Carol Buettger

Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis

Aims/hypothesis. Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia. A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking. Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release. Methods. Recombinant human glucokinase was generated in E. coli. The kcat, glucose S0.5, ATP Km, and Hill number of glucokinase were determined. Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase. A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed. This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database. It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose. Results. Large changes, varying greatly between mutants were found in nearly all variables. Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes. Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model. The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases. Conclusion/interpretation. A kinetics database of wild-type and 20 mutants of glucokinase was developed. Many kinetic differences were found for the mutants. The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations. [Diabetologia 42: 1175–1186]

Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young.

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mm). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of β-cells and hepatocytes.

Chronic effect of fatty acids on insulin release is not through the alteration of glucose metabolism in a pancreatic beta-cell line (βHC9)

Summary Hyperinsulinaemia in the fasting state and a blunted insulin secretory response to acute glucose stimulation are commonly observed in obesity associated non-insulin-dependent diabetes mellitus. Hyperlipidaemia is a hallmark of obesity and may play a role in the pathogenesis of this beta-cell dysfunction because glucose metabolism in pancreatic beta cells may be altered by the increased lipid load. We tested this hypothesis by assessing the chronic effect of oleic acid on glucose metabolism and its relationship with glucose-induced insulin release in βHC9 cells in tissue culture. Our results show: (1) A 4-day treatment with oleic acid caused an enhancement of insulin release at 0–5 mmol/l glucose concentrations while a significant decrease in insulin release occurred when the glucose level was greater than 15 nmol/l; (2) Hexokinase activity was increased and a corresponding left shift of the dose-dependency curve of glucose usage was observed associated with inhibition of glucose oxidation in oleic acid treated βHC9 cells, yet the presumed glucose-related ATP generation did not parallel the change in insulin release due to glucose; (3) The rate of cellular respiration was markedly increased in oleic acid treated βHC9 cells both in the absence of glucose and at all glucose concentrations tested. This enhanced oxidative metabolism may explain the increased insulin release at a low glucose level but is clearly dissociated from the blunted insulin secretion at high glucose concentrations. We conclude that a reduction of oxidative metabolism in pancreatic beta cells is unlikely to be the cause of the dramatic effect that high levels of non-esterified fatty acids have on glucose-induced insulin release. [Diabetologia (1997) 40: 1018–1027]

Extremes of Clinical and Enzymatic Phenotypes in Children With Hyperinsulinism Caused by Glucokinase Activating Mutations

OBJECTIVE Heterozygous activating mutations of glucokinase have been reported to cause hypoglycemia attributable to hyperinsulinism in a limited number of families. We report three children with de novo glucokinase hyperinsulinism mutations who displayed a spectrum of clinical phenotypes corresponding to marked differences in enzyme kinetics. RESEARCH DESIGN AND METHODS Mutations were directly sequenced, and mutants were expressed as glutathionyl S-transferase–glucokinase fusion proteins. Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein. RESULTS Child 1 had an ins454A mutation, child 2 a W99L mutation, and child 3 an M197I mutation. Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2. Expression of the mutant glucokinase ins454A, W99L, and M197I enzymes revealed a continuum of high relative activity indexes in the three children (26, 8.9, and 3.1, respectively; wild type = 1.0). Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation. Estimated thresholds for glucose-stimulated insulin release were more severely reduced by the ins454A than the M197I mutation and intermediate in the W99L mutation (1.1, 3.5, and 2.2 mmol/l, respectively; wild type = 5.0 mmol/l). CONCLUSIONS These results confirm the potency of glucokinase as the pancreatic β-cell glucose sensor, and they demonstrate that responsiveness to diazoxide varies with genotype in glucokinase hyperinsulinism resulting in hypoglycemia, which can be more difficult to control than previously believed.

