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

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (Delta psi M) in pancreatic beta-cells. This built on our previously introduced calculation of the absolute magnitude of Delta psi M in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (Delta psi P) indicator Delta psi M is a central mediator of glucose-stimulated insulin secretion in pancreatic beta-cells Delta psi M 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 Delta psi M and Delta psi P. Glucose addition caused hyperpolarization of Delta psi M and depolarization of Delta psi P. The hyperpolarization was a monophasic step increase, even in cells where the Delta psi P depolarization was biphasic. The biphasic response of Delta psi P was associated with a larger hyperpolarization of Delta psi M than the monophasic response. Analysis of the relationships between Delta psi P and Delta psi M revealed that primary dispersed beta-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with beta-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 Delta psi M and Delta psi P. Finally, we demonstrate a significant problem with using an alternative Delta psi M probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited beta-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.