Mitochondrial O2 Research Articles

Thioredoxin-1 Attenuates Ventricular and Mitochondrial Postischemic Dysfunction in the Stunned Myocardium of Transgenic Mice.

We evaluated the effect of thioredoxin1 (Trx1) system on postischemic ventricular and mitochondrial dysfunction using transgenic mice overexpressing cardiac Trx1 and a dominant negative (DN-Trx1) mutant (C32S/C35S) of Trx1. Langendorff-perfused hearts were subjected to 15 min of ischemia followed by 30 min of reperfusion (R). We measured left ventricular developed pressure (LVDP, mmHg), left ventricular end diastolic pressure (LVEDP, mmHg), and t63 (relaxation index, msec). Mitochondrial respiration, SERCA2a, phospholamban (PLB), and phospholamban phosphorylation (p-PLB) Thr17 expression (Western blot) were also evaluated. At 30 min of reperfusion, Trx1 improved contractile state (LVDP: Trx1: 57.4 ± 4.9 vs. Wt: 27.1 ± 6.3 and DN-Trx1: 29.2 ± 7.1, p < 0.05); decreased myocardial stiffness (LVEDP: Wt: 24.5 ± 4.8 vs. Trx1: 11.8 ± 2.9, p < 0.05); and improved the isovolumic relaxation (t63: Wt: 63.3 ± 3.2 vs. Trx1: 51.4 ± 1.9, p < 0.05). DN-Trx1 mice aggravated the myocardial stiffness and isovolumic relaxation. Only the expression of p-PLB Thr17 increased at 1.5 min R in Wt and DN-Trx1 groups. At 30 min of reperfusion, state 3 mitochondrial O2 consumption was impaired by 13% in Wt and by 33% in DN-Trx1. ADP/O ratios for Wt and DN-Trx1 decrease by 25% and 28%, respectively; whereas the Trx1 does not change after ischemia and reperfusion (I/R). Interestingly, baseline values of complex I activity were increased in Trx1 mice; they were 24% and 47% higher than in Wt and DN-Trx1 mice, respectively (p < 0.01). These results strongly suggest that Trx1 ameliorates the myocardial effects of I/R by improving the free radical-mediated damage in cardiac and mitochondrial function, opening the possibility of new therapeutic strategies in coronary artery disease. Antioxid. Redox Signal. 25, 78-88.

Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Diabetes.

It is generally accepted that mitochondrial dysfunction and endoplasmic reticulum (ER) stress are related to insulin resistance and type 2 diabetes. Mitochondria use substrates from lipid and glucose metabolism in order to generate ATP, and when mitochondrial O2 consumption is decreased due to an altered metabolism there is an increase in reactive oxygen species (ROS) that can impair different types of molecules and cells, especially in β- cells during type 2 diabetes. Furthermore, the maintenance of ER function in insulin-secreting β-cells is crucial, and when ER homeostasis is disrupted, the ER develops an unfolded protein response (UPR) in order to maintain the homeostasis of this organelle. However, when homeostasis fails in mitochondria and ER, these organelles can initiate death signalling pathways. New research has suggested that hyperlipidemia and hyperliglucaemia, known as key factors of type 2 diabetes (T2D), disrupt mitochondrial activity and ER homeostasis, thus triggering a disruption of energy metabolism, unresolvable UPR activation and β-cell death. This review explains the mechanisms of mitochondrial function and ER stress related to the pathological effects of type 2 diabetes in different tissues.

