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

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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.