Membrane potential-independent effect of Diazoxide on insulin secretion
Background and aims: Glucose-induced insulin secretion is mediated by a bifurcating pathway generating “triggering” and “amplifying” signals. The standard protocol to demonstrate the amplifying effect of glucose (and other nutrient secretagogues) is to depolarize the beta cells by raising the external K+ concentration while the KATP channels are clamped open by Diazoxide to ensure the generation of the Nernst equilibrium. Any further increase in secretion which is caused by the addition of glucose is then believed to be mediated by the still incompletely understood amplifying pathway. Diazoxide, however, has been reported to have sites of action in addition to the KATP channel, which may lead to generation of spurious effects.
Methods: All experiments were performed on islets and beta cells from NMRI mice. The membrane potential was measured by the patch clamp technique using the perforated patch configuration. The cytosolic Ca2+ concentration ([Ca2+]i) of islets was measured with the Fura technique. Insulin secretion was quantified by batch perifusion of 50 islets and ELISA of the fractionated efflux. The adenine nucleotide content was measured by the luciferase method after static incubation of islets.
Results: To depolarize the beta cell plasma membrane a K+ concentration was used (15 mM KCl) which mimicked the slow-wave depolarization by glucose (19 – 20 mV) and raised [Ca2+]i to levels as produced by 20 mM glucose. This K+ concentration produced only a modest transient increase of insulin secretion. When glucose was raised from a substimulatory (5 mM) to a moderate stimulatory concentration (10 mM) in the continuing presence of 15 mM KCl, a marked increased of insulin secretion with a biphasic pattern resulted. The addition of 250µM Diazoxide abolished the stimulated secretion. This effect of Diazoxide was unexpected since the beta cell plasma membrane was still depolarized (by 20mV) by KCl and since the presence of 10 mM glucose ought to produce sufficient amplifying signals. Measuring [Ca2+]i using the same protocol showed that the level established by the combined action of 15 mM KCl and 10 mM glucose was diminished by Diazoxide to a level significantly below that established by 15 mM KCl alone. When the glucose concentration was set back to 5 mM in the continuing presence of Diazoxide, [Ca2+]i increased somewhat paradoxically to the same level as seen with 15 mM KCl alone. Compared with the presence of 10 mM glucose plus 15 mM KCl the addition of Diazoxide led to a slight decrease of the ATP/ADP ratio and an equally slight increase of the ATP/AMP ratio.
Conclusions: Diazoxide does not only ensure the establishment of the Nernst equilibrium by opening K+ channels but also affects the balance between the opposing effects (Ca2+ influx vs. internal Ca2+ sequestration) which a stimulatory glucose concentration exerts on beta cell Ca2+ transport. It may not be an appropriate tool to clarify the mechanisms of the amplifying pathway.