We examined the effect of chronic hyperglycemia on phosphoinositide hydrolysis and insulin secretion in isolated perifused rat islets. Rats were infused for 44 h with 40% dextrose in order to raise and maintain the plasma glucose concentration at 350 mg/dl. Control animals were infused with equiosmolar amounts of mannitol. In vivo insulin secretion and rats of glucose disposal were monitored throughout the study. At the end of the infusion, islets were collagenase isolated, and phosphoinositide (PI) hydrolysis (assessed by measuring the increment in [3H]inositol efflux as well as labeled inositol phosphates) and insulin output in response to a 20-mM glucose challenge were quantitated. Plasma insulin concentration and in vivo glucose disposal rates decreased significantly, by 47% and 35% respectively, after 6-8 h of hyperglycemia. In islets perifused immediately after isolation, prior in vivo hyperglycemia markedly altered the pattern of insulin output in response to 20-mM glucose challenge. Compared to mannitol infusion, 20 mM glucose stimulation resulted in an exaggerated first phase insulin secretory response (1121 +/- 88 vs. 467 +/- 75 pg/islets.min) and a blunted second phase insulin secretory response (392 +/- 90 vs. 1249 +/- 205 pg/islet.min). In islets prelabeled with myo-[2-3H]inositol for 2 h, PI hydrolysis, particularly [3H]inositol efflux in response to glucose stimulation was also reduced (0.28 +/- 0.03%/min) compared to that in mannitol-infused animals (0.53 +/- 0.08%/min). Two hours of preincubation in a low glucose medium (2.75 mM) were able to completely reverse the islet defect in both PI hydrolysis and insulin secretion. Our results demonstrate that chronic in vivo hyperglycemia impairs PI hydrolysis in perifused rat islets and suggest that this defect accounts in part for the abnormal pattern of glucose-induced insulin secretion.