Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate

• Published in: Gut Vol. 52; no. 2; pp. 224 - 230
• Main Authors: Clark, E C, Patel, S D, Chadwick, P R, Warhurst, G, Curry, A, Carlson, G L
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group Ltd and British Society of Gastroenterology 01.02.2003; BMJ; BMJ Publishing Group LTD; Copyright 2003 by Gut
• Subjects: Adenosine Triphosphate - analysis; AKG; aminooxyacetate; Aminooxyacetic Acid - pharmacology; AOA; Bacteria; Bacterial Translocation - drug effects; Biological and medical sciences; BSO; Buthionine Sulfoximine - pharmacology; buthionine sulphoximine; Caco-2 cells; Caco-2 Cells - physiology; Caco-2 Cells - ultrastructure; Cell Membrane Permeability - physiology; CFU; colony forming units; Culture Media; cytokines; DFMO; difluoromethylornithine; DMEM; Dulbecco’s modified Eagle’s medium; E coli; Eflornithine - pharmacology; Electric Impedance; Energy Metabolism - physiology; Enterocytes - metabolism; Enzyme Inhibitors - pharmacology; Epithelial Cell Biology; Escherichia coli - physiology; FCS; fetal calf serum; Gangrene; Gastroenterology. Liver. Pancreas. Abdomen; Gln; glutamine; Glutamine - metabolism; Hank’s balanced saline solution; HBSS; Humans; Immune system; intestinal barrier; L-Lactate Dehydrogenase - metabolism; lactate dehydrogenase; LDH; lucifer yellow; Medical research; Medical sciences; Microscopy, Electron; Other diseases. Semiology; Permeability; Polyamines; Rodents; sepsis; Small intestine; Stomach. Duodenum. Small intestine. Colon. Rectum. Anus; Studies; TEER; TNF-α; transepithelial electrical resistance; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-TNF; tumour necrosis factor; tumour necrosis factor α; α-ketoglutarate; Human; Translocation; Cell culture; Intercellular space; Digestive system; Gut; Cell biology; Intestinal wall; Biosynthesis; Permeability; Experimental study; Metabolism; Biological activity; Decrease; Dysfunction; Aminoacid; Bacteria; Mechanism of action; Tumor necrosis factor α; Glutamine
• ISSN: 0017-5749; 1468-3288; 1458-3288
• DOI: 10.1136/gut.52.2.224

Background and aims: 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 α (TNF-α) 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-α (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 α-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-α. 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.