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.

Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects after prolonged culture in a low non-stimulating glucose concentration

“…Using this probe, we found that lowering the glucose concentration from 10 to 5 mmol/l for 18 h increases thiol oxidation in the mitochondrial matrix of dispersed rat islet cells. The increase in mt-roGFP1 fluorescence ratio in G5 is unlikely to have resulted from reduced expression of glutaredoxin without changes in thiol oxidation state, as it also occurs upon acute reduction of glucose from 10 to 2 mmol/l [19]. The increase in mt-roGFP1 fluorescence ratio may also have resulted from a decrease in mitochondrial glutathione content without changes in mt-roGFP1 oxidation state, but we consider this hypothesis unlikely because islet total glutathione content increased [27] and the mitochondrial and cytosolic concentrations of GSH are similar in other cell types (reviewed by Lash [32]).…”

“…Adenoviruses Adenoviruses encoding redox-sensitive green fluorescent protein (roGFP1) and mitochondria-targeted mtroGFP1 were generated and amplified using the pAdEasy system (Stratagene, La Jolla, CA, USA), as described (electronic supplementary material [ESM] Methods) [19].…”

Mechanisms of β-cell dedifferentiation in diabetes: recent findings and future research directions

“…In addition to metabolic genes, physiological glucose stimulation improves and maintains the β-cell differentiated phenotype by modulating several stress responses and prosurvival/proapototic effectors and pathways (Van de Casteele et al 2003, Martens et al 2005, Hou et al 2008a, Bensellam et al 2009, Roma et al 2012, Sarre et al 2012. Among them, the UPR occupies a central place (Elouil et al 2007, Bensellam et al 2009).…”

A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo

“…Wolf et al (14) developed mitochondria-targeted roGFP for assessing changes of redox state, which has been used in Drosophila (15). Two other reporter genes, HyPer and mt-cpYFP, have also been developed to detect H 2 O 2 and superoxide in mitochondria, respectively (16,17). These reagents are extremely useful for real-time measurement in cultured cells as they report instant changes in ROS production and redox state, but cannot be readily used for in vivo measurements because the readouts change instantaneously.…”