The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast.

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Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi

2012-07-30

The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia.

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Hals, Ingrid K; Bruerberg, Simon Gustafson; Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

2015-01-01

To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets

Insulin resistance in HIV-infected youth is associated with decreased mitochondrial respiration.

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Takemoto, Jody K; Miller, Tracie L; Wang, Jiajia; Jacobson, Denise L; Geffner, Mitchell E; Van Dyke, Russell B; Gerschenson, Mariana

2017-01-02

To identify relationships between insulin resistance (IR) and mitochondrial respiration in perinatally HIV-infected youth. Case-control study. Mitochondrial respiration was assessed in perinatally HIV-infected youth in Tanner stages 2-5, 25 youth with IR (IR+) and 50 without IR (IR-) who were enrolled in the Pediatric HIV/AIDS Cohort Study. IR was defined as a homeostatic model of assessment for IR value at least 4.0. A novel, high-throughput oximetry method was used to evaluate cellular respiration in peripheral blood mononuclear cells. Unadjusted and adjusted differences in mitochondrial respiration markers between IR+ and IR- were evaluated, as were correlations between mitochondrial respiration markers and biochemical measurements. IR+ and IR- youth were similar on age, sex, and race/ethnicity. Mean age was 16.5 and 15.6 years in IR+ and IR-, respectively. The IR+ group had significantly higher mean BMI and metabolic analytes (fasting glucose, insulin, cholesterol, triglycerides, and venous lactate and pyruvate) compared with the IR-. Mitochondrial respiration markers were, on average, lower in the IR+ compared with IR-, including basal respiration (417.5 vs. 597.5 pmol, P = 0.074), ATP production (11 513 vs. 15 202 pmol, P = 0.078), proton leak (584.6 vs. 790.0 pmol, P = 0.033), maximal respiration (1815 vs. 2399 pmol, P = 0.025), and spare respiration capacity (1162 vs. 2017 pmol, P = 0.032). Nonmitochondrial respiration did not differ by IR status. The results did not change when adjusted for age. HIV-infected youth with IR have lower mitochondrial respiration markers when compared to youth without IR. Disordered mitochondrial respiration may be a potential mechanism for IR in this population.

Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy

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Anna Czajka

2016-12-01

Full Text Available Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN, a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs and proximal tubular cells (HK-2 were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN.

Oxidative stress negatively affects human sperm mitochondrial respiration.

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Ferramosca, Alessandra; Pinto Provenzano, Sara; Montagna, Daniela Domenica; Coppola, Lamberto; Zara, Vincenzo

2013-07-01

To correlate the level of oxidative stress in serum and seminal fluid and the level of sperm deoxyribonucleic acid (DNA) fragmentation with sperm mitochondrial respiratory efficiency. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. A possible relationship between sperm mitochondrial respiratory efficiency, the level of oxidative stress, and the level of sperm DNA fragmentation was investigated. Sperm motility was positively correlated with mitochondrial respiration but negatively correlated with oxidative stress and DNA fragmentation. Interestingly, sperm mitochondrial respiratory activity was negatively affected by oxidative stress and DNA fragmentation. Our data indicate that sperm mitochondrial respiration is decreased in patients with high levels of reactive oxygen species by an uncoupling between electron transport and adenosine triphosphate synthesis. This reduction in mitochondrial functionality might be 1 of the reasons responsible for the decrease in spermatozoa motility. Copyright © 2013 Elsevier Inc. All rights reserved.

Dynamics of enhanced mitochondrial respiration in female compared with male rat cerebral arteries.

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Rutkai, Ibolya; Dutta, Somhrita; Katakam, Prasad V; Busija, David W

2015-11-01

Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using N(ω)-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I-V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury. Copyright © 2015 the American Physiological Society.

Mitochondrial Respiration and Oxygen Tension.

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Shaw, Daniel S; Meitha, Karlia; Considine, Michael J; Foyer, Christine H

2017-01-01

Measurements of respiration and oxygen tension in plant organs allow a precise understanding of mitochondrial capacity and function within the context of cellular oxygen metabolism. Here we describe methods that can be routinely used for the isolation of intact mitochondria, and the determination of respiratory electron transport, together with techniques for in vivo determination of oxygen tension and measurement of respiration by both CO 2 production and O 2 consumption that enables calculation of the respiratory quotient [CO 2 ]/[O 2 ].

Abnormal mitochondrial respiration in failed human myocardium.

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Sharov, V G; Todor, A V; Silverman, N; Goldstein, S; Sabbah, H N

2000-12-01

Chronic heart failure (HF) is associated with morphologic abnormalities of cardiac mitochondria including hyperplasia, reduced organelle size and compromised structural integrity. In this study, we examined whether functional abnormalities of mitochondrial respiration are also present in myocardium of patients with advanced HF. Mitochondrial respiration was examined using a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles obtained from myocardium of failed explanted human hearts due to ischemic (ICM, n=9) or idiopathic dilated (IDC, n=9) cardiomyopathy. Myocardial specimens from five normal donor hearts served as controls (CON). Basal respiratory rate, respiratory rate after addition of the substrates glutamate and malate (V(SUB)), state 3 respiration (after addition of ADP, V(ADP)) and respiration after the addition of atractyloside (V(AT)) were measured in scar-free muscle bundles obtained from the subendocardial (ENDO) and subepicardial (EPI) thirds of the left ventricular (LV) free wall, interventricular septum and right ventricular (RV) free wall. There were no differences in basal and substrate-supported respiration between CON and HF regardless of etiology. V(ADP)was significantly depressed both in ICM and IDC compared to CON in all the regions studied. The respiratory control ratio, V(ADP)/V(AT), was also significantly decreased in HF compared to CON. In both ICM and IDC, V(ADP)was significantly lower in ENDO compared to EPI. The results indicate that mitochondrial respiration is abnormal in the failing human heart. The findings support the concept of low myocardial energy production in HF via oxidative phosphorylation, an abnormality with a potentially impact on global cardiac performance. Copyright 2000 Academic Press.

Gastrocnemius mitochondrial respiration: are there any differences between men and women?

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Thompson, Jonathan R; Swanson, Stanley A; Casale, George P; Johanning, Jason M; Papoutsi, Evlampia; Koutakis, Panagiotis; Miserlis, Dimitrios; Zhu, Zhen; Pipinos, Iraklis I

2013-11-01

Work on human and mouse skeletal muscle by our group and others has demonstrated that aging and age-related degenerative diseases are associated with mitochondrial dysfunction, which may be more prevalent in males. There have been, however, no studies that specifically examine the influence of male or female sex on human skeletal muscle mitochondrial respiration. The purpose of this study was to compare mitochondrial respiration in the gastrocnemius of adult men and women. Gastrocnemius muscle was obtained from male (n = 19) and female (n = 11) human subjects with healthy lower-extremity musculoskeletal and arterial systems and normal ambulatory function. All patients were undergoing operations for the treatment of varicose veins in their legs. Mitochondrial respiration was determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Complex I-, II-, III-, and IV-dependent respiration was measured individually and normalized to muscle weight, total protein content, and citrate synthase (CS, index of mitochondrial content). Male and female patients had no evidence of musculoskeletal or arterial disease and did not differ with regard to age, race, body mass index, or other clinical characteristics. Complex I-, II-, III-, and IV-dependent respiration normalized to muscle weight, total protein content, and CS did not statistically differ for males compared with females. Our study evaluates, for the first time, gastrocnemius mitochondrial respiration of adult men and women who have healthy musculoskeletal and arterial systems and normal ambulatory function. Our data demonstrate there are no differences in the respiration of gastrocnemius mitochondria between men and women. Copyright © 2013 Elsevier Inc. All rights reserved.

Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

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Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I; Trinity, Joel D; Hyngstrom, John R; Garten, Ryan S; Diakos, Nikolaos A; Ives, Stephen J; Dela, Flemming; Larsen, Steen; Drakos, Stavros; Richardson, Russell S

2014-08-01

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

Microchambers with Solid-State Phosphorescent Sensor for Measuring Single Mitochondrial Respiration.

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Pham, Ted D; Wallace, Douglas C; Burke, Peter J

2016-07-09

It is now well established that, even within a single cell, multiple copies of the mitochondrial genome may be present (genetic heteroplasmy). It would be interesting to develop techniques to determine if and to what extent this genetic variation results in functional variation from one mitochondrion to the next (functional heteroplasmy). Measuring mitochondrial respiration can reveal the organelles' functional capacity for Adenosine triphosphate (ATP) production and determine mitochondrial damage that may arise from genetic or age related defects. However, available technologies require significant quantities of mitochondria. Here, we develop a technology to assay the respiration of a single mitochondrion. Our "micro-respirometer" consists of micron sized chambers etched out of borofloat glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP) mixed with polystyrene. The chambers are sealed with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber to prevent diffusion of oxygen from the environment. As the mitochondria consume oxygen in the chamber, the phosphorescence signal increases, allowing direct determination of the respiration rate. Experiments with coupled vs. uncoupled mitochondria showed a substantial difference in respiration, confirming the validity of the microchambers as single mitochondrial respirometers. This demonstration could enable future high-throughput assays of mitochondrial respiration and benefit the study of mitochondrial functional heterogeneity, and its role in health and disease.

SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia.

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Resseguie, Emily A; Brookes, Paul S; O'Reilly, Michael A

Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness.

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Yu, Emma P K; Reinhold, Johannes; Yu, Haixiang; Starks, Lakshi; Uryga, Anna K; Foote, Kirsty; Finigan, Alison; Figg, Nichola; Pung, Yuh-Fen; Logan, Angela; Murphy, Michael P; Bennett, Martin

2017-12-01

Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE -/- ) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE -/- mice overexpressing the mitochondrial helicase Twinkle (Tw + /ApoE -/- ). Tw + /ApoE -/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw + /ApoE -/- mice had decreased necrotic core and increased fibrous cap areas, and Tw + /ApoE -/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis. Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in

Effect of organic synthetic food colours on mitochondrial respiration.

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Reyes, F G; Valim, M F; Vercesi, A E

1996-01-01

Eleven organic synthetic dyes, currently or formerly used as food colours in Brazil, were tested to determine their effect on mitochondrial respiration in mitochondria isolated from rat liver and kidney. The compounds tested were: Erythrosine, Ponceau 4R, Allura Red, Sunset yellow, Tartrazine, Amaranth, Brilliant Blue, Blue, Fast Red E, Orange GGN and Scarlet GN. All food colours tested inhibited mitochondrial respiration (State III respiration, uncoupled) supported either by alpha-ketoglutarate or succinate. This inhibition varied largely, e.g. from 100% to 16% for Erythrosine and Tartrazine respectively, at a concentration of 0.1 mg food colour per mitochondrial protein. Both rat liver and kidney mitochondria showed similar patterns of inhibition among the food colours tested. This effect was dose related and the concentration to give 50% inhibition was determined for some of the dyes. The xanthene dye Erythrosine, which showed the strongest effect, was selected for further investigation on mitochondria in vivo.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

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Christopher F Bennett

2017-03-01

Full Text Available Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt and extends lifespan. Here we report that transaldolase (tald-1 deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2. We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Effect of Simvastatin, Coenzyme Q10, Resveratrol, Acetylcysteine and Acetylcarnitine on Mitochondrial Respiration.

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Fišar, Z; Hroudová, J; Singh, N; Kopřivová, A; Macečková, D

2016-01-01

Some therapeutic and/or adverse effects of drugs may be related to their effects on mitochondrial function. The effects of simvastatin, resveratrol, coenzyme Q10, acetylcysteine, and acetylcarnitine on Complex I-, Complex II-, or Complex IV-linked respiratory rate were determined in isolated brain mitochondria. The protective effects of these biologically active compounds on the calcium-induced decrease of the respiratory rate were also studied. We observed a significant inhibitory effect of simvastatin on mitochondrial respiration (IC50 = 24.0 μM for Complex I-linked respiration, IC50 = 31.3 μM for Complex II-linked respiration, and IC50 = 42.9 μM for Complex IV-linked respiration); the inhibitory effect of resveratrol was found at very high concentrations (IC50 = 162 μM for Complex I-linked respiration, IC50 = 564 μM for Complex II-linked respiration, and IC50 = 1454 μM for Complex IV-linked respiration). Concentrations required for effective simvastatin- or resveratrol-induced inhibition of mitochondrial respiration were found much higher than concentrations achieved under standard dosing of these drugs. Acetylcysteine and acetylcarnitine did not affect the oxygen consumption rate of mitochondria. Coenzyme Q10 induced an increase of Complex I-linked respiration. The increase of free calcium ions induced partial inhibition of the Complex I+II-linked mitochondrial respiration, and all tested drugs counteracted this inhibition. None of the tested drugs showed mitochondrial toxicity (characterized by respiratory rate inhibition) at drug concentrations achieved at therapeutic drug intake. Resveratrol, simvastatin, and acetylcarnitine had the greatest neuroprotective potential (characterized by protective effects against calcium-induced reduction of the respiratory rate).

Opposite effects of pioglitazone and rosiglitazone on mitochondrial respiration in skeletal muscle of patients with type 2 diabetes

DEFF Research Database (Denmark)

Rabøl, R; Boushel, R; Almdal, T

2010-01-01

mitochondrial respiration per milligram muscle was measured in saponin-treated skinned muscle fibres using high-resolution respirometry. RESULTS: Mitochondrial respiration per milligram muscle was lower in T2DM compared to controls at baseline and decreased during ROSI treatment but increased during PIO...... of ROSI and PIO on mitochondrial respiration, and also show that insulin sensitivity can be improved independently of changes in mitochondrial respiration. We confirm that mitochondrial respiration is reduced in T2DM compared to age- and BMI-matched control subjects....

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

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Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

2001-06-01

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

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Godzik, Katharina; Coleman, Michael P

2015-04-01

The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

Dose Response of Endotoxin on Hepatocyte and Muscle Mitochondrial Respiration In Vitro

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Brandt, Sebastian; Porta, Francesca; Jakob, Stephan M.; Takala, Jukka; Djafarzadeh, Siamak

2015-01-01

Introduction. Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. Methods. Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1–100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. Results. In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). Conclusion. LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner. PMID:25649304

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

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Alba Timón-Gómez

Full Text Available Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

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Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo

2013-01-01

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

Increased platelet mitochondrial respiration after cardiac arrest and resuscitation as a potential peripheral biosignature of cerebral bioenergetic dysfunction.

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Ferguson, Michael A; Sutton, Robert M; Karlsson, Michael; Sjövall, Fredrik; Becker, Lance B; Berg, Robert A; Margulies, Susan S; Kilbaugh, Todd J

2016-06-01

Cardiac arrest (CA) results in a sepsis-like syndrome with activation of the innate immune system and increased mitochondrial bioenergetics. To determine if platelet mitochondrial respiration increases following CA in a porcine pediatric model of asphyxia-associated ventricular fibrillation (VF) CA, and if this readily obtained biomarker is associated with decreased brain mitochondrial respiration. CA protocol: 7 min of asphyxia, followed by VF, protocolized titration of compression depth to systolic blood pressure of 90 mmHg and vasopressor administration to a coronary perfusion pressure greater than 20 mmHg. platelet integrated mitochondrial electron transport system (ETS) function evaluated pre- and post-CA/ROSC four hours after return of spontaneous circulation (ROSC). Secondary outcome: correlation of platelet mitochondrial bioenergetics to cerebral bioenergetic function. Platelet maximal oxidative phosphorylation (OXPHOSCI+CII), P respiration through Complex II (OXPHOSCII, P respiration was not due to uncoupling, as the LEAKCI + CII respiration (mitochondrial respiration independent of ATP-production) was unchanged after CA/ROSC. Larger increases in platelet mitochondrial respiratory control ratio (RCR) compared to pre-CA RCR were significantly correlated with lower RCRs in the cortex (P respiration. Platelet mitochondrial respiration is significantly increased four hours after ROSC. Future studies will identify mechanistic relationships between this serum biomarker and altered cerebral bioenergetics function following cardiac arrest.

Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

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Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

2016-02-01

Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration

International Nuclear Information System (INIS)

Nadanaciva, Sashi; Dykens, James A.; Bernal, Autumn; Capaldi, Roderick A.; Will, Yvonne

2007-01-01

Mitochondrial impairment is increasingly implicated in the etiology of toxicity caused by some thiazolidinediones, fibrates, and statins. We examined the effects of members of these drug classes on respiration of isolated rat liver mitochondria using a phosphorescent oxygen sensitive probe and on the activity of individual oxidative phosphorylation (OXPHOS) complexes using a recently developed immunocapture technique. Of the six thiazolidinediones examined, ciglitazone, troglitazone, and darglitazone potently disrupted mitochondrial respiration. In accord with these data, ciglitazone and troglitazone were also potent inhibitors of Complexes II + III, IV, and V, while darglitazone predominantly inhibited Complex IV. Of the six statins evaluated, lovastatin, simvastatin, and cerivastatin impaired mitochondrial respiration the most, with simvastatin and lovastatin impairing multiple OXPHOS Complexes. Within the class of fibrates, gemfibrozil more potently impaired respiration than fenofibrate, clofibrate, or ciprofibrate. Gemfibrozil only modestly inhibited Complex I, fenofibrate inhibited Complexes I, II + III, and V, and clofibrate inhibited Complex V. Our findings with the two complementary methods indicate that (1) some members of each class impair mitochondrial respiration, whereas others have little or no effect, and (2) the rank order of mitochondrial impairment accords with clinical adverse events observed with these drugs. Since the statins are frequently co-prescribed with the fibrates or thiazolidinediones, various combinations of these three drug classes were also analyzed for their mitochondrial effects. In several cases, the combination additively uncoupled or inhibited respiration, suggesting that some combinations are more likely to yield clinically relevant drug-induced mitochondrial side effects than others

Cisplatin cytotoxicity is dependent on mitochondrial respiration in Saccharomyces cerevisiae

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Santhipriya Inapurapu

2017-01-01

Full Text Available Objective(s: To understand the role of mitochondrial respiration in cisplatin sensitivity, we have employed wild-type and mitochondrial DNA depleted Rho0 yeast cells. Materials and Methods: Wild type and Rho0 yeast cultured in fermentable and non-fermentable sugar containing media, were studied for their sensitivity against cisplatin by monitoring growth curves, oxygen consumption, pH changes in cytosol/mitochondrial compartments, reactive oxygen species production and respiratory control ratio. Results: Wild-type yeast grown on glycerol exhibited heightened sensitivity to cisplatin than yeast grown on glucose. Cisplatin (100 μM, although significantly reduced the growth of wild- type cells, only slightly altered the growth rate of Rho0 cells. Cisplatin treatment decreased both pHcyt and pHmit to a similar extent without affecting the pH difference. Cisplatin dose-dependently increased the oxidative stress in wild-type, but not in respiration-deficient Rho0 strain. Cisplatin decreased the respiratory control ratio. Conclusion: These results suggest that cisplatin toxicity is influenced by the respiratory capacity of the cells and the intracellular oxidative burden. Although cisplatin per se slightly decreased the respiration of yeast cells grown in glucose, it did not disturb the mitochondrial chemiosmotic gradient.

Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

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Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

2017-11-18

Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Is cell aging caused by respiration-dependent injury to the mitochondrial genome

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Fleming, J. E.; Yengoyan, L. S.; Miquel, J.; Cottrell, S. F.; Economos, A. C.

1982-01-01

Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article, the hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells is expanded. It is maintained that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and, therefore, of organismic performance.

Microbiopsies versus Bergström needle for skeletal muscle sampling: impact on maximal mitochondrial respiration rate.

Science.gov (United States)

Isner-Horobeti, M E; Charton, A; Daussin, F; Geny, B; Dufour, S P; Richard, R

2014-05-01

Microbiopsies are increasingly used as an alternative to the standard Bergström technique for skeletal muscle sampling. The potential impact of these two different procedures on mitochondrial respiration rate is unknown. The objective of this work was to compare microbiopsies versus Bergström procedure on mitochondrial respiration in skeletal muscle. 52 vastus lateralis muscle samples were obtained from 13 anesthetized pigs, either with a Bergström [6 gauges (G)] needle or with microbiopsy needles (12, 14, 18G). Maximal mitochondrial respiration (V GM-ADP) was assessed using an oxygraphic method on permeabilized fibers. The weight of the muscle samples and V GM-ADP decreased with the increasing gauge of the needles. A positive nonlinear relationship was observed between the weight of the muscle sample and the level of maximal mitochondrial respiration (r = 0.99, p respiration (r = 0.99, p respiration compared to the standard Bergström needle.Therefore, the higher the gauge (i.e. the smaller the size) of the microbiopsy needle, the lower is the maximal rate of respiration. Microbiopsies of skeletal muscle underestimate the maximal mitochondrial respiration rate, and this finding needs to be highlighted for adequate interpretation and comparison with literature data.

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity

DEFF Research Database (Denmark)

Larsen, Anna Karina; Malinska, Dominika; Koszela-Piotrowska, Izabela

2012-01-01

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time......-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 µg/mL, however, lower PEI levels still exert...... some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 µg/mL PEI) followed by a slower...

Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics.

Science.gov (United States)

Scandurra, Francesca M; Gnaiger, Erich

2010-01-01

When oxygen supply to tissues is limiting, mitochondrial respiration and ATP production are compromised. To assess the bioenergetic consequences under normoxia and hypoxia, quantitative evaluation of mitochondrial oxygen kinetics is required. Using high-resolution respirometry, the "apparent K (m)" for oxygen or p (50) of respiration in 32D cells was determined at 0.05 +/- 0.01 kPa (0.4 mmHg, 0.5 microM, 0.25% air saturation). Close agreement with p (50) of isolated mitochondria indicates that intracellular gradients are small in small cells at routine activity. At intracellular p (O2) respiration is limited by >2% with a p (50) of 0.05 kPa. Over-estimation of p (50) at 0.4 kPa (3 mmHg) would imply significant (>17%) oxygen limitation of respiration under intracellular normoxia. Based on a critical review, we conclude that p (50) ranges from 0.01 to 0.10 kPa in mitochondria and small cells in the absence of inhibitors of cytochrome c oxidase, whereas experimental artefacts explain the controversial >200-fold range of p (50) in the literature on mitochondrial oxygen kinetics.

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol

International Nuclear Information System (INIS)

Hagen, Thilo; D'Amico, Gabriela; Quintero, Marisol; Palacios-Callender, Miriam; Hollis, Veronica; Lam, Francis; Moncada, Salvador

2004-01-01

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1α (HIF1α) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1α in HEK293 cells was found not to be due to an effect on HIF1α synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1α in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho - cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1α stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect

Mitochondrial activity in the regulation of stem cell self-renewal and differentiation.

Science.gov (United States)

Khacho, Mireille; Slack, Ruth S

2017-12-01

Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation by magnesium of potato tuber mitochondrial respiratory activities.

Science.gov (United States)

Vicente, Joaquim A F; Madeira, Vítor M C; Vercesi, Anibal E

2004-12-01

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg2+. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg2+ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg2+. However, respiration of malate, citrate, and alpha-ketoglutarate requires at least 4-mM Mg2+ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg2+ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or alpha-ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg2+. Respiration of succinate is active without external and matrix Mg2+, although stimulated by the cation. Respiration of alpha-ketoglutarate was strictly dependent on external Mg2+ required for substrate transport into mitochondria, and internal Mg2+ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg2+ but, unlike alpha-ketoglutarate, some activity still remains without external Mg2+. All the substrates revealed insensitive to external and internal mitochondrial Ca2+, except the exogenous NADH dehydrogenase, which requires either external Ca2+ or Mg2+ for detectable activity. Calcium is more efficient than Mg2+, both having cumulative stimulation. Unlike Ca2+, Mn2+ could substitute for Mg2+, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg2+.

A regulated response to impaired respiration slows behavioral rates and increases lifespan in Caenorhabditis elegans.

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David Cristina

2009-04-01

Full Text Available When mitochondrial respiration or ubiquinone production is inhibited in Caenorhabditis elegans, behavioral rates are slowed and lifespan is extended. Here, we show that these perturbations increase the expression of cell-protective and metabolic genes and the abundance of mitochondrial DNA. This response is similar to the response triggered by inhibiting respiration in yeast and mammalian cells, termed the "retrograde response". As in yeast, genes switched on in C. elegans mitochondrial mutants extend lifespan, suggesting an underlying evolutionary conservation of mechanism. Inhibition of fstr-1, a potential signaling gene that is up-regulated in clk-1 (ubiquinone-defective mutants, and its close homolog fstr-2 prevents the expression of many retrograde-response genes and accelerates clk-1 behavioral and aging rates. Thus, clk-1 mutants live in "slow motion" because of a fstr-1/2-dependent pathway that responds to ubiquinone. Loss of fstr-1/2 does not suppress the phenotypes of all long-lived mitochondrial mutants. Thus, although different mitochondrial perturbations activate similar transcriptional and physiological responses, they do so in different ways.

Short Communication: HIV Patient Systemic Mitochondrial Respiration Improves with Exercise.

Science.gov (United States)

Kocher, Morgan; McDermott, Mindy; Lindsey, Rachel; Shikuma, Cecilia M; Gerschenson, Mariana; Chow, Dominic C; Kohorn, Lindsay B; Hetzler, Ronald K; Kimura, Iris F

2017-10-01

In HIV-infected individuals, impaired mitochondrial function may contribute to cardiometabolic disease as well as to fatigue and frailty. Aerobic exercise improves total body energy reserves; however, its impact at the cellular level is unknown. We assessed alterations in cellular bioenergetics in peripheral blood mononuclear cells (PBMC) before and after a 12-week aerobic exercise study in sedentary HIV-infected subjects on stable antiretroviral therapy who successfully completed a 12-week aerobic exercise program. In this prospective study, participants underwent supervised 20-40 min of light aerobic exercise (walking or jogging) performed three times per week for 12 weeks, gradually increasing to maintain an intensity of 50%-80% of heart rate reserve. Maximal aerobic capacity (VO 2MAX ) was assessed by a graded exercise test on a cycle ergometer before and after completion of the study. PBMC from compliant subjects (attended at least 70% of exercise sessions) were assessed for mitochondrial respiration using the Seahorse XF24 Bio-Analyzer. Seven of 24 enrolled subjects were compliant with the exercise regimen. In these individuals, a significant increase (p = .04) in VO 2MAX over 12 weeks was found with a median increase of 14%. During the same interval, a 2.45-fold increase in PBMC mitochondrial respiratory capacity (p = .04), a 5.65-fold increase in spare respiratory capacity (p = .01), and a 3.15-fold (p = .04) increase in nonmitochondrial respiration was observed. Aerobic exercise improves respiration at the cellular level. The diagnostic and prognostic value of such improved cellular respiration in the setting of chronic HIV warrants further investigation.

Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice

DEFF Research Database (Denmark)

Kristensen, Jonas Møller; Larsen, Steen; Helge, Jørn Wulff

2013-01-01

signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead a(2) (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism...... and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued...... the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems...

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Science.gov (United States)

Clerc, Pascaline; Polster, Brian M.

2012-01-01

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria. PMID:22496810

The transcriptional co-repressor myeloid translocation gene 16 inhibits glycolysis and stimulates mitochondrial respiration.

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Parveen Kumar

Full Text Available The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4, and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1 was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2 oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

NARCIS (Netherlands)

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

Mitochondrial calcium ([Ca(2+)]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Science.gov (United States)

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

Science.gov (United States)

Vanlerberghe, G C; McIntosh, L

1992-12-01

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

[State of mitochondrial respiration and calcium capacity in livers of rats with different resistance to hypoxia after injections of L-arginine].

Science.gov (United States)

Kurhaliuk, N M

2001-01-01

In experiments on rats with different resistance to hypoxia are investigated processes of mitochondrial respiration, oxidative phosphorylation and calcium capacity in liver under precursor nitric oxide L-arginine (600 mg/kg) and blockator nitric oxide synthase L-NNA (35 mg/kg) injections. We are used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate, 1 mM alpha-ketoglutarate and 2 mM malonic acid. Increasing of ADP-stimulation respiration states under exogenous L-arginine injection, decreasing efficacy of respiration processes (respiration control on Chance and ADP/O) under such substrates oxidation, testify to oxide energy support decreasing and reversing nitric oxide inhibit in such conditions. This will be used as mechanism cell regulation succinate dehydrogenase activity. It has shown that L-arginine injection increase calcium mitochondrial capacity low resistance to hypoxia rats using substrates of oxidation succinate and alpha-ketoglutarate to control meanings of high resistance rats. Effects of nitric oxide precursor influence on this processes limit NO-synthase inhibitor L-NNA.

Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration

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A.T. Pettersson-Klein

2018-03-01

Full Text Available Objective: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1 regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. Methods: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. Results: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. Conclusions: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders. Keywords: Small molecule screening, PGC-1a, PGC-1alpha, PGC-1alpha1, Protein stabilization, UCP1, Mitochondrial respiration, Brown adipose tissue

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Science.gov (United States)

Fiskum, G; Lehninger, A L

1979-07-25

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.

Effect of Hyperglycemia on Mitochondrial Respiration in Type 2 Diabetes

DEFF Research Database (Denmark)

Rabøl, Rasmus; Højberg, Patricia M V; Almdal, Thomas

2009-01-01

AIM: Skeletal muscle mitochondrial content is reduced in type 2 diabetes mellitus (T2DM). Whether hyperglycemia inhibits mitochondrial biogenesis and/or function is unknown. This study examined the effect of different levels of glycemia on skeletal muscle mitochondrial function in patients with T2......DM. PATIENTS AND METHODS: Eleven patients with T2DM [9 males, 2 females; age, 52.8 +/- 2.5 yr (mean +/- se); body mass index, 30.2 +/- 1.1 kg/m(2)] in poor glycemic control were treated with insulin aspart and NPH insulin for a median period of 46 d (range, 31-59). Mitochondrial respiration...... and citrate synthase activity (a marker of mitochondrial content) were measured before and after treatment. Eleven healthy subjects (age, 53.3 +/- 2.7 yr; body mass index, 30.6 +/- 1.1 kg/m(2)) were included as controls. RESULTS: Hemoglobin A1c (9.1 +/- 0.5 to 7.5 +/- 0.3%; P

Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism.

OpenAIRE

Guidot, D M; Repine, J E; Kitlowski, A D; Flores, S C; Nelson, S K; Wright, R M; McCord, J M

1995-01-01

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P 0.05) in all yeast. Seco...

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Energy Technology Data Exchange (ETDEWEB)

Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

International Nuclear Information System (INIS)

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

2010-01-01

Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

Science.gov (United States)

Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

2015-05-04

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults.

Science.gov (United States)

van Schaardenburgh, Michel; Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J R

2016-01-01

Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. ClinicalTrials.gov NCT01842412.

A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function

Directory of Open Access Journals (Sweden)

Mohamad A. Zaini

2018-01-01

Full Text Available Summary: Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα. Protein lysine acetylation is a key post-translational modification (PTM that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT p300 and deacetylated by the lysine deacetylase (KDAC sirtuin1 (SIRT1. SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+ and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. : Zaini et al. show that the transcription factor C/EBPα is acetylated by p300 and deacetylated by the lysine deacetylase SIRT1. Hypoacetylated C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. C/EBPα is a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. Keywords: C/EBPα, SIRT1, p300, lysine acetylation, mitochondrial function, cellular metabolism, NAD+, gene regulation

Regional anatomic differences in skeletal muscle mitochondrial respiration in type 2 diabetes and obesity

DEFF Research Database (Denmark)

Rabøl, R; Larsen, S; Højberg, P M V

2010-01-01

respiration and markers of mitochondrial content in skeletal muscle of arm and leg in patients with T2DM and obese control subjects. Patients: Ten patients with T2DM (age, 52.3 +/- 2.7 yr; body mass index, 30.1 +/- 1.2 kg/m(2)) (mean +/- se) were studied after a 2-wk washout period of oral antihyperglycemic...... agents. Ten control subjects (age, 54.3 +/- 2.8 yr; body mass index, 30.4 +/- 1.2 kg/m(2)) with normal fasting and 2-h oral glucose tolerance test blood glucose levels were also included. Main Outcome Measure: We measured mitochondrial respiration in saponin-treated skinned muscle fibers from biopsies...

Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

International Nuclear Information System (INIS)

Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu; Yoon, Young Eun; Han, Woong Kyu; Choi, Kyung Hwa; Kim, Kyung-Sup

2016-01-01

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Yoon, Young Eun; Han, Woong Kyu [Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Choi, Kyung Hwa [Department of Urology, CHA Bundang Medical Center, CHA University, Seongnam 463-712 (Korea, Republic of); Kim, Kyung-Sup, E-mail: KYUNGSUP59@yuhs.ac [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

2016-06-03

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

Effect of the antitumoral alkylating agent 3-bromopyruvate on mitochondrial respiration: role of mitochondrially bound hexokinase.

Science.gov (United States)

Rodrigues-Ferreira, Clara; da Silva, Ana Paula Pereira; Galina, Antonio

2012-02-01

The alkylating agent 3-Bromopyruvate (3-BrPA) has been used as an anti-tumoral drug due to its anti-proliferative property in hepatomas cells. This propriety is believed to disturb glycolysis and respiration, which leads to a decreased rate of ATP synthesis. In this study, we evaluated the effects of the alkylating agent 3-BrPA on the respiratory states and the metabolic steps of the mitochondria of mice liver, brain and in human hepatocarcinoma cell line HepG2. The mitochondrial membrane potential (ΔΨ(m)), O(2) consumption and dehydrogenase activities were rapidly dissipated/or inhibited by 3-BrPA in respiration medium containing ADP and succinate as respiratory substrate. 3-BrPA inhibition was reverted by reduced glutathione (GSH). Respiration induced by yeast soluble hexokinase (HK) was rapidly inhibited by 3-BrPA. Similar results were observed using mice brain mitochondria that present HK naturally bound to the outer mitochondrial membrane. When the adenine nucleotide transporter (ANT) was blocked by the carboxyatractiloside, the 3-BrPA effect was significantly delayed. In permeabilized human hepatoma HepG2 cells that present HK type II bound to mitochondria (mt-HK II), the inhibiting effect occurred faster when the endogenous HK activity was activated by 2-deoxyglucose (2-DOG). Inhibition of mt-HK II by glucose-6-phosphate retards the mitochondria to react with 3-BrPA. The HK activities recovered in HepG2 cells treated or not with 3-BrPA were practically the same. These results suggest that mitochondrially bound HK supporting the ADP/ATP exchange activity levels facilitates the 3-BrPA inhibition reaction in tumors mitochondria by a proton motive force-dependent dynamic equilibrium between sensitive and less sensitive SDH in the electron transport system.

