Lactobacillus rhamnosus GG decreases lipopolysaccharide-induced systemic inflammation in a gastrostomy-fed infant rat model.

OBJECTIVES: A gastrostomy-fed rat infant "pup-in-a-cup" model was used to test the hypothesis that enterally administered Lactobacillus rhamnosus GG (LGG) decreases the proinflammatory response induced by Escherichia coli lipopolysaccharide (LPS) in the developing infant rat small intestine, plasma, lung and liver. METHODS: Two groups of 6- to 7-day-old pups were fed a rat milk substitute with LPS added via the gastrostomy tube for 6 days. One of the rat milk substitute-fed groups received supplemental LGG; another group received LPS without LGG. Age-matched mother-fed rat pups were used as controls. RESULTS: LPS treatment blunted body growth, but LGG supplementation had no effect on weight increments. LGG decreased LPS-induced inflammation in intestinal tissue; CINC-1 (rodent IL-8 equivalent) production in plasma, liver, lung and distal small intestine; and tumor necrosis factor alpha (TNF-alpha) production in plasma and lung. Cytokine multiplex assay showed lung interleukin (IL)-1beta, IL-6, IL-10, IL-18, growth-related oncogene (GRO)/KC (rat CINC-1) and TNF-alpha were significantly higher in gastrostomy-fed, LPS-treated pups than in mother-reared pups, and LGG significantly blunted the LPS-induced elevation of IL-1beta, IL-10, IL-18, GRO/KC and TNF-alpha; liver GRO/KC was significantly higher in gastrostomy-fed, LPS-treated pups than in mother-reared pups, and LGG significantly blunted the LPS-induced elevation of GRO/KC. CONCLUSIONS: LGG provided by the enteral route is able to downregulate LPS-induced proinflammatory mediators. This effect is not only present in the splanchnic organs, that is, the intestine and the liver, but extends to the plasma and a distal organ, the lung.

Changes in intestinal morphology and permeability in the biobreeding rat before the onset of type 1 diabetes.

OBJECTIVE: Type 1 diabetes is an autoimmune disorder that occurs in genetically susceptible individuals. It has been hypothesized that the disease could be triggered by environmental agents that gain entry into the body through small intestinal absorption. Increased intestinal permeability has been reported both in spontaneous animal models of type 1 diabetes and human type 1 diabetes. In these studies, we examined both the physical and functional permeability characteristics of the small intestine in diabetes-prone and control rats. METHODS: In a series of studies, BioBreeding diabetes-prone(n = 31), BioBreeding diabetes-resistant (n = 20) and control Wistar (n = 25) rats were examined at intervals from 21 to 125 days of age. RESULTS: The percentage of goblet cells and the mucosal crypt depth were significantly greater in BioBreeding diabetes-prone than BioBreeding diabetes-resistant rats (P < 0.001 and P = 0.01, respectively). BioBreeding diabetes-prone and BioBreeding diabetes-resistant rats expressed less of the tight junction protein claudin (P < 0.05) and exhibited greater intestinal permeability (P < 0.001) than did Wistar rats. Intestinal permeability measured both in vivo and ex vivo decreased in all rat strains as age increased (P < 0.001). CONCLUSIONS: In a genetically susceptible rodent model of diabetes, early increased intestinal permeability might allow unregulated passage of environmental antigens that could potentially trigger the autoimmune response leading to type 1 diabetes.

Glutamine modulates LPS-induced IL-8 production through IkappaB/NF-kappaB in human fetal and adult intestinal epithelium.

The intestinal epithelium may serve as a nidus for inflammation that can cause local and systemic organ dysfunction. Relative to the adult, the immature intestine is exquisitely sensitive to inflammatory agents. Glutamine (Gln), an amino acid that is rapidly depleted during critical illness, modulates intestinal inflammation in vitro and in vivo. Here we relate Gln status to activation of the inhibitor of kappaB (IkappaB)/nuclear factor (NF)-kappaB signaling pathway in fetal-derived (H4) and adult (Caco-2) enterocytes. In the absence of Gln with or without LPS, H4 cells expressed more interleukin (IL)-8) than Caco-2 cells. Gln supplementation partially prevented the LPS-induced elevation of IL-8 in both cell types. IkappaBalpha was significantly decreased in both H4 and Caco-2 cells with Gln deprivation, and this was followed by an increase in NF-kappaB p65 in the nucleus. DNA binding of NF-kappaB was increased in both H4 and Caco-2 cells with Gln deprivation. IkappaBalpha phosphorylation was not altered by Gln status in either H4 or Caco-2 cells. Proteasomal inhibition after Gln depletion in Caco-2 cells was associated with an increase in the IkappaB-ubiquitin complex, but a decrease in complex formation in H4 cells, indicating that Gln deprivation alters IkappaBalpha through a pathway that differs from Caco-2 cells. We speculate that a reduced capacity of the immature enterocyte (H4) to respond to Gln deprivation with increased synthesis of IkappaBalpha rather than increased proteolysis as seen in the Caco-2 cells is the underlying mechanism.

