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

Roma, Leticia P;Duprez, Jessica;Takahashi, Hilton;Gilon, Patrick;Jonas, Jean-Christophe;et.al.
(2012) Biochemical Journal — Vol. 441, n° 3, p. 971-978 (2012)

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Abstract
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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Roma, L. P., Duprez, J., Takahashi, H., Gilon, P., Wiederkehr, A., & Jonas, J.-C. (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. Biochemical Journal, 441(3), 971-978. https://doi.org/10.1042/BJ20111770 (Original work published 2012)