Stimulation of mitochondrial respiration induced by laser irradiation in the presence of rhodamine dyes

International Nuclear Information System (INIS)

Krasnikov, B.F.; Zorov, D.B.

1996-01-01

The effect of micromolar concentration of rhodamine 123 (methylrhodamine) and ethyl and amyl esters of unsubstituted rhodamine on oxygen consumption by rat liver mitochondria was studied under irradiation by an argon laser (488 and 514 nm). Irradiation of mitochondria in the presence of rhodamine stimulates their respiration. Light-induced stimulation of respiration is not inhibited by free radical scavenger ionol and by inhibitor of the permeability transition pore cyclosporine A. Stimulation of respiration by moderate doses of radiation is reversed in the dark. Increase in radiation dose resulted in only partial reversal of stimulated respiration in the dark. Rhodamine efficacy in stimulation of mitochondrial respiration depends on its structure (amyl > ethyl > methylrhodamine). 22 refs.; 4 figs

Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

Science.gov (United States)

Nelson, Michael B; Swensen, Adam C; Winden, Duane R; Bodine, Jared S; Bikman, Benjamin T; Reynolds, Paul R

2015-07-01

Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function. Copyright © 2015 the American Physiological Society.

Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage

DEFF Research Database (Denmark)

Sjövall, Fredrik; Ehinger, Johannes K H; Marelsson, Sigurður E

2013-01-01

Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source...... of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further...

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

Science.gov (United States)

Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

2016-09-01

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p ACM (p ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland.

Science.gov (United States)

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

2011-06-01

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman's reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sulfide-inhibition of mitochondrial respiration at very low oxygen concentrations.

Science.gov (United States)

Matallo, J; Vogt, J; McCook, O; Wachter, U; Tillmans, F; Groeger, M; Szabo, C; Georgieff, M; Radermacher, P; Calzia, E

2014-09-15

Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide's inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5-1×10(6) cells in 2 ml of continuously stirred respiration medium at 37 °C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73±0.05 μM, 3.1±0.2 μM, and 6.2±0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2]min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6 [15.5;28.1]min (coupled) and 35.9 [27.4;59.2]min (uncoupled), as well as 42.4 [27.5;42.4]min (coupled) and 51.5 [46.4;51.7]min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching

Chromatin-remodeling SWI/SNF complex regulates coenzyme Q6 synthesis and a metabolic shift to respiration in yeast.

Science.gov (United States)

Awad, Agape M; Venkataramanan, Srivats; Nag, Anish; Galivanche, Anoop Raj; Bradley, Michelle C; Neves, Lauren T; Douglass, Stephen; Clarke, Catherine F; Johnson, Tracy L

2017-09-08

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q 6 (ubiquinone or CoQ 6 ) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ 6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ 6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Zinc and calcium alter the relationship between mitochondrial respiration, ROS and membrane potential in rainbow trout (Oncorhynchus mykiss) liver mitochondria.

Science.gov (United States)

Sharaf, Mahmoud S; Stevens, Don; Kamunde, Collins

2017-08-01

At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨ mt ) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨ mt , and reactive oxygen species (ROS, as hydrogen peroxide (H 2 O 2 )) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨ mt that was associated with relatively slow enhancement of H 2 O 2 release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨ mt . In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and H 2 O 2 release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨ mt , and caused transient and sustained stimulation of respiration and H 2 O 2 release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

International Nuclear Information System (INIS)

Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H.

2010-01-01

Research highlights: → COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q 2 , a synthetic diffusible ubiquinone. → The significance that purified Coq10p contains bound Q 6 was examined by testing over-expression of Coq10p on respiration. → Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. → Respiratory deficiency caused by more Coq10p was specific and restored by Q 2 in mitochondria or by Coq8p in cells. → Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q 2 . Rescue of respiration by Q 2 is a characteristic of mutants blocked in coenzyme Q 6 synthesis. Unlike Q 6 deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q 6 . The physiological significance of earlier observations that purified Coq10p contains bound Q 6 was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q 2 . This suggests that in vivo binding of Q 6 by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

Energy Technology Data Exchange (ETDEWEB)

Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil); Ferreira-Junior, Jose Ribamar [Escola de Artes, Ciencias e Humanidades, Universidade de Sao Paulo, Sao Paulo (Brazil); Tzagoloff, Alexander [Department of Biological Sciences, Columbia University, NY (United States); Barros, Mario H., E-mail: mariohb@usp.br [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil)

2010-11-05

Research highlights: {yields} COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}, a synthetic diffusible ubiquinone. {yields} The significance that purified Coq10p contains bound Q{sub 6} was examined by testing over-expression of Coq10p on respiration. {yields} Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. {yields} Respiratory deficiency caused by more Coq10p was specific and restored by Q{sub 2} in mitochondria or by Coq8p in cells. {yields} Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}. Rescue of respiration by Q{sub 2} is a characteristic of mutants blocked in coenzyme Q{sub 6} synthesis. Unlike Q{sub 6} deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q{sub 6}. The physiological significance of earlier observations that purified Coq10p contains bound Q{sub 6} was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q{sub 2}. This suggests that in vivo binding of Q{sub 6} by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains

Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

Science.gov (United States)

Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B; Khan, Ikhlas A; Nagle, Dale G; Zhou, Yu-Dong

2014-01-24

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

Science.gov (United States)

Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

2014-01-01

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

Regulation of Mitochondrial Function and Cellular Energy Metabolism by Protein Kinase C-λ/ι: A Novel Mode of Balancing Pluripotency

Science.gov (United States)

Mahato, Biraj; Home, Pratik; Rajendran, Ganeshkumar; Paul, Arindam; Saha, Biswarup; Ganguly, Avishek; Ray, Soma; Roy, Nairita; Swerdlow, Russell H.; Paul, Soumen

2014-01-01

Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis is key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing vs. differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-HIF1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts. PMID:25142417

Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

Science.gov (United States)

Bradshaw, Patrick C; Pfeiffer, Douglas R

2013-12-01

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.

[Endoplasmic-mitochondrial Ca(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP3 - sensitive Ca(2+)-channels].

Science.gov (United States)

Velykopols'ka, O Iu; Man'ko, B O; Man'ko, V V

2012-01-01

Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

The Natural Product Osthole Attenuates Yeast Growth by Extensively Suppressing the Gene Expressions of Mitochondrial Respiration Chain.

Science.gov (United States)

Wang, Zhe; Shen, Yan

2017-03-01

The fast growing evidences have indicated that the natural product osthole is a promising drug candidate for fighting several serious human diseases, for example, cancer and inflammation. However, the mode-of-action (MoA) of osthole remains largely incomplete. In this study, we investigated the growth inhibition activity of osthole using fission yeast as a model, with the goal of understanding the osthole's mechanism of action, especially from the molecular level. Microarray analysis indicated that osthole has significant impacts on gene transcription levels (In total, 214 genes are up-regulated, and 97 genes are down-regulated). Gene set enrichment analysis (GSEA) indicated that 11 genes belong to the "Respiration module" category, especially including the components of complex III and V of mitochondrial respiration chain. Based on GSEA and network analysis, we also found that 54 up-regulated genes belong to the "Core Environmental Stress Responses" category, particularly including many transporter genes, which suggests that the rapidly activated nutrient exchange between cell and environment is part of the MoA of osthole. In summary, osthole can greatly impact on fission yeast transcriptome, and it primarily represses the expression levels of the genes in respiration chain, which next causes the inefficiency of ATP production and thus largely explains osthole's growth inhibition activity in Schizosaccharomyces pombe (S. pombe). The complexity of the osthole's MoA shown in previous studies and our current research demonstrates that the omics approach and bioinformatics tools should be applied together to acquire the complete landscape of osthole's growth inhibition activity.

Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan.

Science.gov (United States)

Schroeder, Elizabeth A; Shadel, Gerald S

2014-01-01

Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Glycolysis and mitochondrial respiration in mouse LDHC-null sperm.

Science.gov (United States)

Odet, Fanny; Gabel, Scott; London, Robert E; Goldberg, Erwin; Eddy, Edward M

2013-04-01

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

Enhanced Heme Function and Mitochondrial Respiration Promote the Progression of Lung Cancer Cells

Science.gov (United States)

Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li

2013-01-01

Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer

Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

Energy Technology Data Exchange (ETDEWEB)

Saxena, Saurabh [Experimental Biology Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054 (India); Shukla, Dhananjay [Department of Biotechnology, Gitam University, Gandhi Nagar, Rushikonda, Visakhapatnam-530 045 Andhra Pradesh (India); Bansal, Anju, E-mail: anjubansaldipas@gmail.com [Experimental Biology Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054 (India)

2012-11-01

High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl{sub 2}), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl{sub 2} supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl{sub 2} supplementation in rats augmented the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl{sub 2} supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl{sub 2} for 15 days along with training. ► Co

Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

International Nuclear Information System (INIS)

Saxena, Saurabh; Shukla, Dhananjay; Bansal, Anju

2012-01-01

High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl 2 ), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl 2 supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl 2 supplementation in rats augmented the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl 2 supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl 2 for 15 days along with training. ► CoCl 2 supplementation

The diabetes medication Canagliflozin reduces cancer cell proliferation by inhibiting mitochondrial complex-I supported respiration

Directory of Open Access Journals (Sweden)

Linda A. Villani

2016-10-01

Full Text Available Objective: The sodium-glucose transporter 2 (SGLT2 inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s remain unclear. Methods: Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1 was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin. Results: Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions. Conclusion: These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations. Keywords: AMP

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

International Nuclear Information System (INIS)

Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia; Martinez, Glaucia Regina

2017-01-01

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH_4Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial membrane potential

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

Energy Technology Data Exchange (ETDEWEB)

Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia; Martinez, Glaucia Regina, E-mail: grmartinez@ufpr.br

2017-01-01

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ{sub m}) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH{sub 4}Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial membrane

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

Science.gov (United States)

Barbosa, Marina R; Sampaio, Igor H; Teodoro, Bruno G; Sousa, Thais A; Zoppi, Claudio C; Queiroz, André L; Passos, Madla A; Alberici, Luciane C; Teixeira, Felipe R; Manfiolli, Adriana O; Batista, Thiago M; Cappelli, Ana Paula Gameiro; Reis, Rosana I; Frasson, Danúbia; Kettelhut, Isis C; Parreiras-e-Silva, Lucas T; Costa-Neto, Claudio M; Carneiro, Everardo M; Curi, Rui; Silveira, Leonardo R

2013-10-01

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids. Copyright © 2013 Elsevier B.V. All rights reserved.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

Science.gov (United States)

Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

2016-01-01

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

Science.gov (United States)

Juaristi, Inés; García-Martín, María L; Rodrigues, Tiago B; Satrústegui, Jorgina; Llorente-Folch, Irene; Pardo, Beatriz

2017-07-01

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells. © 2017 International Society for Neurochemistry.

Hyperglycemia decreases mitochondrial function: The regulatory role of mitochondrial biogenesis

International Nuclear Information System (INIS)

Palmeira, Carlos M.; Rolo, Anabela P.; Berthiaume, Jessica; Bjork, James A.; Wallace, Kendall B.

2007-01-01

Increased generation of reactive oxygen species (ROS) is implicated in 'glucose toxicity' in diabetes. However, little is known about the action of glucose on the expression of transcription factors in hepatocytes, especially those involved in mitochondrial DNA (mtDNA) replication and transcription. Since mitochondrial functional capacity is dynamically regulated, we hypothesized that stressful conditions of hyperglycemia induce adaptations in the transcriptional control of cellular energy metabolism, including inhibition of mitochondrial biogenesis and oxidative metabolism. Cell viability, mitochondrial respiration, ROS generation and oxidized proteins were determined in HepG2 cells cultured in the presence of either 5.5 mM (control) or 30 mM glucose (high glucose) for 48 h, 96 h and 7 days. Additionally, mtDNA abundance, plasminogen activator inhibitor-1 (PAI-1), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) transcripts were evaluated by real time PCR. High glucose induced a progressive increase in ROS generation and accumulation of oxidized proteins, with no changes in cell viability. Increased expression of PAI-1 was observed as early as 96 h of exposure to high glucose. After 7 days in hyperglycemia, HepG2 cells exhibited inhibited uncoupled respiration and decreased MitoTracker Red fluorescence associated with a 25% decrease in mtDNA and 16% decrease in TFAM transcripts. These results indicate that glucose may regulate mtDNA copy number by modulating the transcriptional activity of TFAM in response to hyperglycemia-induced ROS production. The decrease of mtDNA content and inhibition of mitochondrial function may be pathogenic hallmarks in the altered metabolic status associated with diabetes

The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells

Energy Technology Data Exchange (ETDEWEB)

Guidarelli, Andrea; Fiorani, Mara; Carloni, Silvia; Cerioni, Liana; Balduini, Walter; Cantoni, Orazio, E-mail: orazio.cantoni@uniurb.it

2016-09-15

We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O{sub 2}{sup .-}) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5 μM, arsenite elicited selective formation of O{sub 2}{sup .-} in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O{sub 2}{sup .-} triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O{sub 2}{sup .-} because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O{sub 2}{sup .-} mediated by NADPH oxidase. Interestingly, extramitochondrial O{sub 2}{sup .-} triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O{sub 2}{sup .-} availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis. - Highlights: • Mitochondrial superoxide mediates arsenite toxicity in respiration-proficient cells. • NADPH-derived superoxide mediates arsenite toxicity in respiration-deficient cells. • Arsenite causes apoptosis

Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

DEFF Research Database (Denmark)

Maynard, Scott; Hejl, Anne-Mette; Dinh, Tran Thuan Son

2015-01-01

AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics...... as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay...... on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD....

Sevoflurane postconditioning improves myocardial mitochondrial respiratory function and reduces myocardial ischemia-reperfusion injury by up-regulating HIF-1.

Science.gov (United States)

Yang, Long; Xie, Peng; Wu, Jianjiang; Yu, Jin; Yu, Tian; Wang, Haiying; Wang, Jiang; Xia, Zhengyuan; Zheng, Hong

2016-01-01

Sevoflurane postconditioning (SPostC) can exert myocardial protective effects similar to ischemic preconditioning. However, the exact myocardial protection mechanism by SPostC is unclear. Studies indicate that hypoxia-inducible factor-1 (HIF-1) maintains cellular respiration homeostasis by regulating mitochondrial respiratory chain enzyme activity under hypoxic conditions. This study investigated whether SPostC could regulate the expression of myocardial HIF-1α and to improve mitochondrial respiratory function, thereby relieving myocardial ischemia-reperfusion injury in rats. The myocardial ischemia-reperfusion rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, postconditioning was performed using sevoflurane alone or in combination with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). The changes in hemodynamic parameters, HIF-1α protein expression levels, mitochondrial respiratory function and enzyme activity, mitochondrial reactive oxygen species (ROS) production rates, and mitochondrial ultrastructure were measured or observed. Compared to the ischemia-reperfusion (I/R) group, HIF-1α expression in the SPostC group was significantly up-regulated. Additionally, cardiac function indicators, mitochondrial state 3 respiratory rate, respiratory control ratio (RCR), cytochrome C oxidase (C c O), NADH oxidase (NADHO), and succinate oxidase (SUCO) activities, mitochondrial ROS production rate, and mitochondrial ultrastructure were significantly better than those in the I/R group. However, these advantages were completely reversed by the HIF-1α specific inhibitor 2ME2 ( P <0.05). The myocardial protective function of SPostC might be associated with the improvement of mitochondrial respiratory function after up-regulation of HIF-1α expression.

Initial brain aging: heterogeneity of mitochondrial size is associated with decline in complex I-linked respiration in cortex and hippocampus.

