Prolonged exposure of islets to fatty acids results in a lowered glucose set-point for insulin secretion. We examined the mechanism in islets cultured for 24 h with 0.25 mmol/l palmitate. As expected, insulin secretion at 2.8 and 8.3 mmol/l glucose was increased in the palmitate-treated islets as opposed to no change at 27.7 mmol/l glucose. Co-culturing with 0.05 microgram/ml Triacsin C, an inhibitor of long chain acyl-CoA synthetase, blocked this effect. Glucose utilization and oxidation showed the same pattern as insulin secretion, with the step-up for both measurements being fully manifest at 2.8 mmol/l glucose. Glucokinase Km and Vmax measured in islet extracts were unaffected by the palmitate. In contrast, hexokinase Vmax was increased by 25-35% in both the cytoplasmic and mitochondrial-bound pools. Our data suggest prolonged exposure to fatty acids increased beta-cell hexokinase activity, thereby modifying the kinetics of glucose entry into the metabolic pathway and glucose-induced insulin secretion. The cellular mediator is likely an increased level of long chain fatty acyl-CoA esters.