Mutations of glutamate dehydrogenase cause the hyperinsulinism/hyperammonemia syndrome by desensitizing glutamate dehydrogenase to allosteric inhibition by GTP. Normal allosteric activation of glutamate dehydrogenase by leucine is thus uninhibited, leading us to propose that children with hyperinsulinism/hyperammonemia syndrome will have exaggerated acute insulin responses to leucine in the postabsorptive state. As hyperglycemia increases beta-cell GTP, we also postulated that high glucose concentrations would extinguish abnormal responsiveness to leucine in hyperinsulinism/hyperammonemia syndrome patients. After an overnight fast, seven hyperinsulinism/hyperammonemia syndrome patients (aged 9 months to 29 yr) had acute insulin responses to leucine performed using an iv bolus of L-leucine (15 mg/kg) administered over 1 min and plasma insulin measurements obtained at -10, -5, 0, 1, 3, and 5 min. The acute insulin response to leucine was defined as the mean increase in insulin from baseline at 1 and 3 min after an iv leucine bolus. The hyperinsulinism/hyperammonemia syndrome group had excessively increased insulin responses to leucine (mean +/- SEM, 73 +/- 21 microIU/ml) compared with the control children and adults (n = 17) who had no response to leucine (1.9 +/- 2.7 microU/ml; P < 0.05). Four hyperinsulinism/hyperammonemia syndrome patients then had acute insulin responses to leucine repeated at hyperglycemia (blood glucose, 150-180 mg/dl). High blood glucose suppressed their abnormal baseline acute insulin responses to leucine of 180, 98, 47, and 28 microU/ml to 73, 0, 6, and 19 microU/ml, respectively. This suppression suggests that protein-induced hypoglycemia in hyperinsulinism/hyperammonemia syndrome patients may be prevented by carbohydrate loading before protein consumption.