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This study considered the role of dietary polyamines in the maturation of intestinal glycoprotein galactosylation during postnatal development. In the rat small intestine, O-glycan: beta-1,3-galactosyltransferase and N-glycan: beta-1,4-galactosyltransferase are, respectively, involved in the glycan chain biosynthesis of mucins and of glycoproteins in the brush border membranes. Their activities increase significantly at weaning, in parallel with a rise in the intestinal content of spermidine and spermine (as determined by high performance liquid chromatography) and in proportion to the polyamine increase in food intake. The oral ingestion of spermidine or spermine (at 0.4 micromol/g body) by immature suckling rats for 4 d reproduced the levels of spermine and spermidine in their intestines at the time of weaning and induced precocious and significant rises in O-glycan: and N-glycan: galactosyltransferase activities to those normally found after weaning. In parallel, more galactose residues (detected in the complex oligosaccharide chains of glycoproteins by specific lectins after electrophoresis and transfer to nitrocellulose membranes) were observed in the brush border membranes of spermidine- and spermine-treated rats. In contrast, the ingestion of putrescine or ornithine had no effect. Diets with different levels of polyamines (milks and commercial diet), when given at weaning, induced variable evolutions of the galactosylation process, partly in relation to the amounts of polyamines ingested. These results indicate that spermidine and spermine are maturation factors that can reproduce, in immature rats, the same increase in intestinal glycoprotein galactosylation that is normally observed during weaning. They also suggest that the maturation of glycoprotein galactosylation may be a multifactorial event in which spermidine and spermine are both involved.

In the rat small intestine, galactosyltransferases are the enzymes implicated in the biosynthesis of glycoproteins of the brush-border membranes and mucins. During postnatal development, the circulating insulin level increased at weaning in parallel with the activities of intestinal galactosyltransferases on O-glycans and N-glycans. This study deals with the role of insulin in the regulation of galactosyltransferase activities during postnatal development. The treatment of immature suckling rats with insulin induced a precocious increase in the activities of the O-glycan and N-glycan galactosyltransferases, partly reproducing the increase in galactosyltransferase activity normally found at weaning, since the O-glycan galactosyltransferase activity increased more quickly than the N-glycan galactosyltransferase activity. The sensitivity of the two galactosyltransferase activities to insulin disappeared after weaning, a period when drastic diet changes occur. In 22-day-old rats submitted to prolonged nursing (high-fat diet), the activities of the O-glycan and N-glycan galactosyltransferases were lower than those found in age-matched normally weaned rats (high-carbohydrate diet), indicating a delay in the maturation of the intestine of prolonged-nursing rats. The circulating insulin level of these animals stayed lower than that of the age-matched weaned rats. When the prolonged-nursing animals were treated with insulin, the O-glycan and N-glycan galactosyltransferase activities reached levels similar to those of the weaned rats. These observations suggest that insulin is one of the maturation factors for intestinal glycoprotein galactosylation and may be partly responsible for the natural enhancement of intestinal galactosyltransferase activities observed during postnatal development in relation to the dietary changes at weaning.

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an alpha-1, 2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in alpha-1,2-fucosyltransferase level and of the decrease in alpha-l-fucosidase level after treatment with spermine, several alpha-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.

In rat small intestine, during postnatal development, the glycoprotein fucosylation is markedly increased at weaning. At the same time, a rise in the intestinal spermidine level was observed, partly due to the increase in the spermidine content of solid food given to animals at this period as compared to the spermidine content of milk. In order to mimic the spermidine increase observed in weanling rat intestines, we had treated suckling rats with spermidine by oral ingestion to study its role as maturation factor of the small intestine. In spermidine-treated suckling rats, the spermidine and N-acetyl-spermidine contents were highly increased. Spermidine treatment induced the rise in alpha-1,2-fucosyltransferase activity and the precocious appearance in the brush-border membrane of some alpha-1,2-fucoproteins in weaned rats. Such results indicate that spermidine could be a maturation factor implicated in the appearance of alpha-1,2-fucoproteins naturally observed at weaning time.

This study deals with the role of insulin in the regulation of the intestinal glycoprotein fucosylation process during postnatal development in the rat. Circulating insulin level was found to increase at weaning time in parallel with alpha-1, 2-fucosyltransferase activity and with the appearance of alpha-1, 2-fucoproteins in brush-border membranes. Insulin treatment of young suckling rats induced a precocious increase in fucosyltransferase activity and in the biosynthesis of its substrate (GDP-fucose), but the sensitivity to insulin disappeared after weaning. The insulin level was lower in 22-day-old rats that received prolonged nursing (on a high-fat diet) compared with age-matched normally weaned rats (on a high-carbohydrate diet), whereas the appearance of alpha-1, 2-fucoproteins and the increase in activity of alpha-1, 2-fucosyltransferase were delayed, as was the decrease in the degradation of GDP-fucose. In 22-day-old animals that received prolonged nursing and insulin treatment, the alpha-1, 2-fucosyltransferase activity reached a level close to that observed in age-matched weaned rats, and several alpha-1,2-fucoproteins appeared in brush-border membranes with a molecular mass similar to that found in weaned rats. These results suggest that changes in insulin levels at weaning time (as caused, in the present case, by dietary modifications) may be responsible for the regulation of the glycoprotein fucosylation process, essentially by increasing fucosyltransferase activity.

Developmental changes in the fucoglycoproteins of the intestinal brush-border membranes were determined by lectin affinoblotting after electrophoresis. Whereas only two alpha(1-6)-fucoglycoproteins were detected in brush-border membranes from suckling rats, a large number of N-fucoglycoproteins with alpha(1-2)- and/or alpha(1-6)-linked fucose residues were detected in rat membranes after weaning. Dietary manipulations at weaning time were used to investigate the effect of nutritional factors in the development of fucosylation in the small intestine of prolonged-nursed rats fed with milk (a high-fat, low-carbohydrate diet) compared to rats weaned normally with a standard high-carbohydrate diet. The fucose content of the mucosa glycoproteins was lower in 22-day-old prolonged-nursed rats than in 22-day-old rats weaned normally with the standard diet. The appearance of fucoglycoproteins in the brush-border membranes, which was delayed by prolonged nursing, was accompanied by a concomitant delay in the increase of intestinal fucosyl-transferase activity and in the decrease of GDP-fucose substrate breakdown. The developmental decrease in the activity of the inhibitory protein which regulates the fucosyl-transferase activity was also delayed by prolonged nursing. The intestinal fucosylation of brush-border membrane glycoproteins (which include many digestive enzymes) displayed ontogenic changes on which were superimposed dietary influences at the time of weaning. The complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, is a developmental event which thus seems to be strongly influenced by the manipulation of nutritional factors during the weaning period.

Antarctic Krill is considered as a valuable protein resource for animal and human nutrition. Due to the high content of long chain polyunsaturated fatty acids of the n-3 family, Krill consumption could be also interesting in cardiovascular diseases. In the search for the demonstration of the absence of toxicity of Krill, we studied the effect of Krill oil, as compared to fish and corn oil, on the rat intestinal fucosylation process at weaning, a very sensitive model of the influence of nutritional factors. Krill oil containing diets were very well tolerated as compared to other currently used oils and induced only slight modification in fucose and mannose proportions in intestinal glycoprotein sugars. These modifications were not reflected in the enzymatic activities involved in the fucosylation pathway. These results confirm the harmlessness of Krill derived products and their possible use in human nutrition.

The aim of this study was to examine the possible role of certain hormones (especially hydrocortisone) in the developmental variations of intestinal fucosyl-transferase activity in rats. Thyroxine and insulin, injected into suckling rats, did not induce significant modifications of the fucosyl-transferase activity, under the conditions used, whereas this enzyme activity was highly enhanced after administration of glucocorticoids (cortisone and hydrocortisone). Hydrocortisone administration to suckling rats induced a precocious and progressive activation of the fucosyl-transferase activity up to adult level as a function of the duration of treatment. The responsiveness of suckling rats to hydrocortisone, as shown by increased fucosyl-transferase activity, disappeared at the end of the third week (corresponding to the weaning time). These physiological periods of responsiveness and unresponsiveness to hydrocortisone could be related to the binding of the hormones to receptors since the antiglucocorticoid RU 38486 counteracted the effect of hydrocortisone in suckling rats but did not prevent the developmental rise of the fucosyl-transferase activity, when administered in the third week of life. These results suggest that the normal developmental rise of the fucosyl-transferase activity is independent of glucocorticoids.

We review the nutritional and developmental variations of the glycosylation processes in digestive organs, since glycoproteins play a prominent part as mucins or digestive enzymes in these tissues. The biosynthesis of the glycannic chains is demonstrated to be largely sensitive to various exogenous (such as nutritional) or endogenous (such as developmental) factors. Although the metabolic regulation by dietary variations appears as rather complex, according to the variety of experimental conditions and the diversity of the organs studied, available data demonstrate that this regulation does exist, depending on the quantity or sometimes the quality of the major or minor components of the diet, which induce significant variations in the glycosylation processes. The synthesis of the internal core of N-glycans is essentially regulated by diet-induced variations of the phosphoryl-dolichol level, whereas the modulation of the biosynthesis of the external part of N-glycans or the biosynthesis of O-glycans is controlled by diet-induced variations in the systems transferring fucose, galactose, sialic acid or hexosamines. Modifications in intestinal glycosylation during post-natal development in the rat control the quality of the glycannic chains of mucins and brush-border enzymes. The post-natal maturation of the intestinal rat tissue is characterized by a shift from sialylation to fucosylation, depending on coordinate changes in glycosyltransferase activities, in sugar-nucleotide breakdown or synthesis or in the activity of regulatory proteins. These activities are largely sensitive to dietary manipulations at weaning and to hormonal stimulations before weaning. However, glucocorticoid hormones do not appear as the triggering signal for the induction of these changes.

During the rat weaning period (about day 19 after birth) the intestinal maturation is accompanied by a drastic increase in the fucose content of mucosal glycoconjugates, concomitant with an increase in fucosyltransferase activities. The regulation of this fucosylation process appears to be a rather complex phenomenon, which involves several systems controlling fucosyltransferase activity or substrate availability. An endogenous protein inhibitor of the fucosyltransferase activities displays an opposite developmental pattern to that of fucosyltransferase activities, since its activity is high before weaning and is decreased 5-fold after weaning. Similarly, the GDP-fucose pyrophosphatase activity markedly decreases at weaning. The transformation of GDP-mannose into GDP-fucose increases early, at day 18, preceding the increase in fucosyltransferase activities. Before weaning, and especially at days 14 and 18, high levels of GDP-4-dehydro-6-deoxymannose, the product of the GDP-mannose 4,6-dehydratase activity, are produced during the transformation of GDP-mannose into GDP-fucose, even in excess of reduced coenzyme. This fact indicates that the second step of the transformation (epimerase-reductase reaction) could be a limiting factor for GDP-fucose availability before weaning, but not after weaning. The inverse relationship between the mucosal fucose content (or the fucosyltransferase activity) and the endogenous protein inhibitor during normal postnatal development supports the hypothesis of a physiological role for this inhibitor.

Activities of rat intestinal fucosyl-transferase (GDP-fucose: glycoprotein fucosyl-transferase; EC 2.4.1.68) and sialyl-transferase (N-acetylneuraminyl-transferase; EC 2.4.99.1) respectively exhibited a significant increase following weaning and a steady decrease between birth and weaning. The variations of the two glycosyl-transferase activities with age could not be explained by the presence of inhibitory factors in microsomes or cytosol, nor were they due to a natural modification of the milk composition at the end of lactation. The increase in fucosyl-transferase activity that followed weaning was prematurely induced by early weaning. Prolonged nursing prevented the normal increase in fucosyl-transferase activity, and late weaning delayed the increase. The sensitivity to the modification of the time of weaning indicated a major effect of dietary changes, related to the introduction of a solid carbohydrate-rich diet, on the developmental pattern of this enzyme. However, a stress response could not be excluded after early weaning since the corticosterone level of the early weaned rats was enhanced as compared to that of suckling rats. For sialyl-transferase, early weaning caused a slightly greater than normal decrease in the activity, whereas prolonged nursing only weakly diminished the normal decrease in the activity. Moreover, late weaning had no effect on the sialyl-transferase activity. The age-related variations of this enzyme likely are due to factors independent of diet. Thus, the developmental variations of the fucosyl- and sialyl-transferases appear to be differently regulated.

Soluble endogenous inhibitory activities for glycoprotein: alpha (1-2) and alpha (1-3) fucosyltransferases are demonstrated in rat small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of poly-nucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the glycoprotein substrate. Inhibition is not due to interfering enzymatic activities (glycosyl-nucleotide pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases.

In order to estimate the effect of a chemically modified casein, glycosylation processes were studied in rats fed a diet containing this protein. Two groups of rats were fed either a methionyl-casein diet or a normal casein diet. The methionyl-casein was enriched in methionine by covalent linkage of this amino-acid. The nutritional data (growth, protein intake...) were not modified by the diet. The microsomal N-acetylgalactosaminyl- and fucosyl-transferase activities were unaffected by the diet. On the contrary, the soluble fucosyl-transferase activity was enhanced and the activation of fucose transfer in cytosol from methionyl-casein-diet fed rats disappeared after enzyme purification by DEAE-cellulose chromatography. The activation was not explained by changes in some interfering reactions (glycosyl-nucleotide pyrophosphatase, oxidase, protease activities).

1. High-starch diet induces an activation of rat pancreatic microsomal mannosyl-transferase activity as compared with a standard diet or a high-fat diet. 2. This increase is found in a mannose-containing lipid which is identified as a dolichyl-phosphoryl-mannose on the criteria of DEAE-cellulose chromatography, alkaline and acid hydrolysis, thin-layer chromatography and identity of this endogenous product with the [14C]mannose-containing product synthesized in presence of exogenous dolichyl-monophosphate added to the incubation medium. 3. Kinetic parameters (Km and Vmax) of the enzyme versus polyprenic acceptor are not modified by the diet. 4. The results indicate that the activation of the mannose transfer is principally due to an increase of the polyprenic endogenous acceptor by the high-starch diet.

High-fat diets decrease microsomic and soluble fucosyl-transferase activities in rat intestinal mucosa. This inhibition is not due to qualitative (pHi) or quantitative (relative activities) changes ion three isoenzymes, nor is it caused by alterations in the kinetic behaviour of these enzymes (Km, V). It is also not due to a direct effect of the fatty acids on the enzyme activities. It seems reasonable to suggest that a decreased biosynthesis of the fucosyl-transferase occurs as a result of the high-fat diets.

Fucosyltransferase activity of rat small intestine microsomes is solubilized by 0.5% Triton X-100. The solubilized activity can be purified up to 8,300-fold using DEAE-cellulose and affinity chromatography on GDP-Sepharose. At this step, chromatography on Sephadex G15 separates different specificities: N-acetylglucosaminide-alpha-(1,3)-fucosyltransferase acting on asialoserotransferrin, and galactoside-alpha-(1,2)-fucosyltransferase acting on O-glycans of asialofetuin. The use of small saccharidic acceptors also indicates the presence of a N-acetylglucosaminide-alpha-(1,4)-fucosyltransferase and of a very weak glucose-alpha-(1,3)-fucosyltransferase activity. These activities are tightly bound to concanavalin A-Sepharose, suggesting that they are supported by N-glycosylproteins.

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.

The soluble fucosyl-transferase of the rat small intestinal mucosa was purified by passing through a Sephadex G-100 column, then resolved in two isoenzymes F1 (pHi = 4,47) and F2 (pHi = 4,96) by isoelectric focusing. The use in the first step of DEAE-cellulose instead of Sephadex G-100 leads to another component F3 (pHi = 8,70). Kinetic parameters (KM et Vm) for the three isoenzymes are given. The enzymatic mechanism seems to be a bi-bi random type for the three isoenzymes, and F3 appears as the most active species.

A culture obtained from rat duodenal smooth muscle layer is described. The cells are isolated by trypsinization (0.2 %) and the medium used for culture is either MEM with glutamine and non essentiel AA, or RPMI, both containing 10 % foetal calf serum. The cell culture contains both smooth muscle cells and fibroblasts in proportions varying with the age of the culture. At day 6, cell differenciation is important. At day 12, when the cells and confluent, the majority of the cells are fibroblasts. Although it is difficult, the transfer of cells is possible at least twice.

The carbohydrate composition of some intestinal glycoproteins has been demonstrated previously to be modified during development. To evaluate the role of the enzymatic mechanisms of glycosylation, the activities of three soluble and microsomal glycosyl-transferases were studied during postnatal development in rat intestinal mucosa. Nonexistent as soluble forms in the cell sap from suckling rats, the membrane-bound N-acetylgalactosaminyl-, sialyl-, and fucosyl-transferases showed different activities in microsomal fractions before weaning. The N-acetylgalactosaminyl-transferase activity was constant whereas the sialyl-transferase activity, which was maintained at a rather high level until the age of 2 wk, decreased just before weaning, while the fucosyl-transferase activity declined progressively from birth to weaning. After weaning, the three enzymatic activities progressively enhanced until adult age. Their variable behaviors on several glycoprotein acceptors may suggest the presence of several sialyl--and fucosyl-transferases with differences in specificities, varying in different ways according to age. Such developmental modifications in intestinal glycosylation patterns may be linked with certain transformation observed in the carbohydrate level of mucus or membrane-bound glycoproteins and related to the profound modifications in nutritional status at the weaning period.

To determine the effect of diets on the biosynthesis of glycoproteins in the intestinal mucosa, male Sprague-Dawley rats were fed, ad libitum, high-carbohydrate high-fat and high-protein diets. In in vivo methodology, the animals from each dietary group received an intraperitoneal injection of [14C]-fucose, or [14C]-galactose or [14C]-glucosamine. Incorporated radioactivity was detected in the different subcellular fractions. In in vitro methodology, five glycosyl-transferases were studied on microsomes and cell sap. In vivo, only the high-fat diet induces a decrease in the incorporation of [14C]-glucosamine in the macromolecules. In vitro, only the fucosyl-transferase activity is modified by several diets: as compared with an increase in the activity of the control with the age of the animals, high-protein diet induces a slight activation after 2 weeks of diet, while high-carbohydrate and particularly high-fat diets inhibit the enzyme activity. The inhibition is the same when high-fat diet consists of either oil mixture or triolein or oleic acid or linoleic acid and becomes significant with a supplementation rate of 10%.

In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.

The glycosyl-transferase activities in the rat pancreas have been previously demonstrated to be modified by the quantity of different dietary components (lipids or proteins). To evaluate the role of the qualitative composition of the diet on such enzymic systems, two groups of rats were fed either a semi-synthetic diet or a commercial diet of very similar quantitative composition but differing in the quality of their components. The two diets induce a quite similar growth of animals, although the pancreas weight of the commercial-diet-fed rats is slightly higher. The galactosyl-, fucosyl- and mannosyl-transferase activities are more or less highly enhanced by the commercial diet according to the enzyme studied. The highest increase is observed for the biosynthesis of mannosyl-phosphoryl-dolichol. This enhancement by the commercial diet disappears when exogenous phosphoryl-dolichol is added. Such results indicate that the mannose transfer is probably modified by an increased level of the pancreatic lipidic acceptor.

Two groups of rats were fed either a commercial (C group) or a semi-synthetic (S group) diet of very similar quantitative composition, which induce similar growth in the animals. Glycosyltransferase activities are very different in the two groups: fucosyltransferase is lower in S group than in C group, regardless of the expression of the results (specific activity versus protein or DNA). The other activities (galactosyl-, N-acetylglucosaminyl-, N-acetylgalactosaminyltransferases) are modified when expressed versus DNA content; N-acetylneuraminyltransferase specific activity is not altered. Since the weight of mucosa is significantly lower in the S group than in the C group, all the total activities are decreased in the S group. The fucosylation process is further characterized by partial purification of the enzyme and study of the synthesis of GDP-fucose. In each case, interfering reactions (glycosyl-nucleotide pyrophosphatases and proteinases) are controlled. The results give evidence that glycosyltransferases are very sensitive to the qualitative composition of the diet.

A culture obtained from rat duodenal smooth muscle layer is described. The cells were isolated by trypsinization (0.2%), and the medium used for culture was either MEM with glutamine and non-essential AA or RPMI, both containing 10% foetal calf serum. The cell culture contained both smooth muscle cells and fibroplasts in proportions varying with the age of the culture. At day 6, cell differentiation is important. At day 12, when the cells are confluent, the majority of the cells are fibroblasts. Although it is difficult, the transfer of cells is possible at least twice.

Sixteen rabbits were killed 12 weeks after sectioning of the right knee anterior cruciate ligament. The left unoperated knee served as a control. The surface area of fibrillated cartilage from femoral condyles and tibial plateau was evaluated and expressed as a percentage of articular surfaces area. Cartilage from the femoro-patellar surfaces was homogenized for the quantification of several degradative activities, based on the release of digested products. Acid phosphatase, several glycosidases and neutral protease activity from the operated joint cartilage were significantly elevated, while collagenolytic activity was unmodified. The percentage of fibrillated cartilage correlated positively with arylsulfatase, glucosidase and neutral protease but negatively with mannosidase and fucosidase. The results may be consistent with the hypothesis of a sequential degradative process leading to cartilage destruction.