期刊
BIOCHEMICAL JOURNAL
卷 441, 期 -, 页码 971-978出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20111770
关键词
glucose; hydrogen peroxide; HyPer; mitochondrion; mt-AlpHi; oxidative stress; pancreatic beta-cell; redox-sensitive green fluorescent protein (roGFP); SypHer
资金
- Fonds de la Recherche Scientifique Medicale (Belgium) [3.4516.09]
- Belgian Science Policy and the Societe Francophone du Diabete [P6/42]
- F.R.I.A. (Fonds pour la formation a la Recherche dans l'Industrie et dans l'Agriculture), (Belgium)
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, beta-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 beta-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in beta-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 beta-cells.
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