title: Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.
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.
authors.affiliation: Buck Institute for Research on Aging, Novato, California, United States of America.|Image Analyst Software, Novato, California, United States of America.
authors.forename: Akos A
authors.initials: AA
authors.lastname: Gerencser
authors.affiliation: Buck Institute for Research on Aging, Novato, California, United States of America.|Touro University California College of Pharmacy, Vallejo, California, United States of America.
authors.forename: Shona A
authors.initials: SA
authors.lastname: Mookerjee
authors.affiliation: Buck Institute for Research on Aging, Novato, California, United States of America.
authors.forename: Martin
authors.initials: M
authors.lastname: Jastroch
authors.affiliation: Buck Institute for Research on Aging, Novato, California, United States of America.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Brand
mesh_terms: D000818:Animals; D002138:Calibration; D045744:Cell Line, Tumor; D048429:Cell Size; D002999:Clone Cells / Q000166:cytology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009840:Oligomycins / Q000494:pharmacology; D061848:Optical Imaging; D009942:Organometallic Compounds / Q000378:metabolism; D011199:Potentiometry; D051381:Rats; D017208:Rats, Wistar; D059010:Single-Cell Analysis / Q000379:methods*; D013997:Time Factors
chemical_list: D009840:Oligomycins; D009942:Organometallic Compounds; C110932:tetramethyl rhodamine ethyl ester; D005947:Glucose