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Together, these results suggest a role for endogenous endocannabinoid signalling in regulation of endocrine secretion in the human pancreas.

In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m 2 , and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, Cpeptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S 0.5 (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k cat was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant.

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]

Glucokinase (GK) is a glycolytic key enzyme that functions as a glucose sensor in the pancreatic ␤-cell, where it governs glucose-stimulated insulin secretion (GSIS). Heterozygous inactivating mutations in the glucokinase gene (GCK) cause a mild form of diabetes (maturityonset diabetes of the young [MODY]2), and activating mutations have been associated with a mild form of familial hyperinsulinemic hypoglycemia. We describe the first case of severe persistent hyperinsulinemic hypoglycemia due to a "de novo" mutation in GCK (Y214C). A baby girl presented with hypoglycemic seizures since the first postnatal day as well as with inappropriate hyperinsulinemia. Severe hypoglycemia persisted even after treatment with diazoxide and subtotal pancreatectomy, leading to irreversible brain damage. Pancreatic histology revealed abnormally large and hyperfunctional islets. The mutation is located in the putative allosteric activator domain of the protein. Functional studies of purified recombinant glutathionyl Stransferase fusion protein of GK-Y214C showed a sixfold increase in its affinity for glucose, a lowered cooperativity, and increased k cat . The relative activity index of GK-Y214C was 130, and the threshold for GSIS predicted by mathematical modeling was 0.8 mmol/l, compared with 5 mmol/l in the wild-type enzyme. In conclusion, we have identified a de novo GCK activating mutation that causes hyperinsulinemic hypoglycemia of exceptional severity. These findings demonstrate that the range of the clinical phenotype caused by GCK mutations varies from complete insulin deficiency to extreme hyperinsulinemia. Diabetes

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Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.