Unregulated elevation of glutamate dehydrogenase activity induces glutamine-stimulated insulin secretion: identification and characterization of a GLUD1 gene mutation and insulin secretion studies with MIN6 cells overexpressing the mutant glutamate dehydrogenase.
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.
Tanizawa
Yukio
Y
Department of Bio-Signal Analysis, Yamaguchi University Graduate School of Medicine, Ube, Japan. tanizawa@yamaguchi-u.ac.jp
Nakai
Kazuaki
K
Sasaki
Terumasa
T
Anno
Takatoshi
T
Ohta
Yasuharu
Y
Inoue
Hiroshi
H
Matsuo
Kiyosato
K
Koga
Mayumi
M
Furukawa
Susumu
S
Oka
Yoshitomo
Y
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D019556:COS Cells; D004252:DNA Mutational Analysis; D005260:Female; D005947:Glucose / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006946:Hyperinsulinism / Q000235:genetics; D007003:Hypoglycemia / Q000235:genetics; D007223:Infant; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D009154:Mutation; D010190:Pancreatic Neoplasms; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D014162:Transfection; D014407:Tumor Cells, Cultured
D001786:Blood Glucose; D007328:Insulin; D005973:Glutamine; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase; D005947:Glucose