Science.gov (United States)

Thomsen, Kirsten; Yokota, Takashi; Hasan-Olive, Md Mahdi; Sherazi, Niloofar; Fakouri, Nima Borhan; Desler, Claus; Regnell, Christine Elisabeth; Larsen, Steen; Rasmussen, Lene Juel; Dela, Flemming; Bergersen, Linda Hildegard; Lauritzen, Martin

2018-01-01

Brain aging is accompanied by declining mitochondrial respiration. We hypothesized that mitochondrial morphology and dynamics would reflect this decline. Using hippocampus and frontal cortex of a segmental progeroid mouse model lacking Cockayne syndrome protein B (CSB m/m ) and C57Bl/6 (WT) controls and comparing young (2-5 months) to middle-aged mice (13-14 months), we found that complex I-linked state 3 respiration (CI) was reduced at middle age in CSB m/m hippocampus, but not in CSB m/m cortex or WT brain. In hippocampus of both genotypes, mitochondrial size heterogeneity increased with age. Notably, an inverse correlation between heterogeneity and CI was found in both genotypes, indicating that heterogeneity reflects mitochondrial dysfunction. The ratio between fission and fusion gene expression reflected age-related alterations in mitochondrial morphology but not heterogeneity. Mitochondrial DNA content was lower, and hypoxia-induced factor 1α mRNA was greater at both ages in CSB m/m compared to WT brain. Our findings show that decreased CI and increased mitochondrial size heterogeneity are highly associated and point to declining mitochondrial quality control as an initial event in brain aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial Energy and Redox Signaling in Plants

Science.gov (United States)

Schwarzländer, Markus

2013-01-01

Abstract Significance: For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression. Recent Advances: Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria. Critical Issues: Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control. Future Directions: Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling. Antioxid. Redox Signal. 18, 2122–2144. PMID:23234467

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates

OpenAIRE

Fendt, Sarah-Maria; Sauer, Uwe

2010-01-01

Abstract Background Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. Results We address this question by linking the functional degree of respiration t...

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity

DEFF Research Database (Denmark)

Lantier, Louise; Fentz, Joachim; Mounier, Rémi

2014-01-01

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle...... diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial...

The ATPase and protease domains of yeast mitochondrial Lon : Roles in proteolysis and respiration-dependent growth

NARCIS (Netherlands)

van Dijl, JM; Kutejova, E; Suda, K; Perecko, D; Schatz, G; Suzuki, CK

1998-01-01

The ATP-dependent Lon protease of Saccharomyces cerevisiae mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth. Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates

Directory of Open Access Journals (Sweden)

Fendt Sarah-Maria

2010-02-01

Full Text Available Abstract Background Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. Results We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. Conclusions Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates.

Science.gov (United States)

Fendt, Sarah-Maria; Sauer, Uwe

2010-02-18

Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential for respiration.

[Research progress on photosynthesis regulating and controlling soil respiration].

Science.gov (United States)

Jing, Yan-Li; Guan, De-Xin; Wu, Jia-Bing; Wang, An-Zhi; Yuan, Feng-Hui

2013-01-01

To understand the mechanisms of soil respiration and accurately estimate its magnitude are the crucial basis of evaluating global carbon balance. However, the previously built soil respiration forecast models usually neglect the physiological processes that photosynthesis supplies substrates for rhizospheric respiration, leading to the defect in evaluating the mechanisms of soil respiration. This paper summarized the research progress on the mechanisms of photosynthetic regulation and control of soil respiration, introduced the related main research methods, and discussed the existing problems and research hotspots.

Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways.

Directory of Open Access Journals (Sweden)

Yao Shen

Full Text Available Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

Response of mitochondrial function to hypothyroidism in normal and regenerated rat skeletal muscle.

Science.gov (United States)

Zoll, J; Ventura-Clapier, R; Serrurier, B; Bigard, A X

2001-01-01

Although thyroid hormones induce a well known decrease in muscle oxidative capacity, nothing is known concerning their effects on mitochondrial function and regulation in situ. Similarly, the influence of regeneration process is not completely understood. We investigated the effects of hypothyroidism on mitochondrial function in fast gastrocnemius (GS) and slow soleus (SOL) muscles either intact or having undergone a cycle of degeneration/regeneration (Rg SOL) following a local injection of myotoxin. Thyroid hormone deficiency was induced by thyroidectomy and propylthiouracyl via drinking water. Respiration was measured in muscle fibres permeabilised by saponin in order to assess the oxidative capacity of the muscles and the regulation of mitochondria in situ. Oxidative capacities were 8.9 in SOL, 8.5 in Rg SOL and 5.9 micromol O2/min/g dry weight in GS and decreased by 52, 42 and 39% respectively (P hypothyroid rats. Moreover, the Km of mitochondrial respiration for the phosphate acceptor ADP exhibited a two-fold decrease in Rg SOL and intact SOL by hypothyroidism (P hypothyroidism markedly altered the sensitivity of mitochondrial respiration to ADP but not to creatine in SOL muscles, suggesting that mitochondrial regulation could be partially controlled by thyroid hormones. On the other hand, mitochondrial function completely recovered following regeneration/degeneration, suggesting that thyroid hormones are not involved in the regeneration process per se.

Variations in dark respiration and mitochondrial numbers within needles of Pinus radiata grown in ambient or elevated CO2 partial pressure

International Nuclear Information System (INIS)

Griffin, K. L.; Anderson, O. R.; Tissue, D. T.; Turnbull, M. H.; Whitehead, D.

2004-01-01

An experiment involving comparison of within-leaf variations in cell size, mitochondrial numbers and dark respiration in the most recently expanded tip, the mid-section and the base of needles of Pinus radiata grown for four years at ambient and elevated carbon dioxide partial pressure, is described. Results showed variation in mitochondrial numbers and respiration along the length of the needle, with the highest number of mitochondria per unit cytoplasm and the highest rate of respiration per unit leaf area at the base of the needle. Elevated carbon dioxide pressure caused the number of mitochondria per unit cytoplasm to double regardless of location (tip, basal or mid sections). Under these conditions, greatest mitochondrial density was observed at the tip. The mean size of mitochondria was not affected by either growth at elevated carbon dioxide pressure or by position on the needle. Respiration per unit leaf area at elevated carbon dioxide pressure was highest at the tip of needles, decreasing towards the middle and basal sections. The observed data supports the hypothesis that the highest number of mitochondria per unit area of cytoplasm occurs at the base of the needle, but does not support the hypothesis that the lowest rate of respiration also occurs at the base. It is suggested that the relationship that determines the association between structure and function in these needles is more complex than previously thought. 33 refs., 4 tabs., 1 fig

Mitochondrial complex III defects contribute to inefficient respiration and ATP synthesis in the myocardium of Trypanosoma cruzi-infected mice.

Science.gov (United States)

Wen, Jian-Jun; Garg, Nisha Jain

2010-01-01

In this study, we conducted a thorough analysis of mitochondrial bioenergetic function as well as the biochemical and molecular factors that are deregulated and contribute to compromised adenosine triphosphate (ATP) production in the myocardium during Trypanosoma cruzi infection. We show that ADP-stimulated state 3 respiration and ATP synthesis supported by pyruvate/malate (provides electrons to complex I) and succinate (provides electrons to complex II) substrates were significantly decreased in left ventricular tissue and isolated cardiac mitochondria of infected mice. The decreased mitochondrial ATP synthesis in infected murine hearts was not a result of uncoupling between the electron-transport chain and oxidative phosphorylation and decreased availability of the intermediary metabolites (e.g., NADH). The observed decline in the activities of complex-I, -IV, and -V was not physiologically relevant and did not contribute to compromised respiration and ATP synthesis in infected myocardium. Instead, complex III activity was decreased above the threshold level and contributed to respiratory-chain inefficiency and the resulting decline in mitochondrial ATP synthesis in infected myocardium. The loss in complex III activity occurred as a consequence of cytochrome b depletion. Treatment of infected mice with phenyl-alpha-tert-butyl nitrone (PBN, antioxidant) was beneficial in preserving the mtDNA-encoded cytochrome b expression, and subsequently resulted in improved complex III activity, mitochondrial respiration, and ATP production in infected myocardium. Overall, we provide novel data on the mechanism(s) involved in cardiac bioenergetic inefficiency during T. cruzi infection.

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.

Science.gov (United States)

Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

2014-11-28

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.

Directory of Open Access Journals (Sweden)

Ulugbek Negmadjanov

Full Text Available Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.Treatment with TGF-β1 (5 ng/mL induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU. TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU, as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control. TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells.TGF-β1 induced

Regulation of the Stress-Activated Degradation of Mitochondrial Respiratory Complexes in Yeast

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Alba Timón-Gómez

2018-01-01

Full Text Available Repair and removal of damaged mitochondria is a key process for eukaryotic cell homeostasis. Here we investigate in the yeast model how different protein complexes of the mitochondrial electron transport chain are subject to specific degradation upon high respiration load and organelle damage. We find that the turnover of subunits of the electron transport complex I equivalent and complex III is preferentially stimulated upon high respiration rates. Particular mitochondrial proteases, but not mitophagy, are involved in this activated degradation. Further mitochondrial damage by valinomycin treatment of yeast cells triggers the mitophagic removal of the same respiratory complexes. This selective protein degradation depends on the mitochondrial fusion and fission apparatus and the autophagy adaptor protein Atg11, but not on the mitochondrial mitophagy receptor Atg32. Loss of autophagosomal protein function leads to valinomycin sensitivity and an overproduction of reactive oxygen species upon mitochondrial damage. A specific event in this selective turnover of electron transport chain complexes seems to be the association of Atg11 with the mitochondrial network, which can be achieved by overexpression of the Atg11 protein even in the absence of Atg32. Furthermore, the interaction of various Atg11 molecules via the C-terminal coil domain is specifically and rapidly stimulated upon mitochondrial damage and could therefore be an early trigger of selective mitophagy in response to the organelles dysfunction. Our work indicates that autophagic quality control upon mitochondrial damage operates in a selective manner.

Impairment of ER-mitochondrial coupling provides neuroprotection in a model of oxytosis

NARCIS (Netherlands)

Honrath, Birgit; Metz, Isabell; Bendridi, Nadia; Rieusset, Jennifer; Culmsee, Carsten; Dolga, Amalia Mihalea

2017-01-01

The crosstalk between the endoplasmic reticulum (ER) and mitochondria facilitates calcium transfer between these organelles, thereby maintaining the driving force for calcium into the mitochondrial matrix to modulate mitochondrial respiration. Glucose-regulated protein 75 (GRP75/mortalin) physically

Adipose tissue mitochondrial respiration and lipolysis before and after a weight loss by diet and RYGB

DEFF Research Database (Denmark)

Hansen, Merethe; Lund, Michael T.; Gregers, Emilie

2015-01-01

OBJECTIVE: To study adipose tissue mitochondrial respiration and lipolysis following a massive weight loss. METHODS: High resolution respirometry of adipose tissue biopsies and tracer determined whole body lipolysis. Sixteen obese patients with type 2 diabetes (T2DM) and 27 without (OB) were...... studied following a massive weight loss by diet and Roux-en-Y gastric bypass (RYGB). RESULTS: The mitochondrial respiratory rates were similar in OB and T2DM, and the mass-specific oxygen flux increased significantly 4 and 18 months post-surgery (P ... 2DM, visceral fat mass was always higher relative to the body fat mass (%) compared to OB. CONCLUSIONS: Adipose tissue mitochondrial respiratory capacity increases with RYGB. Adipocytes adapt to massive weight...

Effect of physical training on mitochondrial respiration and reactive oxygen species release in skeletal muscle in patients with obesity and type 2 diabetes

DEFF Research Database (Denmark)

Hey-Mogensen, Martin; Højlund, K; Vind, B F

2010-01-01

of obese participants with and without type 2 diabetes. METHODS: Type 2 diabetic men (n = 13) and control (n = 14) participants matched for age, BMI and physical activity completed 10 weeks of aerobic training. Pre- and post-training muscle biopsies were obtained before a euglycaemic...... in type 2 diabetic participants. Mitochondrial ROS release tended to be higher in participants with type 2 diabetes. CONCLUSIONS/INTERPRETATION: Aerobic training improves muscle respiration and intrinsic mitochondrial respiration in untrained obese participants with and without type 2 diabetes...

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

Science.gov (United States)

Jones, Charles I; Han, Zhaosheng; Presley, Tennille; Varadharaj, Saradhadevi; Zweier, Jay L; Ilangovan, Govindasamy; Alevriadou, B Rita

2008-07-01

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

Secondary metabolites of the grapevine pathogen Eutypa lata inhibit mitochondrial respiration, based on a model bioassay using the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Kim, Jong H; Mahoney, Noreen; Chan, Kathleen L; Molyneux, Russell J; Campbell, Bruce C

2004-10-01

Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2- isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2- isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2Delta, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1Delta, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H(2)O(2)) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.

In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

Science.gov (United States)

Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

2017-10-01

Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

Idebenone increases mitochondrial complex I activity in fibroblasts from LHON patients while producing contradictory effects on respiration

DEFF Research Database (Denmark)

Angebault, Claire; Gueguen, Naig; Desquiret-Dumas, Valerie

2011-01-01

to impairment in some cases and stimulation in others. Conclusion: These results indicate that idebenone is able to compensate the complex I deficiency in LHON patient cells with variable effects on respiration, indicating that the patients might not be equally likely to benefit from the treatment....... of idebenone in fibroblasts from LHON patients using enzymatic and polarographic measurements. Results: Complex I activity was 42% greater in treated fibroblasts compared to controls (p = 0.002). Despite this complex I activity improvement, the effects on mitochondrial respiration were contradictory, leading...

Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

Science.gov (United States)

Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

2015-08-25

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

Czech Academy of Sciences Publication Activity Database

Špaček, Tomáš; Šantorová, Jitka; Zacharovová, K.; Berková, Z.; Hlavatá, Lydie; Saudek, F.; Ježek, Petr

2008-01-01

Roč. 40, č. 8 (2008), s. 1522-1535 ISSN 1357-2725 R&D Projects: GA MZd(CZ) NR7917 Institutional research plan: CEZ:AV0Z50110509 Keywords : in situ mitochondrial membrane potential * in situ mitochondrial respiration * glucose-stimulated insulin secretion Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.178, year: 2008

PINK1 regulates mitochondrial trafficking in dendrites of cortical neurons through mitochondrial PKA.

Science.gov (United States)

Das Banerjee, Tania; Dagda, Raul Y; Dagda, Marisela; Chu, Charleen T; Rice, Monica; Vazquez-Mayorga, Emmanuel; Dagda, Ruben K

2017-08-01

Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding-deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit β of PKA (PKA/RIIβ) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIβ and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity. © 2017 International Society for Neurochemistry.

Transcriptional regulation of dimethyl sulfoxide respiration in a haloarchaeon, Haloferax volcanii.

Science.gov (United States)

Qi, Qiuzi; Ito, Yoshiyasu; Yoshimatsu, Katsuhiko; Fujiwara, Taketomo

2016-01-01

The halophilic euryarchaeon Haloferax volcanii can grow anaerobically by DMSO respiration. DMSO reductase was induced by DMSO respiration not only under anaerobic growth conditions but also in denitrifying cells of H. volcanii. Deletion of the dmsR gene, encoding a putative regulator for the DMSO reductase, resulted in the loss of anaerobic growth by DMSO respiration. Reporter experiments revealed that only the anaerobic condition was essential for transcription of the dmsEABCD genes encoding DMSO reductase and that transcription was enhanced threefold by supplementation of DMSO. In the ∆dmsR mutant, transcription of the dmsEABCD genes induced by the anaerobic condition was not enhanced by DMSO, suggesting that DmsR is a DMSO-responsive regulator. Transcriptions of the dmsR and mgd genes for Mo-bisMGD biosynthesis were regulated in the same manner as the dmsEABCD genes. These results suggest that the genetic regulation of DMSO respiration in H. volcanii is controlled by at least two systems: one is the DMSO-responsive DmsR, and the other is an unknown anaerobic regulator.

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation.

Science.gov (United States)

Jang, Yunseon; Han, Jeongsu; Kim, Soo Jeong; Kim, Jungim; Lee, Min Joung; Jeong, Soyeon; Ryu, Min Jeong; Seo, Kang-Sik; Choi, Song-Yi; Shong, Minho; Lim, Kyu; Heo, Jun Young; Kweon, Gi Ryang

2015-11-10

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel-Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.

Opposing effects of nitric oxide and prostaglandin inhibition on muscle mitochondrial VO2 during exercise

DEFF Research Database (Denmark)

Boushel, Robert C; Fuentes, Teresa; Hellsten, Ylva

2012-01-01

Nitric oxide (NO) and prostaglandins (PG) together play a role in regulation blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome c oxidase. Indomethacin both uncouples and inhibits the electron transport chain in a concentration......-dependent manner, and thus inhibition of NO and PG may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG blockade (L-NMMA and indomethacin respectively) on mitochondrial respiration in human muscle following knee...... extension (KE) exercise. Mitochondrial respiration was measured ex-vivo by high resolution respirometry in saponin-permeabilized fibers following 6 min KE in control (CON, n=8), arterial infusion of LNMMA (n=4) and Indo (n=4) followed by combined inhibition of NO and PG (L-NMMA + Indo, n=8). ADP...

