Hexose Monophosphate Shunt (Pentose Phosphate Cycle)
Glucose Metabolism

Past contributions of the group

The group has a long-standing interest in the regulation of carbohydrate metabolism and in its inborn errors of metabolism. Previous important achievements have been the discovery of fructose-2,6-bisphosphate (in 1980—the laboratory was still headed by Prof. Henri-Géry Hers, who retired in 1988) and of glucokinase regulatory protein. In the field of inborn errors of metabolism, the group has also contributed the cloning of a glucose-6-phosphate translocase, mutated in glycogen storage disease type Ib and has —in collaboration with Prof J. Jaeken, University of Leuven— identified phosphomannomutase deficiency as the most frequent cause of CDG (congenital disorders of glycosylation). Defects of serine synthesis have also been identified in collaboration with J. Jaeken.

Fructose-2,6-bisphosphate
Role of fructose 2,6-bisphosphate in the control of glycolysis and gluconeogenesis.

Role of fructose 2,6-bisphosphate in the control of glycolysis and gluconeogenesis.

Fructose-2,6-bisphosphate is a powerful stimulator of PFK1 in animals and fungi, of plant PPi-PFK and of trypanosomatid pyruvate kinase
The Structure of Fructose-2,6-bisphosphate

The Structure of Fructose-2,6-bisphosphate

The discovery of fructose 2,6-bisphosphate stems from studies on the mechanism by which glucagon stimulates gluconeogenesis and inhibits glycolysis in liver. Flux measurements and assays of the concentration of intracellular metabolites had shown that glucagon decreases the flux through 6-phosphofructo-1-kinase (PFK1). Further work showed that this is due to the fact that glucagon causes the disappearance of a low-molecular weight stimulator of PFK1 [1]. This stimulator behaved as a non-nucleotidic, non-reducing bisphosphate ester, which was rapidly destroyed in dilute acid at room temperature, and gave then rise to stoichiometric amounts of fructose 6-phosphate and Pi [2]. These properties indicated that the stimulator was fructose 2,6-bisphosphate, which was soon confirmed by chemical synthesis of this “new” phosphate ester.

Fructose-2,6-bisphosphate is not only a potent stimulator of phosphofructokinase but also an inhibitor of fructose-1,6-bisphosphatase. In liver, its synthesis by 6-phosphofructo 2-kinase (PFK2) and hydrolysis by fructose 2,6-bisphosphatase (FBPase2) are controlled by phosphorylation of the bifunctional enzyme PFK2-FBPase 2 by cyclic AMP-dependent protein kinase. In trypanosomatids, fructose-2,6-bisphosphate stimulates pyruvate kinase rather than PFK1.
Key publications of the lab in relation with fructose 2,6-bisphosphate

[1] Van Schaftingen E, Hue L, Hers HG. Control of the fructose-6-phosphate/fructose 1,6-bisphosphate cycle in isolated hepatocytes by glucose and glucagon. Role of a low-molecular-weight stimulator of phosphofructokinase. Biochem J. 1980 192, 887-95.

[2] Van Schaftingen E, Hue L, Hers HG. Fructose 2,6-bisphosphate, the probably structure of the glucose- and glucagon-sensitive stimulator of phosphofructokinase. Biochem J. 1980 15, 192, 897-901

[3] Van Schaftingen E, Hers HG. Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-biphosphate. Proc Natl Acad Sci U S A. 1981, 78, 2861-3.

[4] Van Schaftingen E, Davies DR, Hers HG. Inactivation of phosphofructokinase 2 by cyclic AMP - dependent protein kinase. Biochem Biophys Res Commun. 1981,

[5] Van Schaftingen E, Davies DR, Hers HG. Fructose-2,6-bisphosphatase from rat liver. Eur J Biochem. 1982, 124, 143-9

[6] Van Schaftingen E, Lederer B, Bartrons R, Hers HG. A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate. Eur J Biochem. 1982, 129, 191-5

[7] Van Schaftingen E, Opperdoes FR, Hers HG. Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate. Eur J Biochem. 1985, 153, 403-6.

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Glucokinase regulatory protein

The control of glucokinase activity by its regulatory protein and by metabolites.

The control of glucokinase activity by its regulatory protein and by metabolites.
The regulatory protein inhibits glucokinase; its effect is reinforced by fructose 6-phosphate and antagonized by fructose 1-phosphate. Fructose can therefore stimulate the phosphorylation of glucose by alleviating the inhibition exerted by the regulatory protein.

