Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.
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Research Article|
January 16 2012
Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1
Leticia P. Roma;
Leticia P. Roma
*Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 55, B1.55.06 (UCL 55.30), Brussels B-1200, Belgium
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Jessica Duprez;
Jessica Duprez
*Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 55, B1.55.06 (UCL 55.30), Brussels B-1200, Belgium
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Hilton K. Takahashi;
Hilton K. Takahashi
*Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 55, B1.55.06 (UCL 55.30), Brussels B-1200, Belgium
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Patrick Gilon;
Patrick Gilon
*Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 55, B1.55.06 (UCL 55.30), Brussels B-1200, Belgium
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Andreas Wiederkehr;
Andreas Wiederkehr
1
†Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
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Jean-Christophe Jonas
Jean-Christophe Jonas
2
*Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 55, B1.55.06 (UCL 55.30), Brussels B-1200, Belgium
2To whom correspondence should be addressed (email jean-christophe.jonas@uclouvain.be).
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Publisher: Portland Press Ltd
Received:
October 04 2011
Revision Received:
November 01 2011
Accepted:
November 03 2011
Accepted Manuscript online:
November 03 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 441 (3): 971–978.
Article history
Received:
October 04 2011
Revision Received:
November 01 2011
Accepted:
November 03 2011
Accepted Manuscript online:
November 03 2011
Citation
Leticia P. Roma, Jessica Duprez, Hilton K. Takahashi, Patrick Gilon, Andreas Wiederkehr, Jean-Christophe Jonas; Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1. Biochem J 1 February 2012; 441 (3): 971–978. doi: https://doi.org/10.1042/BJ20111770
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