Exposure to glibenclamide increases rat beta cells sensitivity to glucose

Abstract

An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

Figure 1

Glucose-induced insulin release in control pancreatic islets and in islets exposed for 1 h to 0.1 μmol glibenclamide (pg islet⁻¹ 30 min⁻¹). Data are presented as absolute values in the upper panel, and as per cent of maximal release in the lower panel. Results represent the mean±s.e.mean of 11 separate experiments.

Figure 2

Insulin release in response to α-KIC in control pancreatic islets and in islets exposed for 1 h to 0.1 μmol l⁻¹ glibenclamide (pg islet⁻¹ 30 min⁻¹). Results represent the mean±s.e.mean of three separate experiments.

Figure 3

Glucose-induced insulin release in control pancreatic islets and in islets exposed for 1 h to 100 μmol l⁻¹ tolbutamide (pg islet⁻¹ 30 min⁻¹). Results represent the mean±s.e.mean of three separate experiments.

Figure 4

Subcellular distribution of hexokinase activity in cytosolic and mitochondrial fractions of control rat islets and of islets exposed for 1 h to 0.1 μmol l⁻¹ glibenclamide. Results represent the mean±s.e.mean of eight separate experiments.

Figure 5

Glucose-induced insulin release in control pancreatic islets and in islets exposed for 1 h to 0.1 μmol l⁻¹ glibenclamide in the presence of 20 nmol l⁻¹ dicyclohexylcarbodiimide (DCC), an inhibitor of hexokinase binding to the outer mitochondrial membrane (pg islet⁻¹ 30 min⁻¹). Results represent the mean±s.e.mean of four separate experiments.

Figure 6

Hexokinase protein levels measured by Western blot analysis in cytosolic and mitochondrial fractions of control rat islets and of islets exposed for 1 h to 0.1 μmol l⁻¹ glibenclamide. A representative of three separate experiments is shown. In this experiment, protein aliquots of 20 μg of cytosol and of the mitochondria-enriched fraction were resolved by electrophoresis on a 12% polyacrylamide gel.