Prolactin Regulates Amino Acids Transport Via LAT1 In Mammary Epithelial Cells Of Dairy Cows

Milk and dairy products contain almost all the nutrients needed by humans,and they are indispensable and important foods for humans.Milk protein is the main material basis for the nutritional quality of milk and determines the economic value of milk.Elucidating the molecular mechanism of milk protein synthesis will provide an important theoretical basis for improving the milk quality of dairy cows through molecular biology.Ninety percent of the milk protein in the mammary gland is synthesized de novo using amino acids as raw materials.The mammary gland absorbs amino acids in the blood to synthesize milk protein.This process requires the participation of amino acid transport carriers.L-type amino acid transporter 1(LAT1)is a type of transporter that mainly transports neutral amino acids.It can combine with type II cell membrane glycosylation protein 4F2 hc to form a dimer to assist in completion transport of amino acids across membranes.The results of previous studies in our laboratory showed that LAT1 and 4F2 hc were highly expressed in breast tissue during lactation,suggesting that LAT1 is related to the process of milk protein synthesis.However,in dairy cow mammary epithelial cells,which amino acids can be transported by LAT1 into the cells to participate in the synthesis of milk protein is still unclear.Prolactin(PRL)is a protein hormone secreted by the anterior pituitary gland,which can regulate the initiation and maintenance of lactation.In lactating swine mammary gland tissue,prolactin can increase the uptake of amino acids and the expression of LAT1 in the mammary gland.However,in dairy cow mammary epithelial cells,whether prolactin can regulate the uptake of amino acids by the cell,and whether this process depends on LAT1 has not been reported yet.In order to study the types of amino acids involved in the transport of LAT1 in dairy cow mammary epithelial cells,this study used dairy cow mammary epithelial cells as experimental materials.First,BCH was used to inhibit the activity of LAT1 in the cells,and the amino acid analyzer was used to detect the changes in the types and content of amino acids in the culture medium.The results showed that the content of threonine,valine,methionine,isoleucine,leucine,tyrosine,lysine and histidine in the medium after the LAT1 activity was inhibited was significantly higher than the control group,indicating that LAT1 can transport these amino acids into cow mammary epithelial cells.Moreover,when BCH was used to treat cow mammary epithelial cells and RNAi method interfered with the expression of LAT1 in cow mammary epithelial cells,the expression of 4F2 hc and β-casein in cow mammary epithelial cells was significantly reduced(P<0.05),and cell viability was significantly decreased(P<0.05).These results suggest that the transport of amino acids by LAT1 into cells can up-regulate the ability of milk protein synthesis in cow mammary epithelial cells.In order to study whether prolactin can regulate the uptake of amino acids by dairy cow mammary epithelial cells,and whether this process is carried out via LAT1,this study added prolactin to the dairy cow mammary epithelial cell culture system.The results showed that compared with the control group,the content of threonine,serine,glutamic acid,proline,cysteine,valine,methionine,isoleucine,leucine,tyrosine,lysine and histidine in the medium of prolactin treatment group was significantly reduced;when prolactin was added while adding BCH,the opposite results were obtained,indicating that prolactin can increase the uptake of amino acids by dairy cow mammary epithelial cells,and this process is mediated by LAT1.In order to further study whether the expression of LAT1 in cow mammary epithelial cells is regulated by prolactin,this study used RT-PCR and western blot methods to detect the effect of prolactin on the expression of LAT1 in cow mammary epithelial cells.The results showed that after the addition of prolactin in cow mammary epithelial cells,the expression levels of LAT1 m RNA and protein in the cells increased significantly.Immunofluorescence results show that the effect of prolactin can increase the expression level of LAT1 in the cell membrane and cytoplasm.In order to explore the signaling pathway that prolactin regulates the expression of LAT1,this study used western blot to detect the expression of PI3K-AKT-mTOR signaling pathway molecules in cow mammary epithelial cells under the action of prolactin.The results showed that prolactin can induce the phosphorylation of AKT,mTOR and its downstream signaling molecules P70S6 K and4EBP1(P<0.01).Using LY294002 to inhibit the activity of PI3 K,the phosphorylation level of AKT,mTOR,P70S6 K and 4EBP1 decreased significantly,and the expression of LAT1,4F2 hc and β-casein also decreased significantly(P<0.01).Using rapamycin to inhibit the mTORC1 signaling pathway,the results showed that the phosphorylation level of mTOR and its downstream signaling molecules P70S6 K and 4EBP1 decreased significantly,and the expression of LAT1,4F2 hc and β-casein decreased significantly(P<0.01).These results indicate that prolactin can regulate the expression of LAT1 through the PI3K-AKT-mTOR signaling pathway in dairy cow mammary epithelial cells,thereby affecting milk protein synthesis.In order to study whether prolactin in dairy cow mammary epithelial cells can regulate the transcription activity of LAT1 through the PI3K-AKT-mTOR signaling pathway,this study constructed a luciferase expression vector containing different segments of the LAT1 promoter,using a dual luciferase reporter gene The system screened out that the core region of the promoter of LAT1 was located between-219 and-86 bp.Prolactin treatment can activate the activity of the core promoter region of LAT1(P<0.01).The PI3 K inhibitor LY294002 was used to treat cow mammary epithelial cells.The results of dual luciferase reporter gene showed that the transcription activity of the core promoter region of LAT1 was significantly reduced(P<0.01);and the transcription activity of the core promoter region of LAT1 was increased significantly after the addition of AKT activator SC79 Significant increase(P<0.05).These results prove that prolactin can regulate the transcriptional activity of LAT1 in cow mammary epithelial cells through the PI3KAKT-mTOR signaling pathway.In summary,prolactin regulates the transport of amino acids in dairy cow mammary epithelial cells through LAT1;prolactin regulates the transcription and protein expression of LAT1 in dairy cow mammary epithelial cells by activating the PI3K-AKT-mTOR signaling pathway,thereby regulating the synthesis of milk protein The above research results can provide an experimental basis for in-depth understanding of the regulation mechanism of lactating dairy milk composition,and at the same time are of great significance for improving the nutritional quality of milk and promoting the healthy and sustainable development of my country’s dairy industry.