title: Glycine Regulates Expression and Distribution of Claudin-7 and ZO-3 Proteins in Intestinal Porcine Epithelial Cells.
abstract: 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.
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
authors.forename: Kaiji
authors.initials: K
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
authors.forename: Yun
authors.initials: Y
authors.lastname: Ji
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and bio2046@hotmail.com.
authors.forename: Zhenlong
authors.initials: Z
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
authors.forename: Zhaolai
authors.initials: Z
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and Department of Animal Science, Texas A&M University, College Station, TX.
authors.forename: Guoyao
authors.initials: G
authors.lastname: Wu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D057167:Claudins / Q000378:metabolism*; D019587:Dietary Supplements; D020895:Enterocytes / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D005998:Glycine / Q000494:pharmacology*; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D007421:Intestine, Small / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D010539:Permeability; D013552:Swine; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: D057167:Claudins; D005998:Glycine
keywords: barrier function; glycine; intestinal epithelial cells; permeability; tight junction proteins