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 fructose-6-phosphate-sensitive and fructose-I-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J . Bioclzern. 179, 179-1841 was purified close to homogeneity by a procedure involving poly(ethyleneglyco1) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer.The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecularmash inhibitor.Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1 -phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.Our laboratory has recently reported the occurrence in the liver of a regulatory protein which, in the presence of Fru6P, inhibits glucokinase competitively with respect to glucose [I]. This inhibition is antagonised by FrulP, which acts competitively with Fru6P. The properties of this protein allow one to account for the fact that fructose stimulates the phosphorylation of glucose in isolated rat hepatocytes [2].Several mechanisms could conceivably account for the action of the regulatory protein on glucokinase. The regulator could cause a covalent modification of the enzyme. It could also catalyse the formation of a low-molecular-mass inhibitor, possibly from Fru6P, since this compound greatly reinforces the inhibition exerted by the regulatory protein [I]. Finally the protein could inhibit glucokinase by forming a co...

Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which glucokinase is missing. Fructose 1-phosphate stimulated glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the glucokinase activity was normalized after the same delay. The fact that there appears to be no situation in which glucokinase is expressed without regulatory protein is in agreement with the notion that the regulatory protein forms a functional entity with this enzyme.

The phosphorylation of glucose was measured by the formation of [3H]Hz0 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 pM fructose. Stimulation was maximal 5 niin 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 acylCoA 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 I-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.Glucokinase (hexokinase D) is a high K , hexokinase present in two glucose-sensitive cell types, the liver parenchymal cell and the pancreatic b-cell [l, 21. The activity of this enzyme is regulated in vivo by the intracellular concentration of glucose, which is permanently adjusted to the level of the glycemia in both cell types [3, 41. Kinetic studies have shown that unlike other hexokinases, glucokinase is not inhibited by physiological concentrations of Glc6P 111. Furthermore, although purified glucokinase has been shown to be inhibited by long-chain Fatty acyl-CoAs [5 -71, the physiological significance of this effect has not been demonstrated. Control by substrate concentration represents, therefore, the only type of short-term regulation that is currently considered to play a role under physiological conditions. The literature contains, however, some indications that the activity of glucokinase can be regulated rapidly by other means. Glucose phosphorylation, as measured by the loss of tritium from [2-3H]glucose [8], increases in isolated hepatocytes in response to the addition of 2.5 mM fructose [9] or to the presence of a potassium-rich medium [lo].The purpose of our work was to elucidate the mechanism of the fructose effect. This paper reports studies in which glucose phosphorylation was measured on freshly isolated hepatocytes by the [2-3H]glucose technique and from which we draw the tentative conclusion that the effect of fructose is mediated by Frul P. The accompanying paper [I I] describes the existence in liver of a protein that inhibits glucokinase and whose effect is strengthened by Fru6P and anta...