单一必需氨基酸调控乳蛋白合成机制的研究

发布时间：2018-02-04 21:36

本文关键词： MAC-T 单一EAA 信号蛋白泌乳小鼠仔鼠体增重　出处：《山东农业大学》2016年博士论文　论文类型：学位论文

【摘要】：近年来的研究结果表明,氨基酸不仅是蛋白质合成的底物,而且能够作为生物信号直接参与蛋白质合成的调节。为研究必需氨基酸(EAA)通过细胞内信号途径调控乳蛋白合成的机制,本文以牛乳腺上皮细胞系(MAC-T)为材料,系统研究了10种EAA对调控乳蛋白合成关键信号途径的影响,以泌乳小鼠为模型,以仔鼠体增重衡量蛋白合成速率研究了调控乳蛋白合成潜力EAA对泌乳的影响及机制。单一EAA对调控乳蛋白合成关键信号蛋白磷酸化程度的影响。本试验以MAC-T为试验材料,试验处理为在依次缺失一种EAA的DMEM/F12培养基上培养,以含有全部EAA的DMEM/F12培养基培养为正对照,以缺失全部EAA的DMEM/F12培养基培养为负对照。分别缺失10种EAA对蛋白合成过程中主要信号蛋白磷酸化程度有极显著影响,负对照mTOR磷酸化程度下降58%,缺失Met、Ile或Val使mTOR磷酸化程度分别下降38%、41%、42%;单一EAA缺失降低细胞内4eBP1的磷酸化程度,其中缺失Met、Ile、Thr和Lys分别下降39%、41%、36%和42%;缺失Met、Ile、Leu、Val时S6K1的磷酸化程度分别下降33%、38%、41%、40%;单一EAA缺失显著提高eEF2磷酸化程度,其中缺失Met、Lle、Val磷酸化程度分别提高18%、20%、18%;缺失所有EAA和Met eIF2α磷酸化程度分别提高49%和39%。结果表明,单一EAA主要影响细胞内mTOR和GCN2 2条通路中关键信号蛋白的磷酸化,且关键信号蛋白对完全缺失Met、Leu、Ile、Thr、Val、Lys反应敏感。Met、Leu、Ile、Val对调控乳蛋白合成关键信号蛋白的影响。为研究信号蛋白反应敏感EAA的作用效果,以MAC-T为试验材料,首先研究不同浓度EAA对调控蛋白合成关键信号蛋白的影响以确定细胞内EAA缺乏状态,EAA缺乏时添加Met、Leu、Ile、Val以及这四种EAA两两组合。随着EAA浓度的变化信号蛋白磷酸化程度符合米氏方程变化趋势,EAA浓度20%以下信号蛋白磷酸化程度变化幅度较大,当EAA浓度在30%-90%之间信号蛋白的磷酸化程度变化趋于稳定,据此确定10%为后续研究EAA缺乏状态的浓度。EAA缺乏状态添加Met、Leu、Ile、Val以及这四种EAA两两组合,添加Leu、Ile、Met和Leu、Met和Ile、Leu和Ile mTOR磷酸化程度分别提高47%、69%、187%,200%和165%;单独添加Leu、Ile、Val提高4eBP1的磷酸化程度,且同时添加Met和Leu、Leu和Ile、Val和Ile有加性效果;添加Ile S6K1磷酸化程度提高63%,且Ile与其他三种氨基酸组合有加性作用。结果表明,EAA缺乏时添加Met、Leu、Ile、Val影响提高mTOR通路中信号蛋白的磷酸化程度,且四种氨基酸两两组合对信号蛋白的磷酸化有叠加作用。Leu、Ile、Met、Thr对泌乳小鼠乳蛋白合成的影响。为评估体外试验中乳蛋白合成调控潜力EAA对泌乳的贡献及作用机制,首先确定小鼠蛋白缺乏状态,建立仔鼠体增重和不同蛋白水平的反应曲线,试验选取分娩日期相近的孕鼠,日粮蛋白水平分别为6%、9%、12%、15%、18%、21%、24%,和27%。当蛋白水平低于21%时,随蛋白浓度的增加仔鼠体增重增加,仔鼠体增重随蛋白水平的提高成米氏反应趋势,最大体增重一半时的蛋白水平为14%,最大体增重为113g;随日粮蛋白水平提高泌乳小鼠乳腺组织中S6K1磷酸化程度提高,eEF2的磷酸化程度降低。根据以上结果,确定15%为日粮低蛋白,即负对照,21%为日粮高蛋白,即正对照,低蛋白基础上分别添加Met、Leu、Ile、Thr。低蛋白日粮添加Met、Leu、Ile仔鼠体增重分别提高10%、11%、9%;添加Met、Leu、Ile和Thr后泌乳小鼠乳腺组织中mTOR的磷酸化程度分别提高了55%、34%,、47%和45%;必需氨基酸处理提高泌乳小鼠血浆IGF-1的浓度;仔鼠窝增重和乳腺组织中mTOR,相关系数为0.40。结果表明,低蛋白日粮添加Met、Leu、Ile独立刺激乳蛋白合成速率(仔鼠窝增重),且主要通过mTOR信号通路。Met、Leu诱导内分泌变化对泌乳的影响。初探泌乳潜力EAA(Met、Leu)诱导内分泌的变化对泌乳的影响机制,本试验以泌乳小鼠为模型,以仔鼠体增重衡量蛋白合成速率,低蛋白日粮添加Met、Leu。Met、Leu提高泌乳小鼠仔鼠体增重;添加Met提高泌乳小鼠血浆胰岛素水平,Met、Leu提高血浆中IGF-1水平,提高乳腺组织中Akt的磷酸化程度;Met明显增加泌乳小鼠胰腺中胰岛素分泌和肝脏中IGF-1的分泌,且Met、Leu引起的肝脏差异基因主要富集在胰腺分泌和胰岛素信号途径。综上,在体外,单一EAA主要影响细胞内mTOR和GCN2通路中关键信号蛋白的磷酸化,Met、Leu、Ile、Val对细胞内控制蛋白合成关键信号蛋白的磷酸化有单独和叠加作用;在体内,蛋白缺乏日粮补充Met、Leu、Ile独立刺激乳蛋白合成速率(仔鼠窝增重),可见“木桶理论”不适用于泌乳动物,且Met、Leu、Ile对乳蛋白合成速率的影响主要通过mTOR信号通路,不依赖于GCN2信号通路,Met、Leu诱导激素的变化是EAA调控乳蛋白合成的另一重要途径。
[Abstract]:Recent results show that not only is the amino acid substrates for protein synthesis, and can be used as a biological signal directly involved in the regulation of protein synthesis. For the study of essential amino acids (EAA) through the mechanism of intracellular signal pathway for the synthesis of the regulation of milk protein, milk in the gland epithelial cell line (MAC-T) as material, studied 10 EAA on the regulation of milk protein synthesis of key signaling pathways, in lactating mice with the body weight gain of offspring to measure the rate of protein synthesis on the regulation of milk protein synthesis and potential effects of EAA on lactation mechanism. The effect of single EAA on the regulation of milk protein synthesis key signal protein phosphorylation. This experiment based on MAC-T the test material, test for processing medium in order to loss of a EAA DMEM/F12, which contains all the EAA DMEM/F12 medium as positive control, with the lack of full EAA DMEM/F 12 medium as negative control. Respectively. 