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 µmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 µmol/L Glu (Glu deficiency), 50 µmol/L Glu (Control), or 50 µmol/L Glu plus 100 µmol/L PDC (PDC supplementation). Cell proliferation ( = 24), cell cycle distribution ( = 6), cell apoptosis ( = 6), and expression levels of proteins of interest ( = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited ( < 0.05) by 50, 100, and 200 µmol/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 ( < 0.05) of cell proliferation, a greater ( < 0.05) percentage of cells in the G1 phase, and a lower ( < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced ( < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphor-mammalian 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.
Glutamate, which is one of the most important contributors to oxidative metabolism in the intestinal mucosa, is mainly transported by the excitatory amino acids transporters (EAATs) that are expressed in enterocytes. The objective of this study was to evaluate the effects of in ovo administration of L-trans pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), a potent competitive inhibitor of glutamate uptake by EAATs, on the growth of the small intestine in chicks. Two series of experiments were conducted with hatching eggs; 100 μl of various L-trans-PDC solutions (0, 0.075 or 0.225 mg/egg for the Control group, low-dose L-trans pyrrolidine 2,4-dicarboxylic acid group (L-PDC) or high-dose L-trans pyrrolidine 2,4-dicarboxylic acid group (H-PDC), respectively) was injected into the albumen sac of these hatching eggs before incubation. Hatchlings were sacrificed by cervical dislocation to determine the embryonic development in Experiment I, whereas the birds in Experiment II were raised or sampled at hatching, days 7 and 14 (D7 and D14) for further study. Gene expression in the small intestines was determined by real-time RT-PCR; and serum concentration of free amino acids was determined by an amino acid analyzer. The results showed that the hatchability was decreased by in ovo administration of L-trans-PDC. The small intestinal weights of the H-PDC group were decreased ( P < 0.05) at hatching and increased ( P < 0.05) on D7 and D14 compared with those in the Control group. In addition, the gene expression of EAAT2 in the completed or segmental small intestines was not changed ( P > 0.05); EAAT3 gene expression in the duodenum ( P < 0.05), jejunum ( P = 0.084) and ileum ( P = 0.060) on D14 was lower in the H-PDC group than in the Control group. Furthermore, the serum concentrations of free proline, threonine and phenylalanine but not glutamate or aspartate were increased ( P < 0.06) in H-PDC group. In conclusion, this paper is the first to report that in ovo administration of L-trans-PDC induces small intestinal growth retardation during the embryonic period and catch-up growth after hatching.
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