Reetta Holma

A novel mechanism for gut barrier dysfunction by dietary fat: epithelial disruption by hydrophobic bile acids

Impairment of gut barrier is associated with a fat-rich diet, but mechanisms are unknown. We have earlier shown that dietary fat modifies fecal bile acids in mice, decreasing the proportion of ursodeoxycholic acid (UDCA) vs. deoxycholic acid (DCA). To clarify the potential role of bile acids in fat-induced barrier dysfunction, we here investigated how physiological concentrations of DCA and UDCA affect barrier function in mouse intestinal tissue. Bile acid experiments were conducted in vitro in Ussing chambers using 4- and 20-kDa FITC-labeled dextrans. Epithelial integrity and inflammation were assayed by histology and Western blot analysis for cyclooxygenase-2. LPS was studied in DCA-induced barrier dysfunction. Finally, we investigated in a 10-wk in vivo feeding trial in mice the barrier-disrupting effect of a diet containing 0.1% DCA. DCA disrupted epithelial integrity dose dependently at 1-3 mM, which correspond to physiological concentrations on a high-fat diet. Low-fat diet-related concentrations of DCA had no effect. In vivo, the DCA-containing diet increased intestinal permeability 1.5-fold compared with control (P = 0.016). Hematoxylin-eosin staining showed a clear disruption of the epithelial barrier by 3 mM DCA in vitro. A short-term treatment by DCA did not increase cyclooxygenase-2 content in colon preparations. UDCA did not affect barrier function itself, but it ameliorated DCA-induced barrier disruption at a 0.6 mM concentration. LPS had no significant effect on barrier function at 0.5-4.5 μg/ml concentrations. We suggest a novel mechanism for barrier dysfunction on a high-fat diet involving the effect of hydrophobic luminal bile acids.

Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats.

Background: Galacto-oligosaccharides potentially attenuate colonic inflammation by two mechanisms: through beneficial effects on intestinal microflora and by increasing the colonic short-chain fatty acid concentration. The purpose of this study was to investigate the effects of galacto-oligosaccharides on the development of inflammation and on the growth of bifidobacteria in trinitrobenzene sulphonic acid (TNBS)-induced colitis, a model that has been shown to benefit from short-chain fatty acid administration and to be associated with alterations in the colonic microflora. Methods: Rats were given daily either whey-derived or lactose-derived galacto-oligosaccharides (4 g kg -1 day -1 , p.o.), starting 10 days before colitis induction, or dexamethasone (2 mg kg -1 day -1 , s.c., a positive control), starting at colitis induction. Colon wet weight, macroscopic damage and myeloperoxidase activity were assessed 72 h after the induction of colitis. Faecal bifidobacteria were counted at the beginning of the study, and immediately before and 72 h after colitis induction. Results: Galacto-oligosaccharides increased the colonic levels of bifidobacteria but also the levels of other bacterial species. Neither whey-derived nor lactose-derived galacto-oligosaccharides reduced the severity of inflammation. Conclusions: Galacto-oligosaccharides are able to modify gut microflora in severe TNBS-induced colitis, but unable to attenuate the inflammation.

High-fat-induced intestinal permeability dysfunction associated with altered fecal bile acids

AIM To investigate whether high-fat-feeding is associated with increased intestinal permeability via alterations in bile acid metabolism. METHODS Male C57Bl/6J mice were fed on a high-fat (n = 26) or low-fat diet (n = 24) for 15 wk. Intestinal permeability was measured from duodenum, jejunum, ileum and colon in an Ussing chamber system using 4 kDa FITC-labeled dextran as an indicator. Fecal bile acids were analyzed with gas chromatography. Segments of jejunum and colon were analyzed for the expression of farnesoid X receptor (FXR) and tumor necrosis factor (TNF). RESULTS Intestinal permeability was significantly increased by high-fat feeding in jejunum (median 0.334 for control vs 0.393 for high-fat, P = 0.03) and colon (0.335 for control vs 0.433 for high-fat, P = 0.01), but not in duodenum or ileum. The concentration of nearly all identified bile acids was significantly increased by high-fat feeding (P < 0.001). The proportion of ursodeoxycholic acid (UDCA) in all bile acids was decreased (1.4% ± 0.1% in high-fat vs 2.8% ± 0.3% in controls, P < 0.01) and correlated inversely with intestinal permeability (r = -0.72, P = 0.01). High-fat feeding also increased jejunal FXR expression, as well as TNF expression along the intestine, especially in the colon. CONCLUSION High-fat-feeding increased intestinal permeability, perhaps by a mechanism related to bile acid metabolism, namely a decreased proportion of fecal UDCA and increased FXR expression.