Reprofiling a classical anthelmintic, pyrvinium pamoate, as an anti-cancer drug targeting mitochondrial respiration

Energy Technology Data Exchange (ETDEWEB)

Ishii, Isao [Department of Biochemistry, Keio University Graduate School of Pharmaceutical Sciences, Tokyo (Japan); Harada, Yasuo [Fujii Memorial Research Institute, Otsuka Pharmaceutical Co., Ltd., Shiga (Japan); Kasahara, Tadashi, E-mail: isao-ishii@umin.ac.jp [Department of Biochemistry, Keio University Graduate School of Pharmaceutical Sciences, Tokyo (Japan)

2012-10-02

Pyrvinium pamoate (PP) is an FDA-approved classical anthelmintic, but is now attracting particular attention as an anti-cancer drug after recent findings of its potent cytotoxicity against various cancer cell lines only during glucose starvation, as well as its anti-tumor activity against hypovascular pancreatic cancer cells transplanted in mice. The molecular mechanisms by which PP promotes such preferential toxicity against cancer cells are currently under extensive investigation. PP suppressed the NADH-fumarate reductase system that mediates a reverse reaction of the mitochondrial electron-transport chain complex II in anaerobic organisms such as parasitic helminthes or mammalian cells under tumor microenvironment-mimicking hypoglycemic/hypoxic conditions, thereby inhibiting efficient ATP production. PP also inhibited the unfolded protein response induced by glucose starvation, thereby inhibiting the proliferation of pancreatic cancer cells. Even under normoglycemic/normoxic conditions, PP suppressed the mitochondrial electron-transport chain complex I and thereby STAT3, inhibiting the proliferation of myeloma/erythroleukemia cells. Here, we review accumulating knowledge on its working mechanisms and evaluate PP as a novel anti-cancer drug that targets mitochondrial respiration.

Reprofiling a classical anthelmintic, pyrvinium pamoate, as an anti-cancer drug targeting mitochondrial respiration.

Directory of Open Access Journals (Sweden)

Isao eIshii

2012-10-01

Full Text Available Pyrvinium pamoate (PP is an FDA-approved classical anthelmintic, but is now attracting particular attention as an anti-cancer drug after recent findings of its potent cytotoxicity against various cancer cell lines only during glucose starvation, as well as its anti-tumor activity against hypovascular pancreatic cancer cells transplanted in mice. The molecular mechanisms by which PP promotes such preferential toxicity against cancer cells are currently under extensive investigation. PP suppressed the NADH-fumarate reductase system that mediates a reverse reaction of the mitochondrial electron-transport chain complex II in anaerobic organisms such as parasitic helminthes or mammalian cells under tumor microenvironment-mimicking hypoglycemic/hypoxic conditions, thereby inhibiting efficient ATP production. PP also inhibited the unfolded protein response induced by glucose starvation, thereby inhibiting the proliferation of pancreatic cancer cells. Even under normoglycemic/normoxic conditions, PP suppressed the mitochondrial electron-transport chain complex I and thereby STAT3, inhibiting the proliferation of myeloma/erythroleukemia cells. Here, we review accumulating knowledge on its working mechanisms and evaluate PP as a novel anti-cancer drug that targets mitochondrial respiration.

Reprofiling a classical anthelmintic, pyrvinium pamoate, as an anti-cancer drug targeting mitochondrial respiration

International Nuclear Information System (INIS)

Ishii, Isao; Harada, Yasuo; Kasahara, Tadashi

2012-01-01

Pyrvinium pamoate (PP) is an FDA-approved classical anthelmintic, but is now attracting particular attention as an anti-cancer drug after recent findings of its potent cytotoxicity against various cancer cell lines only during glucose starvation, as well as its anti-tumor activity against hypovascular pancreatic cancer cells transplanted in mice. The molecular mechanisms by which PP promotes such preferential toxicity against cancer cells are currently under extensive investigation. PP suppressed the NADH-fumarate reductase system that mediates a reverse reaction of the mitochondrial electron-transport chain complex II in anaerobic organisms such as parasitic helminthes or mammalian cells under tumor microenvironment-mimicking hypoglycemic/hypoxic conditions, thereby inhibiting efficient ATP production. PP also inhibited the unfolded protein response induced by glucose starvation, thereby inhibiting the proliferation of pancreatic cancer cells. Even under normoglycemic/normoxic conditions, PP suppressed the mitochondrial electron-transport chain complex I and thereby STAT3, inhibiting the proliferation of myeloma/erythroleukemia cells. Here, we review accumulating knowledge on its working mechanisms and evaluate PP as a novel anti-cancer drug that targets mitochondrial respiration.

Formation and Regulation of Mitochondrial Membranes

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Laila Cigana Schenkel

2014-01-01

Full Text Available Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases.

Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

DEFF Research Database (Denmark)

Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro

2011-01-01

those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-¿B pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1...... cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching...

Mitochondrial respiratory control is lost during growth factor deprivation.

Science.gov (United States)

Gottlieb, Eyal; Armour, Sean M; Thompson, Craig B

2002-10-01

The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x(L), restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.

Mitochondrial Respiratory Thresholds Regulate Yeast Chronological Lifespan and its Extension by Caloric Restriction

OpenAIRE

Ocampo, Alejandro; Liu, Jingjing; Schroeder, Elizabeth A.; Shadel, Gerald S.; Barrientos, Antoni

2012-01-01

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases, and determining how this affects chronological lifespan (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS...

Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

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Hagen Thilo

2011-04-01

Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

High Fat Diet-Induced Changes in Mouse Muscle Mitochondrial Phospholipids Do Not Impair Mitochondrial Respiration Despite Insulin Resistance

Science.gov (United States)

Hulshof, Martijn F. M.; van den Berg, Sjoerd A. A.; Schaart, Gert; van Dijk, Ko Willems; Smit, Egbert; Mariman, Edwin C. M.

2011-01-01

Background Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance. Methodology C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR. Principal Findings At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks. Conclusions/Interpretation Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance.

Directory of Open Access Journals (Sweden)

Joris Hoeks

Full Text Available BACKGROUND: Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance. METHODOLOGY: C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD or HFD (45 kcal%. Skeletal muscle mitochondria were isolated and fatty acid (FA composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR. PRINCIPAL FINDINGS: At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9 were decreased (-4.0%, p<0.001, whereas saturated FA (16∶0 were increased (+3.2%, p<0.001 in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6 showed a pronounced increase (+4.0%, p<0.001. Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002 and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks. CONCLUSIONS/INTERPRETATION: Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in

Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes

International Nuclear Information System (INIS)

Downs, R.M.; Hughes, M.A.; Kinsey, S.T.; Johnson, M.C.; Baumgarner, B.L.

2016-01-01

Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. - Highlights: • Caffeine

Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

DEFF Research Database (Denmark)

Satrústegui, Jorgina; Bak, Lasse K

2015-01-01

that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium...

Loss of the SIN3 transcriptional corepressor results in aberrant mitochondrial function

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Hüttemann Maik

2010-07-01

Full Text Available Abstract Background SIN3 is a transcriptional repressor protein known to regulate many genes, including a number of those that encode mitochondrial components. Results By monitoring RNA levels, we find that loss of SIN3 in Drosophila cultured cells results in up-regulation of not only nuclear encoded mitochondrial genes, but also those encoded by the mitochondrial genome. The up-regulation of gene expression is accompanied by a perturbation in ATP levels in SIN3-deficient cells, suggesting that the changes in mitochondrial gene expression result in altered mitochondrial activity. In support of the hypothesis that SIN3 is necessary for normal mitochondrial function, yeast sin3 null mutants exhibit very poor growth on non-fermentable carbon sources and show lower levels of ATP and reduced respiration rates. Conclusions The findings that both yeast and Drosophila SIN3 affect mitochondrial activity suggest an evolutionarily conserved role for SIN3 in the control of cellular energy production.

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

International Nuclear Information System (INIS)

Lee, Heung M.; Reed, Jason; Greeley, George H.; Englander, Ella W.

2009-01-01

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase α subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke

Mitochondrial function and glucose metabolism in the placenta with gestational diabetes mellitus: role of miR-143.

Science.gov (United States)

Muralimanoharan, Sribalasubashini; Maloyan, Alina; Myatt, Leslie

2016-06-01

A predisposing factor for development of the hyperglycaemic state of gestational diabetes mellitus (GDM) is obesity. We previously showed that increasing maternal obesity is associated with significant reductions in placental mitochondrial respiration. MicroRNA (miR)-143 has been previously shown to regulate the metabolic switch from oxidative phosphorylation to aerobic glycolysis in cancer tissues. We hypothesized that mitochondrial respiration is reduced and aerobic glycolysis is up-regulated via changes in miR-143 expression in the placenta of women with GDM. Placental tissue was collected at term from women with A1GDM (controlled by diet), A2GDM (controlled by medication) and body mass index (BMI)-matched controls (CTRL). miR-143 expression was measured by RT-PCR. Expression of mitochondrial complexes, transcription factors peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α) and peroxisome proliferator-activated receptor γ (PPARγ), components of mammalian target of rapamycin (mTOR) signalling, glucose transporter GLUT1 and glycolytic enzymes [hexokinase-2 (HK-2), phosphofructokinase (PFK) and lactate dehydrogenase (LDH)] were measured by Western blot. Trophoblast respiration was measured by XF24 Analyser. Expression of miR-143, mitochondrial complexes, and PPARγ and PGC1α, which act downstream of miR-143, were significantly decreased in A2GDM placentae compared with A1GDM and CTRL (P<0.01). Placental hPL (human placental lactogen) levels, expression of glycolytic enzymes, GLUT1 and mTOR signalling were also significantly increased by more than 2-fold in A2GDM compared with A1GDM and CTRL (P<0.05). There was a 50% reduction in mitochondrial respiration in trophoblast cells isolated from A2GDM placentae. Overexpression of miR-143 was able to increase mitochondrial respiration, increase protein expression of mitochondrial complexes and decrease expression of glycolytic enzymes by 40% compared with A2GDM. Down-regulation of miR-143 mediates

Plant species richness regulates soil respiration through changes in productivity.

Science.gov (United States)

Dias, André Tavares Corrêa; van Ruijven, Jasper; Berendse, Frank

2010-07-01

Soil respiration is an important pathway of the C cycle. However, it is still poorly understood how changes in plant community diversity can affect this ecosystem process. Here we used a long-term experiment consisting of a gradient of grassland plant species richness to test for effects of diversity on soil respiration. We hypothesized that plant diversity could affect soil respiration in two ways. On the one hand, more diverse plant communities have been shown to promote plant productivity, which could increase soil respiration. On the other hand, the nutrient concentration in the biomass produced has been shown to decrease with diversity, which could counteract the production-induced increase in soil respiration. Our results clearly show that soil respiration increased with species richness. Detailed analysis revealed that this effect was not due to differences in species composition. In general, soil respiration in mixtures was higher than would be expected from the monocultures. Path analysis revealed that species richness predominantly regulates soil respiration through changes in productivity. No evidence supporting the hypothesized negative effect of lower N concentration on soil respiration was found. We conclude that shifts in productivity are the main mechanism by which changes in plant diversity may affect soil respiration.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

Science.gov (United States)

Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

2015-08-01

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Fatty acid nitroalkenes induce resistance to ischemic cardiac injury by modulating mitochondrial respiration at complex II

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Jeffrey R. Koenitzer

2016-08-01

Full Text Available Nitro-fatty acids (NO2-FA are metabolic and inflammatory-derived electrophiles that mediate pleiotropic signaling actions. It was hypothesized that NO2-FA would impact mitochondrial redox reactions to induce tissue-protective metabolic shifts in cells. Nitro-oleic acid (OA-NO2 reversibly inhibited complex II-linked respiration in isolated rat heart mitochondria in a pH-dependent manner and suppressed superoxide formation. Nitroalkylation of Fp subunit was determined by BME capture and the site of modification by OA-NO2 defined by mass spectrometric analysis. These effects translated into reduced basal and maximal respiration and favored glycolytic metabolism in H9C2 cardiomyoblasts as assessed by extracellular H+ and O2 flux analysis. The perfusion of NO2-FA induced acute cardioprotection in an isolated perfused heart ischemia/reperfusion (IR model as evidenced by significantly higher rate-pressure products. Together these findings indicate that NO2-FA can promote cardioprotection by inducing a shift from respiration to glycolysis and suppressing reactive species formation in the post-ischemic interval.

Mitochondrial Respiration Is Decreased in Rat Kidney Following Fetal Exposure to a Maternal Low-Protein Diet

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Sarah Engeham

2012-01-01

Full Text Available Maternal protein restriction in rat pregnancy is associated with impaired renal development and age-related loss of renal function in the resulting offspring. Pregnant rats were fed either control or low-protein (LP diets, and kidneys from their male offspring were collected at 4, 13, or 16 weeks of age. Mitochondrial state 3 and state 4 respiratory rates were decreased by a third in the LP exposed adults. The reduction in mitochondrial function was not explained by complex IV deficiency or altered expression of the complex I subunits that are typically associated with mitochondrial dysfunction. Similarly, there was no evidence that LP-exposure resulted in greater oxidative damage to the kidney, differential expression of ATP synthetase β-subunit, and ATP-ADP translocase 1. mRNA expression of uncoupling protein 2 was increased in adult rats exposed to LP in utero, but there was no evidence of differential expression at the protein level. Exposure to maternal undernutrition is associated with a decrease in mitochondrial respiration in kidneys of adult rats. In the absence of gross disturbances in respiratory chain protein expression, programming of coupling efficiency may explain the long-term impact of the maternal diet.

Plant community structure regulates responses of prairie soil respiration to decadal experimental warming.

Science.gov (United States)

Xu, Xia; Shi, Zheng; Li, Dejun; Zhou, Xuhui; Sherry, Rebecca A; Luo, Yiqi

2015-10-01

Soil respiration is recognized to be influenced by temperature, moisture, and ecosystem production. However, little is known about how plant community structure regulates responses of soil respiration to climate change. Here, we used a 13-year field warming experiment to explore the mechanisms underlying plant community regulation on feedbacks of soil respiration to climate change in a tallgrass prairie in Oklahoma, USA. Infrared heaters were used to elevate temperature about 2 °C since November 1999. Annual clipping was used to mimic hay harvest. Our results showed that experimental warming significantly increased soil respiration approximately from 10% in the first 7 years (2000-2006) to 30% in the next 6 years (2007-2012). The two-stage warming stimulation of soil respiration was closely related to warming-induced increases in ecosystem production over the years. Moreover, we found that across the 13 years, warming-induced increases in soil respiration were positively affected by the proportion of aboveground net primary production (ANPP) contributed by C3 forbs. Functional composition of the plant community regulated warming-induced increases in soil respiration through the quantity and quality of organic matter inputs to soil and the amount of photosynthetic carbon (C) allocated belowground. Clipping, the interaction of clipping with warming, and warming-induced changes in soil temperature and moisture all had little effect on soil respiration over the years (all P > 0.05). Our results suggest that climate warming may drive an increase in soil respiration through altering composition of plant communities in grassland ecosystems. © 2015 John Wiley & Sons Ltd.

Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

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Michelle Barbi de Moura

Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

Mitochondrial epigenetics : an overlooked layer of regulation?

NARCIS (Netherlands)

van der Wijst, Monique G. P.; Rots, Marianne G.

Despite decades of research, mitochondrial epigenetics remains a controversial notion. Recent findings, however, indicate that dysfunctional mitochondrial DNA (mtDNA) methylation could underlie aging and disease. Unraveling such a level of regulation will be essential in the understanding of and in

Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

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Xu, Aijun [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Anesthesiology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030 (China); Szczepanek, Karol; Hu, Ying [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Lesnefsky, Edward J. [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298 (United States); McGuire Department of Veterans Affairs Medical Center, Richmond, VA 23249 (United States); Chen, Qun, E-mail: qchen8@vcu.edu [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States)

2013-06-14

Highlights: •Blockade of electron transport prevents the loss of AIF from mitochondria during IR. •Blockade of electron transport decreases caspase-independent cell death during IR. •Mitochondrial AIF content is down-regulated in Harlequin mice. •Blockade of electron transport protects Harlequin mouse hearts during IR. •Amobarbital protection is partially dependent on mitochondrial AIF content. -- Abstract: The transient, reversible blockade of electron transport (BET) during ischemia or at the onset of reperfusion protects mitochondria and decreases cardiac injury. Apoptosis inducing factor (AIF) is located within the mitochondrial intermembrane space. A release of AIF from mitochondria into cytosol and nucleus triggers caspase-independent cell death. We asked if BET prevents the loss of AIF from mitochondria as a mechanism of protection in the buffer perfused heart. BET during ischemia with amobarbital, a rapidly reversible inhibitor of mitochondrial complex I, attenuated a release of AIF from mitochondria into cytosol, in turn decreasing the formation of cleaved and activated PARP-1. These results suggest that BET-mediated protection may occur through prevention of the loss of AIF from mitochondria during ischemia–reperfusion. In order to further clarify the role of mitochondrial AIF in BET-mediated protection, Harlequin (Hq) mice, a genetic model with mitochondrial AIF deficiency, were used to test whether BET could still decrease cell injury in Hq mouse hearts during reperfusion. BET during ischemia protected Hq mouse hearts against ischemia–reperfusion injury and improved mitochondrial function in these hearts during reperfusion. Thus, cardiac injury can still be decreased in the presence of down-regulated mitochondrial AIF content. Taken together, BET during ischemia protects both hearts with normal mitochondrial AIF content and hearts with mitochondrial AIF deficiency. Although preservation of mitochondrial AIF content plays a key role in

Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

International Nuclear Information System (INIS)

Xu, Aijun; Szczepanek, Karol; Hu, Ying; Lesnefsky, Edward J.; Chen, Qun

2013-01-01

Highlights: •Blockade of electron transport prevents the loss of AIF from mitochondria during IR. •Blockade of electron transport decreases caspase-independent cell death during IR. •Mitochondrial AIF content is down-regulated in Harlequin mice. •Blockade of electron transport protects Harlequin mouse hearts during IR. •Amobarbital protection is partially dependent on mitochondrial AIF content. -- Abstract: The transient, reversible blockade of electron transport (BET) during ischemia or at the onset of reperfusion protects mitochondria and decreases cardiac injury. Apoptosis inducing factor (AIF) is located within the mitochondrial intermembrane space. A release of AIF from mitochondria into cytosol and nucleus triggers caspase-independent cell death. We asked if BET prevents the loss of AIF from mitochondria as a mechanism of protection in the buffer perfused heart. BET during ischemia with amobarbital, a rapidly reversible inhibitor of mitochondrial complex I, attenuated a release of AIF from mitochondria into cytosol, in turn decreasing the formation of cleaved and activated PARP-1. These results suggest that BET-mediated protection may occur through prevention of the loss of AIF from mitochondria during ischemia–reperfusion. In order to further clarify the role of mitochondrial AIF in BET-mediated protection, Harlequin (Hq) mice, a genetic model with mitochondrial AIF deficiency, were used to test whether BET could still decrease cell injury in Hq mouse hearts during reperfusion. BET during ischemia protected Hq mouse hearts against ischemia–reperfusion injury and improved mitochondrial function in these hearts during reperfusion. Thus, cardiac injury can still be decreased in the presence of down-regulated mitochondrial AIF content. Taken together, BET during ischemia protects both hearts with normal mitochondrial AIF content and hearts with mitochondrial AIF deficiency. Although preservation of mitochondrial AIF content plays a key role in

The MEF2 gene is essential for yeast longevity, with a dual role in cell respiration and maintenance of mitochondrial membrane potential.

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Callegari, Sylvie; McKinnon, Ross A; Andrews, Stuart; de Barros Lopes, Miguel A

2011-04-20

The Saccharomyces cerevisiae MEF2 gene is a mitochondrial protein translation factor. Formerly believed to catalyze peptide elongation, evidence now suggests its involvement in ribosome recycling. This study confirms the role of the MEF2 gene for cell respiration and further uncovers a slow growth phenotype and reduced chronological lifespan. Furthermore, in comparison with cytoplasmic ρ(0) strains, mef2Δ strains have a marked reduction of the inner mitochondrial membrane potential and mitochondria show a tendency to aggregate, suggesting an additional role for the MEF2 gene in maintenance of mitochondrial health, a role that may also be shared by other mitochondrial protein synthesis factors. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cyclopamine tartrate, an inhibitor of Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration in lung cancer cells

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Alam, Md Maksudul; Sohoni, Sagar; Kalainayakan, Sarada Preeta; Garrossian, Massoud; Zhang, Li

2016-01-01

Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action. The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT’s impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells. Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and

The effect of 2,4-D and ABA on respiration of isolated mitochondria from maize coleoptiles

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Ewa Raczek

2014-01-01

Full Text Available The susceptibility of isolated maize mitochondria to the growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D and abscisic acid (ABA was studied. It was found that 2,4-D (a herbicide inhibits respiration in mitochondria, as do other herbicides or phenoxy-acids. In the entire range of concentrations used (10-3-10-9 M, 2,4-D introduced into the medium before the respiration reaction was begun, or during it, limited the intensity of succinate oxidation. It did not, however, markedly change phosphorylation properties. Uncoupling of oxidative phosphorylation took place only after preincubation of mitochondria with 2,4-D and was the result of the destruction of mitochondrial membranes. ABA (a growth inhibitor of plants caused a similar response in maize mitochondria. Preincubation of mitochondria with ABA lead to the uncoupling of oxidative phosphorylation. Whereas ABA introduced during respiration (state 4 respiration or before its onset, lowered the oxidative potential of mitochondria, it also changed the pattern of state 4-3-4 transition after addition of ADP (it was especially visible at high concentrations, which indicates that the coupling of oxidative phosphorylation with the respiratory chain has faltered. It seems that this negative effect of 2,4-D and ABA on respiration of isolated maize mitochondria is connected with the inhibitory effect of these growth regulators on the growth of maize coleoptiles. Interference in the organization mitochondrial membranes results in a lowered supply of ATP - a source of energy needed in elongation processes.

Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.

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Siamak Djafarzadeh

Full Text Available During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α. Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB. The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis. Our data suggest that

Glycolysis-respiration relationships in a neuroblastoma cell line.

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Swerdlow, Russell H; E, Lezi; Aires, Daniel; Lu, Jianghua

2013-04-01

Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

DMPD: Regulation of mitochondrial antiviral signaling pathways. [Dynamic Macrophage Pathway CSML Database

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Full Text Available 18549796 Regulation of mitochondrial antiviral signaling pathways. Moore CB, Ting J...P. Immunity. 2008 Jun;28(6):735-9. (.png) (.svg) (.html) (.csml) Show Regulation of mitochondrial antiviral ...signaling pathways. PubmedID 18549796 Title Regulation of mitochondrial antiviral signaling pathways. Author

In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress.

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Klöppel, Christine; Michels, Christine; Zimmer, Julia; Herrmann, Johannes M; Riemer, Jan

2010-12-03

The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS. Copyright © 2010 Elsevier Inc. All rights reserved.

Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

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Takayuki Mito

Full Text Available Mitochondrial DNA (mtDNA mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0 mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

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Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.

Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

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Turrens, J F; Bickar, D; Lehninger, A L

1986-06-01

The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

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Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

2017-11-01

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

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Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

2016-04-01

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Deletion of the Mitochondrial Chaperone TRAP-1 Uncovers Global Reprogramming of Metabolic Networks

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Sofia Lisanti

2014-08-01

Full Text Available Reprogramming of metabolic pathways contributes to human disease, especially cancer, but the regulators of this process are unknown. Here, we have generated a mouse knockout for the mitochondrial chaperone TRAP-1, a regulator of bioenergetics in tumors. TRAP-1−/− mice are viable and showed reduced incidence of age-associated pathologies, including obesity, inflammatory tissue degeneration, dysplasia, and spontaneous tumor formation. This was accompanied by global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing deregulated mitochondrial respiration, oxidative stress, impaired cell proliferation, and a switch to glycolytic metabolism in vivo. These data identify TRAP-1 as a central regulator of mitochondrial bioenergetics, and this pathway could contribute to metabolic rewiring in tumors.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

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Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

DEFF Research Database (Denmark)

Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

Redox Regulation of Mitochondrial Function

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Handy, Diane E.

2012-01-01

Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

Mitochondrial Metabolism in Aging Heart

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Lesnefsky, Edward J.; Chen, Qun; Hoppel, Charles L.

2016-01-01

Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area there is an approximate 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction. PMID:27174952

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

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Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

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Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

2012-10-12

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

Impaired mitochondrial function in chronically ischemic human heart

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Stride, Nis Ottesen; Larsen, Steen; Hey-Mogensen, Martin

2013-01-01

, and finally to assess myocardial antioxidant levels. Mitochondrial respiration in biopsies from ischemic and nonischemic regions from the left ventricle of the same heart was compared in nine human subjects. Maximal oxidative phosphorylation capacity in fresh muscle fibers was lower in ischemic compared.......05), and the levels of antioxidant protein expression was lower. Diminished mitochondrial respiration capacity and excessive ROS production demonstrate an impaired mitochondrial function in ischemic human heart muscle. No chronic ischemic preconditioning effect was found....

The Ability of PAS, Acetylsalicylic Acid and Calcium Disodium EDTA to Protect Against the Toxic Effects of Manganese on Mitochondrial Respiration in Gill of Crassostrea virginica.

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Crawford, Sherine; Davis, Kiyya; Saddler, Claudette; Joseph, Jevaun; Catapane, Edward J; Carroll, Margaret A

2011-01-01

Manganese (Mn) is an essential metal that at excessive levels in brain causes Manganism, a condition similar to Parkinson's disease. Previously we showed that Mn had a neurotoxic effect on the dopaminergic, but not serotonergic, innervation of the lateral ciliated cells in the gill of the Eastern Oyster, Crassostrea virginica. While the mechanism of action of Mn toxicity is not completely understood, studies suggest that Mn toxicity may involve mitochondrial damage and resulting neural dysfunction in the brain's dopaminergic system. In this study we utilized micro-batch chambers and oxygen probes to measure oyster gill mitochondrial respiration in the presence of Mn and potential Mn blockers. The addition of Mn to respiring mitochondria caused a dose dependent decrease in mitochondrial O(2) consumption. Pretreating mitochondria with calcium disodium EDTA (caEDTA), p aminosalicylic acid (PAS) or acetylsalicylic acid (ASA) before Mn additions, provided full protection against the toxic effects of Mn. While mitochondrial pretreatment with any of the 3 drugs effectively blocked Mn toxicity, none of the drugs tested was able to reverse the decrease in mitochondrial O(2) consumption seen in Mn treated mitochondria. The study found that high levels of Mn had a toxic effect on gill mitochondrial O(2) consumption and that this effect could be blocked by the drugs caEDTA, PAS and ASA. C. virginica continues to be a good model with which to investigate the mechanism that underlies manganese neurotoxcity and in the pharmacological study of drugs to treat or prevent Manganism.

Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome

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Hwang, Hye Jin [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States); Dornbos, Peter [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Steidemann, Michelle [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Dunivin, Taylor K. [Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Rizzo, Mike [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Cell and Molecular Biology Graduate Program, Michigan State University, East Lansing, MI 48824 (United States); LaPres, John J., E-mail: lapres@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States)

2016-08-01

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes ≥ 2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction. - Highlights: • The mitoAHR is localized in the mitochondrial intermembrane space. • TOMM20 participates in mitoAHR translocation. • AHR contributes to the maintenance of respiratory control ratio following

Dengue virus induces mitochondrial elongation through impairment of Drp1-triggered mitochondrial fission

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Barbier, Vincent; Lang, Diane; Valois, Sierra; Rothman, Alan L.; Medin, Carey L., E-mail: cmedin.uri@gmail.com

2017-01-15

Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associated with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication. - Highlights: •Mitochondrial length and respiration are increased during DENV infection. •DENV inhibits Drp1-triggered mitochondrial fission. •DENV titers are reduced by mitochondrial fragmentation, Drp1 WT and S616D expression. •Viral proteins NS4b and NS3 are associated with subcellular fractions of mitochondria.

Dengue virus induces mitochondrial elongation through impairment of Drp1-triggered mitochondrial fission

International Nuclear Information System (INIS)

Barbier, Vincent; Lang, Diane; Valois, Sierra; Rothman, Alan L.; Medin, Carey L.

2017-01-01

Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associated with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication. - Highlights: •Mitochondrial length and respiration are increased during DENV infection. •DENV inhibits Drp1-triggered mitochondrial fission. •DENV titers are reduced by mitochondrial fragmentation, Drp1 WT and S616D expression. •Viral proteins NS4b and NS3 are associated with subcellular fractions of mitochondria.

Altered Mitochondrial Dynamics and TBI Pathophysiology

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Tara Diane Fischer

2016-03-01

Full Text Available Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS, and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1, which translocates to the mitochondrial outer membrane to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 hours post-injury, followed by a significant decrease in length at 72 hours. Post-TBI administration of Mdivi-1, a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the hippocampus and improved

Varicocele Negatively Affects Sperm Mitochondrial Respiration.

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Ferramosca, Alessandra; Albani, Denise; Coppola, Lamberto; Zara, Vincenzo

2015-10-01

To evaluate the effect of varicocele on oxidative stress, sperm mitochondrial respiratory efficiency, sperm morphology, and semen parameters. A total of 20 patients with varicocele and 20 normozoospermic subjects without varicocele (control group) were recruited from a medical center for reproductive biology. The levels of serum reactive oxygen metabolites and seminal lipid peroxides were assessed for both control and varicocele subjects. Sperm deoxyribonucleic acid fragmentation was measured by sperm chromatin dispersion test. Mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. In this study, varicocele patients were compared with men without varicoceles. Oxidative stress was observed in the serum and seminal fluid of varicocele patients. These patients showed an increase of 59% (P <.05) in serum reactive oxygen metabolites and a 3-fold increase in the level of sperm lipid peroxides. A parallel and significant increase (a 2-fold increase; P <.05) in the degree of sperm deoxyribonucleic acid fragmentation was also observed. Varicocele patients showed a 27% decrease (P <.05) in mitochondrial respiratory activity in comparison to the control group. A 32% increase (P <.05) in sperm midpiece defects and a 41% decrease (P <.05) in sperm concentration and motility were also observed. Men with varicocele have increased markers of oxidative stress and decreased mitochondrial respiratory activity. These results correlated with abnormalities in semen parameters. For morphology, these correlated with midpiece defects. Copyright © 2015 Elsevier Inc. All rights reserved.

The Responses of Tissues from the Brain, Heart, Kidney, and Liver to Resuscitation following Prolonged Cardiac Arrest by Examining Mitochondrial Respiration in Rats.

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Kim, Junhwan; Villarroel, José Paul Perales; Zhang, Wei; Yin, Tai; Shinozaki, Koichiro; Hong, Angela; Lampe, Joshua W; Becker, Lance B

2016-01-01

Cardiac arrest induces whole-body ischemia, which causes damage to multiple organs. Understanding how each organ responds to ischemia/reperfusion is important to develop better resuscitation strategies. Because direct measurement of organ function is not practicable in most animal models, we attempt to use mitochondrial respiration to test efficacy of resuscitation on the brain, heart, kidney, and liver following prolonged cardiac arrest. Male Sprague-Dawley rats are subjected to asphyxia-induced cardiac arrest for 30 min or 45 min, or 30 min cardiac arrest followed by 60 min cardiopulmonary bypass resuscitation. Mitochondria are isolated from brain, heart, kidney, and liver tissues and examined for respiration activity. Following cardiac arrest, a time-dependent decrease in state-3 respiration is observed in mitochondria from all four tissues. Following 60 min resuscitation, the respiration activity of brain mitochondria varies greatly in different animals. The activity after resuscitation remains the same in heart mitochondria and significantly increases in kidney and liver mitochondria. The result shows that inhibition of state-3 respiration is a good marker to evaluate the efficacy of resuscitation for each organ. The resulting state-3 respiration of brain and heart mitochondria following resuscitation reenforces the need for developing better strategies to resuscitate these critical organs following prolonged cardiac arrest.

Mitochondrial fission proteins regulate programmed cell death in yeast.

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Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes.

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Anthony L Luz

Full Text Available Mitochondrial dysfunction has been linked to myriad human diseases and toxicant exposures, highlighting the need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide and oligomycin (ATP-synthase inhibitors, carbonyl cyanide 4-(trifluoromethoxy phenylhydrazone (mitochondrial uncoupler and sodium azide (cytochrome c oxidase inhibitor, we measured the fundamental parameters of mitochondrial respiratory chain function: basal oxygen consumption, ATP-linked respiration, maximal respiratory capacity, spare respiratory capacity and proton leak in the model organism Caenhorhabditis elegans. Since mutations in mitochondrial homeostasis genes cause mitochondrial dysfunction and have been linked to human disease, we measured mitochondrial respiratory function in mitochondrial fission (drp-1-, fusion (fzo-1-, mitophagy (pdr-1, pink-1-, and electron transport chain complex III (isp-1-deficient C. elegans. All showed altered function, but the nature of the alterations varied between the tested strains. We report increased basal oxygen consumption in drp-1; reduced maximal respiration in drp-1, fzo-1, and isp-1; reduced spare respiratory capacity in drp-1 and fzo-1; reduced proton leak in fzo-1 and isp-1; and increased proton leak in pink-1 nematodes. As mitochondrial morphology can play a role in mitochondrial energetics, we also quantified the mitochondrial aspect ratio for each mutant strain using a novel method, and for the first time report increased aspect ratios in pdr-1- and pink-1-deficient nematodes.

Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans.

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Kras, Katon A; Hoffman, Nyssa; Roust, Lori R; Patel, Shivam H; Carroll, Chad C; Katsanos, Christos S

2017-12-01

Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals. To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults. Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions. Eligible participants were healthy lean (body mass index, mass index >30 kg/m2; age 35 ± 3 years; n = 11) subjects. Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions. Mitochondrial respiration and ATP production. AA infusion increased adenosine 5'-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P lean subjects only (P lean or obese subjects (P > 0.05). Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals. Copyright © 2017 Endocrine Society

Epicatechin stimulates mitochondrial activity and selectively sensitizes cancer cells to radiation.

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Hosam A Elbaz

Full Text Available Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (--epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (--epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (--Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (--Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF of 1.7, 1.5, and 1.2, respectively. (--Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (--Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (--epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.

[Biomechanics and regulation of the external respiration in the conditions of 5-day dry immersion].

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Popova, Iu A; Suvorov, A V; D'iachenko, A I; Kolesnikov, V I

2011-01-01

The work was concerned with evaluation of the external respiration function and regulation in healthy human subjects participating in simulation of the microgravity effects by dry immersion (DI). In the baseline data collection period, in DI (days 2 and 4) and after DI completion pulmonary volumes were registered, the ratio of thoracic and abdominal components of quiet breathing and respiratory maneuvers calculated, and parameters of respiration regulation, i.e. length of breath-holding and ability to voluntary control breathing motions, were determined. It was shown that breathing pattern did not undergo gross changes in immersion as compared with pre-DI test data; however, inspiratory reserve volume grew (p immersion. We believe that similar to microgravity, exposure in DI produces regular alterations of pulmonary RV (partly because of changed body position), thoracic-abdominal ratio in breathing motions, and shifts in voluntary respiration regulation.

Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria.

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Agrawal, Anurag; Mabalirajan, Ulaganathan

2016-01-15

Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function are key aspects of pathogenesis in asthma and chronic obstructive pulmonary disease (COPD). This motif is not unique to obstructive airway disease, reported in related airway diseases such as bronchopulmonary dysplasia and parenchymal diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as MitoQ, has substantially improved mitochondrial health and restored respiratory function. Modulation of noncoding RNA or protein regulators of mitochondrial biogenesis, dynamics, or degradation has been found to be effective in models of fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy mitochondria to epithelial cells has been associated with remarkable therapeutic efficacy in models of acute lung injury and asthma. Together, these form a 3R model--repair, reprogramming, and replacement--for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function in lung health and disease, with a focus on asthma and COPD, and provides an overview of mitochondria-targeted strategies for rejuvenating cellular respiration and optimizing respiratory function in lung diseases. Copyright © 2016 the American Physiological Society.

Brain mitochondria from DJ-1 knockout mice show increased respiration-dependent hydrogen peroxide consumption

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Pamela Lopert

2014-01-01

Full Text Available Mutations in the DJ-1 gene have been shown to cause a rare autosomal-recessive genetic form of Parkinson’s disease (PD. The function of DJ-1 and its role in PD development has been linked to multiple pathways, however its exact role in the development of PD has remained elusive. It is thought that DJ-1 may play a role in regulating reactive oxygen species (ROS formation and overall oxidative stress in cells through directly scavenging ROS itself, or through the regulation of ROS scavenging systems such as glutathione (GSH or thioredoxin (Trx or ROS producing complexes such as complex I of the electron transport chain. Previous work in this laboratory has demonstrated that isolated brain mitochondria consume H2O2 predominantly by the Trx/Thioredoxin Reductase (TrxR/Peroxiredoxin (Prx system in a respiration dependent manner (Drechsel et al., Journal of Biological Chemistry, 2010. Therefore we wanted to determine if mitochondrial H2O2 consumption was altered in brains from DJ-1 deficient mice (DJ-1−/−. Surprisingly, DJ-1−/− mice showed an increase in mitochondrial respiration-dependent H2O2 consumption compared to controls. To determine the basis of the increased H2O2 consumption in DJ1−/− mice, the activities of Trx, Thioredoxin Reductase (TrxR, GSH, glutathione disulfide (GSSG and glutathione reductase (GR were measured. Compared to control mice, brains from DJ-1−/− mice showed an increase in (1 mitochondrial Trx activity, (2 GSH and GSSG levels and (3 mitochondrial glutaredoxin (GRX activity. Brains from DJ-1−/− mice showed a decrease in mitochondrial GR activity compared to controls. The increase in the enzymatic activities of mitochondrial Trx and total GSH levels may account for the increased H2O2 consumption observed in the brain mitochondria in DJ-1−/− mice perhaps as an adaptive response to chronic DJ-1 deficiency.

Stimulation of respiration in rat thymocytes induced by ionizing radiation

International Nuclear Information System (INIS)

Gudz, T.I.; Pandelova, I.G.; Novgorodov, S.A.

1994-01-01

The effect of X irradiation on the respiration of rat thymocytes was studied. An increase in the rate of O 2 uptake was observed 1 h after cells were irradiated with doses of 6-10 Gy. The radiation-induced increase in respiration could be blocked by oligomycin, an inhibitor of mitochondrial ATP synthase, suggesting control by increased cytoplasmic ATP turnover. The stimulation of respiration was not associated with changes in the activity of mitochondrial electron transfer enzymes or permeability of the inner membrane. Several inhibitors of processes which used ATP were screened for their effects on the basal respiration rate and on the radiation response. In irradiated thymocytes, an enhancement of inhibition of respiration by ouabain, La 3+ and cycloheximide was observed. These results indicate that the radiation-induced stimulation of respiration is due to changes in ion homeostasis and protein synthesis. The effect of X irradiation was shown to be independent of the redox status of nonprotein thiols and was not associated with detectable changes in some products of lipid peroxidation. The radiation-induced decrease in activity of superoxide dismutase suggests free radical involvement in deleterious effects of radiation. 43 refs., 2 figs., 3 tabs

The Responses of Tissues from the Brain, Heart, Kidney, and Liver to Resuscitation following Prolonged Cardiac Arrest by Examining Mitochondrial Respiration in Rats

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Junhwan Kim

2016-01-01

Full Text Available Cardiac arrest induces whole-body ischemia, which causes damage to multiple organs. Understanding how each organ responds to ischemia/reperfusion is important to develop better resuscitation strategies. Because direct measurement of organ function is not practicable in most animal models, we attempt to use mitochondrial respiration to test efficacy of resuscitation on the brain, heart, kidney, and liver following prolonged cardiac arrest. Male Sprague-Dawley rats are subjected to asphyxia-induced cardiac arrest for 30 min or 45 min, or 30 min cardiac arrest followed by 60 min cardiopulmonary bypass resuscitation. Mitochondria are isolated from brain, heart, kidney, and liver tissues and examined for respiration activity. Following cardiac arrest, a time-dependent decrease in state-3 respiration is observed in mitochondria from all four tissues. Following 60 min resuscitation, the respiration activity of brain mitochondria varies greatly in different animals. The activity after resuscitation remains the same in heart mitochondria and significantly increases in kidney and liver mitochondria. The result shows that inhibition of state-3 respiration is a good marker to evaluate the efficacy of resuscitation for each organ. The resulting state-3 respiration of brain and heart mitochondria following resuscitation reenforces the need for developing better strategies to resuscitate these critical organs following prolonged cardiac arrest.

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

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Sirey, Tamara M; Ponting, Chris P

2016-10-15

The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

VDAC electronics: 4. Novel electrical mechanism and thermodynamic estimations of glucose repression of yeast respiration.

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Lemeshko, Victor V

2017-11-01

Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as "Crabtree effect", has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S. cerevisiae, resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD + . Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD + (1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP. Copyright © 2017 Elsevier B.V. All rights reserved.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

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Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

2018-05-01

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

A mitochondrially targeted compound delays aging in yeast through a mechanism linking mitochondrial membrane lipid metabolism to mitochondrial redox biology

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Michelle T. Burstein

2014-01-01

Full Text Available A recent study revealed a mechanism of delaying aging in yeast by a natural compound which specifically impacts mitochondrial redox processes. In this mechanism, exogenously added lithocholic bile acid enters yeast cells, accumulates mainly in the inner mitochondrial membrane, and elicits an age-related remodeling of phospholipid synthesis and movement within both mitochondrial membranes. Such remodeling of mitochondrial phospholipid dynamics progresses with the chronological age of a yeast cell and ultimately causes significant changes in mitochondrial membrane lipidome. These changes in the composition of membrane phospholipids alter mitochondrial abundance and morphology, thereby triggering changes in the age-related chronology of such longevity-defining redox processes as mitochondrial respiration, the maintenance of mitochondrial membrane potential, the preservation of cellular homeostasis of mitochondrially produced reactive oxygen species, and the coupling of electron transport to ATP synthesis.

Vimar Is a Novel Regulator of Mitochondrial Fission through Miro.

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Lianggong Ding

2016-10-01

Full Text Available As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF. However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila, we incidentally observed that the loss of vimar, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro, we speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, we found that mitochondrial fission played a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro's effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinson's disease (PD, caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, we found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional.

Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

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Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

2013-01-01

Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, prespiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

Quantitative evaluation of the mitochondrial proteomes of Drosophila melanogaster adapted to extreme oxygen conditions.

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Songyue Yin

Full Text Available Mitochondria are the primary organelles that consume oxygen and provide energy for cellular activities. To investigate the mitochondrial mechanisms underlying adaptation to extreme oxygen conditions, we generated Drosophila strains that could survive in low- or high-oxygen environments (LOF or HOF, respectively, examined their mitochondria at the ultrastructural level via transmission electron microscopy, studied the activity of their respiratory chain complexes, and quantitatively analyzed the protein abundance responses of the mitochondrial proteomes using Isobaric tag for relative and absolute quantitation (iTRAQ. A total of 718 proteins were identified with high confidence, and 55 and 75 mitochondrial proteins displayed significant differences in abundance in LOF and HOF, respectively, compared with the control flies. Importantly, these differentially expressed mitochondrial proteins are primarily involved in respiration, calcium regulation, the oxidative response, and mitochondrial protein translation. A correlation analysis of the changes in the levels of the mRNAs corresponding to differentially regulated mitochondrial proteins revealed two sets of proteins with different modes of regulation (transcriptional vs. post-transcriptional in both LOF and HOF. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to extreme oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.

Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins.

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Rey, Benjamin; Halsey, Lewis G; Dolmazon, Virginie; Rouanet, Jean-Louis; Roussel, Damien; Handrich, Yves; Butler, Patrick J; Duchamp, Claude

2008-07-01

In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (Vo2; -33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-alpha) mRNA in pectoralis muscle (-54%, -36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting Vo2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting.

A Heme-Sensing Mechanism in the Translational Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

Science.gov (United States)

Soto, Iliana C.; Fontanesi, Flavia; Myers, Richard S.; Hamel, Patrice; Barrientos, Antoni

2012-01-01

Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N-terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production. PMID:23217259

Skeletal Muscle Mitochondrial Function in Polycystic Ovarian Syndrome

DEFF Research Database (Denmark)

Rabøl, Rasmus; Svendsen, Pernille Maj; Skovbro, Mette

2011-01-01

Hyperinsulinemic euglycemic clamps (40 mU/min/m2) and muscle biopsies were performed on 23 women with PCOS (9 lean (body mass index (BMI) 25 kg/m2)) and 17 age- and weight-matched controls (6 lean and 11 obese). Western blotting and high-resolution respirometry was used to determine mitochondrial function. Results......Objective Polycystic ovarian syndrome (PCOS) is associated with skeletal muscle insulin resistance, which has been linked to decreased mitochondrial function. We measured mitochondrial respiration in lean and obese women with and without PCOS using high-resolution respirometry. Methods...... Insulin sensitivity decreased with PCOS and increasing body weight. Mitochondrial respiration with substrates for complex I and complex I+II were similar in all groups, and PCOS was not associated with a decrease in mitochondrial content as measured by mtDNA/genomicDNA. We found no correlation between...

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

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Guorui Huang

2017-07-01

Full Text Available Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF. Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

DEFF Research Database (Denmark)

Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K

2017-01-01

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration...... and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity......-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate...

Diglycolic acid, the toxic metabolite of diethylene glycol, chelates calcium and produces renal mitochondrial dysfunction in vitro.

Science.gov (United States)

Conrad, Taylor; Landry, Greg M; Aw, Tak Yee; Nichols, Royce; McMartin, Kenneth E

2016-07-01

Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5 mmol/L) than succinate respiration (100 mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to

Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits.

Science.gov (United States)

Masand, Ruchi; Paulo, Esther; Wu, Dongmei; Wang, Yangmeng; Swaney, Danielle L; Jimenez-Morales, David; Krogan, Nevan J; Wang, Biao

2018-03-06

Brown adipose tissue (BAT) thermogenesis is critical for thermoregulation and contributes to total energy expenditure. However, whether BAT has non-thermogenic functions is largely unknown. Here, we describe that BAT-specific liver kinase b1 knockout (Lkb1 BKO ) mice exhibited impaired BAT mitochondrial respiration and thermogenesis but reduced adiposity and liver triglyceride accumulation under high-fat-diet feeding at room temperature. Importantly, these metabolic benefits were also present in Lkb1 BKO mice at thermoneutrality, where BAT thermogenesis was not required. Mechanistically, decreased mRNA levels of mtDNA-encoded electron transport chain (ETC) subunits and ETC proteome imbalance led to defective BAT mitochondrial respiration in Lkb1 BKO mice. Furthermore, reducing mtDNA gene expression directly in BAT by removing mitochondrial transcription factor A (Tfam) in BAT also showed ETC proteome imbalance and the trade-off between BAT thermogenesis and systemic metabolism at room temperature and thermoneutrality. Collectively, our data demonstrate that ETC proteome imbalance in BAT regulates systemic metabolism independently of thermogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

The mitochondrial toxicity of cysteine-S-conjugates: Studies with pentachlorobutadienyl-L-cysteine

International Nuclear Information System (INIS)

Wallin, A.

1990-01-01

Nephrotoxic cysteine conjugates, arising from mercapturate biosynthesis, can perturb the mitochondrial membrane potential and calcium homeostasis in renal epithelial cells. Activation of these cysteine conjugates to reactive species by mitochondrial β-lyases results in covalent binding and mitochondrial damage. PCBC and related cysteine conjugates inhibit ADP-stimulated respiration in mitochondria respiring on alpha-ketoglutrate/malate and succinate indicating that both dehydrogenases may be targets. The respiratory inhibition is blocked by aminooxyacetic acid, an inhibitor of the β-lyase. Hence, metabolic activation is required implying that covalent binding of reactive intermediates may be important to the mitochondrial injury. Binding of 35 S-fragments has been found for 5 conjugates with varying degrees of mitochondrial toxicity. PCBC is more lipophilic and has a higher affinity for cellular membranes than other cysteine conjugates. PCBC rapidly depolarizes the inner membrane potential resulting in an inhibition of mitochondrial oxidative phosphorylation and calcium upon sequestration. Consequently, mitochondria and renal epithelial cells exposed to PCBC show a sudden release of calcium upon exposure to PCBC which is followed by a later increase in state 4 respiration leading to an inhibition of oxidative phosphorylation. The primary effect of other cysteine conjugates is an inhibition of the dehydrogenases, thus inhibiting state 3 respiration

Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer.

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Ajay Kumar

Full Text Available Minnelide/Triptolide (TL has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC. However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS. Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2, along with diminished cellular glutathione (GSH content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC. TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y, restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor.

Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

Science.gov (United States)

Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

2018-05-17

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS mediated cardiomyocyte hypertrophy

NARCIS (Netherlands)

Tigchelaar, Wardit; Yu, Hongjuan; De Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Sillje, Herman H W

2015-01-01

Recently, a genetic variant in the mitochondrial exo/endo nuclease EXOG, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

International Nuclear Information System (INIS)

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh; Godbole, Madan M.

2010-01-01

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1α, NRF-1α and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

Energy Technology Data Exchange (ETDEWEB)

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh [Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014 (India); Godbole, Madan M., E-mail: madangodbole@yahoo.co.in [Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014 (India)

2010-07-02

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.

ABT737 enhances cholangiocarcinoma sensitivity to cisplatin through regulation of mitochondrial dynamics

International Nuclear Information System (INIS)

Fan, Zhongqi; Yu, Huimei; Cui, Ni; Kong, Xianggui; Liu, Xiaomin; Chang, Yulei; Wu, Yao; Sun, Liankun; Wang, Guangyi

2015-01-01

Cholangiocarcinoma responses weakly to cisplatin. Mitochondrial dynamics participate in the response to various stresses, and mainly involve mitophagy and mitochondrial fusion and fission. Bcl-2 family proteins play critical roles in orchestrating mitochondrial dynamics, and are involved in the resistance to cisplatin. Here we reported that ABT737, combined with cisplatin, can promote cholangiocarcinoma cells to undergo apoptosis. We found that the combined treatment decreased the Mcl-1 pro-survival form and increased Bak. Cells undergoing cisplatin treatment showed hyperfused mitochondria, whereas fragmentation was dominant in the mitochondria of cells exposed to the combined treatment, with higher Fis1 levels, decreased Mfn2 and OPA1 levels, increased ratio of Drp1 60 kD to 80 kD form, and more Drp1 located on mitochondria. More p62 aggregates were observed in cells with fragmented mitochondria, and they gradually translocated to mitochondria. Mitophagy was induced by the combined treatment. Knockdown p62 decreased the Drp1 ratio, increased Tom20, and increased cell viability. Our data indicated that mitochondrial dynamics play an important role in the response of cholangiocarcinoma to cisplatin. ABT737 might enhance cholangiocarcinoma sensitivity to cisplatin through regulation of mitochondrial dynamics and the balance within Bcl-2 family proteins. Furthermore, p62 seems to be critical in the regulation of mitochondrial dynamics. - Highlights: • Cholangiocarcinoma may adapt to cisplatin through mitochondrial fusion. • ABT737 sensitizes cholangiocarcinoma to cisplatin by promoting fission and mitophagy. • p62 might participate in the regulation of mitochondrial fission and mitophagy

Seasonal acclimation and latitudinal adaptation are of the same magnitude in Mytilus edulis and Mytilus trossulus mitochondrial respiration

DEFF Research Database (Denmark)

Thyrring, Jakob; Bundgård, Amanda Marie; Sejr, Mikael Kristian

2017-01-01

Species distribution models often assume homogeneous physiological performance within a species distribution range. This assumption potentially underestimates the distribution as it neglects physiological plasticity and adaptation among species and populations. Better knowledge on the physiological......% after cold acclimation. Combined, our results show that seasonal variation in mitochondrial respiration is of the same magnitude as large-scale (>1000 km) latitudinal variation. The high respiratory plasticity in Mytilus spp. improves fitness in changing temperature environments and supports their large...

