Abstract

Although milk biosynthesis relies intricately on amino acid metabolism, the key regulatory amino acids and their molecular mechanisms remain incompletely elucidated. Here, targeted metabolomics identified l-proline (l-Pro) as a key differential serum metabolite between high- and low-yielding dairy cows. In vitro, l-Pro significantly promoted bovine mammary epithelial cell proliferation and milk fat and protein synthesis. Mechanistically, following cellular uptake via the SNAT2 transporter, l-Pro binds to the prolyl 4-hydroxylase subunit alpha 2 (P4HA2). This interaction promotes mTOR lysosomal translocation and activates the mTORC1 signaling pathway. Furthermore, in vivo feeding trials showed that rumen-protected l-Pro significantly increased milk production and quality in midlactation cows. In summary, this study unveils a novel mechanism whereby l-Pro regulates milk synthesis via the SNAT2-P4HA2-mTORC1 axis, identifying potential intervention targets for enhancing the dairy cow production performance.