Oxidative stress and diabetic wound healing

ObjectivesRefractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens. Oxidative stress has been proposed as an important pathogenic factor in diabetic wound complications. The present study was designed to determine the protective effect of the GTP cyclohydrolase I (GTPCH), the rate‐limiting enzyme of BH4 synthesis; soy isoflavone genistein and Ganoderma lucidum polysaccharide (Gl‐PS) on diabetic wound healing.MethodsStreptozotocin‐induced diabetic mice with full‐thickness excisional wounds were used.ResultsThe rate of wound closure in diabetic mice was delayed compared to normal wild‐type (WT) mice, which was rescued in GTPCH transgenic (Tg‐GCH) diabetic mice. In WT diabetic mice, cutaneous eNOS activity and nitrite levels were decreased compared to WT mice, paralleled by increased levels of superoxide anion (O2.−) level. In Tg‐GCH diabetic mice, nitrite levels were potentiated and O2.− level was suppressed. Genistein treatment dose‐dependently rescued the delay of wound closure in diabetic mice and increased the mean perfusion rate. Genistein significantly normalized the elevated O2− production and reversed the attenuated nitrite level by inhibition of forkhead box O transcription factor 1 (FoxO1) levels. Gl‐PS treatment could also rescue the delay of wound closure in diabetic mice. Gl‐PS normalized the increased mitochondrial O2.− production and nitrotyrosine formation by inhibition of MnSOD nitration and the redox enzyme p66Shc expression in diabetic wound tissues.ConclusionGTPCH, genistein and Gl‐PS rescued the delayed wound healing and improved wound angiogenesis in diabetic mice, at least in part, by suppression of oxidative stress.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China No. 81373405 and 30901803, Beijing Higher Education Young Elite Teacher Project (No. YETP0053), Leading Academic Discipline Project of Beijing Education Bureau (No. BMU20110254) and the Fund of Janssen Research Council China (JRCC2011).

Attenuation of 7-ketocholesterol-induced overproduction of reactive oxygen species, apoptosis, and autophagy by dimethyl fumarate on 158 N murine oligodendrocytes

Mitochondrial dysfunctions and oxidative stress are involved in several non demyelinating or demyelinating neurodegenerative diseases. Some of them, including multiple sclerosis (MS), are associated with lipid peroxidation processes leading to increased levels of 7-ketocholesterol (7KC). So, the eventual protective effect of dimethylfumarate (DMF), which is used for the treatment of MS, was evaluated on 7KC-treated oligodendrocytes, which are myelin synthesizing cells. To this end, murine oligodendrocytes 158N were exposed to 7KC (25, 50μM) for 24h without or with DMF (1, 25, 50μM). The biological activities of DMF associated or not with 7KC were evaluated by phase contrast microscopy, crystal violet and MTT tests. The impact on transmembrane mitochondrial potential (ΔYm), O2− and H2O2 production, apoptosis and autophagy was measured by microscopical and flow cytometric methods by staining with DiOC6(3), dihydroethidine and dihydrorhodamine 123, Hoechst 33342, and by Western blotting with the use of specific antibodies raised against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II. DMF attenuates the different effects of 7KC, namely: cell growth inhibition and/or loss of cell adhesion, decrease of ΔΨm, O2− and H2O2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. The ability of DMF to attenuate 7KC-induced reactive oxygen species overproduction, apoptosis, and autophagy on oligodendrocytes reinforces the interest for this molecule for the treatment of MS or other demyelinating diseases.

Mitochondrial ascorbic acid prevents mitochondrial O₂·⁻ formation, an event critical for U937 cell apoptosis induced by arsenite through both autophagic-dependent and independent mechanisms.

A 16 h exposure of U937 cells to 2.5 µM arsenite promotes superoxide (O2(·-)) formation and inhibition of the activity of aconitase, a O2(·-) sensitive enzyme. Both responses were abolished by the complex I inhibitor rotenone, or by the respiration-deficient phenotype. Interestingly, a similar suppressive effect was mediated by a short term pre-exposure to a low concentration of L-ascorbic acid (AA), previously shown to be actively taken up by the cells and by their mitochondria. The mitochondrial origin of O2(·-) was confirmed by fluorescence microscopy studies, whereas different approaches failed to detect a contribution of NADPH oxidase. Under similar conditions, arsenite induced autophagy as well as a decline in mitochondrial membrane potential resulting in delayed (48 h) apoptosis. Importantly, all these events turned out to be sensitive to treatments associated with prevention of O2(·-) formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated by O2(·-)were entirely dependent on its conversion to H2O2. AA-sensitive mitochondrial O2(·-) formation is therefore involved in autophagy and apoptosis induced by arsenite in U937 cells, although part of the lethal response appears mediated by an autophagy-independent mechanism.

