Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats.

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.

Periodate-induced transformation of human peripheral blood lymphocytes and the resultant oxidation of membrane sialyl, galactosyl and fucosyl residues.

Oxidation of human peripheral mononuclear cells with sodium periodate results in lymphocyte activation. Period-date, at optimal mitogenic concentrations, oxidizes membrane sialyl residues (NeuNAc) essentially into the 7 carbon analogue (C7-NeuNAc). Fucosyl and galactosyl residues are also oxidized by periodate, since propane 1,2-diol and glycerol are isolated in acid hydrolysates of lymphocytes oxidized by periodate and reduced by tritiated borohydride. The neuraminidase pretreatment of lymphocytes induces a 40-50% decrease of their response to periodate. Neuraminidase treatment of 108 human peripheral lymphocytes liberated 9.6 microgram NeuNAc (31 nmol), representing 68.5% of the total content. The neuraminidase treatment dramatically enhances the recovery of glycerol in hydrolysates of lymphocytes treated successively with periodate and tritiated borohydride.

Alterations in the carbohydrate moiety of alpha-1-acid glycoprotein purified from human cirrhotic ascitic fluid.

The carbohydrate analysis of alpha 1-AGPc purified from cirrhotic ascitic fluid was performed by immunoaffinity chromatography. It showed a large increase in the fucosyl molar ratio and sugar content (47%). The molar ratio of the oligosaccharides which were released by hydrazinolysis and fractionated by high-performance liquid chromatography confirms the marked increase in fucosyl residues in each fraction. A shift towards fractions with a high degree of branching was also observed. Moreover, the studies of sugar molar ratios and methylation of the tetrasialylated fraction indicated the simultaneous presence of sialyl and fucosyl residues on one of the outer branches.

Modulation of insulin action on 2-deoxyglucose uptake by chloroquine in chick embryo fibroblasts.

Blazar et al. recently found that chloroquine therapy decreased intravenous insulin requirements in a case of extreme insulin resistance. However, no relationship has been shown to exist between insulin degradation and the stimulation of glucose uptake. In this study we investigate the action of insulin on glucose uptake by the ability of this hormone to stimulate 2-deoxyglucose. The effect on alpha-aminoisobutyrate uptake, which is known to be insulin sensitive, is also investigated. Cell-associated 125I-labeled insulin and trichloroacetic acid-soluble and -precipitable substances were measured in parallel. Chloroquine increased insulin-stimulated uptake of 2-deoxyglucose and alpha-aminoisobutyrate. Three hours were required for this effect to appear, and it did not depend on DNA synthesis. Chloroquine also increased cell-associated insulin and slightly decreased the percentage of trichloroacetic acid-soluble products. Methylamine affected neither nutrient uptake processes nor insulin binding and degradation; however, it did abolish the effect of chloroquine on these parameters. These data suggest that in chick embryo fibroblasts a relationship may exist between the increase in undegraded cell-associated insulin and the ability of the hormone to stimulate sugar and amino acid uptake.

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats.

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.

Effect of insulin on aminoisobutyric acid uptake by human non-rheumatoid and rheumatoid synovial cells.

The biological sensitivity of cultured non-rheumatoid human synovial cells (NRSCs) and rheumatoid synovial cells (RSCs) was examined in terms of the ability of insulin to stimulate the uptake of alpha-aminoisobutyrate (AIB). NRSCs, like numerous fibroblastic lines, were sensitive to physiological concentrations of the hormone: half-maximal stimulation was obtained with (4 X 10(-10) M) insulin, while maximum transport was found with a 60-90 min association time. On the contrary, although the basal transport was similar in RSCs, insulin was totally unable to accelerate AIB transport in these cells. Inflammatory processes lead to an insulin resistance which most likely involves a post-receptor step at the cellular level.

Study of hepatic binding protein activity in jejuno-ileal by-passed rat hepatocytes.

The kinetic constants of internalization of asialoorosomucoid were determined for normal and jejuno-ileal by-passed rat hepatocytes. In by-passed rats the maximum velocity of asialoorosomucoid internalization is decreased 3-fold, without any modification of apparant constant of internalization. Moreover, the rate constant of internalization was the same in the two groups of rats. These data suggest that the process of asialoorosomucoid internalization is not altered in by-passed hepatocytes and that the decrease of maximal velocity is only due to a decrease of total uptake receptor number.

