Mechanism Of LAT1 Activating MTORC1 Signaling To Promoting Milk Protein Synthesis In Dairy Cow Mammary Epithelial Cells

Milk protein is an important nutrient in milk that provides humans with various essential amino acids.In-depth research on the molecular mechanism of milk protein synthesis in lactating dairy cows and clarifying the regulation of nutrients on milk protein synthesis can provide a research basis for improving milk production performance through scientific feeding methods.L-type amino acid transporter 1(LAT1)is a Na~+-independent amino acid transporter that can transport neutral amino acids inside and outside cells or organelles by binding with its ligand 4F2 heavy chain(4F2hc).LAT1 provides the essential amino acids needed to m aintain milk protein synthesis in the mammary gland.The early research in our laboratory found that LAT1 expression in the mammary gland of dairy cows was significantly higher than that in pubertal and dry cows.The expression of LAT1 in mammary gland wit h high milk protein content was significantly higher than that in tissue with low milk protein content.Methionine is a limiting amino acid for milk protein synthesis and can induce LAT1 to promote milk protein expression.This process is related to the ac tivation of the mammalian target of rapamycin complex 1(mTORC1)signaling pathway,but the specific molecular mechanism is not yet clear.To clarify the relationship between LAT1 expression in dairy cow mammary epithelial cells and the activation of mTORC1 signaling pathway,this study used LAT1 gene interference and overexpression as well as the addition of the LAT1 inhibitor BCH to treat dairy cow mammary epithelial cells.After 48 hours,the expression changes of LAT1,4F2hc,and mTOR in the cells were detected by western blot.The results showed that LAT1 RNAi or BCH inhibitor treatment significantly reduced the phosphorylation level of mTOR in the cells,and the expression ofβ-CASEIN in the cells also decreased accordingly.Overexpression of the LAT1 gene obtained the opposite result,indicating that LAT1 is involved in regulating the activation of the mTORC1 signaling pathway in dairy cow mammary epithelial cells.The biological function of LAT1 depends on its localization on the plasma membrane.This study investigated the dual role of LAT1 in amino acid transport and mTORC1 signal activation under the action of methionine.The cytoplasm,membrane proteins,and lysosomal proteins of dairy cow mammary epithelial cells were isolated to detect changes in LAT1expression under the action of methionine.Results showed that under the action of methionine,LAT1 expression increased in the cytoplasm and more of it was localized on the plasma membrane.Furthermore,methionine promoted the localization of LAT1 on the lysosome.These results indicate that methionine increases LAT1 expression in dairy cow mammary epithelial cells and induces the translocation of LAT1 to the plasma membrane and lysosome in cells.The activation of mTORC1 mainly occurs on the lysosome,and lysosome-associated protein transmembrane-4B(LAPTM4B)can recruit LAT1 to the lysosome to participate in mTORC1 activation.To verify the interaction between LAT1 and LAPTM4B,this study c o-transfected dairy cow LAT1 and LAPTM4B into He La cells and detected the interaction between exogenous LAT1 and LAPTM4B through immunoprecipitation experiments.In addition,immunoprecipitation was used to detect the interaction between endogenous LAT1and LAPTM4B in dairy cow mammary epithelial cells.The co-localization of LAT1 and LAPTM4B in dairy cow mammary epithelial cells was detected by bimolecular fluorescence complementation experiments.Results showed that the interaction between LAT1 and LAPTM4B was detected in both He La cells and dairy cow mammary epithelial cells.The yellow fluorescence signal produced by the bimolecular fluorescence complementation experiment also confirmed the interaction between LAT1 and LAPTM4B.These results all demonstrate that LAT1 can specifically interact with LAPTM4B in dairy cow mammary epithelial cells.To study the role of LAPTM4B in LAT1-induced mTORC1 signal activation,this study first changed the expression level of LAPTM4B by gene overexpression or interferen ce methods and detected the expression of mTOR,p-mTOR,andβ-CASEIN in dairy cow mammary epithelial cells.The results showed that the overexpression of LAPTM4B did not change the expression level of mTOR,but significantly increased the levels of p-mTOR andβ-CASEIN expression.Furthermore,when LAT1 was overexpressed and LAPTM4B was interfered with simultaneously,the induction effect of LAT1 on the phosphorylation of mTOR and the expression ofβ-CASEIN was inhibited.When LAPTM4B was interfered with under the action of methionine,it was also found that the phosphorylation of mTOR and P70ribosomal protein S6 kinase(P70S6K)induced by methionine and the expression ofβ-CASEIN were inhibited,indicating that LAPTM4B mediates methionine-LAT1-induced mTORC1activation and milk protein synthesis.Vacuolar-type adenosine triphosphatase(v-ATPase)is a necessary molecule for amino acid activation of mTORC1 signaling.To investigate whether v-ATPase is involved in LAPTM4B-mediated mTORC1 activation,two methods,adding the v-ATPase inhibitor Concanamycin A(CCA)or interfering with the v-ATPase component ATPase H~+transporting V0 subunit C(ATP6V0c),were used in this study to detect the effects of v-ATPase on mTORC1activation andβ-CASEIN expression.Results showed that the addition of CCA inhibited the phosphorylation of mTOR and P70 ribosomal protein S6 kinase(P70S6K)induced by methionine and the expression ofβ-CASEIN in normal dairy cow mammary epithelial cells.Inhibiting the expression of LAPTM4B under methionine treatment had the same effect as CCA treatment and significantly inhibited methionine-induced mTORC1 activation.Interfering with ATP6V0c also obtained consistent results with CCA treatment.These results suggest that v-ATPase mediates the activation of the methionine-LAT1-LAPTM4B molecular pathway to activate mTORC1 signaling,thereby inducing the synthesis of milk protein.In summary,in dairy cow mammary epithelial cells,LAT1 and LATPM4B cooperate to activate mTORC1 signaling and milk protein synthesis through the v-ATPase-mediated pathway.