Jean Agneray

Action of ornithine α ketoglutarate on DNA synthesis by human fibroblasts

SummaryOrnithine α ketoglutarate (OKG) is largely used in clinical nutrition for its anabolic effects. However, the mechanism of its action remains questionable. We investigated the effect of OKG on the rate of DNA synthesis in human fibroblasts. The in vitro experimental procedure required to demonstrate in cell culture the anabolic effects of OKG observed in vivo was found to be glutamine-free and serum-poor medium with sparse cells. In these conditions, OKG induced a significant increase in [3H]thymidine incorporation compared to untreated control cells. This effect was dose-dependent and was observed in all the cultures tested. Taken individually, the two constituents of OKG, i.e. αKG and Orn, also showed a stimulatory effect, but did not demonstrate a dose-dependent response. Concomitant analysis of extracellular aminoacids showed in αKG-treated cultures an increase in glutamate and a decrease in aspartate, suggesting a cellular transamination of αKG. Glutamine, which is the preferential energetic substrate of fibroblasts, can be produced from glutamate and might play a role in the action of OKG. Moreover, OKG induced a rise in the cellular polyamine content. This, in association with the inhibitory effect on OKG action of difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suggests a link between the polyamine biosynthesis pathway and the anabolic effect of OKG.

Metabolism and kinetics of parenterally administered ornithine and α-ketoglutarate in healthy and burned animals

Abstract To improve our understanding of the metabolism of ornithine α-ketoglutarate (OKG), used as an adjuvant of parenteral nutrition, we studied the plasma kinetics, localisation of target tissues and metabolism of α ketoglutarate (αKG) and ornithine (Orn) in healthy and burned animals. After parenteral administration, the kinetics of plasma disappearance of the two labelled compounds showed a biphasic decrease reflecting an open two-compartmental model of elimination, with the exception of Orn in burned rats. The appearance of metabolites in the plasma was rapid, particularly with regard to glutamate, proline and, following ornithine administration, glutamine. This witnessed the engagement of the substrates in multiple metabolic pathways. The study of tissular distribution by autoradiography demonstrated certain target tissues in common for αKG and Orn such as the liver, intestinal mucosa, salivary glands, kidney and muscle. This is consistent with a possible synergic action of these two compounds. The identification of labelled amino-acids and aliphatic polyamines was also performed in the tissues. Two major observations were made, which could be of interest in further work concerning cellular mechanisms of OKG action. Firstly, after αKG administration, labelled Glu and αKG were detected simultaneously in muscle; this would render possible the local biosynthesis of the anticatabolic compound α ketoisocaproate. Secondly, the injection of 14 C-ornithine was followed by the appearance in the intestinal mucosa and salivary glands of labelled aliphatic polyamines, which possess an anabolic effect.

Evaluation of the degree of desialylation of serum α1-acid glycoprotein and α1-antitrypsin

Abstract A method for evaluating the degree of desialylation of α 1 -AGP and α 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. (1) No significant desialylation of α 1 -AGP and α 1 -AT occurred in normal subjects. 2. (2) No correlation between desialylation of α 1 -AGP and α 1 -AT and their amounts existed. 3. (3) Desialylation was preferentially observed in patients with severe hepatic damage but also with inflammatory disorders.

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 α-aminoisobutyrate uptake, which is known to be insulin sensitive, is also investigated. Cell-associated 125l-labeled insulin and trichloroacetic acid-soluble and -precipitable substances were measured in parallel. Chloroquine increased insulin-stimulated uptake of 2-deoxyglucose and a-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.

Sensitivity of cultured fibroblasts to human, bovine and porcine insulins

We have studied the effects of human, bovine and porcine insulin on sugar transport by cultured chicken embryo fibroblast monolayers. For a 30 min. association time, human and bovine insulin at a concentration of 5.10(-8) M stimulated 2-deoxy-D-glucose uptake. (respectively by an average 58 p.cent and 55 p.cent over basal). Porcine insulin was less potent since a concentration of 5.10(-7) M was necessary to obtain similar stimulation. Moreover, the maximal effect of porcine insulin occur only after 60 min. association time instead of 30 min. for the other peptides. The differences between the effects of insulin from different sources is related to species-dependent differences in their structure.

