Mechanism On The Regulation Of Milk Casein Synthesis In Mammary Epithelial Cells Of Dairy Cows By Phenylalanine

The "Quality Milk Project" is a development strategy proposed by China’s dairy industry at a critical period of transformation and efficiency enhancement,and its core concept is to provide consumers with truly healthy and safe milk products that are rich in nutrients and of excellent quality.Through extensive practice,researchers have made certain achievements in the nutritional regulation of dairy cows.However,there are still many biologically active components in milk and their potential nutritional value has not yet been explored in depth.Amino acids are not only substrates for milk protein synthesis,but also act as active molecules in many biological reactions,and a full understanding of the multiple roles of amino acids is key to current research.L-phenylalanine（Phe）,a physiologically active aromatic amino acid,is one of the potential limiting amino acids for lactating cows.It has been shown that the infusion of a Phe-free amino acid mixture into the wrinkled stomach of cows reduces milk protein production compared to controls,suggesting that Phe may be an important factor in milk protein synthesis.However,the mechanism by which Phe regulates milk protein synthesis remains unclear.The authors proposed the hypothesis that Phe regulates protein synthesis through a signal transduction pathway involved in protein synthesis.Based on this,this experiment was conducted on the mammary epithelial cell line MAC-T in dairy cows,focusing on the effect of Phe on casein synthesis in MAC-T,focusing on the response of key genes and proteins in the signalling pathway to Phe,using proteomics,isotope labelling and other molecular biology techniques to analyse the casein synthesis network,with a view to laying a theoretical foundation for the application of Phe to improve the quality of bovine milk.Experiment 1: Effects of phenylalanine on casein synthesis in mammary epithelial cells.To investigate the effect of Phe on casein synthesis in mammary epithelial cells,five concentration gradients of Phe medium（using the Phe concentration in cow serum as a reference）were set up:-Phe（0 m M Phe,Control）,Phe（0.22 m M Phe）,2Phe（0.44 m M Phe）,4Phe（0.88 m M Phe）,8Phe（1.76 m M Phe）and growth medium as positive controls（Normal）.The cell viability,m RNA and protein expression levels of amino acid transport vectors and casein synthesis genes were measured.The results showed that Phe treatment had no significant effect on cell viability（P > 0.05）.The m RNA expression of amino acid transport carriers ASCT2 and LAT1 was increased in2 Phe and 4Phe treated groups compared to 0Phe,but not significantly（P > 0.05）.m RNA expression of casein synthesis-related genes CSN1S1,CSN2 and CSN3 was significantly increased in 4Phe group（P < 0.05）.m RNA expression of ASCT2 was significantly decreased in 8Phe treated group（P < 0.05）.m RNA expression of ASCT2 was significantly lower under 8Phe treatment（P < 0.05）.Compared with-Phe,4Phe significantly increased the expression of α-casein（P < 0.05）.In summary,the concentration of Phe in the medium significantly affected the m RNA expression levels of the casein synthesis genes CSN1S1,CSN2 and CSN3 and the synthesis ofα-casein,but not β-casein,with the 4Phe treatment group significantly increasing the synthesis and expression of casein.Experiment 2: Mechanism of phenylalanine on casein synthesis in mammary epithelial cells.To further investigate the mechanism by which Phe affects casein synthesis in mammary epithelial cells,based on the results of experiment 1,three treatment groups,-Phe,4Phe and Control,were selected for Label-free proteomics analysis to screen and validate key signalling pathways.The results were as follows: 418,503 and 606 proteins were identified in the three treatment groups,respectively.KEGG enrichment analysis showed that the pathways related to ribosome function were enriched with more differential proteins,and some of the proteins related to lactation performance were differentially altered,including RPS6,which was up-regulated in 4Phe,a protein functionally annotated to be related to the m TOR signalling pathway.The phosphorylation levels of m TOR-related factors were further examined and it was found that Phe and 4Phe significantly increased the expression of 4EBP1 compared to-Phe（P < 0.05）;S6k1 phosphorylation levels did not show significant changes（P >0.05）.e IF2α showed the opposite trend,with the lack of Phe promoting its phosphorylation and increasing Phe concentration having no effect on its activity（P >0.05）,indicating that e IF2α is not normally activated when there are sufficient available amino acids.The combined proteomic and m TOR pathway factor phosphorylation results indicate that Phe can enhance m TOR signalling pathway activity and promote α-casein synthesis in dairy mammary epithelial cells by regulating the phosphorylation levels of 4EBP1 and e IF2α.Experiment 3: Metabolism of phenylalanine in mammary epithelial cells.The proteomic results from experiment two showed that the Phe metabolic pathway was enriched for a few differential proteins.Based on this,the metabolism of AA within the medium was measured in this experiment using liquid chromatography.The results showed that increasing the concentration of Phe gradually increased the uptake of Phe by the cells and reached a maximum at 8Phe treatment,when the uptake of Ile was also promoted（P < 0.05）.In combination with the results of experiment 1,excess Phe instead inhibited casein synthesis.To further reveal the metabolic regulation of Phe in cells,we assayed intracellular phenylalanine hydroxylase（PAH）concentrations and incubated cells with stable isotope-labelled Phe（13C9;15N-L-Phenylalanine）to identify the biometabolic pathways in which Phe is involved.The results showed that PAH concentrations were significantly reduced in the Phe and 4Phe groups.A total of 1542 proteins were identified in the stable isotope assay,of which 940 peptides were doped with relabelled phenylalanine,enriched in245 pathways.Apart from most unknown pathways,carbon metabolism,purine metabolism,glycolysis,tricarboxylic acid cycle,oxidative phosphorylation and Cys-Met metabolism were the pathways in which Phe was most involved in synthesis.It is thus clear that the regulation of casein synthesis by Phe in mammary epithelial cells involves the synergistic action of multiple pathways.In summary,the results of this study suggest that: Phe enhances α-casein synthesis by regulating the phosphorylation levels of 4EBP1 and e IF2α and promoting the formation of the m TOR-centred casein translation initiation complex.