10 EAA had significant effect on protein synthesis in the process of the main signal protein phosphorylation, negative control mTOR phosphorylation decreased 58%, deletion of Met, Ile or Val to mTOR phosphorylation decreased 38%, respectively, 41%, 42%; single EAA deficiency reduced intracellular the phosphorylation of 4eBP1, including Ile, Thr and loss of Met and Lys were decreased by 39%, 41%, 36% and 42%; Ile, Leu, Met deletion, Val phosphorylation of S6K1 were decreased by 33%, 38%, 41%, 40%; single EAA deletion significantly increased eEF2 phosphorylation level in the absence of Met. Lle, phosphorylation of Val were increased by 18%, 20%, 18%; all EAA and Met lack of eIF2 alpha phosphorylation were increased by 49% and 39%. results showed that EAA mainly affects the key single intracellular signal protein mTOR and GCN2 2 pathways of phosphorylation and protein of key signal complete absence of Met, Leu. Ile, Thr, Val, Lys Sensitive.Met, Leu, Ile, Val impact on the regulation of milk protein synthesis of key signaling proteins. For the effect of signal protein was sensitive to EAA, with MAC-T as test material, first study the effects of different concentrations of EAA on regulation of protein synthesis key signaling proteins to determine intracellular EAA deficiency, add Met, EAA the lack of Leu, Ile, Val and the four EAA 22. With the combination of signal protein phosphorylation of EAA concentration changes consistent with Michaelis equation change trend, EAA concentration below 20% signal protein phosphorylation changes greatly, when the concentration of EAA in 30%-90% phosphorylation signal protein changes tended to be stable, to determine the 10% for further study of EAA lack of concentration.EAA state condition of lack of adding Met, Leu, Ile, Val and the four EAA 22, adding Leu, Ile, Met and Leu, Met and Ile, Leu and Ile phosphorylation of mTOR were increased 47%, 69%, 187%, 200% and 165%; Ile Val, adding Leu alone, increased the phosphorylation of 4eBP1, and the addition of Met and Leu, Leu and Ile, Val and Ile had additive effect; adding Ile S6K1 phosphorylation increased 63%, and the Ile and other three kinds of amino acids in combination with additive effects. The results indicate that the addition of Met, EAA Leu Ile, Val deficiency, affecting the increase of signal protein in mTOR pathway phosphorylation, phosphorylation of 22 amino acids and four kinds of combination of signaling proteins have additional effects on.Leu, Ile, Met, Thr on the synthesis of lactation mice milk protein. For contribution and mechanism in vitro evaluation of milk protein synthesis control potential of EAA on lactation, first determine the mouse