Effects of Leucine and H.istidine on Milk Protein Synthesis via Mammalian Target of Rapamycin Signaling Pathway in the Bovine Mammary Epithelial Cells

GAO Haina, HU Han, WANG Jiaqi, ZHENG Nan

doi:10.3969/j.issn.1006-267x.2015.04.016

2015 , Vol. 27 >Issue 4: 1124 - 1134

DOI: https://doi.org/10.3969/j.issn.1006-267x.2015.04.016

Ruminant Nutrition

Effects of Leucine and H.istidine on Milk Protein Synthesis via Mammalian Target of Rapamycin Signaling Pathway in the Bovine Mammary Epithelial Cells

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1. Ministry of Agriculture-Milk Risk Assessment Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China;
3. Ministry of Agriculture-Milk and Dairy Product Inspection Center, Beijing 100193, China;
4. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2014-11-25

Online published: 2015-04-21

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Abstract

This study employed the immortalized bovine mammary epithelial cells (CMECs-H) as a model to clarify the regulatory effects of single supplement of leucine (Leu) and histidine (His) on casein synthesis molecular mechanism via mammalian target of rapamycin (mTOR) signaling pathway. The casein and expression of genes encoding mTOR signaling were detected by added single supplement of different concentrations of Leu and His. The Earle's balanced salts solution without amino acids was set as negative control. After added single supplement of different concentrations of Leu and His, CMECs-H proliferation of 12 and 24 h was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) colorimetry, and mRNA expression amounts of 4 casein related genes and 8 mTOR signaling pathway related genes were detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed as follows: after the addition of different concentrations of Leu or His for 12 and 24 h, the trends of cell proliferation were the same. Compared with negative control group, CMECs-H proliferation increased with added of Leu in the range of 0.15 to 5.40 mmol/L and His in the range of 0.15 to 9.60 mmol/L, respectively. Compared with the negative control group, the expression amounts of α_s1-casein (CSN1S1), α_s2-casein (CSN1S2) and κ-casein (CSN3) genes measured at 6 h with Leu in the range of 0.45 to 10.80 mmol/L were significantly up-regulated (P<0.05), and the expression amounts of CSN1S1, β-casein (CSN2) and CSN3 genes measured at 6 h with His in the range of 0.15 to 4.80 mmol/L were significantly up-regulated (P<0.05). The expression amounts of mTOR signaling pathway related genes, which were mTOR, a binding partner of target of rapamycin (raptor), binds to the kinase domain of mTOR, stabilizes protein of raptor (GβL), eukaryotic initiation factor 4E binding protein (4EBP1) and eukaryotic elongation factor 2 (eEF2), were highest in the experimental groups with the addition of 1.35 mmol/L Leu, while the expression amount of the ribosomal protein S6 kinase 1 (S6K1) gene reduced along with the increasing of Leu concentration. When adding His to the negative medium, the expression amounts of 4EBP1, eEF2, eukaryotic initiation factor 4E (EIF4E) and ribosomal protein S6 (rps6) genes increased along with the increasing of His concentration, while the expression amount of mTOR gene reduced along with the increasing of His concentration. GβL gene acquired its highest expression amount at 4.80 mmol/L and S6K1 gene at 1.20 mmol/L of His during the experimental groups. These results demonstrate that Leu or His supplements can promote the expression of genes related casein via mTOR signaling pathway related genes in mammary epithelial cells.

Key words： Leu; His; casein; mTOR; cell proliferation; gene expression

Cite this article

GAO Haina, HU Han, WANG Jiaqi, ZHENG Nan . Effects of Leucine and H.istidine on Milk Protein Synthesis via Mammalian Target of Rapamycin Signaling Pathway in the Bovine Mammary Epithelial Cells[J]. Chinese Journal of Animal Nutrition, 2015 , 27(4) : 1124 -1134 . DOI: 10.3969/j.issn.1006-267x.2015.04.016

References

[1] 杨金勇.蛋氨酸、赖氨酸及其二肽对奶牛乳腺上皮细胞酪蛋白αs1基因表达的影响[D].硕士学位论文.杭州:浙江大学, 2006:9-18.

[2] SINGH K, ERDMAN R A, SWANSON K M, et al.Epigenetic regulation of milk production in dairy cows[J].Journal of Mammary Gland Biology and Neoplasia, 2010, 15(1):101-112.

[3] 李庆章.奶牛乳腺发育与泌乳生物学[M].北京:科学出版社, 2014:251-252.

[4] BRODERICK G A, STEVNSON M J, PATTON R A, et al.Effect of supplementing rumen-protected methionine on production and nitrogen excretion in lactating dairy cows[J].Journal of Dairy Science, 2008, 91(3):1092-1102.

[5] SOCHA M T, PUTNAM D E GARTHWAITE B D, et al.Improving intestinal amino acid supply of pre- and postpartum dairy cows with rumen-protected methionine and lysine[J].Journal of Dairy Science, 2005, 88(3):1113-1126.

