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-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 ... -  《-》

Cinnamicaldehyde regulates the expression of tight junction proteins and amino acid transporters in intestinal porcine epithelial cells.

Sun K ，Lei Y ，Wang R ，Wu 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 ... -  《-》

[Effects of short chain fatty acid on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide and the related mechanism].

Feng YH ，Huang YL ，Wang P ，Wang FJ ... -  《-》