Mechanism of pure glucose-dependent insulinotropic activity of a novel imidazoline compound BL11282

Abstract

We developed a novel, pure glucose-dependent, insulinotropic imidazoline compound, BL11282, which directly affects the insulin exocytotic machinery and does not block the KATP channel activity. BL11282 does not induce insulin secretion at basal glucose concentrations, whereas it stimulates insulin secretion at an elevated glucose level. Therefore this imidazoline should not provoke hypoglycemic episodes as it has been observed for the sulfonylureas. However, so far, the detailed biochemical and pharmacological mechanisms underlying the insulinotropic effects of BL11282 are not fully established.

The overall objective of this study was to investigate signaltransduction pathways involved in the pure glucose-dependent activity of BL11282 on insulin release. Using SUR1(-/-) mice, we unambiguously confirmed the previous notion that the insulinotropic activity of BL11282 is unrelated to its interaction with ATPdependent K+ channels. We have also shown that previously described targets for imidazoline compounds like alpha2-adrenoreceptors, imidazoline I1-receptors and monomeric G-protein Rhes are not involved in the mechanisms of the insulinotropic action of BL11282.

To clarify the molecular mechanisms underlying the effects of BL11282 on insulin secretion, we have used an approach involving desensitization of beta-cells to the insulinotropic activity of BL11282 by prolonged incubation with the compound. The data obtained show that overnight pretreatment of pancreatic islets with BL11282 desensitizes the subsequent islet response to this imidazoline. Islets pretreated for the same time with efaroxan, another insulinotropic imidazoline, are unresponsive to subsequent addition of efaroxan but preserve their response to BL11282. The effect of pretreatment with BL11282 is accompanied by an increased insulinotropic response to subsequent high glucose concentration. Desensitization of islet response to BL11282 does not eliminate subsequent islet response to GLP-1, but significantly decreases the fold potentiation of insulin release by this peptide. The latter effect points to the importance of GLP-1 stimulated signal-transduction pathways for the insulinotropic activity of BL11282. Our results support the involvement of the cAMP-GEFII·Rim2 pathway in BL11282 stimulated insulin secretion. Indeed, expression of dominant negative cAMP-GEFII and Rim2 mutant proteins in MIN6 cells lead to a significant reduction in insulin secretion stimulated by the imidazoline. To further investigate this direct mechanism of BL11282 on insulin release, we turned our attention to calcium-independent PLA2 isoform iPLA2beta, which is predominantly expressed in pancreatic islets and plays an important role in insulin secretion in pancreatic islets.

Our observations indicate a deficiency in iPLA2beta isoform expression in diabetic Goto-Kakizaki (GK) rat islets compared to Wistar rat islets, this effect being in agreement with an impairment in the glucose-stimulated insulin response in GK rat islets. Pharmacological inhibition of iPLA2 and cytochrome P-450 enzymes completely abolished the insulinotropic effect of BL11282. BL11282 stimulated arachidonic acid release from the islets in the presence of high glucose concentration and this effect was fully blocked by iPLA2 inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, independent of concomitant changes in cytoplasmic free Ca2+ concentration, involves the release of arachidonic acid by iPLA2 and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway.