Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

Akos A Gerencser,Martin D Brand,Martin Jastroch,Shona A Mookerjee

doi:10.1371/journal.pone.0159199

Abstract

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

Highlights

In healthy pancreatic β-cells insulin is secreted when elevated glucose availability increases mitochondrial energy metabolism, hyperpolarizing the mitochondrial membrane potential (ΔψM), raising the cytoplasmic ATP/ADP ratio, closing ATP-sensitive K+-channels (KATP), depolarizing the plasma membrane potential (ΔψP), activating Ca2+ entry and triggering exocytosis
The ΔψP indicator (PMPI; #R8042 FLIPR Membrane Potential Assay Explorer Kit) was from Molecular Devices (Sunnyvale, CA); Accutase, Geltrex, tetramethylrhodamine methyl ester (TMRM) and other fluorescence probes were from Life Technologies (Carlsbad, CA); zosuquidar was from MedKoo Biosciences (Chapel Hill, NC), and other fine chemicals were from Sigma-Aldrich
We modeled the effect of a ±2.4% simultaneous change in fluorescence emission of TMRM and PMPI for the duration of glucose addition on ΔψP and ΔψM (Fig 6C and 6D, diagonal lines)

Summary

Introduction

In healthy pancreatic β-cells insulin is secreted when elevated glucose availability increases mitochondrial energy metabolism, hyperpolarizing the mitochondrial membrane potential (ΔψM), raising the cytoplasmic ATP/ADP ratio, closing ATP-sensitive K+-channels (KATP), depolarizing the plasma membrane potential (ΔψP), activating Ca2+ entry and triggering exocytosis. This is the canonical or triggering pathway of glucose-stimulated insulin secretion (GSIS). Most secondary coupling factors may feedback-regulate energy metabolism, and this property is currently seriously overlooked, the regulation of ΔψM in GSIS requires further scrutiny. This paper describes the β-cell specific optimization and application of the absolute and unbiased ΔψM assay technology that will enable these questions to be addressed in the future

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Conclusion