Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.
Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.
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DOI：
10.1371/journal.pone.0159199
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Brand MD
中科院分区：
文献类型：
--
作者：
Gerencser AA;Mookerjee SA;Jastroch M;Brand MD
本研究的目的是简化、改进和验证胰腺β细胞线粒体膜电位（ΔψM）的定量测量。这建立在我们之前介绍的完整细胞中ΔψM绝对大小的计算基础上，使用四甲基罗丹明甲酯的非猝灭模式荧光的延时成像和双氧醇质膜电位（ΔψP）指示器。ΔψM是胰腺β细胞中葡萄糖刺激胰岛素分泌的中心介质。ΔψM位于细胞能量产生和需求的十字路口，因此精确测定其大小是研究这些过程如何在胰岛素分泌中相互作用的有价值的工具。分散的胰岛细胞培养允许对ΔψM和ΔψP的细胞间异质性进行细胞类型特异性的单细胞观察。葡萄糖的加入引起ΔψM的超极化和ΔψP的去极化。即使在ΔψP去极化为双相的细胞中，超极化也是单相的阶跃增加。与单相反应相比，ΔψP的双相反应与ΔψM的更大的超极化有关。对ΔψP和ΔψM之间关系的分析表明，原代分散β-细胞对葡萄糖的反应是异质性的，由能量代谢的不同激活驱动。校准的敏感性分析与β-细胞在线粒体数量上具有实质性的细胞间变化是一致的，并且预计这不会损害ΔψM和ΔψP相对变化测定的准确性。最后，我们演示了使用另一种ΔψM探针罗丹明123的一个重要问题。在葡萄糖刺激和寡霉素抑制的β-细胞中，罗丹明123试验的原理被破坏，导致误导性的结论。
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.