From Clinicogenetic Studies of Maturity-Onset Diabetes of the Young to Unraveling Complex Mechanisms of Glucokinase Regulation

Glucokinase functions as a glucose sensor in pancreatic β-cells and regulates hepatic glucose metabolism. A total of 83 probands were referred for a diagnostic screening of mutations in the glucokinase (GCK) gene. We found 11 different mutations (V62A, G72R, L146R, A208T, M210K, Y215X, S263P, E339G, R377C, S453L, and IVS5 + 1G>C) in 14 probands. Functional characterization of recombinant glutathionyl S-transferase–G72R glucokinase showed slightly increased activity, whereas S263P and G264S had near-normal activity. The other point mutations were inactivating. S263P showed marked thermal instability, whereas the stability of G72R and G264S differed only slightly from that of wild type. G72R and M210K did not respond to an allosteric glucokinase activator (GKA) or the hepatic glucokinase regulatory protein (GKRP). Mutation analysis of the role of glycine at position 72 by substituting E, F, K, M, S, or Q showed that G is unique since all these mutants had very low or no activity and were refractory to GKRP and GKA. Structural analysis provided plausible explanations for the drug resistance of G72R and M210K. Our study provides further evidence that protein instability in combination with loss of control by a putative endogenous activator and GKRP could be involved in the development of hyperglycemia in maturity-onset diabetes of the young, type 2. Furthermore, based on data obtained on G264S, we propose that other and still unknown mechanisms participate in the regulation of glucokinase.

A Glucose Sensor Role for Glucokinase in Anterior Pituitary Cells

Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells.

Sulfonylurea-Binding Sites and ATP-Sensitive K+ Channels in α-TC Glucagonoma and β-TC Insulinoma Cells

α-Cells secrete glucagon in a fuel-dependent fashion. We tested the hypothesis that α-cells contain sulfonylurea- and ATP-sensitive K+ channels. We studied two clonal lines of α-TC cells (simian virus 40 T-antigen induced glucagonoma cells) and for reference purposes, similarly transformed β-TC insulinoma cells. α-TC cells each contained ∼3000 high-affinity binding sites for the sulfonylurea [3H]glyburide. Whole-cell ATP- and tolbutamide-sensitive K+ currents of α-TC and β-TC cells, relative to cell surface area, were comparable. In cell-attached membrane patches of α-TC cells, two types of K+ channels were observed. They had slope conductances of ∼63 and 33 pS when the electrode contained 151 mM K+. Tolbutamide and diazoxide decreased and enhanced, respectively, the open probability of these channels. The membrane of α-TC cells depolarized periodically. This electrical activity was inhibited by diazoxide. A physiological mixture of amino acids enhanced glucagon release, and high glucose partially inhibited this release. Tolbutamide also enhanced glucagon release, whereas diazoxide inhibited it. Thus, α-TC glucagonoma cells contain ATP-sensitive K+ channels that regulate glucagon release, yet allow inhibition of hormone release by glucose.

Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1-/-) and control mouse islets with d-[U-13C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and γ-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1-/- islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-β-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1-/- and glyburide-treated SUR1+/+ islets. Glucose oxidation and pyruvate cycling was increased in SUR1-/- islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1-/- islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1-/- islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.

Clinical Heterogeneity in Monogenic Diabetes Caused by Mutations in the Glucokinase Gene (GCK-MODY)

OBJECTIVE To evaluate the heterogeneity in the clinical expression in a family with glucokinase mature-onset diabetes of the young (GCK-MODY). RESEARCH DESIGN AND METHODS Members (three generations) of the same family presented either with overt neonatal hyperglycemia, marked postprandial hyperglycemia, or glucosuria. Homeostasis model assessment of insulin resistance (HOMAIR) and insulinogenic and disposition indexes were calculated. Oral glucose tolerance test (OGTT) results in the GCK mutation carriers from this family were compared with those from other subjects with GCK mutations in the same codon (GCK261), with other missense and other types of GCK mutations in different codons from the European MODY Consortium database (GCKm). RESULTS Mutation G261R was found in the GCK gene. During the OGTT, glucose (P = 0.02) and insulin (P = 0.009) response at 2 h as well as at the 2-h glucose increment (GCK261 versus other missense GCK mutations, P = 0.003) were significantly higher in GCK261 than in GCKm carriers. CONCLUSIONS Differing from other GCKm carriers, the glucose and insulin response to oral glucose was significantly higher in GCK261 carriers, indicating clinical heterogeneity in GCK-MODY.

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.