Abstract

L-Theanine regulates the synthesis of milk in BMECs via TAS1R1. • L-Theanine can regulate the protein expression levels of FRMD6 through TAS1R1 to promote milk synthesis in BMECs. • L-Theanine can enhance the interaction between FRMD6 and mTOR, promote the translocation of mTOR from the cytoplasm to the lysosomal surface, and then activate the mTORC1 signaling pathway to promote milk synthesis. L-Theanine (LT), a characteristic amino acid found in tea, possesses diverse biological activities, including antioxidant, growth-promoting, and immune-enhancing effects, and is widely used in supplements. However, the regulatory role of LT in milk synthesis in dairy cows remains unclear. This study aimed to elucidate the effects of LT on milk synthesis and its underlying molecular mechanisms in primary bovine mammary epithelial cells (BMECs). We isolated primary BMECs and investigated the effects of LT treatment (100 μM), utilizing overexpression and knockdown techniques for TAS1R1, FERM domain containing 6 (FRMD6), and mTOR. Results showed that 100 μM LT significantly promoted BMECs proliferation and the synthesis of milk protein and fat. Knockdown of its putative receptor, TAS1R1, reversed these stimulatory effects of LT. Transcriptome sequencing analysis revealed FRMD6 as a key target gene mediating the LT-induced promotion of BMEC proliferation and milk synthesis. Mechanistic studies further indicated that LT enhanced the interaction between FRMD6 and mTOR, promoted mTOR translocation from the cytoplasm to the lysosomal surface, subsequently leading to the activation of the mTORC1 signaling pathway, ultimately promoting milk synthesis. The clinical experiment results on dairy cows show that Rumen-Protected LT can significantly increase the milk production of mid-lactation cows and improve the quality of their milk. In conclusion, our research provides strong preclinical evidence for LT as a potential functional feed additive to promote milk synthesis. Download: Downlo