Glucose-Dependent Modulation of Insulin Secretion and Intracellular Calcium Ions by GKA50, a Glucokinase Activator

Abstract

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca²⁺ homeostasis ([Ca²⁺]_i) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 μmol/l GKA50 was found to stimulate insulin secretion and raise [Ca²⁺]_i in the presence of glucose (2–10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 μmol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC₅₀) values for glucose were shifted by 3 mmol/l in rat islets and ∼10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca²⁺]_i (1 μmol/l GKA50) or to stimulate insulin release (30 nmol/l–10 μmol/l GKA50). At 5 mmol/l glucose, the EC₅₀ for GKA50 in MIN6 cells was ∼0.3 μmol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca²⁺]_i but not the actions of tolbutamide. Finally, the ATP-sensitive K⁺ channel agonist diazoxide (200 μmol/l) inhibited GKA50-induced insulin release and its elevation of [Ca²⁺]_i. We show that GKA50 is a glucose-like activator of β-cell metabolism in rodent and human islets and a Ca²⁺-dependent modulator of insulin secretion.