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Biomedical subjects

J Picard

Publications and source records attributed to J Picard.

At least 109 records · Page 6Linked to original sources
PubMed1984

Evidence for diabetes-induced alterations in the sulfation of heparin sulfate intestinal epithelial cells.

35S-heparan sulfate (HS) metabolism by intestinal epithelial cells isolated from streptozotocin-diabetic and control rats was studied. In diabetic cells, a greater amount of 35S-radioactivity was incorporated into HS, however specific radioactivity of this polysaccharide was decreased. Studies into the distribution of sulfate residues in HS after selective deamination of the glucosamine units within the glycosaminoglycan (GAG)-chain, demonstrated that O-sulfate groups are preferentially located in relatively small deamination products: tetrasaccharides and disaccharides. A lower amount of radioactivity related to N-sulfate groups was found in HS from diabetic cells compared to that of control cells demonstrating that, in diabetes, less glucosamine residues within HS chains are subjected to N-sulfation. An increase in the percentage of 35-sulfate and in the percentage amount of uronic acid in tetrasaccharides of HS of diabetic cells indicated that a greater number of tetrasaccharides were generated by deaminative degradation of this HS. Since a decrease in the specific activity of uronic acid in disaccharides as in tetrasaccharides from HS of diabetic cells was observed, it is clear that the degree of O-sulfation of this HS is reduced. It is suggested, that "in vivo" changes in HS metabolism in diabetic intestinal epithelial cells lie in a disturbance in the degree of N- and O-sulfation of disaccharide units within the HS macromolecule.

Animals↗
PubMed1984

Insulin receptor processing in hepatoma cells: some steps involved in both basal receptor turnover and insulin-induced down-regulation.

Insulin receptor processing was studied in cultured Zajdela hepatoma cells (ZHC). The basal receptor turnover was estimated in the presence of tunicamycin (TM), which inhibited the insertion into plasma membranes of newly synthesized underglycosylated receptors. After a lag phase of 4 h, the surface receptor number decreased, with a t 1/2 of 7 h, for up to 24 h. This process was markedly slowed down when cells were either briefly preincubated with dansylcadaverine, chloroquine, or cycloheximide or treated for 24 h with TM. The effects of these agents on the insulin-induced receptor down-regulation process was then tested. When cells were treated with chloroquine or dansylcadaverine or placed in calcium-free medium, this process was impeded; similarly, it was inhibited by actinomycin D or cycloheximide, but was not affected by TM after a brief incubation. However, after a 24-h treatment with TM, it disappeared, although receptors remained functional when testing insulin's action upon glycogen synthesis. These results indicate that receptor degradation, both basal and activated by insulin leading to the down-regulated state, was altered under similar experimental conditions. The effects of dansylcadaverine, chloroquine, or the absence of calcium reveal that endocytotic pathways were involved in these processes. The results obtained when mRNA, protein, or glycoprotein synthesis was inhibited indicate that cellular glycoprotein(s) and short-lived protein(s) were necessary for the receptor processing in both cases. These data led us to postulate that basal and insulin-activated receptor degradation may occur in the same way within the cell.

Animals↗
PubMed1983

Evidence for surface glycoprotein involvement in the intracellular bioactivity of insulin in rat adipocytes.

Lectins specific for D-mannose (concanavalin A), N-acetyl-D-glucosamine (wheat-germ agglutinin) or D-galactose (Ricinus communis agglutinin I) inhibited insulin binding and activated glucose transport in rat adipocytes [Cherqui, Caron, Capeau & Picard (1982) Mol. Cell. Endocrinol. 28, 627-643]. In the present investigation, the intracellular activities of insulin and lectins on lipogenesis and protein synthesis were studied under conditions where neither agent had an effect on membrane transport processes. (1) When glucose transport was rate-limiting (0.5 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 2.4-3-fold. (2) When passive diffusion of glucose was amplified (10 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 1.6-1.8-fold even in the presence of 50 microM-cytochalasin B, which completely blocked glucose transport. (3) Insulin (6 ng/ml), concanavalin A and wheat-germ agglutinin (40 micrograms/ml) stimulated the incorporation of L-[U-14C]leucine into fat-cell protein 1.5-fold but did not modify alpha-aminoisobutyric acid uptake or 14C-labelled protein degradation. (4) Peanut and soya-bean agglutinins (specific for O-glycosidically-linked oligosaccharides), known not to alter insulin binding, were ineffective. (5) Lectin effects were dose-dependent and were markedly inhibited by specific monosaccharides (50 mM). (6) Insulin and lectin maximal effects were not additive and were completely abolished by neuraminidase treatment of fat-cells (0.05 unit/ml). These data indicate involvement of surface sialylated glycoproteins of the complex N-linked type in the insulin stimulation of glucose and amino acid intracellular metabolic processes. They suggest, together with our previous results, that the transmission of the insulin signal for both membrane and intracellular effects occurs via glycosylated effector entities of, or closely linked to, the insulin-receptor complex.

Adipose Tissue↗
PubMed1983

The structural characterization of proteoglycans of cultured aortic smooth muscle cells and arterial wall of the pig.

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (rho av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (rho av = 1.60 g/ml) fraction and three fifths (rho av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83-90%) with lower proportions of heparan sulfate (5-11%) and hyaluronic acid (3-6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11-13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with Kav 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column Vo and the rest at a Kav of 0.34. The majority of subunit macromolecules eluted at the Vo fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4-7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.

Animals↗
PubMed1983

Further evidence for a role of carbohydrates in insulin binding: studies in lectin-purified receptors.

