Annick Vandercammen

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta‐cells differ in their individual rates of glucose‐induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta‐cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta‐cells were 2‐ to 4‐fold higher than in low responsive beta‐cells, whereas their leucine and glutamine oxidation was only 10–50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13–19 pmol/min/10(3) beta‐cells) were at least 50‐fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3–0.7 pmol/min/10(3) beta‐cells) ranged within those of total glucose utilization (0.2–0.4 pmol/min/10(3) beta‐cells). High responsive beta‐cells exhibited a 60% higher glucokinase activity than low responsive beta‐cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta‐cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta‐cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

The regulatory protein of liver glucokinase

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several phosphate esters, mainly polyol-phosphates, were found to mimick the effect of fructose 6-phosphate. The most potent is sorbitol 6-phosphate, suggesting that fructose 6-phosphate is recognized by the regulatory protein in its open-chain configuration. Other phosphate esters and Pi have a fructose 1-phosphate-like effect. The stimulatory effect of fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.