title: L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and.
authors: Bin
authors: B
authors: Wang
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and bio2046@hotmail.com.
authors: Zhenlong
authors: Z
authors: Wu
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and.
authors: Yun
authors: Y
authors: Ji
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and.
authors: Kaiji
authors: K
authors: Sun
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and.
authors: Zhaolai
authors: Z
authors: Dai
authors: State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and Department of Animal Science, Texas A&M University, College Station, TX.
authors: Guoyao
authors: G
authors: Wu
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D062506:Claudin-4 / Q000235:genetics / Q000378:metabolism; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D005973:Glutamine / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D007422:Intestines / Q000166:cytology / Q000187:drug effects; D062726:Junctional Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D062793:Occludin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013552:Swine; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism; D062827:Zonula Occludens-2 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D062506:Claudin-4; D062726:Junctional Adhesion Molecules; D062793:Occludin; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D005973:Glutamine; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases
keywords: AMPK; CaMKK2; intestinal epithelial cells; l-glutamine; tight junction