[6] 曹洋, 艾阳, 张源淑.乳蛋白合成的信号通路与营养调控[J].畜牧与兽医, 2014, 6(7):133-136.

[7] TOERIEN C A, TROUT D R, CANT J P.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].The Journal of Nutrition, 2010, 140(2):285-292.

[8] TOERIEN C A, CANT J P.Abundance and phosphorylation state of translation initiation factors in mammary glands of lactating and nonlactating dairy cows[J].Journal of Dairy Science, 2007, 90(6):2726-2734.

[9] 袁国铖.不同类型日粮对泌乳奶牛乳腺组织胰岛素通路及JAK-STAT信号通路的影响[D].硕士学位论文.兰州:甘肃农业大学, 2013 .

[10] GERMAN T, BARASH I.Characterization of an epithelial cell line from bovine mammary gland[J].In Vitro Cellular & Developmental Biology:Animal, 2002, 38(5):282-292.

[11] 陈洪菊, 屈艺, 母得志.mTOR信号通路的生物学功能[J].生命的化学, 2010, 30(4):555-561.

[12] DEGUIL J, PERAULT-POCHAT M C, CHAVANT F, et al.Activation of the protein p70S6K via ERK phosphorylation by cholinergic muscarinic receptors stimulation in human neuroblastoma cells and in mice brain[J].Toxicology Letters, 2008, 182(1/2/3):91-96.

[13] 曾波航, 庄莹, 陈静琦, 等.雷帕霉素对肺癌细胞mTOR信号通路相关蛋白表达的作用[J].肿瘤防治研究, 2011, 38(10):1105-1108.

[14] 庞学燕, 季昀, 田青, 等.亮氨酸对κ-酪蛋白合成的影响及相关信号通路的研究[J].中国畜牧杂志, 2013, 49(9):38-41.

[15] KIMBALL S R, JEFFERSON L S.New functions for amino acids:effects on gene transcription and translation[J].The American Journal of Clinical Nutrition, 2006, 83(2):500S-507S.

[16] MABJEESH S J, KYLE C E, MACRAE J C, et al.Vascular sources of amino acids for milk protein synthesis in goats at two stages of lactation[J].Journal of Dairy Science, 2002, 85(4):919-929.

[17] KIMBALL S R, JEFFERSON L S.Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis[J].The Journal of Nutrition, 2006, 136(1S):227S-231S.

[18] APPUHAMY J A D R N, KNOEBEL N A, NAYANANJALIE W A D, et al.Isoleucine and leucine independently Regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices[J].The Journal of Nutrition, 2012, 142(3):484-491.

[19] PRIZANT R L, BARASH I.Negative effects of the amino acids lys, his, and thr on s6k1 phosphorylation in mammary epithelial cells[J].Journal of Cellular Biochemistry, 2008, 105(4):1038-1047.

[20] HU H, WANG J Q, BU D P, et al.In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow[J].PLoS Ooe, 2009, 4(11):e7636.

[21] 徐柏林.精氨酸对乳腺上皮细胞中酪蛋白合成的影响及其调控机制[D].硕士学位论文.扬州:扬州大学, 2012:2-55.

[22] SIGL T, MEYER H H D, WIEDEMANN S.Gene expression analysis of protein synthesis pathways in bovine mammary epithelial cells purified from milk during lactation and short-term restricted feeding[J].Journal of Animal Physiology and Animal Nutrition, 2014, 98(1):84-95.

[23] HAYASHI A A, NONES K, ROY N C, et al.Initiation and elongation steps of mRNA translation are involved in the increase in milk protein yield caused by growth hormone administration during lactation[J].Journal of Dairy Science, 2009, 92(5):1889-1899.

[24] 李喜艳.奶牛乳腺上皮细胞中赖氨酸蛋氨酸配比模式对酪蛋白合成的影响及机理研究[D].硕士学位论文.北京:中国农业科学院, 2011:1-32.

[25] 胡菡, 王加启, 李发弟, 等.高温诱导体外培养奶牛乳腺上皮细胞的应激响应[J].农业生物技术学报, 2011, 19(2):287-293.

[26] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J].Methods, 2001, 25(4):402-408.

[27] EHMANN U K, PETERSON W D, Jr, MISFELDT D S.To grow mouse mammary epithelial cells in culture[J].Journal of Cell Biology, 1984, 98(3):1026-1032.

[28] RAGGIO G, PACHECO D, BERTHIAUME R, et al.Effect of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows[J].Journal of Dairy Science, 2004, 87(10):3461-3472.

[29] MERCIER J C, GAYE P.Early events in secretion of main milk proteins:occurrence of precursors[J].Journal of Dairy Science, 1982, 65(2):299-316.

[30] APPUHAMY J A D R, BELL A L, NAYANANJALIE W A D, et al.Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices[J].The Journal of Nutrition, 2011, 141(6):1209-1215.

[31] APELO S I A, SINGER L M, LIN X Y, et al.Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue[J].Journal of Dairy Science, 2014, 97(2):1047-1056.

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