The influence of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in human colon

The bacterial enzymes beta-glucosidase, beta-glucuronidase, and urease may contribute to the development of colon cancer by generating carcinogens. A reduction in the activity of these enzymes by certain lactic acid bacteria is considered to be beneficial. This study examined fecal beta-glucosidase, beta-glucuronidase, and urease activities during administration of Lactobacillus rhamnosus LC705 (LC705) together with Propionibacterium freudenreichii ssp shermanii JS (PJS). Thirty-eight healthy men participated in this randomized, double-blind, placebo-controlled, two-period crossover study with treatment periods of 4 weeks. Subjects consumed daily bacterial or placebo capsules. Bacterial capsules contained viable LC705 and PJS (2x10(10) CFU of each strain daily). The activities of beta-glucosidase, beta-glucuronidase and urease, recovery of LC705 and PJS, and counts of total lactobacilli and propionibacteria were determined from feces. The mean fecal counts of total lactobacilli and propionibacteria as well as strains LC705 and PJS were significantly increased during the administration of bacteria (3.5-, 13-, 80- and 11-fold, respectively). beta-glucosidase activity decreased by 10% (P=0.18) and urease activity by 13% (P=0.16) during bacterial supplementation versus placebo. The change in beta-glucosidase activity was negatively correlated with the change in propionibacteria counts (R=-0.350, P=0.039), being -2.68 versus 0.94 nmol/min/mg protein in subjects with increased and unchanged/decreased propionibacteria, respectively (P=0.003). To conclude, the administration of LC705 and PJS was followed by an increase in the fecal counts of lactobacilli and propionibacteria and a decrease in the activity of beta-glucosidase with increasing counts of propionibacteria.

Lactobacillus rhamnosus LC705 Together with Propionibacterium freudenreichii ssp shermanii JS Administered in Capsules Is Ineffective in Lowering Serum Lipids

Objective: The aim of this study was to examine the effect of the administration of Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp shermanii strain JS in capsules on serum cholesterol and triglyceride levels in mildly or moderately hypercholesterolemic men. Methods: Thirty-eight basically healthy men, mean age 42 years (range 24–55), mean cholesterol 6.2 mmol/L (5.3–8.2 mmol/L), participated in this double-blind, randomised, placebo-controlled, two-period crossover study with 4-week treatment periods. The subjects consumed daily two probiotic capsules containing viable Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp shermanii JS (2 × 1010 colony forming units of each strain daily) or two placebo capsules. Serum lipids were assessed before the intervention, at the end of both 4-week treatment periods, and 2 weeks after the second treatment period. Dietary and lifestyle habits were carefully monitored. Results: All the subjects completed the study, and the probiotic capsules were well tolerated. Dietary habits and the intake of energy and nutrients, such as saturated fatty acids and cholesterol, did not differ between the treatment groups. No changes in total cholesterol, HDL cholesterol, LDL cholesterol or triglyceride levels were observed during the consumption of the probiotics compared to placebo. Conclusions: The administration of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp shermanii JS did not affect serum lipids.

Acute effects of the cys-leukotriene-1 receptor antagonist, montelukast, on experimental colitis in rats

Cysteinyl leukotrienes play a part in inflammatory reactions such as inflammatory bowel diseases. The aim of the present study was to evaluate the acute effects of a cys-leukotriene-1 receptor antagonist, montelukast, on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. Montelukast (5, 10 or 20 mg kg(-1) day(-1)), a 5-lipoxygenase inhibitor, zileuton (50 or 100 mg kg(-1) day(-1), a positive control), or the vehicle was administered intracolonically to the rats twice daily throughout the study, starting 12 h before the induction of colitis with TNBS. The severity of colitis (macroscopic and histological assessment, as well as myeloperoxidase activity), the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, and eicosanoid production in colonic tissue incubation were assessed 24 and 72 h after colitis induction. Montelukast increased prostaglandin E(2) production at 24 h and tended to reduce the cyclooxygenase-2 protein expression at 72 h, but did not influence the severity of colitis. Zileuton failed to decrease the inflammatory reaction in spite of reduced leukotriene B(4) production at 72 h. The results suggest that drugs that block cysteinyl leukotriene receptors have limited potential to ameliorate acute TNBS-induced colitis, but that they exert some beneficial effects which make them capable of modulating the course of colitis.

