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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.
MedLine Citation:
PMID:  22050124     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Using the 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. Here, we used the ratiometric fluorescent proteins HyPer and roGFP1 targeted to mitochondria (mt-HyPer/mt-roGFP1) to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration (mt-HyPer) and glutathione redox state (mt-roGFP1) 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 hydrogen peroxide, 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, 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 hydrogen peroxide in β-cells. In contrast, mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference by pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.
Authors:
Leticia P Roma; Jessica Duprez; Hilton K Takahashi; Patrick Gilon; Andreas Wiederkehr; Jean-Christophe Jonas
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-11-3
Journal Detail:
Title:  The Biochemical journal     Volume:  -     ISSN:  1470-8728     ISO Abbreviation:  -     Publication Date:  2011 Nov 
Date Detail:
Created Date:  2011-11-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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