Unregulated Insulin Secretion by Pancreatic Beta Cells in Hyperinsulinism/Hyperammonemia Syndrome: Role of Glutamate Dehydrogenase, ATP-Sensitive Potassium Channel, and Nonselective Cation Channel

Abstract

ABSTRACT:The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca 2ϩ firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (K ATP channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the K ATP channel, but also by opening of the NSCC. The resultant activation of the voltagegated Ca 2ϩ channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome. T he HI/HA syndrome is an autosomal dominant disorder characterized by symptomatic hypoglycemia and persistent high plasma ammonia levels (1,2). Inappropriate secretion of insulin in pancreatic beta cells and defective nitrogen metabolism in the liver are caused by defects of the gene encoding GDH. The pathogenesis of concurrent hyperinsulinism and hyperammonemia is dysregulation, in other words, "gain of function," of GDH. Missense mutations were first discovered in 15 amino acid sequences between F440 and H454 (exons 11 and 12), within the pivot helix of GDH (site 1) (3,4). Recently, another two sites exhibiting gain of function have been identified: the site 2 mutation (exons 6 and 7) in the GTP-binding region (5,6) and the site 3 mutation (exon 10) in the ␣-helix of the antenna-like structure on the other side from the hinge (7,8). Although mitochondrial GDH is found in all organisms, this antenna region does not exist in bacterial or fungal GDH. So far, only two mutations in site 3 mutations have been reported: L413V (7) and N410T (8). We previously reported two GDH mutations in Japanese patients [L413V (site 3) and G446D (site 1)] and characterized a...

Clinical Management and Gene Mutation Analysis of Children with Congenital Hyperinsulinism in South China

“…Both low blood glucose and insufficient treatment increased the risk of neurodevelopmental impairment in CHI (38). HI/HA syndrome is caused by activating mutations in the GLUD1 gene, which encodes the intramitochondrial enzyme glutamate dehydrogenase (GDH) (39). The epilepsy and developemental problems in HI/HA syndrome are thought to be a result of recurrent hypoglycaemia, chronic hyperammonaemia or decreased brain concentrations of the neurotransmitter GABA due to increased GDH activity (27).…”

Branched-chain Amino Acid Metabolon

“…The main allosteric activators are leucine and ADP, which act by destabilizing the abortive complex of GDH1. GTP is a potent inhibitor that acts through stabilization of the abortive complexes (41)(42)(43)(44). Changes in the allosteric conformation of GDH1 can be analyzed using CD spectroscopy (45,46).…”

Hyperinsulinaemic hypoglycaemia: genetic mechanisms, diagnosis and management

“…(b) Enzyme Defects I.Hyperinsulinism-Hyperammonaemia Syndrome (HI/HA): HI/HA syndrome, the second most common form of CHI, is caused by activating mutations in the GLUD1 gene, which encodes for the intramitochondrial enzyme glutamate dehydrogenase (GDH) (12,51,52,53,54,55,56). GDH activity is regulated by allosteric inhibitors (GTP) and activators (ADP and leucine).…”