Effects of Lactobacillus rhamnosus GG and Lactobacillus reuteri R2LC on Acetic Acid-Induced Colitis in Rats

Background: Certain lactobacilli reduce the severity of experimental colitis. The aim of this study was to compare the effects of a human strain Lactobacillus rhamnosus GG and a rat strain Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats. Methods:Lactobacillus rhamnosus GG, Lactobacillus reuteri R2LC or sulphasalazine were given orally to the rats. Colitis was assessed 72 h after induction with acetic acid. Results:Lactobacillus reuteri R2LC significantly antagonized body weight loss caused by inflammation compared with Lactobacillus rhamnosus GG and sulphasalazine, and oedema formation in the colon compared with sulphasalazine. Lactobacillus reuteri R2LC reduced the median value of macroscopic ulceration and the protein content of inducible nitric oxide synthase by 50% and the median of the protein content of inducible cyclooxygenase by 30% compared with that of the colitis control group, and Lactobacillus rhamnosus GG reduced the median of inducible nitric oxide protein content by 40% and increased the median of inducible cyclooxygenase protein content by 30% compared with the median value of the colitis control group, but these differences were not statistically significant. Conclusions: The rat strain Lactobacillus reuteri R2LC, but not the human strain Lactobacillus rhamnosus GG, is of benefit in reducing the severity of acetic acid-induced colitis in rats. These results suggest that it is not the total amount of Lactobacillus but the particular species or strain of Lactobacillus that is important in attenuating experimental colitis.BACKGROUND Certain lactobacilli reduce the severity of experimental colitis. The aim of this study was to compare the effects of a human strain Lactobacillus rhamnosus GG and a rat strain Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats. METHODS Lactobacillus rhamnosus GG, Lactobacillus reuteri R2LC or sulphasalazine were given orally to the rats. Colitis was assessed 72 h after induction with acetic acid. RESULTS Lactobacillus reuteri R2LC significantly antagonized body weight loss caused by inflammation compared with Lactobacillus rhamnosus GG and sulphasalazine, and oedema formation in the colon compared with sulphasalazine. Lactobacillus reuteri R2LC reduced the median value of macroscopic ulceration and the protein content of inducible nitric oxide synthase by 50% and the median of the protein content of inducible cyclooxygenase by 30% compared with that of the colitis control group, and Lactobacillus rhamnosus GG reduced the median of inducible nitric oxide protein content by 40% and increased the median of inducible cyclooxygenase protein content by 30% compared with the median value of the colitis control group, but these differences were not statistically significant. CONCLUSIONS The rat strain Lactobacillus reuteri R2LC, but not the human strain Lactobacillus rhamnosus GG, is of benefit in reducing the severity of acetic acid-induced colitis in rats. These results suggest that it is not the total amount of Lactobacillus but the particular species or strain of Lactobacillus that is important in attenuating experimental colitis.

Constipation Is Relieved More by Rye Bread Than Wheat Bread or Laxatives without Increased Adverse Gastrointestinal Effects

Rye bread and lactobacilli modify the colonic environment and have the potential to relieve constipation and could be a safe and convenient alternative to laxatives. The effects of rye bread and cultured buttermilk with Lactobacillus rhamnosus GG (LGG) on bowel function and colon metabolism were investigated and compared with laxatives in 51 constipated adults. They were randomized to receive whole-grain rye bread (minimum 240 g/d), LGG (2 x 10(10) colony-forming units/d), whole-grain rye bread (minimum 240 g/d) + LGG (2 x 10(10) colony-forming units/d), white wheat bread (maximum 192 g/d), or laxatives (as usual for a participant) for 3 wk. Participants recorded their dietary habits, bowel function, and gastrointestinal symptoms. Fecal weight, pH, SCFA and bacterial enzyme activities, total intestinal transit time (TITT), and breath hydrogen were determined. Rye bread, compared with white wheat bread, shortened TITT by 23% (P = 0.040), increased weekly defecations by 1.4 (P = 0.014), softened feces [odds ratio (OR) 3.98; P = 0.037], eased defecation (OR 5.08; P = 0.018), increased fecal acetic acid and butyric acid contents by 24% (P = 0.044) and 63% (P <0.001), respectively, and reduced fecal beta-glucuronidase activity by 23% (P = 0.014). Compared with laxatives, rye bread reduced TITT by 41% (P = 0.006), fecal beta-glucuronidase activity by 38% (P = 0.033), and fecal pH by 0.31 units (P = 0.006). LGG did not relieve constipation or significantly affect colonic metabolism. Gastrointestinal adverse effects did not significantly differ among the study groups. In conclusion, rye bread relieves mild constipation and improves colonic metabolism compared with white wheat bread and commonly used laxatives without increasing gastrointestinal adverse effects.