Symmorphosis and skeletal muscle V̇O2 max : in vivo and in vitro measures reveal differing constraints in the exercise-trained and untrained human.

The concept of symmorphosis postulates a matching of structural capacity to functional demand within a defined physiological system, regardless of endurance exercise training status. Whether this concept applies to oxygen (O2 ) supply and demand during maximal skeletal muscle O2 consumption (V̇O2 max ) in humans is unclear. Therefore, in vitro skeletal muscle mitochondrial V̇O2 max (Mito V̇O2 max , mitochondrial respiration of fibres biopsied from vastus lateralis) was compared with in vivo skeletal muscle V̇O2 max during single leg knee extensor exercise (KE V̇O2 max , direct Fick by femoral arterial and venous blood samples and Doppler ultrasound blood flow measurements) and whole-body V̇O2 max during cycling (Body V̇O2 max , indirect calorimetry) in 10 endurance exercise-trained and 10 untrained young males. In untrained subjects, during KE exercise, maximal O2 supply (KE Q̇O2max ) exceeded (462 ± 37 ml kg(-1) min(-1) , P < 0.05) and KE V̇O2 max matched (340 ± 22 ml kg(-1) min(-1) , P > 0.05) Mito V̇O2 max (364 ± 16 ml kg(-1) min(-1) ). Conversely, in trained subjects, both KE Q̇O2max (557 ± 35 ml kg(-1) min(-1) ) and KE V̇O2 max (458 ± 24 ml kg(-1) min(-1) ) fell far short of Mito V̇O2 max (743 ± 35 ml kg(-1) min(-1) , P < 0.05). Although Mito V̇O2 max was related to KE V̇O2 max (r = 0.69, P < 0.05) and Body V̇O2 max (r = 0.91, P < 0.05) in untrained subjects, these variables were entirely unrelated in trained subjects. Therefore, in untrained subjects, V̇O2 max is limited by mitochondrial O2 demand, with evidence of adequate O2 supply, whereas, in trained subjects, an exercise training-induced mitochondrial reserve results in skeletal muscle V̇O2 max being markedly limited by O2 supply. Taken together, these in vivo and in vitro measures reveal clearly differing limitations and excesses at V̇O2 max in untrained and trained humans and challenge the concept of symmorphosis as it applies to O2 supply and demand in humans.

A novel SOD mimic with a redox-modulating mn (II) complex, ML1 attenuates high glucose-induced abnormalities in intracellular Ca2+ transients and prevents cardiac cell death through restoration of mitochondrial function

A key contributor to the pathophysiology of diabetic cardiomyopathy, mitochondrial superoxide can be adequately countered by Mn-superoxide dismutase, which constitutes the first line of defense against mitochondrial oxidative stress. Our group has recently synthesized low molecular weight SOD mimics, demonstrating superior protection against oxidative damages to kidney cells. In the current study, we sought to evaluate the protective effect of the SOD mimic ML1 against high glucose induced cardiomyopathy in diabetes. Mechanistic studies using rat cardiac myoblast H9c2 showed that ML1 markedly inhibited High Glucose (HG) induced cytotoxicity. This was associated with increased Mn-SOD expression along with decreased mitochondrial O2·-, ONOO- and Ca2+ accumulation, unveiling its anti-oxidant potentials. ML1 also attenuated HG-induced loss of mitochondrial membrane potential (ΔΨm) and release of cytochrome c, suggesting that ML1 effectuates its cytoprotective action via the preservation of mitochondrial function. In an ex-vivo model normal adult rat ventricular myocytes (ARVMs) were isolated and cultured in either normal glucose (5.5mmol/l glucose) or HG (25.5mmol/l glucose) conditions and the efficiency of ML-1 was analyzed by studying contractile function and calcium indices. Mechanical properties were assessed using a high-speed video-edge detection system, and intracellular Ca2+ transients were recorded in fura-2–loaded myocytes. Pretreatment of myocytes with ML1 (10nM) ameliorated HG induced abnormalities in relaxation including depressed peak shortening, prolonged time to 90% relenghthening, and slower Ca2+ transient decay. Thus, ML1 exhibits significant cardio protection against oxidative damage, perhaps through its potent antioxidant action via activation of Mn-SOD.

