Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Specific amino acids increase mucin synthesis and microbiota in dextran sulfate sodium-treated rats.

During the anabolic response associated with inflammation, mucin synthesis and colonic protection may be compromised by the limited availability of specific amino acids. We therefore determined the effect of dietary amino acid supplementation on the microbiota, mucin status, and mucosal damage in dextran sulfate sodium (DSS)-treated rats. From 8 d before to 28 d after colitis induction, male Sprague-Dawley rats (10 mo old, n = 8/group) were fed a control diet supplemented or not with 2 different doses of an amino acid cocktail containing L-threonine, L-serine, L-proline, and L-cysteine. All diets were isonitrogenous (adjusted with L-alanine). The higher dose of amino acids increased the number of Muc2-containing goblet cells in the surface epithelium of the ulcerated area, stimulated mucin production in the colon, and restored the mucin amino acid composition and mucosal content to healthy, control values. The colonic mucin synthesis rate was specifically stimulated by 95%, whereas the protein turnover was unchanged. All bacterial populations, markedly altered by the DSS treatment, were promoted. In conclusion, in inflammatory situations, an increase in threonine, serine, proline, and cysteine dietary supply can promote mucin synthesis, reequilibrate the gut microbiota, and thus favor colonic protection and mucosal healing.

Dietary threonine restriction specifically reduces intestinal mucin synthesis in rats.

We determined whether the steady-state levels of intestinal mucins are more sensitive than total proteins to dietary threonine intake. For 14 d, male Sprague-Dawley rats (158 +/- 1 g, n = 32) were fed isonitrogenous diets (12.5% protein) containing 30% (group 30), 60% (group 60), 100% (control group), or 150% (group 150) of the theoretical threonine requirement for growth. All groups were pair-fed to the mean intake of group 30. The mucin and mucosal protein fractional synthesis rates (FSR) did not differ from controls in group 60. By contrast, the mucin FSR was significantly lower in the duodenum, ileum, and colon of group 30 compared with group 100, whereas the corresponding mucosal protein FSR did not differ. Because mucin mRNA levels did not differ between these 2 groups, mucin production in group 30 likely was impaired at the translational level. Our results clearly indicate that restriction of dietary threonine significantly and specifically impairs intestinal mucin synthesis. In clinical situations associated with increased threonine utilization, threonine availability may limit intestinal mucin synthesis and consequently reduce gut barrier function.

Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats.

The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum).

Short-term dietary conjugated linoleic acid supplementation does not enhance the recovery of immunodepleted dexamethasone-treated rats.

BACKGROUND: Conjugated linoleic acid (CLA) has been reported to decrease fat deposition, and increase lean body mass. This has been broadly inferred to mean that CLA alters protein turnover. However, data to test the effects of CLA on protein turnover are lacking. An enhancement in immune responses by CLA has also been demonstrated. AIM OF THE STUDY: The objective of this study was to determine the potential for dietary CLA and protein intervention to improve nutritional and functional recovery in an animal model of catabolic stress and immunodepletion. METHODS: Diets varying in their protein levels in the presence or absence of CLA were tested for their effects on the recovery of glucocorticoid (intraperitoneal injection of dexamethasone, 120 mg/kg) treated rats. Following steroid injection, rats were fed 4 dietary treatments for 4 d. The diets contained 10 or 20 g/100 g protein with or without 0.5 g/100 g CLA. RESULTS: Dexamethasone treatment resulted in a decreased food intake and loss of weight, independent of dietary treatment. A higher number of blood monocytes occurred in rats fed the high CLA diets. The protein fractional synthesis rate in spleens of rats fed the diets containing either high proteins or CLA were higher compared to those fed diets with low protein content or without CLA, respectively. CLA, consumed post-dexamethasone treatment, did not improve protein turnover in the other tissues studied, including gut mucosa, liver, muscle and thymus. CONCLUSIONS: The present study was performed to determine the effect of CLA in acute conditions, as opposed to a preventive approach, on the recovery from a catabolic stress with immunodepletion. Overall, no effect of short-term feeding CLA on the recovery from dexamethasone-mediated immunodepletion was observed.