Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic beta cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K+ channels and electrical spiking. The latter results in oscillatory Ca2+ influx through voltage-gated Ca2+ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca2+](cyt) and [Ca2+](mit), respectively) during these cycles is poorly understood. Importantly, the activation of Ca2+-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca2+ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary beta cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca2+ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca2+ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca2+ oscillation frequency as stable increases in [Ca2+](mit) and cytosolic ATP/ADP; (3) mitochondrial Ca2+ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation ("plasticity") and (4) the relationship between [Ca2+](cyt) and [Ca2+](mit) is essentially unaffected by changes in endoplasmic reticulum Ca2+ ([Ca2+](ER)). Our findings thus highlight new aspects of Ca2+ signalling in beta cells of relevance to the actions of both glucose and sulphonylureas.