Simon Dufrane

Clustering of gangliosides in phospholipid bilayers

Gangliosides have been implicated in a variety of cell surface functions as receptor candidates for toxins [ 1,2], hormones [3] and lectins [4]. Their structures contrast strongly with those of classical lipids, since the head groups are very large as compared to the hydrocarbon tails. Little is known about their organization in the lipid bilayers. A possible approach to this problem is to use model systems [5-S]. Here, we report evidence from electron spin resonance (ESR) measurements that gangliosides incorporated in lipid vesicles can interact and form clusters. Our results show that this clustering process is modulated by the lipid fluidity. Above the lipid phase transition of dipalmitoyl phosphatidyl choline, the model membrane can be viewed as a fluid system in which gangliosides are randomly distributed. Below the phase transition, the model membrane consists of a mosaic structure composed of ganglioside clusters embedded in the lipid matrix. Moreover, we demonstrated that the g~~io~de org~~ation modulates the permeab~ity of liposomes to carboxyfluorescein. This result is briefly discussed in regard of the membrane action of biochemical ligands for which gangliosides are postulated as receptors.

Glucose metabolism in insulin-producing tumoral cells

Homogenates of insulin-producing tumoral cells catalyzed the phosphorylation of glucose, mannose, and fructose. The kinetics of phosphorylation at increasing glucose concentrations, the inhibitory effect of glucose 6-phosphate, and the comparison of results obtained with distinct hexoses indicated the presence of both low-Km hexokinase-like and high-Km enzymatic activities, the results being grossly comparable to those collected in normal pancreatic islets. Relative to protein content, the glucose-phosphorylating enzymatic activity was higher in tumoral than normal islet cells. The activity of other enzymes was either lower (glutamate dehydrogenase), moderately higher (phosphoglucomutase, lactate dehydrogenase) or considerably greater (ornithine decarboxylase) in tumoral than in normal islet cells. In intact tumoral cells, incubated under increasing glucose concentrations, the oxidation of D-[U-14C]glucose and the output of lactic and pyruvic acids reached a close-to-maximal value at 2.8 mM glucose. The ratios for glucose oxidation/utilization and lactate/pyruvate output were much lower in tumoral than in normal islet cells. Although glucose caused a modest increase in insulin output from the tumoral cells, this effect was saturated at a low glucose concentration (2.8 mM) and less marked than that of other secretagogues (e.g., L-leucine, L-ornithine, or forskolin). Thus, despite a close-to-normal enzymatic equipment for glucose phosphorylation, the tumoral cells displayed severe abnormalities in the metabolism and secretory response to this hexose. These findings point to regulatory mechanisms distal to glucose phosphorylation in the control of glucose metabolism in insulin-producing cells.

Inhibitory effect of clonidine upon adenylate cyclase activity, cyclic AMP production, and insulin release in rat pancreatic islets.

Conflicting opinions were recently expressed concerning the possible effect of α2-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca2+), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.

A micromethod for the assay of aldose reductase, its application to pancreatic islets

A micromethod for the assay of aldose reductase is described. The method, which is based on the fluorometric measurement of the NADP+ formed when an aldose is converted to its corresponding polyol, was applied to lens and pancreatic islet crude homogenates, as well as semipurified lens aldose reductase. The fluorometric method has proved to be reproducible, more rapid, and more sensitive than the classical spectrophotometric procedure, and should find ready application in the screening of potential aldose reductase inhibitors.

Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets

Exposure of rat pancreatic islet membranes to [alpha-32P]-NAD+ in the presence of Bordetella Pertussis toxin (islet-activating protein) reveals the ADP-ribosylation of a peptide with a Mr close to 41 kDa, which corresponds to the alpha-subunit of the guanine nucleotide regulatory protein Ni. Islets removed from rats pretreated with the Bordetella Pertussis toxin display a specific increase in adenylate cyclase responsiveness to GTP and are characterized by a resistance to the inhibitory action of alpha2-adrenergic agonists upon either adenylate cyclase activity or glucose-induced insulin release.

Fasting-induced impairment of glucose-1, 6-bisphosphate synthesis in pancreatic islets

In pancreatic islets removed from rats fasted for 48 hours, the insulin secretory response to glucose is decreased. Although the activity of phosphoglucomutase is unaffected by fasting, the decrease in glucose-stimulated insulin release coincides with a suppression of the glucose-induced increment in both glucose-1,6-P2 content and lactate or pyruvate output. These findings are compatible with a regulatory role of glucose-1,6-P2 in the control of glycolysis in pancreatic islets.

Anomeric specificity of mammalian hexokinase

The anomeric specificity of hexokinase was examined in crude homogenates of rat parotid gland, erythrocytes and pancreatic islets. At 8 degrees C, the alpha/beta ratio in maximal velocity averaged 0.73, 0.66 and 0.75 in the parotid, erythrocytes and pancreatic islets, respectively. Hexokinase displayed a greater affinity for alpha- than beta-D-glucose as judged from three criteria: the Km value, the reaction velocity measured with mixtures of the two anomers and their effect upon the phosphorylation of D-[U-14C] glucose in anomeric equilibrium. The latter procedure yielded an alpha/beta ratio in Km close to 0.51, 0.49 and 0.39 in parotid, erythrocytes and pancreatic islets, respectively. Within the limits of this study, the anomeric specificity of mammalian hexokinase would appear to be a mirror image of that of yeast hexokinase.

The coupling of metabolic to secretory events in pancreatic islets: Inhibition by 2-cyclohexene-1-one of the secretory response to cyclic AMP and cytochalasin B

In rat pancreatic islets perifused in the presence of 2-cyclohexene-1-one (CHX; 1.0 mM), the secretory response to either D-glucose or 2-ketoisocaproate, but not that evoked by the association of L-leucine and L-glutamine, was severely decreased. This coincided with a decreased stimulation of [45Ca] efflux from prelabelled islets, whereas the inhibitory action of D-glucose or 2-ketoisocaproate upon both [86Rb] and [45Ca] efflux appeared little or not affected. In the presence of D-glucose, the islets exposed to CHX were virtually unresponsive to either forskolin, theophylline or cytochalasin B. A severe decrease in the secretory response to forskolin was also observed in CHX-treated islets exposed to L-leucine and L-glutamine. Except for a somewhat lower sensitivity to NaF, no major change in adenylate cyclase activity or cyclic AMP production was observed in CHX-treated islets. The activity of protein kinase A was decreased in such islets but its responsiveness to cyclic AMP appeared unaltered. Transglutaminase activity was severely decreased in homogenates derived from CHX-treated islets. These findings suggest that CHX, possibly by lowering the GSH content of islet cells, impairs the functional capacity of the effector system for insulin release, in addition to and independently of any effect that it may exert upon nutrient catabolism and cationic fluxes in the islet cells.