L-Glutamate deficiency can trigger proliferation inhibition via down regulation of the mTOR/S6K1 pathway in pig intestinal epithelial cells

Abstract

The objective of this study was to investigate the effects of L-glutamate (Glu) deficiency or L-trans pyrrolidine-2,4-dicarboxylic acid (PDC) supplementation on the proliferation of pig intestinal epithelial cells (IPEC-1). First, IPEC-1 cells were cultured in normal growing medium supplemented with 0 (Control), 50, 100, or 200 mmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 mmol/L Glu (Glu deficiency), 50 mmol/L Glu (Control), or 50 mmol/L Glu plus 100 mmol/L PDC (PDC supplementation). Cell proliferation (n = 24), cell cycle distribution (n = 6), cell apoptosis (n = 6), and expression levels of proteins of interest (n = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited (P < 0.05) by 50, 100, and 200 mmol/L PDC supplementation at 24 and 48 h after treatment. Variance analysis was performed using the GLM procedure, and the results demonstrated that Glu deficiency or PDC supplementation led to the inhibition (P < 0.05) of cell proliferation, a greater (P < 0.05) percentage of cells in the G1 phase, and a lower (P < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced (P < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphormammalian target of rapamycin (p-mTOR; Ser2448), p-ribosomal protein S6 kinase 1 (S6K1; Thr389), and p-S6 (Ser235/236). This study demonstrates that Glu deficiency or PDC supplementation inhibits proliferation of IPEC-1 cells via downregulation of the mTOR/ S6K1 pathway and EAAT3 expression indicating that Glu deficiency may lead to the disturbances of intestinal epithelial renewal in pigs, particularly in neonates.