Mechanism Of Arf1-mTORC1 Signaling Network Sensing Glutamine To Promote Proliferation Of Porcine Intestinal Epithelial Cells

Glutamine is the preferred energy substrate for intestinal epithelial cells,which plays an important role in intestinal development and homeostasis.However,the mechanism by which intestinal epithelial cells respond to glutamine remains to be explored.In this study,IPEC-J2 cells were used as materials,CCK8,immunofluorescence,Tandem Mess Tags(TMT)phosphorylated proteomics,Western Blotting(WB),real-time quantitative PCR,and knockdown plasmid construction were used to investigate the molecular mechanism of glutamine on ADP-ribosylation factor 1(ADP-ribosylation factor 1,Arf1)-mammalian target protein Complex of Rapamycin 1(Mammalian target of rapamycin complex 1,mTORC1)to promote proliferation of porcine intestinal epithelial cells.The main research results are as follows:1.CCK8 method and immunofluorescence staining were used to detect the effect of glutamine on the proliferation of pig intestinal epithelial cells.It was found that in amino acid starvation treatment,compared with control group(0 mmol/L glutamine),the addition of 0.5 mmol/L,1 mmol/L,2 mmol/L,4 mmol/L and 5 mmol/L glutamine in IPEC-J2 for 6 h could significantly improve the activity of cells.The 1 mmol/L,2mmol/L and 4 mmol/L glutamine groups were significantly higher than those treated with 0.5 mmol/L and 5 mmol/L,but there was no significant difference between the 1mmol/L,2 mmol/L and 4 mmol/L glutamine groups.The results of different concentrations of glutamine treated IPEC-J2 cells for 24 h were basically consistent with those treated for 6 h.Therefore,1 mmol/L glutamine for 6 h used for next experiments.2.Phosphorylated proteome analysis showed that glutamine significantly upregulated the phosphorylation of S6 at serine 235/236,suggesting that glutamine activated the mTORC1 signaling pathway.Western Blotting results confirmed that the protein expression level of p-S6(Ser235/236)was significantly up-regulated,and the immunofluorescence staining results of p-S6 were consistent with the results of Western Blotting.In addition,the expression levels of p-mTOR and p-S6K1,key targets of the mTORC1 signaling pathway,were significantly up-regulated.The mTORC1 inhibitor Rapamycin(Rapamycin,Rap)test further verified that mTORC1 participated in IPEC-J2 cell proliferation by glutamine.3.Real-time quantitative PCR results showed that glutamine significantly increased the mRNA abundance of Arf1 and Rag A,but had no significant effect on the mRNA abundance of Rag B.The results of Western Blotting were consistent with those of real-time fluorescence quantitative PCR.Brefeldin A(Brefeldin A,BFA)is a specific inhibitor of Arf1 function,and BFA inhibitor tests have shown that it reduces the protein expression levels of p-S6K1 and p-S6 induced by glutamine.And Arf1 interference assay have shown that it reduces the protein expression levels of pmTOR and p-S6K1 induced by glutamine.It was confirmed that Arf1 was involved in glutamine-induced activation of mTORC1 to promote the proliferation of IPEC-J2 cells.In addition,Rag A knockout did not alter glutamine induce mTORC1 activation.Thus,glutamine activation of the mTORC1 pathway is dependent on Arf1 but not Rag A/B GTPases.In summary,glutamine promotes cell proliferation in IPEC-J2 cells through the Arf1-mTORC1 signaling pathway,independent of Rag GTPase.In this study,we elucidate the molecular pathway of mTORC1 induced glutamine to promote the proliferation of pig intestinal epithelial cells,enrich the theory of amino acid nutrition regulation,and provide a theoretical basis for the better application of glutamine in production.