[Relationship between the structure and activity of a histamine-blocking serum glycoprotein]. / Relation entre structure et activité biologique d'une glycoprotéine sérique histamino-bloquante

A plasmatic glycoprotein is submitted to a mild periodate oxydation and its pharmacological activity is studied. This glycoprotein contains much N acetyl Neuraminic Acid (NANA = 15 p. cent), and it reduces the biological activity of histamine on smooth muscle such as guinea pig ileum. See article. We also identify the 8 NANA and 7 NANA derivaties. Th only 8 carbon derivative is obtained when about one mole of m-periodate is consumed for one mole of NANA. The 7 carbon derivative appears as soon as the consumption of a second mole leads ta a second cleavage. These results prove that the oxydation islimited to the sole N acetyl neuraminic acid and more precisely to the lateral polyhydroxylic chain. Under these conditions, pharmacological activity gradually decreases, it disappears as soon as the lateral polyhydroxylic chain is completely cut off.

Comparative effects of diabetes and monensin on the lectin asialoglycoprotein receptor: biosynthesis, structure and function in rats.

The hepatic asialoglycoprotein receptor is the first studied mammalian lectin. Modulations in vivo by diabetes and in vitro by the carboxylic ionophore monensin gave rise to similar apparent alterations on its biosynthesis, structure and ligand binding capacity. In normal rats, the receptor (whether purified by ligand or antibody-affinity chromatography) presented a similar pattern in SDS-PAGE analysis, with a major 42-kDa band and two minor ones (49 and 52-54 kDa). In diabetic rats, a new 38-kDa band appeared, but only after antibody-affinity purification. In vitro biosynthesis of the receptor by normal hepatocytes in the presence of 35S-methionine showed that this 38-kDa band was present at the end of a 30-min pulse but decreased during a 180-min chase, in association with an increase in the major 42-kDa band. In diabetic cells, this evolution was retarded. Using a 30-min pulse followed by a 120-min chase in the presence of 100 microM monensin, we showed that this carboxylic ionophore had similar effects on diabetes, leading to a delay in the maturation process of the 42-kDa band and the persistent emergence of the 38-kDa species. Allowing incubation in the presence of 25 or 100 microM monensin, we observed a decrease in the number of ligand binding sites both at the surface (40%) and within the cell (28%). In hepatocytes from diabetic rats, monensin showed no additional effect on the partial diabetes-induced inactivation.

Insulin-like growth factor I/somatomedin C action on 2-deoxyglucose and alpha-amino isobutyrate uptake in chick embryo fibroblasts.

Maximum 125I-IGF-I/Sm-C total binding to chick embryo fibroblasts was 3% at +37 degrees C and decreased to less than 1% in presence of 2.8 X 10(-9) M unlabelled IGF-I/Sm-C. Insulin did not compete with IGF-I/Sm-C for the binding to cells. Biological action of IGF-I/Sm-C was evaluated on 2-deoxyglucose and alpha-aminoisobutyrate uptake. Results are compared with those obtained with insulin. Maximal peptide effects on the two transport processes were obtained at a 0.65 X 10(-7) M concentration and for a 120 minute association time, whereas cells were markedly less sensitive to insulin and time response curves were different. These results suggest that insulin action on nutrient uptake in chick embryo fibroblasts is not mediated by the binding of the hormone to IGF-I/Sm-C receptors.

A macrophage-derived factor induced by alpha 1-acid glycoprotein that inhibits IL-1 comitogenic activity.

After exposure to a concanavalin A (Con A)-unreactive variant of alpha 1-acid glycoprotein (AGP), macrophages released an inhibitor of interleukin-1 (IL-1) proliferative activity in the thymocyte comitogenic assay. This effect was observed with AGP concentrations above 100 micrograms/ml in the macrophage supernatant and would appear to be mediated by the macrophages, since native AGP had no activity on thymocyte proliferation. Preliminary physicochemical characterization showed that the factor was partially resistant to heating, undialyzable, and eluted with an apparent molecular mass of 50-100 kDa when subjected to Sephacryl S-200 chromatography. Murine IL-1 and human (h) recombinant (r) IL-1 were affected by this factor to the same extent. IL-1 and IL-2 co-induced thymocyte proliferation, which is mitogen-independent, was also inhibited, whereas hrIL-2 activity was not suppressed when assayed in thymocytes with PHA at a submitogenic concentration or in CTLL cells. The factor did not interfere with TNF alpha or hrIL-6 activity when tested against their specific cell line. These data indicate that the inhibitor may act specifically against IL-1 activity and further elucidate the possible role of AGP in the modulation of IL-1 activity via the secretion of an inhibitor.

