Glucose and NAADP trigger elementary intracellular β-cell Ca2+ signals

Abstract

Pancreatic β-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in β-cells leads to closure of cell membrane ATP-gated potassium (K_ATP) channels, plasma membrane depolarisation, Ca²⁺ influx, and finally the exocytosis of insulin granules. However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the K_ATP channel alone may not be sufficient to explain all β-cell responses to glucose elevation. We have previously proposed that NAADP-evoked Ca²⁺ release is an important step in stimulus-secretion coupling in pancreatic β-cells. Here we show using total internal reflection fluorescence (TIRF) microscopy that glucose as well as the Ca²⁺ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in β-cells, lead to highly localised elementary intracellular Ca²⁺ signals. These were found to be obscured by measurements of global Ca²⁺ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). Building on our previous work demonstrating that NAADP-evoked Ca²⁺ release is an important step in stimulus-secretion coupling in pancreatic β-cells, we provide here the first demonstration of elementary Ca²⁺ signals in response to NAADP, whose occurrence was previously suspected. Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca²⁺ signalling events, inositol trisphosphate (IP₃) receptor mediated blips, and ryanodine receptor mediated quarks. We propose that a mechanism based on these highly localised intracellular Ca²⁺ signalling events mediated by NAADP may initially operate in β-cells when they respond to elevations in blood glucose.