L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells.

The tight junctions (TJs) are essential for maintenance of the intestinal mucosal barrier integrity. Results of our recent work show that dietary l-glutamine (Gln) supplementation enhances the protein abundance of TJ proteins in the small intestine of piglets. However, the underlying mechanisms remain largely unknown. This study was conducted to test the hypothesis that Gln regulates TJ integrity through calcium/calmodulin-dependent kinase 2 (CaMKK2)-AMP-activated protein kinase (AMPK) signaling which, in turn, contributes to improved intestinal mucosal barrier function. Jejunal enterocytes isolated from a newborn pig were cultured in the presence of 0-2.0 mmol Gln/L for indicated time points. Cell proliferation, monolayer transepithelial electrical resistance (TEER), paracellular permeability, expression and distribution of TJ proteins, and phosphorylated AMPK were determined. Compared with 0 mmol Gln/L, 2.0 mmol Gln/L enhanced (P < 0.05) cell growth (by 31.9% at 48 h and 11.1% at 60 h). Cells treated with 2 mmol Gln/L increased TEER by 32.2% at 60 h, and decreased (P < 0.05) TJ permeability by 20.3-40.0% at 36-60 h. In addition, 2.0 mmol Gln/L increased (P < 0.05) the abundance of transmembrane proteins, such as occludin, claudin-4, junction adhesion molecule (JAM)-A, and the plaque proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 by 1.8-6 times. In contrast, 0.5 mmol Gln/L had a moderate effect on TJ protein abundance (20.2-70.5%; P < 0.05) of occludin, claudin-3, claudin-4, JAM-A, and ZO-1. 2.0 mmol Gln/L treatment led to a greater distribution of claudin-1, claudin-4, and ZO-1 at plasma membranes compared with 0 mmol Gln/L. This effect of Gln was mediated by the activation of CaMKK2-AMPK signaling, because either depletion of calcium from the medium or the presence of an inhibitor of CaMKK2 abrogated the effect of Gln on epithelial integrity. Our findings indicate that activation of CaMKK2-AMPK signaling by Gln is associated with improved intestinal mucosal barrier function through enhancing the abundance of TJ proteins and altering their intracellular localization in intestinal porcine epithelial cells.

Wang B ，Wu Z ，Ji Y ，Sun K ，Dai Z ，Wu G ... -  《-》

Glycine Regulates Expression and Distribution of Claudin-7 and ZO-3 Proteins in Intestinal Porcine Epithelial Cells.

Glycine traditionally is classified as a nutritionally nonessential amino acid in humans and animals. Because of its abundance in the body and its extensive use via multiple pathways, requirements for glycine are particularly high in neonates. Our recent studies show that dietary glycine supplementation is needed for optimal intestinal development in piglets. Importantly, reduced concentrations of glycine in the lumen of the small intestine are associated with gut dysfunction in low-birth-weight piglets. However, the mechanisms responsible for the beneficial effects of glycine on the intestinal mucosal barrier are largely unknown. This study tested the hypothesis that glycine may regulate the expression and distribution of tight junction (TJ) proteins, thereby contributing to intestinal mucosal barrier function. Enterocytes isolated from the jejunum of a healthy newborn pig were propagated to establish a stable cell line. The cells were cultured with 0.05 mmol glycine/L (control; concentration in the small intestinal lumen of low-birth-weight piglets) or 0.25 or 1.0 mmol glycine/L for the indicated periods of time. Epithelial barrier integrity and expression and localization of TJ proteins were analyzed by using monolayer transepithelial electrical resistance (TEER) and paracellular permeability, Western blot, and immunofluorescence imaging. Compared with controls, cells cultured with 0.25 or 1.0 mmol glycine/L increased TEER (P < 0.05) by 46-53% and 80-111%, respectively, at 60-72 h. Correspondingly, paracellular permeability was reduced (P < 0.05) by 6-21% and 18-27% for 0.25 or 1.0 mmol glycine/L treatment, respectively, at 36-72 h. Compared with controls, protein abundances for claudin-3, claudin-7, and zonula occludens (ZO) 3 were enhanced (25-33%, P < 0.05) by 0.25 and 1.0 mmol glycine/L at 8 h, whereas those for occludin, claudin-1, claudin-4, and ZO-2 were not affected. Compared with controls, 1.0 mmol glycine/L reduced the protein abundance of ZO-1 by 20% at 8 h (P < 0.05), but 0.25 mmol glycine/L had no effect. A glycine concentration of 0.25 mmol/L sustained the localization of claudin-7 and ZO-3 to the interface between enterocytes. Interestingly, 1 mmol glycine/L promoted the distribution of claudin-4 and claudin-7 to the cytosol and nucleus, and the localization of ZO-3 to the plasma membranes, while decreasing the distribution of ZO-1 at cell-cell contact sites, compared with control cells. Physiologic concentrations of glycine support intestinal mucosal barrier function by regulating the abundance and distribution of claudin-7 and ZO-3 in enterocytes. Supplementation with glycine may provide an effective nutritional strategy to improve intestinal integrity in piglets.

