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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: 3673

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Nan Zheng

http://orcid.org/0000-0002-8469-4243

Hai-na Gao

http://orcid.org/0000-0003-1327-4003

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

http://doi.org/10.1631/jzus.B1400337


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


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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.

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%T Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway
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T1 - Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway
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DOI - 10.1631/jzus.B1400337


Abstract: 
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.

亮氨酸和组氨酸通过mTOR信号通路调控奶牛腺上皮细胞中酪蛋白的合成

目的:以体外培养的永生化奶牛乳腺上皮细胞(CMEC-H)为模型,利用蛋白免疫印迹法(Westernblotting)检测酪蛋白和哺乳动物雷帕霉素靶蛋白(mTOR)信号路径元件的蛋白磷酸化表达。进一步探索亮氨酸和组氨酸通过mTOR信号途径调控酪蛋白合成机制的重要意义。
创新点:首次在CMEC-H模型中研究不同浓度的亮氨酸和组氨酸通过mTOR复合物1(mTORC1)中mTOR(Ser2481)、mTOR调控蛋白(Raptor,Ser792)和G蛋白β亚基样蛋白(GβL)对酪蛋白表达调控的影响。研究证明mTOR(Ser2448)并不是在乳腺上皮细胞中激活mTOR信号通路的唯一磷酸化位点,mTOR(Ser2481)同样可作为激活mTOR信号通路的生物标记。
方法:以厄尔平衡溶液代替培养基,设为阴性对照,分别添加不同浓度的亮氨酸或组氨酸,利用Westernblotting检测酪蛋白和mTOR信号路径元件的蛋白表达。
结论:与阴性对照组相比,当在CMEC-H细胞中添加0.45~10.80mmol/L亮氨酸6h时,4种酪蛋白的表达和mTOR(Ser2481)、Raptor(Ser792)、真核翻译起始因子4E(eIF4E,Ser209)和真核细胞翻译延伸因子(eEF2,Thr56)的磷酸化表达均显著上调(P<0.01)。而当亮氨酸浓度在10.80mmol/L时,真核翻译起始因子4E结合蛋白1(4EBP1,Thr37)的磷酸化表达被抑制;在5.40~10.80mmol/L时,核糖体蛋白S6(RPS6,Ser235/236)的磷酸化被抑制。当添加0.15~9.60mmol/L组氨酸6h时,αs2-酪蛋白、β-酪蛋白、κ-酪蛋白的表达和mTOR(Ser2481)、Raptor(Ser792)、核糖体S6蛋白激酶(S6K1,Thr389)、4EBP1(Thr37)、eIF4E(Ser209)和eEF2(Thr56)的磷酸化表达均显著上调(P<0.01)。而当组氨酸浓度在9.60mmol/L时,αs1-酪蛋白的表达量降低;在0.15和9.60mmol/L时,GβL被抑制;在4.80~9.60mmol/L时,RPS6磷酸化被抑制。线性回归模型显示,当添加亮氨酸时,αs1-酪蛋白的表达与mTOR、S6K1和eEF2的磷酸化表达成显著的正相关(P<0.01;表4);当添加组氨酸,β-酪蛋白和κ-酪蛋白均与eEF2磷酸化表达成显著的正相关(P<0.01;表5)。综上所述,在乳腺上皮细胞中,亮氨酸和组氨酸能通过mTOR信号通路促进酪蛋白基因的表达。

关键词:奶牛乳腺上皮细胞;亮氨酸;组氨酸;蛋白免疫印迹;mTOR;酪蛋白

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

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