Identification of the molecular dysfunction caused by glutamate dehydrogenase S445L mutation responsible for hyperinsulinism/hyperammonemia.

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.