Fibronectin and retinyl acetate effects on attachment and spreading of normal and rheumatoid human synovial cells

The action of two effectors - fibronectin (FN) and retinyl acetate (RA) - on cell attachment and spreading of human synoviocytes was investigated by adding these two drugs to the cell culture medium. No relationship was observed between the level of the effectors (FN = 20-80 micrograms/well, RA = 0.50-2 micrograms/well) and the biological effects studied. For normal human synoviocytes, fibronectin was less effective on the adhesion than fetal calf serum (FCS) present in the control culture medium; retinyl acetate, a drug acting on glycoprotein synthesis, led to similar effects to those observed for FCS-treated cells. In the case of rheumatoid synovial cells, the degree of adhesion was similar for drug- and FCS-treated cultures. Moreover, FN and RA had little effect on the spreading compared to FCS. Given these results, it would appear that synoviocytes differ in their behaviour from usual fibroblastic models.

Isolement du sérum humain normal d'une protéine capable de diminuer l'activité biologique de l'histamine sur l'iléon isolé de Cobaye: Étude préliminaire du mode d'action

Summary We have isolated from human serum a chemically well-defined compound which is able to decrease the biological activity of histamin on Guinea-pig isolated ileon. Modern protein partition technique made it possible to isolate a highly purified protein which reproduces the total effect. The first part of the study describes the techniques used to isolate the active principle and states the physico-chemical characteristics of this molecule. The purified protein enabled us to study its mode of action. The pharmacological activity is partly linked to the large affinity of this factor for histamin. We give the first prove for the formation of a stable combination between histamin and the protein causing this effect.

Utilisation du fructose par I'hépatocyte isolé de rat nourri Zucker obèse fa/fa.

The utilization of fructose by isolated hepatocytes was investigated in fed obese Zucker fa/fa rats compared with their lean littermates (Fa/?) and Sprague-Dawley rats. Hepatocytes were incubated duriThe utilization of fructose by isolated hepatocytes was investigated in fed obese Zucker fa/fa rats compared with their lean littermates (Fa/?) and Sprague-Dawley rats. Hepatocytes were incubated during 3 h using U14C fructose (20 mM). Our results show: a significant increase of fructose consumption, glucose, lactate and pyruvate production and faster turnover of glycogen by fa/fa rats. In these animals, synthesis of acylglycerol was also significantly enhanced. Our results suggest that fructose in fa/fa rats was used preferentially as precursor for lipid synthesis not only by the liver but also by the adipose tissue after a prior transformation into glucose by hepatocytes. All these abnormalities result in an accumulation of acylglycerols maintaining an obesity state in fa/fa rats.

'Abnormality of 2-deoxyglucose uptake kinetics in fibroblasts at low concentrations.

2-deoxyglucose uptake rates at low sugar concentrations (less than 500 microM) appeared to be lower than those predicted by the Michaelis-Menten model which correctly described higher concentrations. This phenomenon which we will call concentration-dependent transport lag, was also observed for L-glucose uptake which suggest that this phenomenon is carrier-independent. A model involving the perimembrane space is developed which, for L-glucose, gives k1 = 0.931 +/- 0.072 X 10(-6) l X mg protein-1 X minute-1, k2 = 2.97 +/- 0.19 X 10(-7) l X mg protein-1 X minute-1 and So = 88,8 +/- 4,3 microM; where k1 is the diffusion constant in the cell membrane, k2 is the diffusion constant in the perimembrane space and So the sugar concentration required in the external medium in order to provide an équivalent sugar concentration in the transport carrier area.