Milk Protein Synthesis Is Regulated By G Protein-coupled Receptor T1R1/T1R3 Through The MTOR Pathway

Milk protein is always a concern because of its high nutritional value and the importance in the agricultural production.Improve milk protein synthesis in animals,perfect the composition of milk protein had been the hot spot of the animal nutritionists.Thus to reveal the regulatory mechanism of milk protein synthesis is important to solve the above problems.In present study,siRNA was used to interfere with T1R1 in mouse mammary epithelial cell lines HC11 cells.It was found that T1R1 involved in HC11 cells amino acids sense and activation of the mTOR pathway.Essential amino acids transported into cells by amino acid transporters.mRNAs coding three important amino acid transporters(SLC1A5,SLC3A2,and SLC7A5)were increased by T1R1 knockdown in HC11 cells.The amounts of most amino acids including glutamine and the nonessential amino acids concentrations were similar,which indicated meaningful cross-talk that transporters mRNA was increased when T1R1 was knocked down in H9C2 cells and in HC11 cells(present study)without affecting intracellular amino acid concentrations.By using the amino acid transporter inhibitor BCH and GPNA,we confirmed that the amino acid transporter also involved in the regulation of mTOR pathway.Activation of the mTOR pathway can be partly repressed by T1R1 siRNA or SLC7A5/SLC3A2 inhibitor,and the combination of these two treatments further repressed the activity of this pathway.In vivo experiments were performed by using the model of T1R1 knockout mouse.The results showed that T1R1 KO could significantly down-regulate the expression of ?-casein and the phosphorylation of S6 K in 2-3 days before parturition.As expected,the knockout of T1R1 not only reduced the total milk yield in the mice mammary glands,but also repressed ?-casein synthesis.Additionally,the phosphorylation of 4EBP1 and S6 K was decreased in T1R1 knockout mice.Similarly,knockout of T1R1 increased the expression of amino acid transporter,consistent with the results of cytology experiments.By using the T1R3 inhibitor Lactisole,we observed that T1R3 inhibition decreased mTOR activity.Interestingly,in the present study indicated that heterodimer of T1R2/T1R3 also taken part in the regulation of the mTOR pathway,the underlying molecular mechanisms of which need further study.Taken together,the results of cytology and in vivo experiments indicated that T1R1/T1R3 is involved in the regulation of extracellular amino acids and the synthesis of milk protein in mice,by mediating the mTOR signaling pathway.The study of T1R1/T1R3 not only enriches the understanding of mTOR upstream pathway,but also provides a new theoretical basis for mammalian milk control,milk protein production and molecular design of dairy cows.