牛乳腺上皮细胞SNAT2对氨基酸调节乳合成的影响

doi:10.16431/j.cnki.1671-7236.2018.08.010

摘要/Abstract

摘要：

试验旨在研究氨基酸转运体钠离子依赖的中性氨基酸转运蛋白（the sodium-dependent neutral amino acid transporter 2，SNAT2）在牛乳腺上皮细胞（bovine mammary epithelial cell，BMEC）中对氨基酸调节乳合成的影响。利用组织块法成功培养原代BMEC，添加不同氨基酸（蛋氨酸（Met）、赖氨酸（Lys）和亮氨酸（Leu）刺激BMEC后，通过实时荧光定量PCR、Western blotting技术和甘油三酯试剂盒检测SNAT2、酪蛋白（β-casein）基因的表达量和BMEC培养液上清甘油三酯的分泌量；将N-flag-SNAT2真核表达载体及SNAT2 siRNA分别转入细胞中进行SNAT2基因的过表达和敲低试验；通过Western blotting和甘油三酯试剂盒分别检测SNAT2、哺乳动物雷帕霉素靶蛋白（the mammalian target of rapamycin，mTOR）、β-casein蛋白表达量和BMEC培养液上清的甘油三酯含量。结果显示，3种氨基酸（Met、Lys、Leu）均能显著促进BMEC分泌乳蛋白和乳脂，并激活mTOR信号途径，其中Met、Lys还能够显著上调SNAT2基因表达；SNAT2能够正向调节BMEC乳蛋白和乳脂肪的合成，并激活mTOR信号通路，说明氨基酸激活mTOR信号通路是通过SNAT2基因介导完成的，进而调节了BMEC乳蛋白和乳脂肪合成。

关键词: 氨基酸; SNAT2基因; mTOR; 乳蛋白; 乳脂肪

Abstract:

This experiment was aimed to investigate the effect of amino acid transporter the sodium-dependent neutral amino acid transporter 2 (SNAT2) on amino acid-stimulated milk protein and milk fat synthesis in bovine mammary epithelial cell (BMEC).After stimulating BMEC with methionine (Met),lysine (Lys) and leucine (Leu),the expression level of SNAT2 and β-casein gene was detected by Real-time quantitative PCR and Western blotting,and the secretion of triglyceride in culture supernatant was detected using BMEC triglyceride kit.The N-flag-SNAT2 eukaryotic expression vector and SNAT2 siRNA were transfected into the cells for SNAT2 overexpression and knockdown experiments,respectively.The expression level of SNAT2,β-casein,the mammalian target of rapamycin (mTOR),and the content of triglyceride in the culture supernatant of BMEC were detected by Western blotting and triglyceride kit,respectively.The results showed that all three amino acids (Met,Lys and Leu) could significantly increase the content of milk protein and milk fat secreted by BMEC and activate the mTOR signaling pathway.Met and Lys also up-regulated significantly the expression of SNAT2 gene.SNAT2 regulated positively the synthesis of milk protein and milk fat in BMEC,and activated the mTOR signaling pathway,indicating that amino acid activation of the mTOR signaling pathway was mediated through SNAT2,which in turn regulated BMEC milk protein and milk fat synthesis.

Key words: amino acids; SNAT2 gene; mTOR; milk protein; milk fat

中图分类号:

S823

孟春雨, 黄鑫, 刘丽杰, 陈东莹, 高学军. 牛乳腺上皮细胞SNAT2对氨基酸调节乳合成的影响[J]. 《中国畜牧兽医》, 2018, 45(8): 2119-2127.

MENG Chunyu, HUANG Xin, LIU Lijie, CHEN Dongying, GAO Xuejun. Effects of SNAT2 on Amino Acid-stimulated Milk Protein and Milk Fat Synthesis in Bovine Mammary Epithelial Cells[J]. , 2018, 45(8): 2119-2127.