In Vitro Study on the Response of Fibroblast Cellular Respiration to Lipoic acid, Thiamine and Carnitine in Patients with Dihydrolipoyl Dehydrogenase Deficiency

Objectives: This study examined in vitro responses of fibroblast cellular respiration to lipoic acid, thiamine and carnitine in patients with dihydrolipoyl dehydrogenase (DLD) deficiency. This disorder impairs cellular bioenergetics and these compounds are used to improve clinical manifestations of the disease. The study aimed to utilize mitochondrial O2 consumption as a surrogate biomarker for examining cellular responses to metabolic therapies. Methods: Cultured fibroblasts from three patients were treated with therapeutic concentrations of the compounds for 24 hours. Cells were then harvested and processed for measuring respiration using phosphorescence oxygen analyzer. Patients 1 and 2 were severely symptomatic infants with homozygous c.1436A>T mutation in the DLD gene. Patient-3 was a mildly symptomatic adolescent with homozygous c.685G>T mutation. Results: The rate of respiration (mean ± SD, n=6, μM O2 min-1/107 cells) in fibroblasts from a normal infant was 9.3 ± 1.6, in fibroblasts from Patient-1 was 5.1 ± 0.9 (p=0.001), in fibroblasts from Patient-2 was 7.4 ± 1.4 (p=0.051), and in fibroblasts from Patient-3 was 10.3 ± 3.3 (p=0.836). In normal fibroblasts, respiration decreased by the thiamine (p=0.012) and carnitine (p=0.023) treatments. In Patient-1, respiration increased by the lipoic acid (p<0.002), thiamine (p<0.001), and carnitine (p=0.018) treatments; this patient clinically responded to thiamine. In Patient-2, respiration decreased by the thiamine (p=0.026) and carnitine (p=0.008) treatments; this patient did not respond to these drugs. In Patient-3, respiration increased by the carnitine (p=0.012) treatment; the patient clinically responded to carnitine. Conclusions: The results show cellular respiration is a suitable biomarker for the disease. The significance of using this tool to assess responses to therapies requires further studies.

Mitochondrial physiology and reactive oxygen species production are altered by hypoxia acclimation in killifish (Fundulus heteroclitus).

Many fish encounter hypoxia in their native environment, but the role of mitochondrial physiology in hypoxia acclimation and hypoxia tolerance is poorly understood. We investigated the effects of hypoxia acclimation on mitochondrial respiration, O2kinetics, emission of reactive oxygen species (ROS), and antioxidant capacity in the estuarine killifish ( ITALIC! Fundulus heteroclitus). Killifish were acclimated to normoxia, constant hypoxia (5 kPa O2) or intermittent diel cycles of nocturnal hypoxia (12 h:12 h normoxia:hypoxia) for 28-33 days and mitochondria were isolated from liver. Neither pattern of hypoxia acclimation affected the respiratory capacities for oxidative phosphorylation or electron transport, leak respiration, coupling control or phosphorylation efficiency. Hypoxia acclimation also had no effect on mitochondrial O2kinetics, but ITALIC! P50(the O2tension at which hypoxia inhibits respiration by 50%) was lower in the leak state than during maximal respiration, and killifish mitochondria endured anoxia-reoxygenation without any impact on mitochondrial respiration. However, both patterns of hypoxia acclimation reduced the rate of ROS emission from mitochondria when compared at a common O2tension. Hypoxia acclimation also increased the levels of protein carbonyls and the activities of superoxide dismutase and catalase in liver tissue (the latter only occurred in constant hypoxia). Our results suggest that hypoxia acclimation is associated with changes in mitochondrial physiology that decrease ROS production and may help improve hypoxia tolerance.

