title: Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate.
abstract: OBJECTIVE
  Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes.
  
  
  METHODS
  The effect of tumour necrosis factor alpha (TNF-alpha) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy.
  
  
  RESULTS
  Exposure to basolateral TNF-alpha (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of non-glutamine containing amino acids. Inhibition of glutamine conversion to alpha-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-alpha. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space.
  
  
  CONCLUSIONS
  In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.
authors.affiliation: Gut Barrier Research Group, and Department of Surgery, University of Manchester, Hope Hospital, Manchester, UK.
authors.forename: E C
authors.initials: EC
authors.lastname: Clark
authors.forename: S D
authors.initials: SD
authors.lastname: Patel
authors.forename: P R
authors.initials: PR
authors.lastname: Chadwick
authors.forename: G
authors.initials: G
authors.lastname: Warhurst
authors.forename: A
authors.initials: A
authors.lastname: Curry
authors.forename: G L
authors.initials: GL
authors.lastname: Carlson
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D018988:Bacterial Translocation / Q000187:drug effects*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D018938:Caco-2 Cells / Q000502:physiology / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D003470:Culture Media; D000518:Eflornithine / Q000494:pharmacology; D017097:Electric Impedance; D004734:Energy Metabolism / Q000502:physiology; D020895:Enterocytes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000502:physiology*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; D005973:Glutamine; D000625:Aminooxyacetic Acid; D019328:Buthionine Sulfoximine; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D000518:Eflornithine