Effects of protein deprivation on growth and small intestine morphology are not improved by glutamine or glutamate in gastrostomy-fed rat pups.

OBJECTIVES: Critically ill neonates often have their enteral intake severely limited shortly after birth. Whether glutamine (Gln) or glutamate (Glu) can preserve intestinal structure and function in the neonate undergoing limited enteral feeding is not clear. We hypothesize that Gln and Glu can similarly preserve intestinal structure in the developing small intestine of infant rats fed a low protein diet. METHODS: Using a gastrostomy-fed "pup-in-a-cup" rat model, the effects of Gln and Glu on the developing rat small intestine were examined. Four groups of 6- to 7-day-old pups were fed rat milk substitute (RMS) via gastrostomy tube. One group was provided 100% and three were provided 25% of the protein normally received from their mothers. Two of the groups fed 25% protein received additional Gln or Glu for 6 days. RESULTS: Pups receiving the 100% protein RMS were larger than pups receiving the 25% protein RMS with or without Gln/Glu supplementation (P < 0.001). Average villus height (P < 0.01) and area (P < 0.01) were greater in pups receiving 100% protein RMS than in pups given 25% protein RMS formula. There was no significant difference among the groups in mucosal maltase or alkaline phosphatase activities. Tight junction protein claudin-1 was significantly higher in the group fed 100% protein RMS diet, while occludin did not differ among the 4 groups. Neither Gln nor Glu increased claudin-1 or occludin in rats fed 25% protein. CONCLUSIONS: These results suggest that neither Gln nor Glu supplementation can substitute effectively for whole protein in the developing rat small intestine for the outcomes that were evaluated.

Glutamine regulates Caco-2 cell tight junction proteins.

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.

Mechanism of glutamine-mediated amelioration of lipopolysaccharide-induced IL-8 production in Caco-2 cells.

The mechanism of glutamine (Gln)-mediated down-regulation of inflammation in the intestine is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated IL-8 production in intestinal epithelial cells via transcription factors that counteract the effect of LPS-mediated increase in IL-8. Caco-2 cells were incubated with different doses of Gln with or without methionine sulfoximine (MS), an inhibitor of glutamine synthetase for 24 h before stimulation by LPS (100 microg/ml for 24 h). Inhibitors of the mitogen activated protein kinase (MAPK) family were added to cells for 1.5 h following stimulation by LPS. The p38 inhibitor SB 203580 resulted in a significant decrease in IL-8 peptide production (p < 0.01). However, p38 MAPK activity increased with Gln (p < 0.05), suggesting that this was not involved with Gln-mediated down-regulation of IL-8. Screening of 54 transcription factors demonstrated that STAT-4 was the only inflammation-related transcription factor that was up-regulated by Gln depletion and down-regulated with Gln supplementation (2-fold increase), paralleling IL-8 production. EMSA analysis confirmed these findings (3.5-fold increase). These results indicate that Gln deprivation enhances IL-8 production by Caco-2 cells after LPS stimulation and that down-regulation of IL-8 production with Gln is associated with alterations in STAT-4 transcription factor binding.

Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats.

Using a gastrostomy-fed (GF) rat infant "pup-in-a-cup" model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprived, non-Gln-treated group compared with mother-fed pups (P < 0.05). Gln blunted intestinal CINC mRNA (P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated approximately 13-fold compared with the mother-reared pups (P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide (P < 0.05). Gln but not Glu decreased plasma CINC peptide (P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% (P < 0.001) and 54% (P < 0.05), respectively. Intestinal and plasma TNF-alpha were increased in GF, LPS-treated pups (P < 0.01), and Gln and Glu both blunted this increase (P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.

Glutamine decreases lipopolysaccharide-induced IL-8 production in Caco-2 cells through a non-NF-kappaB p50 mechanism.

Glutamine (Gln) supplementation has been shown to decrease production of pro-inflammatory cytokines by the human intestinal mucosa. The mechanism of this is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated pro-inflammatory cytokine production in Caco-2 cells by nuclear factor-kappa B (NF-kappaB). Caco-2 cells were incubated with different concentrations of Gln with or without methionine sulfoximine (MS, an inhibitor of glutamine synthetase) before stimulation with LPS. IL-6, IL-8, IL-10 and TNF-alpha protein and mRNA level were determined. NF-kappaB translocation was determined using an ELISA-based kit. IL-8 was the only detectable cytokine/chemokine. The largest amount of IL-8 was secreted by cells in the presence of MS with no Gln in the medium after exposure to LPS. LPS increased IL-8 production, peaking 10h after LPS administration. The addition of Gln (0.5 or 5.0mM) decreased IL-8 peptide and mRNA expression. LPS increased NF-kappaB nuclear translocation in the presence or absence of MS. Neither Gln nor MS altered NF-kappaB nuclear translocation. These results indicate that the lack of glutamine increases IL-8 production by Caco-2 cells after LPS stimulation. However, the glutamine-mediated decrease in LPS-stimulated IL-8 production is not associated with NF-kappaB p50 nuclear binding.