protein deficiency, establish rat body weight and increase the reaction curve of different protein levels, were selected to the date of delivery is similar in pregnant rats. Dietary protein levels were 6%, 9%, 12%, 15%, 18%, 21%, 24% when the eggs, and 27%. The white level is less than 21%, with the body weight gain of offspring increased protein concentration increased, the body weight gain of rats with increased levels of protein into the Michaelis Menten reaction trend, most generally increase protein levels for the 14% half of weight, the gross weight of 113g; S6K1 phosphorylation level of lactating mammary gland in mice with increased the level of dietary protein, phosphorylation of eEF2 decreased. According to the above results, determine the 15% low dietary protein, negative control, 21% for high protein diets, positive control, low protein basis were added to Met, Leu, Ile, Thr. low protein diets adding Met, Leu, weight increased 10%, Ile rats increase 11%, 9%; Leu, add Met, mTOR Ile and Thr lactating mouse mammary gland after phosphorylation were increased by 55%, 34%, 47%, and 45%; essential amino acid treatment increased the concentration of IGF-1 in plasma of lactating rats; nest mTOR weight gain and mammary gland tissue, the correlation coefficient is 0.40.. The results show that low protein diets containing Met, Leu, Ile independent stimulation of milk protein synthesis rate (rat nest weight), and mainly through the mTOR.Met pathway and effects of Leu induced endocrine changes on lactation of lactation. The potential of EAA (Met, Leu) changes induced by endocrine influence mechanism on lactation, the in order to test lactating mice with the body weight gain of offspring to measure the rate of protein synthesis, low protein diet supplemented with Met, Leu.Met, Leu increase the milk of mice body weight gain; improve the level of insulin, mouse plasma Met Met Leu add milk, improve the level of IGF-1 in plasma, improve the breast tissue of the phosphorylation of Akt Met; increased secretion of pancreatic insulin secretion in lactating mice and IGF-1 in liver and Met, Leu induced differences in gene expression of liver mainly enriched in pancreatic secretion and insulin signaling pathway. In conclusion, the main effect of EAA in vitro, single cell mTOR and GCN2 The key signal pathway in protein phosphorylation, Met, Leu, Ile, Val control protein synthesis key signaling proteins to intracellular phosphorylation alone and superposition; in vivo, lack of protein diets supplemented with Met, Leu, Ile independent stimulation of milk protein synthesis rate (rat nest weight), visible "cask the theory does not apply to lactating animal, and Met, Leu, Ile influence on milk protein synthesis rate mainly through the mTOR pathway, does not depend on the GCN2 signaling pathway, Met, Leu induced changes in hormone is another important way to EAA regulation of milk protein synthesis.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.2

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