Purification of liver membrane insulin receptors on concanavalin A- and ricin I-lectin columns gave a 15-fold enrichment in the insulin binding capacity per milligram of protein. Final receptor and protein recoveries were 53 and 3.8% respectively. Lectin-purification increased the receptor affinity for insulin, as indicated by a left-ward shift in the binding competition curve and a steeper slope in the Scatchard plot. Lectin-purification increased the receptor sensitivity towards the glycosidic probes. The maximal effects of beta-galactosidase, ricin I (galactose-binding lectin) and alpha-mannosidase were markedly amplified: 80, 90 and 60% inhibition, versus 45, 40 and 15% with particulate membranes. The limulus polyphemus (LPA) and wheat germ (WGA) agglutinins (sialic acid- and N-acetyl-glucosaminyl-binding lectins) became effective in modifying the insulin binding: 45 and 80% inhibition, respectively. The effects were dose-dependent, reversed by the monosaccharide competitors (lectin effects) and unrelated to the state of receptor occupancy. These findings indicate that, within the hormone recognition area, peptide chains containing galactose, mannose and N-acetyl-glucosamine are strictly required for insulin-receptor interaction and suggest that change in the receptor affinity is related to the role of carbohydrate in insulin binding.

Animals↗
PubMed1982

Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end.

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.

ABO Blood-Group System↗
PubMed1982

[Alpha-N-Acetylgalactosaminyl transferase activity in mucous ovarian tumors obtained from blood group O2 patients (author's transl)].

We have demonstrated, by the transfer of (1-14C) GalN Ac from exogenous UDP[ (1-14C) GalN Ac into a glycopeptide H-acceptor, the presence of an alpha-N-acetylgalactosaminyltransferase in the wall and fluid of ovarian tumors. The transfer occurs in the absence of exogenous acceptor but to a greater degree in the tumor wall and fluid obtained from blood group O patients, thereby indicating the presence of preferential endogenous acceptor(s) in tumors of these subjects.

ABO Blood-Group System↗
PubMed1982

Insulin-induced receptor regulation in cultured Zajdela rat hepatoma cells and relationship to the stimulation of glycogen synthesis.

Insulin receptors were measured in cultured Zajdela rat hepatoma cells (ZHC cells), a stable cell line which presents differentiated hepatic functions. The number of sites was 50,000/cell at 2 C, and the dissociation constant for high affinity binding was 1.6 x 10(-10) M. Down-regulation of receptors occurred rapidly when cells were treated with insulin; this process was related to ambient insulin concentrations and led to a decrease in the number of insulin receptors from 50,000 to 30,000/cell. Cycloheximide prevented part of this regulation. When down-regulated cells were incubated in standard medium devoid of insulin, the number of receptor sites gradually increased and attained control values within 7 h; cyclohexamide inhibited this process. Insulin markedly enhanced glycogen synthesis in ZHC cells, with an ED50 of 1.0 x 10(-9) M, leading to an increase in the total cell glycogen content. In addition, the predicted righthand shift of the dose-response curve was observed for insulin-treated cells. These findings provide evidence of insulin-induced receptor regulation in cultured ZHC cells which is related to the biological effect of the hormone on glycogen synthesis.

Animals↗
PubMed1981

Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding.

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I] insulin binding to soluble receptors from mouse liver was inhibited by digestion with beta-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H] hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10-7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified with two galactoglycoprotein of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with beta-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10-7 M proinsulin slightly hindered labeling, while pretreatment with 10-7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate that glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.

Animals↗
PubMed1981

Action of insulin on glycogen metabolism in cultured hepatoma cells.

ZHC cells, an established hepatoma cell line characterized by its capacity to synthesize and store glycogen, retain responsiveness to insulin. Sensitivity to insulin is correlated with culture development and is maximal in the confluent monolayer cultures. Insulin induces, within 2-3 h, an increase of glycogen content by stimulating the net synthesis of new glycogen molecules and without affecting their breakdown. Insulin directly acts on glycogen metabolism, and does not modify total cell protein or DNA synthesis. The ZHC cell line can provide a new model for the study of insulin regulation of glycogen metabolism, in the absence of other hormones that modulate the same pathway.

Animals↗
PubMed1981

Further characterization of the insulin receptor glycosidic moiety in rat adipocytes.

Simultaneous or sequential treatment of rat adipocytes with neuraminidase plus beta-galactosidase decreased insulin binding by 43%. No modification was observed with either enzyme individually. alpha-Mannosidase enhanced insulin binding (38%), whereas beta-N-acetylglucosaminidase and alpha-L-fucosidase were ineffective. Lectins that interact with galactose (Ricinus communis I, RCAI), mannose, Lens culinaris agglutinin (LCA), Concanavalin A (Con A) or N-acetylglucosamine (wheat-germ agglutinin, WGA) decreased insulin binding by 43, 57, 59 and 85% respectively. Lectin inhibition was dose-dependent, saturable and prevented by specific monosaccharides. RCAI, LCA, Con A and WGA decreased the insulin dissociation process by 45, 90, 78 and 84% respectively. Lectins specific for sialic acid, terminal galactose, N-acetylgalactosamine or fucose (Limulus polyphemus, peanut, soybean and Ulex I agglutinins) did not modify either insulin binding or dissociation. These results indicate involvement of penultimate D-galactose, internal N-acetyl-D-glucosamine and D-mannose residues in both processes. They suggest that, in rat adipocytes, a glycosidic moiety participates in the insulin-receptor interaction through N-linked oligosaccharides of the 'complex type'.

Acetylglucosaminidase↗