Nitric oxide metabolism in heart mitochondria

Normal cardiac function is accomplished through a continuous energy supply provided by mitochondria. Heart mitochondria are the major source of reactive oxygen and nitrogen species: superoxide anion (O2-) and nitric oxide (NO). NO production by mitochondrial NOS (mtNOS) is modified by metabolic state and shows an exponential dependence on Δψ. The interaction between mtNOS and complexes I and IV might be a mechanism involved in the regulation of mitochondrial NO production. NO exerts a high affinity, reversible and physiological inhibition of cytochrome c oxidase activity. A second effect of NO on the respiratory chain is accomplished through its interaction with ubiquinol-cytochrome c oxidoreductase. The ability of mtNOS to regulate mitochondrial O2 uptake and O2- and H2O2 productions through the interaction of NO with the respiratory chain is named mtNOS functional activity. Together, heart mtNOS allows NO to optimize the balance between cardiac energy production and utilization, and to regulate the steady-state concentrations of other oxygen and nitrogen species.

Combination therapy with metformin and coenzyme Q10 in murine experimental autoimmune arthritis

Metformin (Met) and coenzyme Q10 (CoQ10) are reported to have therapeutic functions in several inflammatory diseases. These drugs have shown anti-inflammatory effects and have been utilized in mouse models of rheumatoid arthritis (RA). However, there is no evidence of the additive effect of Met and CoQ10 in RA. Although Met and CoQ10 may be involved in the improvement of mitochondrial dysfunction, limited information is available regarding whether this effect can improve mitochondrial dysfunction in RA in particular. In this study, we sought to determine whether Met and CoQ10 attenuate the severity of collagen-induced arthritis (CIA) and show an additive effect in a mouse model. The combination of Met and CoQ10 improved CIA, reducing joint inflammation, Th17 differentiation and IgG production. In contrast, the combination of Met and CoQ10 induced Treg differentiation. Osteoclastogenesis was reduced by the combination of Met and CoQ10. The protein expression of interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in mice splenocytes exposed to lipopolysaccharide decreased after drug combination therapy. We also found that the expression of JC-1 and COX IV were enhanced by treatment with the combination of Met and CoQ10. Moreover, the combination of Met and CoQ10 promoted mitochondrial O2 consumption. These findings suggest that the combination of Met and CoQ10 reduced CIA severity, improving mitochondrial dysfunction compared to Met or CoQ10 alone. These results present a novel, significant preventive targets in RA and may enhance our understanding of its pathogenesis.

Thyroid Hormone Stimulation of Autophagy Is Essential for Mitochondrial Biogenesis and Activity in Skeletal Muscle.

