Stimulation of insulin secretion independently from changes in cytosolic free Ca²+-concentration : studies with imidazolines and inositol polyphosphates

Insulin secretion from the islets of Langerhans is regulated by changes in glucose concentration. Glucose stimulates secretion by two mechanisms. First, it increases cytosolic free Ca2+ concentration ([Ca2+]i) by inhibition of ATP-dependent K+-channels (KATP) and induction of membrane depolarization. Second, glucose directly promotes insulin exocytosis. A number of other physiological and pharmacological stimuli, which modulate insulin secretion, can also influence either one or both of the two mentioned pathways. This study concentrates on mechanisms by which insulinotropic agents stimulate insulin exocytosis. For this purpose we have used two groups of compounds, imidazoline derivatives and inositol polyphosphates. The imidazoline compound, RX871024, stimulated insulin secretion through interaction with several molecular targets. First, the compound inhibited activity of KATP-channels as well as voltage-gated K+-channels, which in turn led to membrane depolarization and increase in [Ca2+]i, Elevated levels of [Ca2+]i stimulated insulin exocytosis. Second, RX871024 induced an increase in insulin secretion in cells with clamped [Ca2+]i, This effect required high both ATP and Ca2+ concentrations inside the cell. Third, the compound increased diacylglycerol (DAG) concentration in the islets. Finally, inhibition of protein kinase C (PKC) and cytochrome P-450 abolished the insulinotropic effect of RX871024. We interpreted these findings as indicating that the direct effect of RX871024 on insulin exocytosis was at least in part mediated by rise in DAG concentration, by the PKC inhibitor, and InsP6 also stimulated PKC activity in HIT T15 cell homogenate which then activated PKC and increased generation of arachidonic acid (AA) metabolites. Both PKC and AA pathway activation resulted in potentiation of insulin secretion. Comparison of the effects of RX871024 and the classical antidiabetic agent glibenclamide on insulin secretion demonstrated that the imidazoline exerted stronger stimulation of insulin secretion than glibenclamide. RX871024 also produced a potent increase in insulin secretion in islets from diabetic GK rats. In non-diabetic and diabetic rats, as well as non-diabetic humans, insulinotropic activity of RX871024 was highly dependent on glucose concentration and was stronger than that of glibenclamide. Inositol polyphosphates: myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5) and myoinositol 1,2,3,4,5,6-hexakisphosphate (InsP6) stimulated basal (Ca2+-independent) insulin exocytosis in permeabilized insulin secreting HIT T15 cells. The effect of inositol polyphosphates was observed only in the presence of low free Ca2+ concentrations (30 nM) as InsP6 did not potentiate Ca2+-induced insulin exocytosis. However, when added before the concentration of Ca2+ was increased, InsP6 exerted a potent priming effect on Ca2+-induced insulin exocytosis. The effects of InsP6 on insulin exocytosis have been blocked. Thus, stimulation of Ca2+-independent insulin exocytosis and priming of Ca2+-dependent insulin exocytosis by inositol polyphosphates are mediated through activation of PKC. In addition to its effects on exocytosis, InsP6 increased rates of endocytosis in HIT T15 cells at both basal and elevated free Ca2+ concentrations. It is concluded that stimulation of protein kinases with subsequent phosphorylation of cellular proteins is responsible for the effects of imidazolines and inositol polyphosphates on insulin secretion. Compounds with effects on transport and fusion of insulin containing granules are therefore potential candidates for treatment of type 2 diabetes