Mechanism of action of the regulatory protein of glucokinase

Mechanism of action of the regulatory protein of glucokinase

The regulatory protein exists under two different conformations, one ® able to bind fructose 6-phosphate and glucokinase, and the other (R’), able to bind fructose 1-phosphate but not glucokinase. The glucokinase-regulatory protein complex is inactive. In the absence of metabolite, the R’ form predominates. Fructose 6-phosphate shifts the equilibrium towards R, hence reinforcing inhibition. Fructose 1-phosphate sequesters the R’ form and has therefore the opposite effect.

Glucokinase is an enzyme that displays a low affinity for glucose, and can therefore play the role of “glucose sensor” in liver and in ß-cells of pancreatic islets. In liver, the activity of this enzyme is controlled not only by the glucose concentration, but also by a fructose 6-phosphate- and fructose-1-phosphate-sensitive protein, termed ‘regulatory protein of glucokinase’ (GKRP). The latter was identified while trying to understand the mechanism by which fructose stimulates the phosphorylation of glucose in liver [1,2]. Glucokinase regulatory protein behaves as an inhibitor of glucokinase in the presence of fructose-6-phosphate, and this inhibition is suppressed by fructose-1-phosphate, accounting for the effect of fructose to stimulate glucose phosphorylation. Both proteins form a one-to-one complex in the presence of fructose 6-phosphate, but dissociate in the presence of fructose 1-phosphate [3]. GKRP is distantly homologous to sugar phosphate-isomerases and binds both fructose 6-phosphate and fructose 1-phosphate to a single site [9]. Work by other groups has shown that GKRP controls the subcellular localization of glucokinase.

Selected publications of the lab

[1] Van Schaftingen E, Vandercammen A. Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes. Eur J Biochem. 1989, 179, 173-7.

[2] Van Schaftingen E. A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem. 1989, 179, 179-84.

[3] Vandercammen A, Van Schaftingen E. The mechanism by which rat liver glucokinase is inhibited by the regulatory protein. Eur J Biochem. 1990, 191, 483-9.

[4] Van Schaftingen E, Davies DR. Fructose administration stimulates glucose phosphorylation in the livers of anesthetized rats. FASEB J. 1991, 5, 326-30.

[5] Detheux M, Vandekerckhove J, Van Schaftingen E. Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase. FEBS Lett. 1993, 321, 111-5.

[6] Vandercammen A, Van Schaftingen E. Species and tissue distribution of the regulatory protein of glucokinase. Biochem J. 1993, 294, 551-6.

[7] Veiga-da-Cunha M, Courtois S, Michel A, Gosselain E, Van Schaftingen E. Amino acid conservation in animal glucokinases. Identification of residues implicated in the interaction with the regulatory protein. J Biol Chem. 1996, 271, 6292-7.

[8] Moukil MA, Veiga-da-Cunha M, Van Schaftingen E. Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose. Diabetes. 2000, 49, 195-201.

[9] Veiga-da-Cunha M, Van Schaftingen E. Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase. J Biol Chem. 2002, 277, 8466-73.

[10] Veiga-da-Cunha M, Delplanque J, Gillain A, Bonthron DT, Boutin P, Van Schaftingen E, Froguel P. Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians. Diabetologia. 2003, 46, 704-11.

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Glucose 6-phosphate translocase

Glucose-6-phosphatase is the enzyme that catalyzes the last step of glycogenolysis and gluconeogenesis in liver and kidneys, i.e. the hydrolysis of glucose 6-phosphate to inorganic phosphate and free glucose. Its genetic deficiency causes glycogen storage disease type I, a disease in which glycogen accumulation in liver and kidneys, hypoglycemia and accumulation of lactic acid in blood are observed. Glucose 6-phosphatase, which is known to be associated with the endoplasmic reticulum, was postulated by Arion et al. (1975) to consist of a hydrolase whose catalytic site faces the lumen of the organelle and of translocases required for the transport of glucose 6-phosphate, Pi and glucose. The cDNA of the hydrolase was cloned by Shelly et al., 1993 and shown to be mutated in the patients with glycogen storage disease type Ia (deficiency in the catalytic component). Our group cloned the glucose 6-phosphate translocase in 1997 and showed that it is mutated in patients with glycogen storage disease type Ib [1,2], a disease in which the classical metabolic symptoms of glucose 6-phosphatase deficiency are associated with neutropenia and neutrophil dysfunction. Other work provided additional evidence for the fact that glucose 6-phosphate must enter the endoplasmic reticulum before being hydrolysed by glucose 6-phosphatase or oxidized by hexose-6-phosphate dehydrogenase.