[Incidence of varying factors on the immunochemical behavior of alpha 1-acid glycoprotein]. / Incidence de divers facteurs sur le comportement immunochimique de l'alpha 1-glycoprotéine acide.

Electroimmunodiffusion methods of Laurell and radial immunodiffusion method of Mancini are compared for the qualitative and quantitative analysis of native and desialylated alpha 1-acid glycoprotein. Samples are incubated under different conditions at decreasing pH (3.5 to 0.5 pH units), with increasing ionic strength and with neuraminidase during different time intervals. Results show a pronounced decrease in electrophoretic mobility of alpha 1-acid glycoprotein treated either with acidic reagents or with neuraminidase (ionic strength has no effect). Such a procedure might involve chemical or enzymatic hydrolysis by which sialyl residues are removed. This hydrolysis implicates lower results in the estimation of the desialylated glycoprotein by electroimmunodiffusion. On the other hand, the amounts of alpha 1-acid glycoprotein evaluated by radial immunodiffusion are not modified after incubation. This is expected since diffusion and antigenic properties are not related to the sialic acid content. The data suggest that radial immunodiffusion, less accurate and sensitive than electroimmunodiffusion, is nevertheless more adequate for estimating native and desialylated alpha 1-acid glycoprotein.

Changes in human high density lipoproteins in patients with extra-hepatic biliary obstruction.

Plasma high density lipoproteins (HDL) from patients with obstruction of the common bile duct were studied by crossed immunoelectrophoresis and isoelectric focusing. All cholestatic HDL fractions were rich in phospholipids (51.5 +/- 9%) with high proportions of free cholesterol (13.8 +/- 1.5%). Moreover, crossed immunoelectrophoresis of sera against anti-Apo A revealed the presence of multiple immunoprecipitates sharply contrasting with the pattern formed by normal sera. Tandem crossed immunoelectrophoresis against anti-Apo A and anti-Apo B was performed with whole serum and with the HDL fraction from various cholestatic subjects. Crossed identity was observed for two of these precipitates, which could be explained by the decrease in HDL stability due to the detergent effect of bile salts. The most noteworthy changes found in cholestatic patients appeared to be the apolipoprotein pattern of HDL. Both Apo AI (48%) and Apo AII (5.5%) were greatly diminished and Apo E was present in remarkably high amounts (39%) with two additional isoforms (Apo E'1 and Apo E'2).

Evaluation of the degree of desialylation of serum alpha 1-acid glycoprotein and alpha 1-antitrypsin.

A method for evaluating the degree of desialylation of alpha 1-AGP and alpha 1-AT has been developed. It consists in their simultaneous determination by radial immunodiffusion (RID) and electroimmunodiffusion (EID). When a desialylation exists, an underestimation by EID relative to RID is found. (1) No significant desialylation of alpha 1-AGP and alpha 1-AT occurred in normal subjects. (2) No correlation between desialylation of alpha 1-AGP and alpha 1-AT and their amounts existed. (3) Desialylation was preferentially observed in patients with severe hepatic damage but also with inflammatory disorders.

Lipogenesis from U14C lactate in obese Zucker rat hepatocytes. Effect of albumin-bound oleate.

Lipogenesis from U(14C) lactate was studied in hepatocytes isolated from obese Zucker rats (fa/fa) their lean littermates (Fa/?) and Sprague Dawley rats. The distribution of radioactive carbon between the glycerol and the fatty acid moieties of the acylglycerols were studied. Radioactive lactate was better utilized for glycerol formation than it was for fatty acid formation in the obese rats. However, when oleate was added to the hepatocytic incubation medium, radioactive lactate was preferentially incorporated into the fatty acid moiety of the acylglycerols. Zucker obesity classified as a "metabolic obesity" by Meyer (1) depends upon abnormalities in carbohydrate metabolism associated with increased lipogenesis. This might be explained by biochemical shifts in the utilization of nutrients (2). Among the nutrients, lactate seems to be a better source of carbon than glucose for lipid synthesis (3). It has been shown that there is an increased hepatic portal blood concentration of lactate several hours after eating: about 4 mM in Wistar rats (4) and 10-15 mM in obese Zucker rats (3). We are interested in determinating the incorporation of carbon from lactate either into glycerol or into fatty acyl moieties of hepatic acylglycerols, and in determining the influence of exogenous fatty acids on acylglycerol synthesis, since a high level of circulating fatty acids in Zucker obese rats has been reported (5). Our purpose was to determine the incorporation of lactate into glycerol and fatty acyl moieties of acylglycerols, under the influence of oleate. Hepatocytes were isolated from ad libitum fed obese Zucker rats (fa/fa), their lean littermates (Fa/?) and Sprague-Dawley rats (SD). Incorporation of lactate was studied for three hours, in order to exclude short-term regulation effects and to allow oleate to be distributed into all cellular compartments.

