title: 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.
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.
authors: 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.
authors: Leticia P
authors: LP
authors: Roma
authors: Jessica
authors: J
authors: Duprez
authors: Hilton K
authors: HK
authors: Takahashi
authors: Patrick
authors: P
authors: Gilon
authors: Andreas
authors: A
authors: Wiederkehr
authors: Jean-Christophe
authors: JC
authors: Jonas
mesh_terms: D000818:Animals; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050417:Insulin-Secreting Cells / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D005978:Glutathione