20,986 research outputs found

    Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress

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    In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H2O2) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings

    The role of reactive oxygen species in insulin resistance

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    Insulin resistance and type II diabetes mellitus are major public health issues in the U.S.; more specifically insulin resistance is strongly correlated with obesity. Multiple factors influence insulin resistance such as hyperglycemic conditions and increased levels of reactive oxygen species (ROS). As oxygen radicals accumulate in adipocytes from increased glucose oxidation, they interfere with insulin signaling and affect glucose uptake. To determine if insulin sensitivity can be prolonged with the aid of antioxidants, adipocytes were maintained in a high glucose medium supplemented with antioxidants ascorbic acid or &alpha-tocopherol and then tested for insulin sensitivity. Our studies reveal that ROS levels fluctuate in insulin resistant adipocytes (Day 11-21). We show for the first time that there is a decrease in ROS levels at Day 13, which then significantly increase at Day 15 and this trend continues every other day till Day 21. At Day 10 and 15 the ROS levels are high, but adipocytes have high GLUT4 expression at Day 10 and decreased expression on Day 15. By Day 15 there is increased phosphorylation of AKT but no change in IRS-1 phosphorylation. The levels of ROS were significantly decreased with the application of antioxidants. With decreased ROS levels there was overall increased phosphorylation of IRS-1 and AKT. The lipid content and distribution was not affected by ROS levels. In conclusion, the development of insulin resistance is effected by ROS, however the data indicates that there is a mechanism independent of AKT, or a target downstream of AKT that is affected and leading to the development of insulin resistance. ROS levels also regulate the expression of GLUT4 and thus GLUT4-mediated glucose uptake resulting in resistance to insulin signaling. Insulin resistance is affected by ROS levels differently if it is added exogenously, or if it is amassed endogenously. Identification of factors that interfere with insulin signaling and thus type II diabetes, like ROS levels, can lead to the development of possible therapeutic treatments

    Escaping death: Mitochondrial redox homeostasis in cancer cells

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    Reactive oxygen species (ROS) are important signaling molecules that act through the oxidation of nucleic acids, proteins, and lipids. Several hallmarks of cancer, including uncontrolled proliferation, angiogenesis, and genomic instability, are promoted by the increased ROS levels commonly found in tumor cells. To counteract excessive ROS accumulation, oxidative stress, and death, cancer cells tightly regulate ROS levels by enhancing scavenging enzymes, which are dependent on the reducing cofactor nicotinamide adenine dinucleotide phosphate (NADPH). This review focuses on mitochondrial ROS homeostasis with a description of six pathways of NADPH production in mitochondria and a discussion of the possible strategies of pharmacological intervention to selectively eliminate cancer cells by increasing their ROS levels

    Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation

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    Bone remodeling is a continuous physiological process that requires constant generation of new osteoblasts from mesenchymal stem cells (MSCs). Differentiation of MSCs to osteoblast requires a metabolic switch from glycolysis to increased mitochondrial respiration to ensure the sufficient energy supply to complete this process. As a consequence of this increased mitochondrial metabolism, the levels of endogenous reactive oxygen species (ROS) rise. In the current study we analyzed the role of forkhead box O3 (FOXO3) in the control of ROS levels in human MSCs (hMSCs) during osteogenic differentiation. Treatment of hMSCs with H2O2 induced FOXO3 phosphorylation at Ser294 and nuclear translocation. This ROS-mediated activation of FOXO3 was dependent on mitogen-activated protein kinase 8 (MAPK8/JNK) activity. Upon FOXO3 downregulation, osteoblastic differentiation was impaired and hMSCs lost their ability to control elevated ROS levels. Our results also demonstrate that in response to elevated ROS levels, FOXO3 induces autophagy in hMSCs. In line with this, impairment of autophagy by autophagy-related 7 (ATG7) knockdown resulted in a reduced capacity of hMSCs to regulate elevated ROS levels, together with a reduced osteoblast differentiation. Taken together our findings are consistent with a model where in hMSCs, FOXO3 is required to induce autophagy and thereby reduce elevated ROS levels resulting from the increased mitochondrial respiration during osteoblast differentiation. These new molecular insights provide an important contribution to our better understanding of bone physiology

    Effects of EGb 761® Ginkgo biloba extract on mitochondrial function and oxidative stress

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    As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria

    Duox is the primary NADPH oxidase responsible for ROS production during adult caudal fin regeneration in zebrafish

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    Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for regeneration in many organisms. This has been shown primarily via the use of pharmacological inhibitors targeting the family of NADPH oxidases (NOXes). To identify the specific NOXes involved in ROS production during adult caudal fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4) and crossed these lines with a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels. Homozygous duox mutants had the greatest effect on ROS levels and rate of fin regeneration among the single mutants. However, duox:cyba double mutants showed a greater effect on fin regeneration than the single duox mutants, suggesting that Nox1-4 also play a role during regeneration. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate with a circadian rhythm.S

    Linking ROS Levels to Autophagy: The Key Role of AMPK

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    Oxygen reactive species (ROS) are a group of molecules generated from the incomplete reduction of oxygen. Due to their high reactivity, ROS can interact with and influence the function of multiple targets, which include DNA, lipids, and proteins. Among the proteins affected by ROS, AMP-activated protein kinase (AMPK) is considered a major sensor of the intracellular energetic status and a crucial hub involved in the regulation of key cellular processes, like autophagy and lysosomal function. Thanks to these features, AMPK has been recently demonstrated to be able to perceive signals related to the variation of mitochondrial dynamics and to transduce them to the lysosomes, influencing the autophagic flux. Since ROS production is largely dependent on mitochondrial activity, through the modulation of AMPK these molecules may represent important signaling agents which participate in the crosstalk between mitochondria and lysosomes, allowing the coordination of these organelles' functions. In this review, we will describe the mechanisms through which ROS activate AMPK and the signaling pathways that allow this protein to affect the autophagic process. The picture that emerges from the literature is that AMPK regulation is highly tissue-specific and that different pools of AMPK can be localized at specific intracellular compartments, thus differentially responding to altered ROS levels. For this reason, future studies will be highly advisable to discriminate the specific contribution of the activation of different AMPK subpopulations to the autophagic pathway

    Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification

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    Chondrocyte hypertrophy during endochondral ossification is a well-controlled process in which proliferating chondrocytes stop proliferating and differentiate into hypertrophic chondrocytes, which then undergo apoptosis. Chondrocyte hypertrophy induces angiogenesis and mineralization. This step is crucial for the longitudinal growth and development of long bones, but what triggers the process is unknown. Reactive oxygen species (ROS) have been implicated in cellular damage; however, the physiological role of ROS in chondrogenesis is not well characterized. We demonstrate that increasing ROS levels induce chondrocyte hypertrophy. Elevated ROS levels are detected in hypertrophic chondrocytes. In vivo and in vitro treatment with N-acetyl cysteine, which enhances endogenous antioxidant levels and protects cells from oxidative stress, inhibits chondrocyte hypertrophy. In ataxia telangiectasia mutated (Atm)–deficient (Atm−/−) mice, ROS levels were elevated in chondrocytes of growth plates, accompanied by a proliferation defect and stimulation of chondrocyte hypertrophy. Decreased proliferation and excessive hypertrophy in Atm−/− mice were also rescued by antioxidant treatment. These findings indicate that ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes
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