L-Arginine Affects Arginine Transportation, Cell Proliferation And Gene Expression In Intestinal Porcine Epithelial Cells (IPEC-1)

Arginine is a semi-essential amino acid that serve as an important role in diverse physiological function ranging from regulation of animal nutrition metabolism to the process of growth and development. Dietary arginine can promote the growth of piglets as well as the development of intestine. It can also improve the expression of VEGF gene. However, it is not clear that the regulatory mechanism of arginine transporter in the regulation of cell growth, the effect of L-Arg-NO pathway in cell proliferation and the connection between arginine transporter and mTOR signaling pathway.Therefore, we used a cell model of IPEC-1cells which were grown in DMEM-F12containing different concentrations of arginine, to investigate IPEC-1cell growth, proliferation, apoptosis and related signaling pathway. Meanwhile, we have inhibited NOS in using L-NAME to discuss the role of Arg-NO pathway in cell growth and proliferation. The study was designed to elucidate the molecular mechanism of the regulations of arginine in intestinal porcine epithelial cells growth and proliferation, and then to lay the foundation of the research of arginine functions in pig. The results shows that (1)306genes like AKR1C4、PTGFR、GHRL、ITGB2、 IL15were up-regulation while208genes like SLC25A25、COL4A3、C7、TLR1were down-regulation in supplementation of350μM Arg. Through KEGG Pathway analysis, genetic changes of arginine regulation were mostly found in pathways associated with cell adhesion molecules, type I diabetes, cell communication, calcium signaling pathway.(2)Supplementation of arginine had no effect on cell arginine transfer rate, but increased gene and protein levels of y+LAT1and y+CAT1. Addition of L-NAME to suppress NO metabolic pathway had no difference on arginine transfer rate and the levels of y+CAT1and y+CAT2, but reduced the level of y+LAT1.(3) Supplementation of arginine improved cell proliferation, reduced the cell apoptosis, increased the cell number at S phase, while inhibition of NO pathway decreased cell prolifeartion, increased the cell apopotosis. Compared with arginine, L-NAME significantly decreased NO synthesis and expression of eNOS, Arginine significantly increased relative protein levels of p-mTOR、p-4EBP1、p-p70S6K、p-PI3K、Akt、 p-Akt and Bcl-2, yet L-NAME had inhibitory effect on above proteins.In conclusion, these findings indicate that arginine improves cell growth and proliferation through mTOR signaling pathway, while regulates cell cycle and apoptosis through PI3K-Akt-Bcl2pathway. L-NAME, as a NOS inhibitor, has no competitive inhibition with arginine transporters of intestinal porcine epithelial cells, but can regulate cell growth and proliferation by inhibiting mTOR pathway and promoting PI3K-Akt-Bcl2pathway. NO metabolic pathway plays a great important role in arginine regulation of intestinal porcine epithelial cell growth and proliferation. These results provide effective theoretical basis of arginine application in piglets nutrition and also provide reference for using arginine to remedy human intestinal disease.