A Probiotic Mixture Including Galactooligosaccharides Decreases Fecal β-Glucosidase Activity but Does Not Affect Serum Enterolactone Concentration in Men during a Two-Week Intervention

A high serum concentration of enterolactone, an enterolignan produced by colonic microbiota from precursors in cereals, vegetables, and fruits, is associated with reduced risk of acute coronary events. Probiotics and prebiotics modify colonic metabolism and may affect the serum enterolactone concentration. The effects of a probiotic mixture alone and with galactooligosaccharides (GOS) on serum enterolactone concentration and fecal metabolism were investigated in 18 healthy men. Participants received 3 interventions, each for 2 wk: 1) probiotics [Lactobacillus rhamnosus strains GG (LGG) and LC705, Propionibacterium freudenreichii ssp. shermanii JS, and Bifidobacterium breve Bb99, for a total amount of 2 × 10(10) CFU/d]; 2) probiotics and GOS 3.8 g/d; 3) probiotics, GOS, and rye bread (minimum 120 g/d). Serum enterolactone and fecal dry weight, enzyme activities, pH, SCFA, lactic acid bacteria, bifidobacteria, propionibacteria, and the strains LGG and LC705 were determined. The serum enterolactone concentration (nmol/L) tended to be decreased from baseline [mean (95% CI) 18.6 (10.8-26.4)] by probiotics alone [15.2 (7.8-22.7); P = 0.095], was not significantly affected by probiotics with GOS [21.5 (13.2-29.8)], and was increased by probiotics with GOS and rye bread [24.6 (15.4-33.7); P < 0.05]. Probiotics alone did not affect fecal β-glucosidase activity and bifidobacteria, but probiotics with GOS decreased β-glucosidase activity and increased bifidobacteria compared with baseline (P < 0.05) and with probiotics alone (P < 0.01). In conclusion, this probiotic mixture with or without GOS does not significantly affect serum enterolactone concentration. Because probiotics with GOS decreased fecal β-glucosidase activity but not serum enterolactone, the reduced fecal β-glucosidase, within the range of activities measured, does not seem to limit the formation of enterolactone.

Genetically obese mice do not show increased gut permeability or faecal bile acid hydrophobicity.

Gut barrier dysfunction may lead to metabolic endotoxaemia and low-grade inflammation. Recent publications have demonstrated gut barrier dysfunction in obesity induced by a diet high in fat, and a pathogenetic role for luminal bile acids has been proposed. We aimed to investigate whether genetically obese mice develop increased gut permeability and alterations in luminal bile acids on a diet with a regular fat content. We used seven obese male ob/ob mice of C57BL/6J background and ten male wild-type (WT) mice of the same strain. Faeces were collected for bile acid analysis. Intestinal permeability was measured in an Ussing chamber upon euthanasia, using 4 kDa fluorescein isothiocyanate dextran, as per mille (‰, 1/1000) of translocated dextran. We analysed the liver expression of lipopolysaccharide-binding protein (LBP), as well as serum LBP (ELISA). Intestinal permeability was not affected by genetic obesity (jejunum: 0·234 (sem 0·04) ‰ for obese v. 0·225 (sem 0·03) ‰ for WT, P= 0·93; colon: 0·222 (sem 0·06) ‰ for obese v. 0·184 (sem 0·03) ‰ for WT, P= 0·86), nor was liver LBP expression (relative expression: 0·55 (sem 0·08) for obese v. 0·55 (sem 0·13) for WT, P= 0·70). Serum LBP was 2·5-fold higher in obese than in WT mice (P= 0·001). Obese mice had increased daily excretion of total bile acids, but their faecal bile acid hydrophobicity was unchanged. In conclusion, genetic obesity did not impair gut barrier function in mice on a regular chow diet, nor was faecal bile acid hydrophobicity affected.