Role of mTORC1 in regulation of milk protein synthesis by amino acids and insulin in the bovine mammary gland

我的研究方向主要是了解奶牛膳食和生理因素调节乳腺蛋白质代谢和乳蛋白合成的分子机制。氨基酸是蛋白质的组成部分，牛奶蛋白质的合成需要氨基酸的充足供应。除了作为底物的作用外，多种氨基酸还可以激活雷帕霉素复合体1 （mTORC1）的机制靶点，促进牛乳腺细胞的蛋白质合成。雷帕霉素复合体1是细胞生长的主要调节剂。其中，亮氨酸和异亮氨酸是体外培养的牛乳腺组织和细胞中最有效的mTORC1激活剂。然而，补充亮氨酸和异亮氨酸对奶牛乳腺mTORC1活性和乳蛋白产量的影响尚不清楚。最近发现了几个在mTORC1上游感知细胞内氨基酸水平的蛋白质，但它们在乳蛋白合成调节中的相关性尚不清楚。除了氨基酸外，内分泌信号也会影响牛奶蛋白的产生。特别是，胰岛素输注能有效地提高奶牛的乳蛋白产量，但其分子机制尚不清楚。胰岛素激活牛乳腺细胞中的mTORC1信号，提示其可能介导奶牛胰岛素诱导的乳蛋白反应。本提案的目的是确定mTORC1在氨基酸和胰岛素对乳蛋白合成的调节中的作用。为此，我们将1)确定静脉输注亮氨酸和异亮氨酸对奶牛乳蛋白产量和乳腺mTORC1激活的影响。为了深入了解氨基酸调节乳蛋白合成的机制，我们将应用我们新开发的牛乳腺乳蛋白合成细胞模型和创新的基因组编辑工具来表征氨基酸传感器在乳腺细胞mTORC1激活和乳蛋白合成中的作用。2)为了明确胰岛素调节奶牛乳蛋白合成的分子机制，我们将使用复杂的胰岛素钳法来评估胰岛素诱导的乳蛋白产量激活与乳腺mTORC1激活之间的关系。为了提供mTORC1在胰岛素诱导的乳蛋白合成中的作用的明确证据，我们将使用两种互补的方法：a)奶牛乳腺内输注特异性mTORC1抑制剂雷帕霉素，b)敲除乳腺细胞中mTORC1的重要成分。这些研究结果可能为提高奶牛乳蛋白生产效率和减少氮排泄的策略和技术的发展提供指导。该计划将为自然科学领域的职业生涯培训至少15名HQP。因此，该计划将为加拿大带来经济和环境效益。

My research program focuses on understanding the molecular mechanisms by which dietary and physiological factors regulate mammary protein metabolism and milk protein synthesis in dairy cows. Amino acids are the building blocks of proteins and their adequate supply is required for synthesis of milk proteins. In addition to their role as substrates, multiple amino acids can activate the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cell growth, to promote protein synthesis in bovine mammary cells. Among these, leucine and isoleucine are the most potent activators of mTORC1 in cultured bovine mammary tissue and cells in vitro. However, the effect of supplementation of leucine and isoleucine on mammary mTORC1 activity and milk protein production in dairy cows is not clear. Several proteins that sense intracellular amino acid levels upstream of mTORC1 were recently discovered, but their relevance in the regulation of milk protein synthesis is not known. In addition to amino acids, endocrine signals can influence milk protein production. In particular, infusion of insulin potently increases milk protein yield in dairy cows, but the molecular mechanisms are not known. Insulin activates mTORC1 signaling in bovine mammary cells, suggesting that it may mediate the insulin-induced milk protein response in dairy cows. The objective of this proposal is to determine the role of mTORC1 in the regulation of milk protein synthesis by amino acids and insulin. To this end, we will 1) determine the effect of intravenous infusion of leucine and isoleucine on milk protein yield and mammary mTORC1 activation in dairy cows. To gain insight into the mechanisms by which amino acids regulate milk protein synthesis, we will apply our newly developed milk protein-synthesizing cellular model of the bovine mammary gland and innovative genomic editing tools to characterize the role of amino acids sensors on activation of mTORC1 in mammary cells and synthesis of milk proteins. 2) To define the molecular mechanisms by which insulin regulates milk protein synthesis in dairy cows, we will use sophisticated insulin clamp methodologies to assess the association between activation of insulin-induced milk protein yield and mammary mTORC1 activation. To provide definitive evidence for a role of mTORC1 on insulin-induced milk protein synthesis, we will use two complimentary approaches: a) intra-mammary infusion of the specific mTORC1 inhibitor rapamycin in cows and b) knockout of an essential component of mTORC1 in mammary cells. The results of these studies may lead to the development of strategies and technologies to increase the efficiency of milk protein production and reduce nitrogen excretion by dairy cows. The proposed program will train at least 15 HQP for careers in the natural sciences. Thus, the program will lead to economic and environmental benefits to Canada.