Denis Moënnoz

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).

Mucin Production and Composition Is Altered in Dextran Sulfate Sodium-Induced Colitis in Rats

We evaluated the small and large intestinal mucin production in a rat model of human ulcerative colitis by measuring the in vivo fractional synthesis rate (FSR) and the expression of mucins. A chronic colitis was induced by oral administration of 5% dextran sulfate sodium (DSS) for 9 days followed by 2% DSS for 18 days. DSS-treated rats showed increased colonic MUC2,3 mRNA levels compared pair-fed controls. The mucin FSR was unaffected while mucin-containing goblet cells were depleted in the vicinity of lesions. In the small intestine, no inflammatory lesions were observed but ileal MUC2 mRNA levels and mucin FSR were decreased by 46% and 21%, respectively. Finally, DSS-treated rats showed a marked decrease in mucins threonine + serine content all along the gut, which may lead to a reduction of potential O-glycosylation sites. Our data indicate that the chronic colitis may impair the mucus layer protective function all along the gut.

The Chronic Colitis Developed by HLA-B27 Transgenic Rats Is Associated with Altered In Vivo Mucin Synthesis

HLA-B27 transgenic rats spontaneously developing a chronic inflammation mainly involving the colon are recognized as a powerful animal model for IBD. We investigated the mucin production in 6-month-old HLA-B27 rats by measuring in vivo fractional synthesis rate (FSR) and expression of mucins. In the inflamed colon of HLA-B27 rats, the mucin FSR was stimulated by 75% compared to F-344 controls, while MUC2,3 mRNA expression was unchanged. A local depletion in mucus-containing goblet cells was observed, suggesting a rapid mucin production/release and/or a real global decrease in goblet cell number. In the noninflamed jejunum of HLA-B27 rats, the mucin FSR was reduced by 35% compared to controls, while MUC2,3 mRNA expression was unchanged. Different alterations in mucin metabolism and expression are observed between HLA-B27 rats and a model of chemically induced chronic colitis (DSS-treated rats), suggesting that mucin alterations may be dependent on the animal model and colitis underlying mechanism.

Effects of diet-induced hyperthreoninemia. I). Amino acid levels in the central nervous system and peripheral tissues.

Rats were fed four levels of threonine (Thr, 0.4, 0.6, 0.8, and 5.8 g/100 g diet). After two weeks, Thr, serine (Ser), and glycine (Gly) levels were measured in plasma, liver, muscle, and central nervous system. The diet containing 5.8 g/100 g of Thr elevated Thr and Gly concentrations in plasma and nervous tissue in comparison with a standard diet. In muscle and liver, Thr concentrations were also raised but Gly levels did not change. The hepatic Thr dehydratase activity was enhanced. Diets containing moderate Thr quantities (0.6 and 0.8 g/100 g) induced slight elevations of Thr levels in all tissues. Gly concentrations were not modified. The activity of hepatic Thr dehydratase was diminished. Our results show that a high dietary content of Thr (15 times the normal levels) elevates Gly levels in various tissues, including the brain. On the contrary, diets containing 2 to 4 times the normal levels of Thr induce a weak hyperthreoninemia insufficient to modify brain Gly.

Effect of threonine on the behavioural development of the rat

Rats received different levels of threonine (Thr), one, 1.7 and four times the normal dietary intake, from conception to adulthood. The mothers were fed the experimental diets before and during pregnancy. Their offspring received a daily oral load of Thr or placebo until weaning. Thereafter, the juveniles were fed the same diet as their mothers. Morphologic development, ingestive behaviour, homing, and locomotion were observed before weaning. Exploration and spontaneous alternation were studied thereafter. Animals exposed during gestation to 1.7 times the normal Thr intake consumed more food during the test of independent ingestion. Grooming showed inconsistent variations between days 12 and 29 in pups fed 1.7 times the normal Thr intake. Rats performed equally well on the other behavioural tasks independently of the dietary treatment. We conclude that Thr intake as much as four times higher than the levels found in normal diets does not impair the behavioural ontogenesis of the rat.