泌乳奶牛乳腺氨基酸吸收、激素分泌和mTOR信号途径对单一EAA缺乏的响应模式研究

Lactation responses to essential amino acid deficiency in lactating ruminants are relatively insensitive, which is the major predicament in amino acid requirement research in these animals. Essential amino acid (EAA) deficiency depresses milk protein synthesis mainly through two pathways. First, restricted EAA supply decreased mammary uptakes of the limiting EAA, which elevate the amount of uncharged tRNA that activate phosphorylation of eIF2 as part of the integrated stress response (ISR) and slows the initiation of mRNA translation. Second, endocrine system and the mTOR signal transduction pathway can detect and response to changed circulating amino acid concentration and then modulate milk protein synthesis in the mammary gland. Previous studies indicated that insulin, glucagon, growth hormone (GH), and insulin-like growth hormone (IGF-I) were the major circulating hormones that regulate milk protein synthesis. GH stimulates the release of hepatic IGF-I, which in turn inhibits pituitary GH release through a feedback mechanism. Therefore, their concentrations in plasma may be not responsive to EAA deficiency but there were evidences that their hepatic receptor expression were decreased in facing certain EAA deficiency. Changes in mammary amino acid extraction, plasma concentrations of relevant hormones and their hepatic and mammary receptor expression, and phosphorylation status of signal factors in the mTOR pathway in mammary epithelium cells during degraded individual EAA deficiency in lactating cows will be investigated in the present study. The aim is to evaluate their responsive sensitivity to individual EAA deficiency and if can be used as candidate indicators of EAA deficiency in lactating ruminants. Previous studies revealed that mammary blood flow and extraction efficiency of the deficient EAA in lactating ruminants were increased in facing certain EAA deficiency, which constituted a buffering mechanism to EAA deficiency by circulating amino acid pool and contributed to the relative insensitive lactational responses of lactating ruminants. There is as yet no evaluation on the capacity of this mechanism. For most of the EAA, their deficiency may induce depressed milk protein synthesis through the endocrine and mTOR pathways before their respective mammary uptakes are deceased. Except for Arg, it has been observed that decreases in mammary uptake of all the other investigated EAA occurred after milk protein yield was significantly dropped. Arg may have metabolic requirement other the milk protein synthesis in the mammary gland as its mammary uptakes in two times more than its secretion in milk protein. Endocrine pathway may act through mTOR to regulate milk protein synthesis and the two pathways may have coordination effect in EAA deficiency induced milk protein depression. The second aim of the present study is to verify the above speculations. Lys, Met, and Arg are the candidate EAA and lactating cows are used in the present project.

奶牛对必需氨基酸（EAA）缺乏的泌乳反应不敏感，影响了其氨基酸需要量研究。EAA缺乏通过两条途径抑制乳蛋白合成。一是缺乏EAA的乳腺吸收量减少，泌乳细胞内空载tRNA增加，抑制mRNA翻译起始。二是EAA缺乏引起内分泌和细胞mTOR信号途径反应。与乳蛋白合成相关激素主要有胰岛素、胰高血糖素、GH和IGF-I等。GH与IGF-I的分泌经反馈机制相互调节，两种激素的血浆浓度对EAA缺乏不敏感，但肝脏受体表达量的反应敏感。在EAA缺乏时，mTOR途径信号因子磷酸化发生变化，调控乳蛋白合成。本研究的目的之一是观察乳腺EAA吸收、内分泌和mTOR途径对单一EAA缺乏的反应，以期找到对缺乏反应敏感的特征指标。 EAA缺乏引起乳腺血流量和缺乏EAA乳腺提取率升高，形成对EAA缺乏的缓冲作用，还不清楚这种缓冲能力的评估方法。研究体液氨基酸对特定EAA缺乏的缓冲能力，是课题的研究目的之二。

单一限制性氨基酸理论不能很好地解释必需氨基酸（EAA）构成对乳蛋白合成的影响，制约了泌乳反刍动物氨基酸平衡日粮技术的发展。已有研究表明，不同EAA在肝脏代谢、乳腺上皮细胞mTOR信号途径调控及胰腺激素分泌刺激上存在差异，这很可能是EAA构成影响乳蛋白合成的生物学基础。为此，本项目进行了三个方面的研究。一是选取在肝脏代谢上有明显差异的Lys和Met，观察其供给量变化对乳蛋白产量、循环胰腺激素和氨基酸乳腺代谢的影响；二是研究在体外试验中发现能够刺激乳腺上皮细胞mTOR信号途径的四种EAA（Met、Leu、Ile、Thr）是否在活体试验中仍有相似作用；三是研究是否可利用此类mTOR信号途径的EAA有效提高泌乳奶牛日粮蛋白的转化效率。. 以泌乳奶山羊为试验动物，分别研究了Lys和Met梯度缺乏对乳蛋白合成的影响。研究结果表明，Lys梯度缺乏线性降低乳蛋白产量，血浆胰高血糖素显著升高，胰岛素浓度升高接近有趋势水平，乳腺组织对Lys的亲和力没有显著变化，乳腺Lys提取量显著降低，但乳腺Lys提取量与乳蛋白产量无显著相关，Lys供给量对乳腺上皮细胞mTOR信号途径无明显影响。综合以上结果，Lys缺乏抑制乳蛋白合成的主要原因是胰腺激素分泌变化。Met梯度缺乏线性降低乳蛋白产量，血浆胰高血糖素、胰岛素浓度无显著变化，乳腺组织对Met的亲和力线性升高，乳腺Met提取量无显著变化，Met供给量对乳腺上皮细胞mTOR信号途径有显著影响。综上所述，Met缺乏抑制乳蛋白合成的主要原因是乳腺上皮细胞mTOR途径活性变化。. 以泌乳小鼠为试验动物，体内研究证实了Met、Leu、Ile和Thr的mTOR信号途径刺激作用。随后用60头奶牛进行的试验证实，添加四种信号氨基酸，日粮蛋白水平下降2个百分点不影响乳蛋白产量，即使用信号具有提高日粮蛋白泌乳转化效率的可行性。