Glucose and NAADP trigger Elementary Intracellular Ca 2+ Signals in Pancreatic β -Cells

PM Heister, T Powell, A Galione. University of Oxford, Oxford, UK

Pancreatic beta cells release insulin upon rise in blood glucose. The exact mechanism of this stimulus-secretion coupling, and its failure in diabetes mellitus type 2, remain to be elucidated. The consensus model, as well as currently prescribed anti-diabetic drugs, are based around the observation that glucose-triggered ATP production in the beta cell leads to closure of cell membrane KATP channels, depolarisation, calcium influx, and exocytosis (1; 4). However it has been demonstrated by the activation and inactivation of this pathway using genetic and pharmacological means that other mechanisms are likely to be involved (5; 10). Here we show using total internal reflection fluorescence (TIRF) microscopy (2) that glucose as well as novel calcium mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to be present in beta cells (6; 7), lead to elementary intracellular calcium signals. Optical quantal analysis of these reveals a unitary event amplitude equivalent to that of known elementary calcium signals IP3 receptor mediated blips (8; 9) and ryanodine receptor mediated quarks (3). This suggests that an alternative stimulus-secretion mechanism based on intracellular calcium signalling via NAADP may be at work in beta cells when they react to glucose.

Figure 1 Intensity profiles of a β-cell cluster (a) at baseline, (b) in the presence of an elementary calcium event, (c) during global calcium influx. (d) Representative trace of a β-cell showing calcium events triggered by NAADP-AM. Maximum intensity change after normalising to baseline is plotted against time. Red line denotes baseline average. Extracellular calcium was re-admitted at the end of the experiment; leading to a global calcium response. (e) Quantal analysis of elementary calcium events. Frequency histogram of event amplitude across areas of interest within a single representative cell (n=51). Note: Histogram was cropped for better resolution (maximum intensity bin is 2.09-2.095). The peaks of the putative modal distribution were selected to fit a quintuple poly-Gaussian function (sum of five Gaussian functions with means, SDs: 0.143, 0.007; 0.171, 0.008; 0.201, 0.0089; 0.235, 0.0092; 0.270, 0.0103, respectively). The putative unitary event amplitude thus lies at ∼0.03 ΔF/F0.

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