Redox Imaging Research Articles

Gelsemium low doses protect against serum deprivation-induced stress on mitochondria in neuronal cells

Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.

In vivo redox imaging of plasma-induced skin-inflammation in mice

Cold atmospheric plasma (CAP) generates reactive oxygen species (ROS) which induce biological effects on living cells. CAP has potential applications in medicine, but its highly reactive nature can lead to adverse skin complications. A noninvasive technique to examine redox changes in skin is needed for monitoring the treatment process. This study was conducted to develop a skin-inflammation model triggered by CAP-derived ROS and to monitor its progression noninvasively by in vivo dynamic nuclear polarization-MRI (DNP-MRI). The model was successfully developed by exposing the skin to both direct and remote modes of CAP. In vivo DNP-MRI imaging revealed faster reduction rates of TEMPOL in plasma-irradiated skin-inflammation areas, particularly in the remote mode plasma-irradiated skin. MRI revealed high-intensity areas in both the superficial and deep layers of the plasma-irradiated skin. The study highlights the potential importance of DNP-MRI in imaging skin-inflammation models and could improve the use of CAP in medical treatments.

Optical redox imaging to screen synthetic hydrogels for stem cell-derived cardiomyocyte differentiation and maturation

Optical redox imaging to screen synthetic hydrogels for stem cell-derived cardiomyocyte differentiation and maturation

Optical redox imaging to screen synthetic hydrogels for stem cell-derived cardiomyocyte differentiation and maturation.

Heart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling. Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs. We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts. Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage. We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.

Use of Optical Redox Imaging to Quantify Alveolar Macrophage Redox State in Infants: Proof of Concept Experiments in a Murine Model and Human Tracheal Aspirates Samples.

Emerging data indicate that lung macrophages (LM) may provide a novel biomarker to classify disease endotypes in bronchopulmonary dysplasia (BPD), a form of infant chronic lung disease, and that augmentation of the LM phenotype may be a potential therapeutic target. To contribute to this area of research, we first used Optical Redox Imaging (ORI) to characterize the responses to H2O2-induced oxidative stress and caffeine treatment in an in vitro model of mouse alveolar macrophages (AM). H2O2 caused a dose-dependent decrease in NADH and an increase in FAD-containing flavoproteins (Fp) and the redox ratio Fp/(NADH + Fp). Caffeine treatment did not affect Fp but significantly decreased NADH with doses of ≥50 µM, and 1000 µM caffeine treatment significantly increased the redox ratio and decreased the baseline level of mitochondrial ROS (reactive oxygen species). However, regardless of whether AM were pretreated with caffeine or not, the mitochondrial ROS levels increased to similar levels after H2O2 challenge. We then investigated the feasibility of utilizing ORI to examine macrophage redox status in tracheal aspirate (TA) samples obtained from premature infants receiving invasive ventilation. We observed significant heterogeneity in NADH, Fp, Fp/(NADH + Fp), and mitochondrial ROS of the TA macrophages. We found a possible positive correlation between gestational age and NADH and a negative correlation between mean airway pressure and NADH that provides hypotheses for future testing. Our study demonstrates that ORI is a feasible technique to characterize macrophage redox state in infant TA samples and supports further use of this method to investigate lung macrophage-mediated disease endotypes in BPD.

Quantitative Optical Redox Imaging of Melanoma Xenografts with Different Metastatic Potentials.

To develop imaging biomarkers for tumors aggressiveness, our previous optical redox imaging (ORI) studies of the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp, containing flavin adenine dinucleotide, i.e., FAD) in tumor xenografts of human melanoma associated the high optical redox ratio (ORR = Fp/(Fp + NADH)) and its heterogeneity to the high invasive/metastatic potential, without having reported quantitative results for NADH and Fp. Here, we implemented a calibration procedure to facilitate imaging the nominal concentrations of tissue NADH and Fp in the mouse xenografts of two human melanoma lines, an indolent less metastatic A375P and a more metastatic C8161. Images of the redox indices (NADH, Fp, ORR) revealed the existence of more oxidized areas (OAs) and more reduced areas (RAs) within individual tumors. ORR was found to be higher and NADH lower in C8161 compared to that of A375P xenografts, both globally for the whole tumors and locally in OAs. The ORR in the OA can differentiate xenografts with a higher statistical significance than the global averaged ORR. H&E staining of the tumors indicated that the redox differences we identified were more likely due to intrinsically different cell metabolism, rather than variations in cell density.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which inturn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later findingdemonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.

Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Optical redox imaging of ANT1-deficient muscles.

Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3-5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups (p = 0.0004 and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group (p = 0.004). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future.

Redox imaging of tumors with a novel compact and portable EPRI device and its potential for multimodal imaging

Redox imaging of tumors with a novel compact and portable EPRI device and its potential for multimodal imaging

P-272 - Synthesis of [64Cu]Cu+ complex for redox imaging and evaluation of its reactivity with reactive oxygen species

P-272 - Synthesis of [64Cu]Cu+ complex for redox imaging and evaluation of its reactivity with reactive oxygen species

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Engineering Alignment Has Mixed Effects on Human Induced Pluripotent Stem Cell Differentiated Cardiomyocyte Maturation.

The potential of human induced pluripotent stem cell differentiated cardiomyocytes (hiPSC-CMs) is greatly limited by their functional immaturity. Strong relationships exist between cardiomyocyte (CM) structure and function, leading many in the field to seek ways to mature hiPSC-CMs by culturing on biomimetic substrates, specifically those that promote alignment. However, these in vitro models have so far failed to replicate the alignment that occurs during cardiac differentiation. We show that engineered alignment, incorporated before and during cardiac differentiation, affects hiPSC-CM electrochemical coupling and mitochondrial morphology. We successfully engineer alignment in differentiating human induced pluripotent stem cells (hiPSCs) as early as day 4. We uniquely apply optical redox imaging to monitor the metabolic changes occurring during cardiac differentiation. We couple this modality with cardiac-specific markers, which allows us to assess cardiac metabolism in heterogeneous cell populations. The engineered alignment drives hiPSC-CM differentiation toward the ventricular compact CM subtype and improves electrochemical coupling in the short term, at day 14 of differentiation. Moreover, we observe the glycolysis to oxidative phosphorylation switch throughout differentiation and CM development. On the subcellular scale, we note changes in mitochondrial morphology in the long term, at day 28 of differentiation. Our results demonstrate that cellular alignment accelerates hiPSC-CM maturity and emphasizes the interrelation of structure and function in cardiac development. We anticipate that combining engineered alignment with additional maturation strategies will result in improved development of mature CMs from hiPSCs and strongly improve cardiac tissue engineering.

Effects of X-ray irradiation on the redox status of mouse colorectal tissues

The effects of X-ray irradiation on the redox status of colorectal tissues were investigated using magnetic resonance redox imaging.The pyrrolidine-ring nitroxyl contrast agent, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP), was used as a suitable redox-sensitive contrast agent for colorectal tissues. The lower abdomen of an 8-week-old female C3H/HeSlc mouse was X-ray irradiated with a single 10-Gy dose under anesthesia. X-rays were collimated through a 19-mm slit made by a pair of 5-cm-thick lead blocks. MRI experiments using CmP as the contrast agent were conducted on mice irradiated by X-rays at different times, i.e., 2‒8 h, 1, 2, 4, 8, 12, and 16 day(s) after irradiation. Dynamic datasets of multi-slice T1-weighted images were obtained by repeating scans every 20 s for 22 min. The time course of the CmP-induced T1-weighted signal in colorectal tissues was analyzed. The relationship between mitochondrial damage and redox responses was also investigated.The k1 decay rate of CmP, which mainly reflects the biological reduction of nitroxyl radicals in tissue, was significantly suppressed on day 2, increased on day 8, and then recovered to the control level. The decrease in the k1 value on day 2 indicated the inhibition of biological activity in injured tissue. Further animal studies are required to verify the relationship between the redox response of the colorectum tissue and the mitochondrial damages. This magnetic resonance redox imaging technique has the potential to visualize radio-biological responses in the tissues of living animals.

Expression of the H2O2 Biosensor roGFP-Tpx1.C169S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

Glutathione Transferases are Involved in Salicylic Acid-Induced Transcriptional Reprogramming

Salicylic acid (SA) plays a crucial role not only in defence against pathogen attacks, but also in abiotic stress responses. Recently, some key steps of SA signalling outlined the importance of redox state-dependent processes. This study explores the role of glutathione transferases (GSTs) in the transcriptional reprogramming of redox status-related genes in seven-day-old wild type and Atgst mutant Arabidopsis thaliana plants. The timing of redox changes, detected by the redox-sensitive green fluorescent protein (roGFP2), differed in wild type roots treated with 10 μM or 100 μM SA. Our results verified how the applied SA concentrations had different effect on the expression of oxidative stress- and redox-related genes, among them on the expression of AtGSTF8 and AtGSTU19 genes. Lower vitality and less negative EGSH values were specific characteristics of the Atgst mutants compared to the wild type plants throughout the experiment. Changes in the redox potential were only modest in the mutants after SA treatments. A slightly modified gene expression pattern was observed in control conditions and after 1 h of SA treatments in Atgst mutants compared to Col-0 roots. These data originating from the whole roots provide indirect evidence for the role of the investigated AtGSTF8 and AtGSTU19 isoenzymes in the transduction of the redox signal. Our results demonstrate that the investigated Arabidopsis GSTs have a role in maintaining the levels of reactive oxygen species- and redox homeostasis and are involved in transcriptional reprogramming in the roots.

Empagliflozin prevents oxidative stress induced by acute hyperglycemia in cardiac myocytes.

Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, has shown to improve cardiovascular outcomes in patients with diabetes and heart failure. Empagliflozin has also been shown to provide advantageous effects in excised hearts and isolated cardiomyocytes despite SGLT-2 expression lacking in cardiac myocytes. This suggests empagliflozin may have important off-target effects. Previous studies suggested that empagliflozin effects may include attenuation of both reactive oxygen species (ROS) and Ca/calmodulin-dependent kinase II activation, but exact signaling details remained unclear. Using transgenic mice expressing glutaredoxin 1-coupled redox-sensitive green fluorescent protein 2 (Grx1-roGFP2) sensor localized to either the cytosol or mitochondria, we tested for empagliflozin effect on oxidative stress in myocytes bathed in high-glucose (30 mmol/L) containing Tyrode's solution. To assess the changes in redox potential, we exposed each cell to H2O2 and dithiothreitol to calibrate the sensor. We found acute hyperglycemia induced significant increases in ROS over a 3-5min of perfusion, specifically in the cytosolic compartment. This is in line with previous report showing that hyperglycemia activates CaMKII by intracellular O-GlcNAcylation, then the active CaMKII promotes cytosolic ROS production via NAPDH oxidase 2 (NOX2). Importantly, cells preincubated with empagliflozin were fully protected against the hyperglycemia-induced increase in ROS. We also extended our studies on angiotensin II (AngII)-dependent ROS production because AngII activates NOX2, and the excess ROS promotes oxidation- dependent CaMKII activation. Myocytes pretreated with the selective CaMKII inhibitor autocamtide-2-relative inhibitory peptide (cell-permeable myristoylated form) were used to test the role of CaMKII in ROS production by either hyperglycemia or Ang-II. These studies could reveal whether empagliflozin acts on glucose uptake, CaMKII activation, or via direct antioxidant mechanisms. Our findings may provide a mechanistic basis for the positive effects of empagliflozin in myocytes under conditions relevant to heart disease.

Early detection of late blight in potato by whole-plant redox imaging.

Late blight caused by the oomycete Phytophthora infestans is a most devastating disease of potatoes (Solanum tuberosum). Its early detection is crucial for suppressing disease spread. Necrotic lesions are normally seen in leaves at 4 days post-inoculation (dpi) when colonized cells are dead, but early detection of the initial biotrophic growth stage, when the pathogen feeds on living cells, is challenging. Here, the biotrophic growth phase of P. infestans was detected by whole-plant redox imaging of potato plants expressing chloroplast-targeted reduction-oxidation sensitivegreen fluorescent protein (chl-roGFP2). Clear spots on potato leaves with a lower chl-roGFP2 oxidation state were detected as early as 2dpi, before any visual symptoms were recorded. These spots were particularly evident during light-to-dark transitions, and reflected the mislocalization of chl-roGFP2 outside the chloroplasts. Image analysis based on machine learning enabled systematic identification and quantification of spots, and unbiased classification of infected and uninfected leaves in inoculated plants. Comparing redox with chlorophyll fluorescence imaging showed that infected leaf areas that exhibit mislocalized chl-roGFP2 also showed reduced non-photochemical quenching and enhanced quantum PSII yield (ΦPSII) compared with the surrounding leaf areas. The data suggest that mislocalization of chloroplast-targeted proteins is an efficient marker of late blight infection, and demonstrate how it can be utilized for non-destructive monitoring of the disease biotrophic stage using whole-plant redox imaging.

Effects of selenium deficiency on biological results of X-ray and carbon-ion beam irradiation in mice.

The impact of radiation-induced hydrogen peroxide (H2O2) on the biological effects of X-rays and carbon-ion beams was investigated using a selenium-deficient (SeD) mouse model. Selenium is the active center of glutathione peroxidase (GSH-Px), and SeD mice lack the ability to degrade H2O2. Male and female SeD mice were prepared by feeding a torula yeast-based SeD diet and ultrapure water. Thirty-day survival rates after whole-body irradiation, radiation-induced leg contracture, and MRI-based redox imaging of the brain were assessed and compared between SeD and normal mice. Thirty-day lethality after whole-body 5.6 Gy irradiation with X-rays or carbon-ion beams was higher in the SeD mice than in the normal mice, while SeD did not give the notable difference between X-rays and carbon-ion beams. SeD also did not affect the maximum leg contracture level after irradiation with carbon-ion beams, but delayed the leg contraction rate. In addition, no marked effects of SeD were observed on variations in the redox status of the brain after irradiation. Collectively, the present results indicate that SeD slightly altered the biological effects of X-rays and/or carbon-ion beams. GSH-Px processes endogenous H2O2 generated through mitochondrial respiration, but does not have the capacity to degrade H2O2 produced by irradiation.

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress

BackgroundOxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes. MethodsWe constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo. ResultsHepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress. ConclusionsThis study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed. General significanceRoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.