guanosine-triphosphate and Hypoglycemia

The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second-most common form of congenital hyperinsulinism, has been associated with dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP.. To identify the underlying genetic etiology in 2 siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation.. The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated.. A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26-aa in-frame deletion in the first repeat domain of the protein, which abolishes transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content, which likely leads to a hyperactivation of glutamate dehydrogenase in our patients.. We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.

Topics: Congenital Hyperinsulinism; Glutamate Dehydrogenase; Guanosine Triphosphate; HeLa Cells; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Mutation; Nucleotides

Mammalian glutamate dehydrogenase (GDH) has complex allosteric regulation and the loss of GTP inhibition causes the hyperinsulinism/hyperammonemia syndrome (HHS) where insulin is hypersecreted upon consumption of protein. The archetypical HHS lesion is H454Y and lies in the GTP binding pocket. To better understand the mechanism of HHS, we determined the crystal structure of H454Y. When the bovine GDH crystal structures were minimized to prepare for further computational analysis, unusually large deviations were found at the allosteric NADH binding site due to chemical sequence errors. Notably, 387 lies in an allosteric where several activators and inhibitors bind and should be lysine rather than asparagine. All structures were re-refined and the consequence of this sequence error on NADH binding was calculated using free energy perturbation. The binding free energy penalty going from the correct to incorrect sequence found is +5 kcal/mol per site and therefore has a significant impact on drug development. BROADER AUDIENCE ABSTRACT: Glutamate dehydrogenase is a key enzyme involved in amino acid catabolism. As such, it is heavily regulated in animals by a wide array of metabolites. The importance of this regulation is most apparent in a genetic disorder called hyperinsulinism/hyperammonemia (HHS) where patients hypersecrete insulin upon the consumption of protein. We determined the atomic structure of one of these HHS mutants to better understand the disease and also analyzed an allosteric regulatory site.

Topics: Allosteric Regulation; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperinsulinism; Hypoglycemia; Models, Molecular; Mutant Proteins; Mutation; Protein Conformation

Mitochondrial GTP (mtGTP)-insensitive mutations in glutamate dehydrogenase (GDH(H454Y)) result in fasting and amino acid-induced hypoglycemia in hyperinsulinemia hyperammonemia (HI/HA). Surprisingly, hypoglycemia may occur in this disorder despite appropriately suppressed insulin. To better understand the islet-specific contribution, transgenic mice expressing the human activating mutation in β-cells (H454Y mice) were characterized in vivo. As in the humans with HI/HA, H454Y mice had fasting hypoglycemia, but plasma insulin concentrations were similar to the controls. Paradoxically, both glucose- and glutamine-stimulated insulin secretion were severely impaired in H454Y mice. Instead, lack of a glucagon response during hypoglycemic clamps identified impaired counterregulation. Moreover, both insulin and glucagon secretion were impaired in perifused islets. Acute pharmacologic inhibition of GDH restored both insulin and glucagon secretion and normalized glucose tolerance in vivo. These studies support the presence of an mtGTP-dependent signal generated via β-cell GDH that inhibits α-cells. As such, in children with activating GDH mutations of HI/HA, this insulin-independent glucagon suppression may contribute importantly to symptomatic hypoglycemia. The identification of a human mutation causing congenital hypoglucagonemic hypoglycemia highlights a central role of the mtGTP-GDH-glucagon axis in glucose homeostasis.

Topics: Amino Acids; Animals; Glucagon; Glucagon-Secreting Cells; Glucose Clamp Technique; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mice; Mice, Transgenic; Mitochondria; Mutation; Syndrome

Topics: Amino Acids; Animals; Glucagon; Glucagon-Secreting Cells; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mitochondria

Topics: Brain Damage, Chronic; Child; Dietary Proteins; Enzyme Activation; Glucose Transporter Type 1; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Leucine; Liver; Mutation, Missense; Neurologic Examination; Pancreas

Hyperinsulinism-hyperammonaemia syndrome (HHS) is a rare cause of congenital hyperinsulinism, due to missense mutations in the GLUD1 gene, resulting in glutamate dehydrogenase (GDH) overactivity. The aim of this study was to document the spectrum of neurological disturbances associated with HHS and to identify possible phenotype-genotype correlations. We retrospectively analyzed the neurological outcomes of 22 consecutive patients (12 males, 10 females) aged from 18 months to 40 years and diagnosed with HHS. We analyzed demographic and clinical features and neuroradiological, biochemical, and genetic findings. Fourteen patients had childhood-onset epilepsy. Learning disability was found in 17 patients. Two patients had pyramidal involvement and one had generalized dystonia. Seizures were observed in 11 of 19 patients with documented GLUD1 mutations, and nine of these 11 patients had a mutation in the guanosine triphosphate (GTP) binding site. Our data demonstrate that neurological disorders in HHS are more frequent than previously thought and might suggest that mutations in the GTP binding site of GDH could be associated with more frequent epilepsy.

Topics: Adolescent; Adult; Alleles; Brain; Brain Damage, Chronic; Child; Child, Preschool; DNA Mutational Analysis; Electroencephalography; Enzyme Activation; Epilepsies, Myoclonic; Epilepsy, Absence; Epilepsy, Generalized; Epilepsy, Tonic-Clonic; Female; Genotype; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Infant; Liver; Magnetic Resonance Imaging; Male; Mutation, Missense; Neurologic Examination; Pancreas; Phenotype; Retrospective Studies; Young Adult

Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to alpha-ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8--25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic beta-cells.

Topics: Adenosine Diphosphate; Animals; Blood Glucose; COS Cells; DNA Mutational Analysis; Female; Glucose; Glutamate Dehydrogenase; Glutamine; Guanosine Triphosphate; Humans; Hyperinsulinism; Hypoglycemia; Infant; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Mutation; Pancreatic Neoplasms; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Transfection; Tumor Cells, Cultured