参考文献

[1] ROBINSON P H,CHALUPA W,SNIFFEN C J,et al.Influence of postruminal supplementation of methionine and lysine,isoleucine,or all three amino acids on intake and chewing behavior,ruminal fermentation,and milk and milk component production[J].Journal of Animal Science,1999,77(10):2781-2792.
[2] HANSEN C G,NG Y L D,LAM W L M,et al.The Hippo pathway effectors YAP and TAZ promote cell growth by modulating amino acid signaling to mTORC1[J].Cell Research,2015,25(12):1299.
[3] XIAO H,TAN B,MIAOMIAO W U,et al.Regulatory mechanism of mTOR signaling pathway on cell energy metabolism[J].Scientia Sinica,2015,45(11):1124-1131.
[4] YAO D,MACKENZIE B,MING H,et al.A novel system A isoform mediating Na⁺/neutral amino acid cotransport[J].Journal of Biological Chemistry,2000,275(30):22790-22797.
[5] SHAFFER P L,GOEHRING A,SHANKARANARAYANAN A, et al.Structure and mechanism of a Na⁺-independent amino acid transporter[J].Science,2009,325(5943):1010-1014.
[6] PINILLA J,ALEDO J C,CWIKLINSKI E,et al.SNAT2 transceptor signalling via mTOR:A role in cell growth and proliferation[J].Frontiers in Bioscience,2011,3(4):1289-1299.
[7] ROSARIO F J,KANAI Y,POWELL T L,et al.Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells[J].Journal of Physiology,2013,591(3):609-625.
[8] LUO J Q,CHEN D W,YU B.Upregulation of amino acid transporter expression induced by L-leucine availability in L6 myotubes is associated with ATF4 signaling through mTORC1-dependent mechanism[J].Nutrition,2013,29(1):284-290.
[9] AYE I L,JANSSON T,POWELL T L.TNF-α stimulates system A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling[J].Physiological Reports,2015,3(10):e12594.
[10] 陈建晖.奶牛乳腺上皮细胞系的建立[J].畜牧兽医学报,2009,40(5):743-747.CHEN J H.Establishment of dairy cow mammary gland epithelial cell line[J].Acta Veterinaria et Zootechnica Sinica,2009,40(5):743-747.(in Chinese)
[11] 史琳琳,赵锋,高学军,等.催乳复合物、胰岛素样生长因子-Ⅰ对奶牛乳腺上皮细胞中AKT-1和mTOR表达的作用[J].中国畜牧兽医,2013,40(3):23-27.SHI L L,ZHAO F,GAO X J,et al.Effection of DIP and IGF-Ⅰ on the expression of AKT-1 and mTOR genes in bovine mammary epithelial cells[J].China Animal Husbandry & Veterinary Medicine,2013,40(3):23-27.(in Chinese)
[12] 王佳丽,高学军,李庆章,等.蛋氨酸对体外培养奶牛乳腺上皮细胞泌乳能力的影响[J].乳业科学与技术,2012,35(1):8-10.WANG J L,GAO X J,LI Q Z,et al.Effect of methionine on lactation ability of dairy cow mammary epithelial cells cultured in vitro[J].Journal of Dairy Science and Technology,2012,35(1):8-10.(in Chinese)
[13] BUSSOLATI O,DALL'ASTA V,FRANCHI-GAZZOLA R,et al.The role of system A for neutral amino acid transport in the regulation of cell volume[J].Membrane Biochemistry,2001,18(1):27-38.
[14] TANG Y,TAN B,LI G,et al.The regulatory role of MeAIB in protein metabolism and the mTOR signaling pathway in porcine enterocytes[J].International Journal of Molecular Sciences,2018,19(3):E714.
[15] ORITIZ V,ALEMAN G,ESCAMILLA-DEL-ARENAL M,et al.Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene[J].American Journal of Physiology Endocrinology and Metabolism,2011,300(6):E1092-E1102.
[16] TALUKDER J.TNF alpha requires cAMP/calcium response elements (CRE) to inhibit slc38a2 transcription for SNAT2 mediated Met uptake in enterocytes[J].FASEB Journal,2015,29(Suppl 1):970.
[17] WOLFSON R L,SABATINI D M.The dawn of the age of amino acid sensors for the mTORC1 pathway[J].Cell Metabolism,2017,26(2):301-309.
[18] EFEYAN A,ZONCU R,SABATINI D M.Amino acids and mTORC1:From lysosomes to disease[J].Trends in Molecular Medicine,2012,18(9):524-533.
[19] MORDIER S,DEVAL C,BACHER D,et al.Leucine limitation induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through an mTOR-independent signaling pathway[J].Journal of Biology Chemistry,2000,275(38):29900-29906.
[20] OSORIO J S,LOHAKARE J,BIONAZ M.Biosynthesis of milk fat,protein,and lactose:Roles of transcriptional and posttranscriptional regulation[J].Physiol Genomics,2016,48(4):231-256.
[21] CHENG Q,MOSTAFA N,BROWN J,et al.The system-L amino acid transporter-1 is essential for leucine-dependent insulin secretion and growth factor activation of mTORC1[J].Diabetic Medicine,2013,30:32-32.
[22] YE J,GAO C,LI X,et al.EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells[J].Oncotarget,2016,7(25):38681.