Exposure of rat pancreatic islets to 20 mM leucine for 24 h reduced insulin release in response to glucose (16.7 and 22.2 mM). Insulin release was normal when the same islets were stimulated with leucine (40 mM) or glyburide (1 microM). To investigate the mechanisms responsible for the different effect of these secretagogues, we studied several steps of glucose-induced insulin secretion. Glucose utilization and oxidation rates in leucine-precultured islets were similar to those of control islets. Also, the ATP-sensitive K(+) channel-independent pathway of glucose-stimulated insulin release, studied in the presence of 30 mM K(+) and 250 microM diazoxide, was normal. In contrast, the ATP-to-ADP ratio after stimulation with 22.2 mM glucose was reduced in leucine-exposed islets with respect to control islets. The decrease of the ATP-to-ADP ratio was due to an increase of ADP levels. In conclusion, prolonged exposure of pancreatic islets to high leucine levels selectively impairs glucose-induced insulin release. This secretory abnormality is associated with (and might be due to) a reduced ATP-to-ADP ratio. The abnormal plasma amino acid levels often present in obesity and diabetes may, therefore, affect pancreatic islet insulin secretion in these patients.