Thyroid hormone (TH) and autophagy share similar functions in regulating skeletal muscle growth, regeneration, and differentiation. Although TH recently has been shown to increase autophagy in liver, the regulation and role of autophagy by this hormone in skeletal muscle is not known. Here, using both in vitro and in vivo models, we demonstrated that TH induces autophagy in a dose- and time-dependent manner in skeletal muscle. TH induction of autophagy involved reactive oxygen species (ROS) stimulation of 5'adenosine monophosphate-activated protein kinase (AMPK)-Mammalian target of rapamycin (mTOR)-Unc-51-like kinase 1 (Ulk1) signaling. TH also increased mRNA and protein expression of key autophagy genes, microtubule-associated protein light chain 3 (LC3), Sequestosome 1 (p62), and Ulk1, as well as genes that modulated autophagy and Forkhead box O (FOXO) 1/3a. TH increased mitochondrial protein synthesis and number as well as basal mitochondrial O2 consumption, ATP turnover, and maximal respiratory capacity. Surprisingly, mitochondrial activity and biogenesis were blunted when autophagy was blocked in muscle cells by Autophagy-related gene (Atg)5 short hairpin RNA (shRNA). Induction of ROS and 5'adenosine monophosphate-activated protein kinase (AMPK) by TH played a significant role in the up-regulation of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), the key regulator of mitochondrial synthesis. In summary, our findings showed that TH-mediated autophagy was essential for stimulation of mitochondrial biogenesis and activity in skeletal muscle. Moreover, autophagy and mitochondrial biogenesis were coupled in skeletal muscle via TH induction of mitochondrial activity and ROS generation.

Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O2−), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O2−mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O2− in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O2− production by mitochondria. Both rotenone and PQ, which increase O2− in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O2− into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O2− emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O2−, specifically within the matrix of mitochondria when O2− is in adequate supply. Our results also show that O2− amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein.

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide.

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•-). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•- blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•- production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.

Abstract 120: Increased Mitochondrial Superoxide in the Brain, but not Periphery, Sensitizes Mice to Angiotensin II-Mediated Hypertension

Mitochondrial superoxide (O 2 [[Unable to Display Character: &amp;#9679;]]- ) is a critical signaling intermediate in angiotensin II (AngII)-dependent hypertension. However, it remains unknown if increased mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- flux in the absence of hypertensive stimuli is sufficient to affect blood pressure. We hypothesized that elevated levels of systemic mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- leads to increased blood pressure and exacerbates AngII-induced hypertension. To test this, we utilized a conditional mouse model of manganese superoxide dismutase knock-out (MnSOD lox/lox ), which we have shown amplifies steady-state mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- levels in all cell types examined. When combining the MnSOD lox/lox mouse with a tamoxifen-inducible cre recombinase expressed by the systemic ROSA26 promoter, MnSOD was knocked-down (30-98%, p&lt;0.05) in peripheral organs after intraperitoneal tamoxifen administration. However, no changes in MnSOD protein levels were observed in the brain. Interestingly, mean arterial pressure (MAP) and heart rate were unaffected by the loss of MnSOD in these peripheral tissues, and moreover, upon subcutaneous infusion with AngII (400 ng/kg/min) both wild-type and MnSOD knock-down mice exhibited a similar increase in MAP. Due to these unexpected results, we examined the role of elevated mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- levels specifically in the brain subfornical organ (SFO) by targeting the loss of MnSOD to this critical AngII-sensitive region. We observed a 60% decrease of MnSOD (p&lt;0.05) with concomitant increase in mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- , as measured by MitoSox Red fluorescence, in the SFO following adenovirus-mediated gene transfer of cre recombinase to the SFO in MnSOD lox/lox mice. Intriguingly, these mice demonstrated no change in baseline MAP (92.8 ± 0.4 mmHg in knock-down vs. 93.1 ± 0.4 mmHg in control mice), but did show a significant elevation in MAP upon peripheral AngII infusion (MAP max = 137.8 ± 2.7 mmHg in knock-down vs. 128.3 ± 3.3 mmHg in control mice, p&lt;0.05). Taken together, our data suggest that increased mitochondrial O 2 [[Unable to Display Character: &amp;#9679;]]- in the absence of hypertensive stimuli is not sufficient to alter baseline hemodynamics, but dysregulation of mitochondrial redox status in the SFO may be a predisposition to increased responsiveness to hypertensive stimuli, such as AngII.

Effects of metformin on mitochondrial function of leukocytes from polycystic ovary syndrome patients with insulin resistance.

Oxidative stress and mitochondrial dysfunction are implicated in polycystic ovary syndrome (PCOS). The present study assesses the effect of metformin treatment on mitochondrial function in polymorphonuclear cells from PCOS subjects. Additionally, we evaluate endocrine parameters and levels of interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα). Our study population was comprised of 35 women of reproductive age diagnosed with PCOS and treated with metformin for 12 weeks, and their corresponding controls (n=41), adjusted by age and BMI. We evaluated the alteration of endocrinological and anthropometrical parameters and androgen levels. Mitochondrial O2 consumption (using a Clark-type O2 electrode), membrane potential, mitochondrial mass, and levels of reactive oxygen species (ROS) and glutathione (GSH) (by means of fluorescence microscopy) were assessed in poymorphonuclear cells. H2O2 was evaluated with the Amplex Red(R) H2O2/Peroxidase Assay kit. IL6 and TNFα were measured using the Luminex 200 flow analyser system. Metformin had beneficial effects on patients by increasing mitochondrial O2 consumption, membrane potential, mitochondrial mass and glutathione levels, and by decreasing levels of reactive oxygen species and H2O2. In addition, metformin reduced glucose, follicle-stimulating hormone, IL6 and TNFα levels and increased dehydroepiandrosterone sulfate levels. HOMA-IR and mitochondrial function biomarkers positively correlated with ROS production (r=0.486, P=0.025), GSH content (r=0.710, P=0.049) and H2O2 (r=0.837, P=0.010), and negatively correlated with membrane potential (r=-0.829, P=0.011) at baseline. These differences disappeared after metformin treatment. Our results demonstrate the beneficial effects of metformin treatment on mitochondrial function in leukocytes of PCOS patients.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2−) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2− production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2− levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression

Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.

Remodeling of Afferent Arterioles From Mice With Oxidative Stress Does Not Account for Increased Contractility but Does Limit Excessive Wall Stress.

Because superoxide dismutase (SOD) knockout enhances arteriolar remodeling and contractility, we hypothesized that remodeling enhances contractility. In the isolated and perfused renal afferent arterioles from SOD wild type (+/+) and gene-deleted mice, contractility was assessed from reductions in luminal diameter with perfusion pressure from 40 to 80 mm Hg (myogenic responses) or angiotensin II (10(-6) mol/L), remodeling from media:lumen area ratio, superoxide (O2 (·-)) and hydrogen peroxide (H2O2) from fluorescence microscopy, and wall stress from wall tension/wall thickness. Compared with +/+ strains, arterioles from SOD1-/-, SOD2+/-, and SOD3-/- mice developed significantly (P<0.05) more O2 (·-) with perfusion pressure and angiotensin II and significantly increased myogenic responses (SOD1-/-: -20.7±2.2% versus -12.7±1.6%; SOD2+/-: -7.4±1.3% versus -12.6±1.4%; and SOD3-/-: -9.1±1.9% versus -15.8±2.2%) and angiotensin II contractions and ≈2-fold increased media:lumen ratios. Media:lumen ratios correlated with myogenic responses (r(2) =0.23; P<0.01), angiotensin II contractions (r(2)=0.57; P<0.0001), and active wall tension (r(2) =0.19; P<0.01), but not with active wall stress (r(2)=0.08; NS). Differences in myogenic responses among SOD3 mice were abolished by bath addition of SOD and were increased 3 days after inducing SOD3 knockout (-26.9±1.7% versus -20.1±0.7%; P<0.05), despite unchanged media:lumen ratios (2.01±0.09 versus 2.02±0.03; NS). We conclude that cytosolic, mitochondrial, or extracellular O2 (·-) enhance afferent arteriolar contractility and remodeling. Although remodeling does not enhance contractility, it does prevent the potentially damaging effects of increased wall stress.

LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF-1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF-1α. A clear anti-tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF-1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF-1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel-Lindau protein. In addition, at this concentration, LW6 induced hypoxia-selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6-treated hypoxic A549 cells and LW6 induced a hypoxia-selective increase of mitochondrial O2•−. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.