E Van Schaftingen

Short-term control of glucokinase activity: role of a regulatory protein.

Glucokinase is one of the four hexokinases present in mammalian tissues. It is expressed in two cell types that have to respond to changes in the blood glucose concentration, the liver parenchymal cell and the β‐cells of pancreatic islets. The former are responsible for the metabolism and storage of an important part of the ingested glucose, whereas the latter secrete insulin in response to an increase in the blood glucose level. One major characteristic of glucokinase is that it has a relatively low affinity for glucose and displays positive cooperativity for this substrate, despite the fact that it is a monometic enzyme. Furthermore, unlike other hexokinases, it is not inhibited by micromolar (physiological) concentrations of glucose 6‐phosphate but by a regulatory protein that transduces the effect of fructose 6‐phosphate and of fructose 1‐phosphate. The purpose of this review is to describe these aspects of the regulation of glucokinase.— Van Schaftingen, E., Detheux, M., Veiga da Cunha, M. Short‐term control of glucokinase activity; role of a regulatory protein. FASEB J. 8: 414‐419; 1994.

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.

3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.

Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man.

Carbohydrate-deficient glycoprotein syndromes become congenital disorders of glycosylation: an updated nomenclature for CDG. First International Workshop on CDGS

During the last few years, progress in identifying the molecular defects of the carbohydrate-deficient glycoprotein syndromes has been very rapid. Up to this date, six different gene defects have been elucidated. The plethora of defects that will eventually be identified makes it indispensable to use a simple and straightforward nomenclature for this group of diseases.A group of specialists in this field met for a round-table discussion at the “First International Workshop on CDGS” in Leuven, Belgium, November 12–13, 1999, and came up with the following recommendations.1. CDG stands for “Congenital Disorders of Glycosylation”.2. The disorders are divided into groups, based on the biochemical pathway affected: group I refers to defects in the initial steps of N-linked protein glycosylation. These deficiencies affect the assembly of dolichylpyrophosphate linked oligosaccharide and/or its transfer to asparagine residues on the nascent polypeptides; group II refers to defects in the processing of protein-bound glycans or the addition or other glycans to the protein. This grouping no longer refers directly to the isoelectric focusing pattern of serum transferrins or other serum glycoproteins.3. CDG types are assigned to one of the groups and will be numbered consecutively as they are identified: Ia, Ib,...[emsp4 ], IIa, IIb,...[emsp4 ], etc. The currently distinguished types are: CDG-Ia (PMM2[emsp4 ]), CDG-Ib (MPI[emsp4 ]), CDG-Ic (ALG6[emsp4 ]), CDG-Id (ALG3[emsp4 ]), CDG-Ie (DPM1), CDG-IIa (MGAT2[emsp4 ]).4. No new designations will be made unless the genetic defect is established. Untyped cases are considered “x” cases (CDG-x) until the genetic defect is known.

l-2-Hydroxyglutaric aciduria, a disorder of metabolite repair

SummaryThe neurometabolic disorder l-2-hydroxyglutaric aciduria is caused by mutations in a gene present on chromosome 14q22.1 and encoding l-2-hydroxyglutarate dehydrogenase. This FAD-linked mitochondrial enzyme catalyses the irreversible conversion of l-2-hydroxyglutarate to alpha-ketoglutarate. The formation of l-2-hydroxyglutarate results from a side-activity of mitochondrial l-malate dehydrogenase, the enzyme that interconverts oxaloacetate and l-malate, but which also catalyses, very slowly, the NADH-dependent conversion of alpha-ketoglutarate to l-2-hydroxyglutarate. l-2-Hydroxyglutarate has no known physiological function in eukaryotes and most prokaryotes. Its accumulation is toxic to the mammalian brain, causing a leukoencephalopathy and increasing the susceptibility to develop tumours. l-2-Hydroxyglutaric aciduria appears to be the first disease of ‘metabolite repair’.

l-2-Hydroxyglutaric aciduria, a defect of metabolite repair

Summaryl-2-hydroxyglutaric aciduria is a metabolic disorder in which l-2-hydroxyglutarate accumulates as a result of a deficiency in FAD-linked l-2-hydroxyglutarate dehydrogenase, a mitochondrial enzyme converting l-2-hydroxyglutarate to α-ketoglutarate. The origin of the l-2-hydroxyglutarate, which accumulates in this disorder, is presently unknown. The oxidation–reduction potential of the 2-hydroxyglutarate/α-ketoglutarate couple is such that l-2-hydroxyglutarate could potentially be produced through the reduction of α-ketoglutarate by a NAD- or NADP-linked oxidoreductase. In fractions of rat liver cytosolic extracts that had been chromatographed on an anion exchanger we detected an enzyme reducing α-ketoglutarate in the presence of NADH. This enzyme co-purified with cytosolic l-malate dehydrogenase (cMDH) upon further chromatography on Blue Sepharose. Mitochondrial fractions also contained an NADH-linked, ‘α-ketoglutarate reductase’ which similarly co-purified with mitochondrial l-malate dehydrogenase (mMDH). Purified mMDH catalysed the reduction of α-ketoglutarate to l-2-hydroxyglutarate with a catalytic efficiency that was about 107-fold lower than that observed with oxaloacetate. For the cytosolic enzyme, this ratio amounted to 108, indicating that this enzyme is more specific. Both cMDH and mMDH are highly active in tissues and α-ketoglutarate is much more abundant than oxaloacetate and more concentrated in mitochondria than in the cytosol. As a result of this, the weak activity of mMDH on α-ketoglutarate is sufficient to account for the amount of l-2-hydroxyglutarate that is excreted by patients deficient in FAD-linked l-2-hydroxyglutarate dehydrogenase. The latter enzyme appears, therefore, to be responsible for a ‘metabolite repair’ phenomenon and to belong to the expanding class of ‘house-cleaning’ enzymes.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

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.

Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes.

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet. 19, 88-92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

Phosphoserine phosphatase deficiency in a patient with Williams syndrome.

Decreased serine levels were found in plasma and cerebrospinal fluid (CSF) of a boy with pre- and postnatal growth retardation, moderate psychomotor retardation, and facial dysmorphism suggestive of Williams syndrome. Fluorescence in situ hybridisation with an elastin gene probe indicated the presence of a submicroscopic 7q11.23 deletion, confirming this diagnosis. Further investigation showed that the phosphoserine phosphatase (EC 3.1.3.3.) activity in lymphoblasts and fibroblasts amounted to about 25% of normal values. Oral serine normalised the plasma and CSF levels of this amino acid and seemed to have some clinical effect. These data suggest that the elastin gene and the phosphoserine phosphatase gene might be closely linked. This seems to be the first report of phosphoserine phosphatase deficiency.