Frequency-dependent mitochondrial Ca ²⁺ accumulation regulates ATP synthesis in pancreatic β cells

Abstract

Pancreatic β 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 Ca ²⁺ influx through voltage-gated Ca ²⁺ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca ²⁺] _cyt and [Ca ²⁺] _mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca ²⁺-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca ²⁺ 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 β 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 Ca ²⁺ and cytosolic ATP/ADP ( Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca ²⁺ 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 Ca ²⁺ oscillation frequency as stable increases in [Ca ²⁺] _mit and cytosolic ATP/ADP; (3) mitochondrial Ca ²⁺ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca ²⁺] _cyt and [Ca ²⁺] _mit is essentially unaffected by changes in endoplasmic reticulum Ca ²⁺ ([Ca ²⁺] _ER). Our findings thus highlight new aspects of Ca ²⁺ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.

Electronic supplementary material

The online version of this article (doi:10.1007/s00424-012-1177-9) contains supplementary material, which is available to authorized users.