Study of carbohydrate material isolated from ultrafiltrates in patients with chronic renal failure.

We have isolated substances of molecular weight ranging between 350 and 2,000 daltons from ultrafiltrates of 3 patients treated by maintenance haemodialysis for chronic renal failure. Such substances might have a role in the genesis of uraemic toxicity. We have chiefly studied their carbohydrate content. Material was fractionated according to a procedure previously used to urine in healthy controls. Consecutive ion exchange, charcoal Celite and paper chromatography lead to the isolation and purification of oligosaccharides, glycopeptides, glucuronoconjugates and peptides. The different classes of carbohydrate material present in dialysis fluids from uraemic patients are close to those formed in normal urines. All the oligosaccharides in renal failure urine had have identified in normal urine. In a previous studies we have demonstrated that the levels of glucuronoconjugates are higher in the blood of uraemic patients. The glucuronoconjugates and their aglycones could have a toxic effect but a great part of them is removed by hemodialysis.

Fate of enterally administered ornithine in healthy animals: interactions with alpha-ketoglutarate.

In order to improve our understanding of the metabolic interactions between alpha-ketoglutarate (alpha KG) and ornithine (Orn), which constitute the two parts of ornithine alpha-ketoglutarate (OKG) used as an adjuvant in enteral nutrition, we have investigated the plasma appearance and tissue distribution (qualitative and quantitative) of enterally administered 14C-Orn and 14C-alpha KG in healthy mice and rats. The influence of unlabelled alpha KG or Orn on 14C-Orn or 14C-alpha KG metabolism, respectively, was also studied. Unlabelled alpha KG was able to reduce strongly the rate of intestinal absorption of 14C-Orn, whereas the inverse was not true. This alpha KG-induced loss in plasma radioactivity after a load of Orn was associated with a decrease of radioactivity in tissue with no modification of the qualitative distribution in organs. In this study, a direct interaction between alpha KG and Orn was demonstrated at the intestinal level. The mechanisms involved in this phenomenon probably involve the regulation of metabolic conversions among alpha KG, Glu, pyrroline-5-carboxylate, and Orn. This is of importance in the therapeutic use of ornithine salts in clinical nutrition.

Free fatty acid microdetermination by gas-liquid chromatography without transmethylating effects of methylation procedure.

A fast and practicable gas-liquid chromatographic method for the quantitation of non-esterified fatty acids (C14:0-C18:2) from 100 microliter plasma is described. This technique includes extraction, purification using the solvents n-heptane and 0.5 M Na2CO3, and methylation by 0.1 M HCl-methanol. Extraction, quantification and optimal methylation conditions without transmethylation have been investigated. Reproducibility is good and results agree with values found in the literature.

Prevalence of tri- and tetraantennary glycans of human alpha 1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity.

Based on the affinity for concanavalin A (Con A), human alpha 1-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the mannosyl-fucosyl receptor.

Comparative proliferation of non-rheumatoid and rheumatoid human synovial cells.

While normal synovial membrane cells have a very long doubling time, rheumatoid arthritis increases cell turn-over leading to the formation of a pannus. We studied the comparative proliferative behaviour in culture of synoviocytes of rheumatoid (RA) and non-rheumatoid (NR) origin in order to evaluate the usefulness of this model to investigate the drugs used in the treatment of inflammatory diseases. First-passage cultures of cells from patients with clinically defined non-inflammatory joint disease or rheumatoid arthritis were observed for 8 days. In the presence of various combinations of supplemented media, 3H-thymidine incorporation, protein content and cell density were assessed. In addition to a relationship between the fetal calf serum (FCS) concentration and cell growth, it was found that RA cells proliferated more rapidly than NR cells. In 1% FCS, protein content and cell density increased in RA cultures whereas NR synoviocytes accumulated in the quiescent phase. In 5% and 10% FCS, RA cells responded more strongly than NR in terms of protein and DNA synthesis and cell division. After 48 hours of relative FCS deprivation, NR cells abruptly started to proliferate; the response of RA cultures was delayed, but the synoviocytes quickly reached preconfluence.