Li W ，Sun K ，Ji Y ，Wu Z ，Wang W ，Dai Z ，Wu G ... -  《-》

l-Tryptophan Activates Mammalian Target of Rapamycin and Enhances Expression of Tight Junction Proteins in Intestinal Porcine Epithelial Cells.

Besides serving as a substrate for protein synthesis, L-tryptophan (L-Trp) is used via serotonin-, kynurenine-, and niacin-synthetic pathways to produce bioactive compounds crucial for whole-body homeostasis. It is unknown whether L-Trp itself can regulate metabolic pathways in animal cells. This study tested the hypothesis that L-Trp may activate mammalian target of rapamycin (mTOR) complex 1 and enhance expression of tight junction (TJ) proteins in intestinal porcine epithelial cells. Jejunal enterocytes, intestinal porcine epithelial cell line 1 (IPEC-1) isolated from newborn pigs, were cultured in customized Dulbecco's modified Eagle medium (DMEM) supplemented with or without L-Trp for the indicated time periods. Cell proliferation, L-Trp metabolism, protein turnover, mRNA abundance for L-Trp transporters [solute carrier family 3 member 1 (SLC3A1), solute carrier family 6 member 14 (SLC6A14), solute carrier family 6 member 19 (SLC6A19), and Na(+)/K(+) ATPase subunit-α1 (ATP1A1)], abundance of proteins involved in mTOR signaling, and TJ proteins were determined. L-Trp was not degraded in IPEC-1 cells. Compared with basal medium containing 0.04 mmol/L L-Trp, 0.4 and 0.8 mmol/L L-Trp enhanced (P < 0.05) protein synthesis by 45-52% and cell growth by 17% and 25% on day 1 and 72% and 51% on day 2, respectively, while reducing (P < 0.05) protein degradation by 12% and 22%, respectively. These effects of L-Trp were associated with mTOR activation and increased (P < 0.05) mRNA abundance for L-Trp transporters (SLC6A19, SLC6A14, and SLC3A1) by 1.5-2.7 fold and ATP1A1 by 3 fold. L-Trp also upregulated (P < 0.05) the abundance of occludin, claudin-4, zonula occludens (ZO) 1 and 2 by 0.5-2 fold but did not affect expression of claudin-1 or ZO-3 in IPEC-1 cells. L-Trp is not catabolized by pig small intestinal epithelial cells but can regulate intracellular protein turnover and expression of TJ proteins in these cells.

Wang H ，Ji Y ，Wu G ，Sun K ，Sun Y ，Li W ，Wang B ，He B ，Zhang Q ，Dai Z ，Wu Z ... -  《-》

Naringenin enhances intestinal barrier function through the expression and cytoskeletal association of tight junction proteins in Caco-2 cells.

We have previously reported that naringenin promotes the tight junction (TJ) integrity in intestinal Caco-2 cells. This study investigated the naringenin-mediated effect in Caco-2 cells with a particular focus on the modulation of TJ structure and claudin-4 transcriptional regulation. Naringenin (10~100 μM) dose-dependently enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance and FITC-dextran flux. Immunoblot analysis showed that naringenin increased the cytoskeletal association of TJ proteins, zonula occludens-2, occludin, claudin-1, and claudin-4, simultaneously with increased occludin phosphorylation. The total expression of claudin-4 was also increased by naringenin. Quantitative RT-PCR and luciferase reporter assay revealed that naringenin transcriptionally increased the claudin-4 expression with activation of claudin-4 promoter. The mutation of the binding site of the transcriptional factor Sp1 in the claudin-4 promoter sequence and the pharmacological inhibition of Sp1 partially suppressed the naringenin-mediated activation of the claudin-4 promoter. Further, naringenin induced the heat shock protein 70 expression in the cells. Naringenin enhances barrier integrity through the assembly and expression of TJ proteins in intestinal epithelial cells. Naringenin-mediated claudin-4 expression occurs, at least partially, through Sp1-dependent transcriptional regulation. The induction of heat shock protein 70 may be also involved in the increased claudin-4 expression.

Noda S ，Tanabe S ，Suzuki T 《-》

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells.

Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo.

Beutheu S ，Ghouzali I ，Galas L ，Déchelotte P ，Coëffier M ... -  《-》