mTOR signalling in anabolic function. Biochem. Soc. Trans., 35(5):1187-1190.

[39]Stipanuk, M.H., 2007. Leucine and protein synthesis: mTOR and beyond. Nutr. Rev., 65(3):122-129.

[40]Toerien, C.A., Trout, D.R., Cant, J.P., 2010. Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal S6 kinase 1. J. Nutr., 140(2):285-292.

[41]Wang, C., Liu, H.Y., Wang, Y.M., et al., 2010. Effects of dietary supplementation of methionine and lysine on milk production and nitrogen utilization in dairy cows. J. Dairy Sci., 93(8):3661-3670.

[42]Wang, X., Proud, C.G., 2006. The mTOR pathway in the control of protein synthesis. Physiology (Bethesda), 21:362-369.

[43]Wilde, C.J., Quarrie, L.H., Tonner, E., et al., 1997. Mammary apoptosis. Livest. Prod. Sci., 50(1-2):29-37.

[44]Yang, Q., Inoki, K., Kim, E., et al., 2006. TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity. PNAS, 103(18):6811-6816.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE