Developmental changes in intestinal glycosylation: nutrition-dependent multi-factor regulation of the fucosylation pathway at weaning time.

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.

Reaction of metformin with dicarbonyl compounds. Possible implication in the inhibition of advanced glycation end product formation.

Dicarbonyl compounds such as methylglyoxal and glyoxal are extremely reactive glycating agents involved in the formation of advanced glycation end products (AGEs), which in turn are associated with diabetic vascular complications. Guanidino compounds such as aminoguanidine appear to inhibit AGE formation by reacting with alpha-dicarbonyl compounds. The aim of this work was to study whether the antihyperglycemic agent metformin (a guanidine-like compound) might react with reactive alpha-dicarbonyls. Metformin was incubated at pH 7.4 and 37 degrees in the presence of either methylglyoxal or glyoxal and reaction products analysed by HPLC coupled to mass tandem spectrometry. AGE formation on albumin by methylglyoxal and glyoxal in the presence or absence of metformin was also studied by measuring the fluorescence at 370/440 nm after albumin-AGE isolation by ultrafiltration. As a standard for mass spectra analysis, a metformin-methylglyoxal adduct was chemically synthesised and characterised as a triazepinone (2-amino-4-(dimethyl-amino)-7-methyl-5,7-dihydro-6H-[1,3,5]triazepin+ ++-6-one). The results obtained showed that metformin strongly reacted with methylglyoxal and glyoxal, forming original guanidine-dicarbonyl adducts. Reaction kinetic studies as well as mass fragmentation spectra of the reaction products were compatible with the presence of triazepinone derivatives. In the presence of metformin, AGE-related fluorescence after albumin incubation with either glyoxal or methylglyoxal was decreased by 37% and 45%, respectively. These results suggest that besides its known antihyperglycemic effect, metformin could also decrease AGE formation by reacting with alpha-dicarbonyl compounds. This is relevant to a potential clinical use of metformin in the prevention of diabetic complications by inhibition of carbonyl stress.

Metformin inhibition of glycation processes.

A number of studies have shown that metformin is beneficial in reducing diabetes associated vascular risk beyond the benefits expected from its antihyperglycaemic effect. One of the main pathogenic mechanisms leading to chronic complications of diabetes is non-enzymatic glycation where damage is mediated through increased production of highly chemically reactive glucose and alpha-dicarbonyl compounds which lead to production of advanced glycation products (AGEs). We present laboratory and clinical data supporting the hypothesis that one important explanation of metformin's effect on diabetic complications could be its ability to reduce toxic dicarbonyls and AGEs. This effect could be related either to the binding of the alpha-dicarbonyls, methylglyoxal (MG) or 3-deoxyglucosone, or to an increase in enzymatic detoxification. Our studies presented in this manuscript document extracellular binding of MG by metformin to form a specific product (triazepinone) in vivo. This condensation product appears to be only one of several inactive end products resulting from this chemical reaction and we discuss the possibility that these or other condensation products (hydroimidazolones) could be indicative of inactivation of MG by metformin. Additional studies of other possible condensation products, as well as other potential cellular effects of metformin on MG production, will help to clarify this potentially important effect of metformin and provide a further rationale for using metformin to prevent long-term complications.

Growth modulation of retinal microvascular cells by early and advanced glycation products.

To investigate the possible implication of non-enzymatic glycosylation in the etiopathogenesis of the diabetic retinopathy, we studied the effect of early and advanced glycation products on the growth of retinal microvascular cells. Glucose modified products were obtained by incubating bovine serum albumin or fetal bovine serum with 0.5 M glucose for 10 (early glycation products: EG-BSA and EG-FBS, respectively) or 60 days (advanced glycation end products: AGE-BSA and AGE-FBS, respectively). Cell growth was assessed by cell counting and DNA content determination. EG-BSA or AGE-BSA significantly decreased pericyte proliferation after 8 days of culture (33 and 13% inhibition, respectively). Concerning endothelial cells, EG-BSA reduced proliferation to 40% whereas AGE-BSA increased it to 156% after 4 days of culture. The glucose-treated sera didn't exhibit the same growth effects, neither the EG-FBS nor the AGE-FBS significantly affected endothelial cell proliferation. Only the AGE-FBS showed a significant inhibitory effect on pericyte proliferation (40% inhibition). We conclude that retinal microvascular cell growth in vitro could be differently modulated by early and advanced glycation products. The inhibitory effect of AGEs observed on pericyte growth, suggests that glycoxidation could be implicated in the pericyte loss observed in diabetic retinopathy.

In vitro and in vivo alterations of enzymatic glycosylation in diabetes.

Carbohydrate composition changes of glycoconjugates constituting the glycocalix of microvascular cells could be involved in the alterations of cell-cell interactions observed in diabetic retinopathy. In this field, we have recently reported that advanced glycation end products (AGEs) modify galactose, fucose and sialic acid contents of specific cellular glycoproteins. To better understand the mechanisms involved in glycoprotein modifications in diabetes, we now investigate whether glucose and AGEs could affect the activities of enzymes involved in galactose, fucose and sialic acid metabolism : glycosyltransferases (synthesis) and glycosidases (catabolism). For this, bovine retinal endothelial cells (BREC) and pericytes (BRP) were cultured in the presence of high glucose concentration or AGEs, and cell glycosidase and glycosyltransferase activities were measured. The same enzymatic activities were studied in the whole retina from streptozotocin-treated rats. The results show that high glucose concentration did not affect glycosidases and glycosyltransferases neither in BRP nor in BREC except for galactosyltransferase activities in BREC. Concerning BRP, only galactosyltransferase activities were altered by AGEs. In contrast, in BREC, AGEs increased beta-D galactosidase, alpha-L fucosidase and neuraminidase activities (+37%, +56%, 36% respectively) whereas galactosyltransferase, fucosyltransferase and sialyltransferase activities were decreased (-11%, -24% and -23% respectively). In the retina from diabetic rats, beta-D galactosidase, alpha-L fucosidase and neuraminidase activities increased (+70%, +57%, +78% respectively) whereas fucosyl and sialyltransferase decreased (-7% and -15% respectively). The possible consequence of these enzymatic activity changes could be a defect in the carbohydrate content of some glycoproteins that might participate in the endothelial cell dysfunctions in diabetic microangiopathy.

[Characterization and postnatal evolution of an endogenous protein inhibitor of intestinal fucosyltransferase activities]. / Caractérisation et évolution post-natale d'un inhibiteur protéique endogène des fucosyl-transférases intestinales.

An endogenous protein inhibitor was shown to act in vitro on small intestinal fucosyl-transferase activities. In order to support the hypothesis of a physiological role in vivo for this inhibitor in the regulation of the fucosylation process, we characterized this inhibitor and studied its postnatal development. This inhibitor acts differently upon the enzymes of intestinal fucosylation processes. Among the different organs analyzed, the inhibitor was found only in the intestine and the pancreas. Intestinal inhibitory activity decreased 10 fold between the 7th and the 24th day of rat postnatal development; this decrease was inversely correlated with intestinal fucosyl-transferase activities. Two mechanisms of action by which the inhibitor could exert a physiological regulatory role are discussed.

A nondenaturing preparative gel electrophoresis system for the recovery of functional proteins. Application to the identification of an endogenous protein inhibitor of fucosyl-transferase activities.

Electroelution of protein bands resolved by nondenaturing polyacrylamide gel electrophoresis was performed to identify an endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, through its biological activity. After electrophoresis protein bands were negatively stained with zinc acetate, indicating that this staining technique can be also applied to nondenaturing polyacrylamide gels. However, even under appropriate electroelution conditions, a strong fucosyl-transferase inhibitory activity was eluted from the polyacrylamide gel itself that impeded the measure of fuctinin activity. As an alternative to electroelution, collection of proteins resolved by nondenaturing polyacrylamide gel electrophoresis as they are electrophoresed off the end of the gel was assayed. In the commercially available preparative electrophoretic systems, an elution chamber is limited by a semipermeable membrane on which proteins were adsorbed in the low-ionic-strength buffer used in nondenaturing electrophoresis. We demonstrate that the simple and inexpensive electrofractionation system described by Shain et al. (Anal. Biochem. 200, 47-51, 1992) can be successfully applied under nondenaturing conditions to purify and identify fuctinin through its biological activity. However, caution must be taken in the design of the apparatus in order to avoid local heating and thermal denaturation of proteins. The utility of this nondenaturing preparative electrophoresis system for the study of functional proteins is also demonstrated by the recovery of enzymatic activities of alkaline phosphatase and beta-galactosidase.

Effects of endogenous soluble beta-galactoside binding lectins and protein inhibitor of fucosyltransferase on the enzymes involved in the intestinal fucosylation process.

Soluble beta-galactoside binding lectins were prepared from the rat small intestinal mucosa by chromatography on asialofetuin-Sepharose. The lectin fraction exhibits 3 bands with Mr of 21,5 kDa, 19 kDa and 17 kDa on SDS-PAGE. This fraction inhibits a partially purified soluble alpha(1-2)-fucosyltransferase by interaction with the glycoprotein substrate asialofetuin, whereas the inhibition is non competitive for the donor GDP-fucose. It has no effect on other enzymes of the fucosylation system, namely glycosyl-nucleotide pyrophosphatase and the system synthesizing GDP-fucose from GDP-mannose. A different and specific soluble protein inhibitor of fucosyltransferase activity inhibits this activity by a competitive mechanism for GDP-fucose and a non competitive one for asialofetuin. Unlike the lectins, this inhibitor also inhibits the action of pyrophosphatase and the formation of GDP-fucose by different mechanisms. The possible extension of these in vitro results to the in vivo regulation of glycosylation is discussed.

Participation of an endogenous inhibitor of fucosyltransferase activities in the developmental regulation of intestinal fucosylation processes.

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.

Purification and partial amino acid sequence of fuctinin, an endogenous inhibitor of fucosyltransferase activities.

A powerful endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, was purified to homogeneity from rat small-intestinal mucosa. The purification scheme involved DEAE-cellulose ion-exchange chromatography, ammonium sulfate fractionation, hexyl-agarose hydrophobic chromatography and size-exclusion HPLC. Active native fuctinin has an isoelectric point of 4.55 and apparent molecular mass approximately 66 kDa, whereas a single protein band with a molecular mass of approximately 24 kDa was obtained by denaturing polyacrylamide gel electrophoresis, suggesting that fuctinin is an oligomeric protein. Two-dimensional polyacrylamide gel electrophoresis displayed eight spots in this single band. Comparisons of the N-terminal amino acid sequences of each spot support the idea of the existence of three related polypeptides and suggest a proteolytic N-terminal cleavage despite the use of an efficient protease inhibitor throughout the purification. In spite of the presence of an N-glycosylation site, fuctinin is not glycosylated. One of the three polypeptides, peptide 3, possesses two consensus sequences for phosphorylation and a consensus sequence for myristoylation. The sequences of functinin-related peptides, especially peptide 3, exhibit high similarity to the N-terminal domain of the Set protein and a putative human leukocyte antigen-associated protein. The possible implications of these results are discussed.

Evidence for the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activities.

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.

Comparative effects of dietary corn, fish and Krill oils on intestinal glycosylation.

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.