Aims/hypothesis. SUR1(ABCC8) −/− mice lacking functional K ATP channels are an appropriate model to test the significance of K ATP channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. Methods. Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca 2+ concentrations ([Ca 2+ ] c ) and mitochondrial membrane potential (∆Ψ) were measured using fluorescent dyes. Results. In contrast to the controls, SUR1 −/− beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca 2+ -dependent action potentials were detected.
The membrane potential (V (m)) of beta-cells oscillates at glucose concentrations between ~6 and 25 mM, i.e. burst phases with action potentials alternate with silent interburst phases generating so-called slow waves. The slow waves drive oscillations of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and insulin secretion. The length of the bursts correlates with the amount of insulin release. Thus, the fraction of plateau phase (FOPP), i.e. the percentage of time with burst activity, is an excellent marker for beta-cell function and metabolic integrity. Extracellular voltage changes of mouse islets were measured using a microelectrode array (MEA) allowing the detection of burst and interburst phases. At a non-stimulating glucose concentration (3 mM) no electrical activity was detectable while bursting was continuous at 30 mM. The glucose concentration-response (determined as FOPP) curve revealed half-maximal stimulation at 12 ± 1 mM (Hill equation fit). The signal was sensitive to K(ATP) channel modulators, e.g. tolbutamide or diazoxide. Simultaneous recordings of electrical activity and [Ca(2+)](c) revealed congruent bursts and peaks, respectively. The extracellular recordings are in perfect agreement with more time-consuming intracellular electrical recordings. The results provide a 'proof-of-principle' for detection of beta-cell slow waves and determination of the FOPP using extracellular electrodes in a MEA-based system. The method is facile and provides the capability to study the effects of modulators of beta-cell function including possible anti-diabetic drugs in real time. Moreover, the method may be useful for checking the metabolic integrity of human donor islets prior to transplantation.
Aims/hypothesis: Islets or beta cells from Sur1 −/− mice were used to determine whether changes in plasma membrane potential (V m
Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.
KATP channel activity influences beta cell Ca2+ homeostasis by regulating Ca2+ influx through L-type Ca2+ channels. The present paper demonstrates that loss of KATP channel activity due to pharmacologic or genetic ablation affects Ca2+ storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca2+ release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca2+ ATPases by cyclopiazonic acid abolished the FCCP-induced Ca2+ transient. In beta cells treated with KATP channel inhibitors FCCP elicited a significantly larger Ca2+ transient. Cyclopiazonic acid did not abolish this Ca2+ transient suggesting that non-ER compartments are recruited as additional Ca2+ stores in beta cells lacking KATP channel activity. Genetic ablation of KATP channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca2+-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca2+ evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of KATP channel activity conveys Ca2+ release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca2+ it is suggested that mitochondria are the recruited store. The change in Ca2+ sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.
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