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 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.
Subject(s)
Cell Proliferation/physiology , Epithelial Cells/drug effects , Glutamic Acid/deficiency , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Swine , TOR Serine-Threonine Kinases/metabolism , Animals , Apoptosis/drug effects , Cell Cycle , Cell Proliferation/drug effects , Dicarboxylic Acids/administration & dosage , Dicarboxylic Acids/pharmacology , Down-Regulation , Epithelial Cells/physiology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Glutamic Acid/pharmacology , Intestines/drug effects , Pyrrolidines/administration & dosage , Pyrrolidines/pharmacology , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/geneticsABSTRACT
The search for new and improved antipsychotic agents has escalated during the past five years. The era of searching for non-toxic copies of clozapine has been followed by several different lines of research, some of which pursue the traditional dopamine track, although at a higher level of sophistication, whereas others focus on other neurotransmitters, such as serotonin and glutamate. Emerging knowledge about the interactions between different neurotransmitters in complex neurocircuits opens up possibilities for achieving antipsychotic activity by interfering with many different neurotransmitters. Most intriguing is the finding in animal experimental models, indicating that it should be possible to alleviate psychotic conditions by stabilizing rather than paralyzing neurocircuits, thus avoiding the risk of motor and mental side effects of the currently used drugs. Among these new classes dopaminergic stabilizers and 5-HT2A receptor antagonists seem to offer most promise at present. In a longer perspective, drugs interfering with glutamate function via different mechanisms may also turn out to be useful, especially in the control of negative symptoms.