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Open Access Gold as soon as Publ. Version/Full Text is submitted to ZB.
Measurement of the absolute magnitude and time courses of mitochondrial membrane potential in primary and clonal pancreatic beta-cells.
PLoS ONE 11:e0159199 (2016)
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.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Stimulated Insulin-secretion; Oxidative-phosphorylation; Glucose Sensitivity; Ca2+ Concentration; Mouse Islets; B-cells; Calcium; Atp; Metabolism; Plasma
ISSN (print) / ISBN
1932-6203
Journal
PLoS ONE
Quellenangaben
Volume: 11,
Issue: 7,
Article Number: e0159199
Publisher
Public Library of Science (PLoS)
Publishing Place
Lawrence, Kan.
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Diabetes and Obesity (IDO)