UCP2 Regulates Mitochondrial Fission and Ventromedial Nucleus Control of Glucose Responsiveness.

Science.gov (United States)

Toda, Chitoku; Kim, Jung Dae; Impellizzeri, Daniela; Cuzzocrea, Salvatore; Liu, Zhong-Wu; Diano, Sabrina

2016-02-25

The ventromedial nucleus of the hypothalamus (VMH) plays a critical role in regulating systemic glucose homeostasis. How neurons in this brain area adapt to the changing metabolic environment to regulate circulating glucose levels is ill defined. Here, we show that glucose load results in mitochondrial fission and reduced reactive oxygen species in VMH neurons mediated by dynamin-related peptide 1 (DRP1) under the control of uncoupling protein 2 (UCP2). Probed by genetic manipulations and chemical-genetic control of VMH neuronal circuitry, we unmasked that this mitochondrial adaptation determines the size of the pool of glucose-excited neurons in the VMH and that this process regulates systemic glucose homeostasis. Thus, our data unmasked a critical cellular biological process controlled by mitochondrial dynamics in VMH regulation of systemic glucose homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of respiration buffer composition on mitochondrial metabolism and function

OpenAIRE

Wollenman, Lucas C.; Vander Ploeg, Matthew R.; Miller, Mackinzie L.; Zhang, Yizhu; Bazil, Jason N.

2017-01-01

Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffer...

Methylene blue improves mitochondrial respiration and decreases oxidative stress in a substrate-dependent manner in diabetic rat hearts.

Science.gov (United States)

Duicu, Oana M; Privistirescu, Andreea; Wolf, Adrian; Petruş, Alexandra; Dănilă, Maria D; Raţiu, Corina D; Muntean, Danina M; Sturza, Adrian

2017-11-01

Diabetic cardiomyopathy has been systematically associated with compromised mitochondrial energetics and increased generation of reactive oxygen species (ROS) that underlie its progression to heart failure. Methylene blue is a redox drug with reported protective effects mainly on brain mitochondria. The purpose of the present study was to characterize the effects of acute administration of methylene blue on mitochondrial respiration, H 2 O 2 production, and calcium sensitivity in rat heart mitochondria isolated from healthy and 2 months (streptozotocin-induced) diabetic rats. Mitochondrial respiratory function was assessed by high-resolution respirometry. H 2 O 2 production and calcium retention capacity were measured spectrofluorimetrically. The addition of methylene blue (0.1 μmol·L -1 ) elicited an increase in oxygen consumption of mitochondria energized with complex I and II substrates in both normal and diseased mitochondria. Interestingly, methylene blue elicited a significant increase in H 2 O 2 release in the presence of complex I substrates (glutamate and malate), but had an opposite effect in mitochondria energized with complex II substrate (succinate). No changes in the calcium retention capacity of healthy or diabetic mitochondria were found in the presence of methylene blue. In conclusion, in cardiac mitochondria isolated from diabetic and nondiabetic rat hearts, methylene blue improved respiratory function and elicited a dichotomic, substrate-dependent effect on ROS production.

Patients with sepsis exhibit increased mitochondrial respiratory capacity in peripheral blood immune cells

DEFF Research Database (Denmark)

Sjövall, Fredrik; Morota, Saori; Persson, Johan Mikael

2013-01-01

to 7). Mitochondrial DNA (mtDNA), cytochrome c (Cyt c), and citrate synthase (CS) were measured as indicators of cellular mitochondrial content. RESULTS: In intact PBICs with endogenous substrates, a gradual increase in cellular respiration reached 173% of controls after 1 week (P = 0......INTRODUCTION: In sepsis, mitochondria have been associated with both initial dysfunction and subsequent upregulation (biogenesis). However, the evolvement of mitochondrial function in sepsis over time is largely unknown, and we therefore investigated mitochondrial respiration in peripheral blood.......001). In permeabilized cells, respiration using substrates of complex I, II, and IV were significantly increased days 1 to 2, reaching 137%, 130%, and 173% of controls, respectively. In parallel, higher levels of CS activity, mtDNA, and Cyt c content in PBICs (211%, 243%, and 331% of controls for the respective...

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

Science.gov (United States)

Kathiresan, Meena; Martins, Dorival; English, Ann M.

2014-01-01

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

Science.gov (United States)

Kathiresan, Meena; Martins, Dorival; English, Ann M

2014-12-09

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

Nutrition controls mitochondrial biogenesis in the Drosophila adipose tissue through Delg and cyclin D/Cdk4.

Directory of Open Access Journals (Sweden)

Claudia Baltzer

Full Text Available MITOCHONDRIA ARE CELLULAR ORGANELLES THAT PERFORM CRITICAL METABOLIC FUNCTIONS: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. Here, we demonstrate that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, we show that Delg (CG6338, the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. Our data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity.

Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration.

Science.gov (United States)

Gamberi, Tania; Fiaschi, Tania; Modesti, Alessandra; Massai, Lara; Messori, Luigi; Balzi, Manuela; Magherini, Francesca

2015-08-01

Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

DEFF Research Database (Denmark)

Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

2009-01-01

,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

Targeting Mitochondrial Dysfunction with L-Alpha Glycerylphosphorylcholine.

Directory of Open Access Journals (Sweden)

Gerda Strifler

Full Text Available We hypothesized that L-alpha-glycerylphosphorylcholine (GPC, a deacylatedphosphatidylcholine derivative, can influence the mitochondrial respiratory activity and in this way, may exert tissue protective effects.Rat liver mitochondria were examined with high-resolution respirometry to analyze the effects of GPC on the electron transport chain in normoxic and anoxic conditions. Besides, Sprague-Dawley rats were subjected to sham operation or standardized liver ischemia-reperfusion (IR, with or without GPC administration. The reduced glutathione (GSH and oxidized glutathione disulfide (GSSG, the tissue myeloperoxidase, xanthine oxidoreductase and NADPH oxidases activities were measured. Tissue malondialdehyde and nitrite/nitrate formation, together with blood superoxide and hydrogen-peroxide production were assessed.GPC increased the efficacy of complex I-linked mitochondrial oxygen consumption, with significantly lower in vitro leak respiration. Mechanistically, liver IR injury was accompanied by deteriorated mitochondrial respiration and enhanced ROS production and, as a consequence, by significantly increased inflammatory enzyme activities. GPC administration decreased the inflammatory activation in line with the reduced oxidative and nitrosative stress markers.GPC, by preserving the mitochondrial complex I function respiration, reduced the biochemical signs of oxidative stress after an IR episode. This suggests that GPC is a mitochondria-targeted compound that indirectly suppresses the activity of major intracellular superoxide-generating enzymes.

Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

Directory of Open Access Journals (Sweden)

William Dott

2014-01-01

Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes.

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Patrick, Ping; Price, Tulin O; Diogo, Ana L; Sheibani, Nader; Banks, William A; Shah, Gul N

Hyperglycemia in diabetes mellitus causes oxidative stress and pericyte depletion from the microvasculature of the brain thus leading to the Blood-Brain Barrier (BBB) disruption. The compromised BBB exposes the brain to circulating substances, resulting in neurotoxicity and neuronal cell death. The decline in pericyte numbers in diabetic mouse brain and pericyte apoptosis in high glucose cultures are caused by excess superoxide produced during enhanced respiration (mitochondrial oxidative metabolism of glucose). Superoxide is precursor to all Reactive Oxygen Species (ROS) which, in turn, cause oxidative stress. The rate of respiration and thus the ROS production is regulated by mitochondrial carbonic anhydrases (mCA) VA and VB, the two isoforms expressed in the mitochondria. Inhibition of both mCA: decreases the oxidative stress and restores the pericyte numbers in diabetic brain; and reduces high glucose-induced respiration, ROS, oxidative stress, and apoptosis in cultured brain pericytes. However, the individual role of the two isoforms has not been established. To investigate the contribution of mCA VA in ROS production and apoptosis, a mCA VA overexpressing brain pericyte cell line was engineered. These cells were exposed to high glucose and analyzed for the changes in ROS and apoptosis. Overexpression of mCA VA significantly increased pericyte ROS and apoptosis. Inhibition of mCA VA with topiramate prevented increases both in glucose-induced ROS and pericyte death. These results demonstrate, for the first time, that mCA VA regulates the rate of pericyte respiration. These findings identify mCA VA as a novel and specific therapeutic target to protect the cerebromicrovascular bed in diabetes.

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

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Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus microplus Embryos

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Carlos Logullo

2011-06-01

Full Text Available The physiological roles of polyphosphates (polyP recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5’-triphosphate synthesis in hard tick embryos.

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus) microplus Embryos

Science.gov (United States)

Campos, Eldo; Façanha, Arnoldo R.; Costa, Evenilton P.; Fraga, Amanda; Moraes, Jorge; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

2011-01-01

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5′-triphosphate synthesis in hard tick embryos. PMID:21747692

Maintaining ancient organelles: mitochondrial biogenesis and maturation.

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Vega, Rick B; Horton, Julie L; Kelly, Daniel P

2015-05-22

The ultrastructure of the cardiac myocyte is remarkable for the high density of mitochondria tightly packed between sarcomeres. This structural organization is designed to provide energy in the form of ATP to fuel normal pump function of the heart. A complex system comprised of regulatory factors and energy metabolic machinery, encoded by both mitochondrial and nuclear genomes, is required for the coordinate control of cardiac mitochondrial biogenesis, maturation, and high-capacity function. This process involves the action of a transcriptional regulatory network that builds and maintains the mitochondrial genome and drives the expression of the energy transduction machinery. This finely tuned system is responsive to developmental and physiological cues, as well as changes in fuel substrate availability. Deficiency of components critical for mitochondrial energy production frequently manifests as a cardiomyopathic phenotype, underscoring the requirement to maintain high respiration rates in the heart. Although a precise causative role is not clear, there is increasing evidence that perturbations in this regulatory system occur in the hypertrophied and failing heart. This review summarizes current knowledge and highlights recent advances in our understanding of the transcriptional regulatory factors and signaling networks that serve to regulate mitochondrial biogenesis and function in the mammalian heart. © 2015 American Heart Association, Inc.

The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

International Nuclear Information System (INIS)

Park, Jeehye; Lee, Gina; Chung, Jongkyeong

2009-01-01

The two Parkinson's disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.

Protein Carbonylation and Adipocyte Mitochondrial Function*

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Curtis, Jessica M.; Hahn, Wendy S.; Stone, Matthew D.; Inda, Jacob J.; Droullard, David J.; Kuzmicic, Jovan P.; Donoghue, Margaret A.; Long, Eric K.; Armien, Anibal G.; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J.; Bernlohr, David A.

2012-01-01

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte. PMID:22822087

Protein carbonylation and adipocyte mitochondrial function.

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Curtis, Jessica M; Hahn, Wendy S; Stone, Matthew D; Inda, Jacob J; Droullard, David J; Kuzmicic, Jovan P; Donoghue, Margaret A; Long, Eric K; Armien, Anibal G; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J; Bernlohr, David A

2012-09-21

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.

Endotoxin-induced basal respiration alterations of renal HK-2 cells: A sign of pathologic metabolism down-regulation

Energy Technology Data Exchange (ETDEWEB)

Quoilin, C., E-mail: cquoilin@ulg.ac.be [Laboratory of Biomedical Spectroscopy, Department of Physics, University of Liege, 4000 Liege (Belgium); Mouithys-Mickalad, A. [Center of Oxygen Research and Development, Department of Chemistry, University of Liege, 4000 Liege (Belgium); Duranteau, J. [Department of Anaesthesia and Surgical ICU, CHU Bicetre, University Paris XI Sud, 94275 Le Kremlin Bicetre (France); Gallez, B. [Biomedical Magnetic Resonance Group, Louvain Drug Research Institute, Universite catholique de Louvain, 1200 Brussels (Belgium); Hoebeke, M. [Laboratory of Biomedical Spectroscopy, Department of Physics, University of Liege, 4000 Liege (Belgium)

2012-06-29

Highlights: Black-Right-Pointing-Pointer A HK-2 cells model of inflammation-induced acute kidney injury. Black-Right-Pointing-Pointer Two oximetry methods: high resolution respirometry and ESR spectroscopy. Black-Right-Pointing-Pointer Oxygen consumption rates of renal cells decrease when treated with LPS. Black-Right-Pointing-Pointer Cells do not recover normal respiration when the LPS treatment is removed. Black-Right-Pointing-Pointer This basal respiration alteration is a sign of pathologic metabolism down-regulation. -- Abstract: To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) both by high-resolution respirometry and electron spin resonance spectroscopy. These two complementary methods have shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells.

Interactive effects of pH and metals on mitochondrial functions of intertidal bivalves Crassostrea virginica and Mercenaria mercenaria

International Nuclear Information System (INIS)

Ivanina, Anna V.; Sokolova, Inna M.

2013-01-01

Highlights: •Interactive effects of trace metals Cd and Cu and pH were studied in mitochondria of clams and oysters. •Mitochondrial respiration and membrane potential of bivalves were robust to pH variation (6.6–7.8). •Elevated levels of Cd and Cu inhibited mitochondrial respiration in the pH-dependent manner but did not affect the membrane potential. •Negative effects of Cd and Cd on mitochondrial respiration were alleviated at low pH (7.0 and below). •Moderate acidosis may protect molluscan mitochondria from metal toxicity. -- Abstract: Intertidal bivalves experience broad fluctuations of environmental temperature, pH and oxygen content which could change their intracellular pH. They are also exposed to trace metals such as cadmium (Cd) and copper (Cu) that accumulate in their tissues and may negatively affect mitochondrial functions and bioenergetics. We determined the interactive effects of pH and trace metals (25 μM Cd or Cu) on mitochondrial functions (including respiration and membrane potentials in both ADP-stimulated (state 3) and resting (state 4) states) of two common marine bivalves, the hard clams (Mercenaria mercenaria) and eastern oysters (Crassostrea virginica). In the absence of the trace metals, mitochondrial functions of C. virginica and M. mercenaria were insensitive to pH in a broad physiologically relevant range (6.6–7.8). Mitochondrial respiration was generally suppressed by 25 μM Cd or Cu (with the stronger effects observed for ADP-stimulated compared to the resting respiration) while the mitochondrial membrane potential was unaffected. pH modulated the effects of Cu and Cd on mitochondrial respiration of the bivalves. In oysters, Cu suppressed ADP-stimulated mitochondrial respiration at high and low pH values (6.6 and 7.8, respectively), but had no effect in the intermediate pH range (7.0–7.4). In clams, the negative effect of Cu on ADP-stimulated respiration was only observed at extremely high pH (7.8). A decrease in p

Interactive effects of pH and metals on mitochondrial functions of intertidal bivalves Crassostrea virginica and Mercenaria mercenaria

Energy Technology Data Exchange (ETDEWEB)

Ivanina, Anna V.; Sokolova, Inna M., E-mail: isokolov@uncc.edu

2013-11-15

Highlights: •Interactive effects of trace metals Cd and Cu and pH were studied in mitochondria of clams and oysters. •Mitochondrial respiration and membrane potential of bivalves were robust to pH variation (6.6–7.8). •Elevated levels of Cd and Cu inhibited mitochondrial respiration in the pH-dependent manner but did not affect the membrane potential. •Negative effects of Cd and Cd on mitochondrial respiration were alleviated at low pH (7.0 and below). •Moderate acidosis may protect molluscan mitochondria from metal toxicity. -- Abstract: Intertidal bivalves experience broad fluctuations of environmental temperature, pH and oxygen content which could change their intracellular pH. They are also exposed to trace metals such as cadmium (Cd) and copper (Cu) that accumulate in their tissues and may negatively affect mitochondrial functions and bioenergetics. We determined the interactive effects of pH and trace metals (25 μM Cd or Cu) on mitochondrial functions (including respiration and membrane potentials in both ADP-stimulated (state 3) and resting (state 4) states) of two common marine bivalves, the hard clams (Mercenaria mercenaria) and eastern oysters (Crassostrea virginica). In the absence of the trace metals, mitochondrial functions of C. virginica and M. mercenaria were insensitive to pH in a broad physiologically relevant range (6.6–7.8). Mitochondrial respiration was generally suppressed by 25 μM Cd or Cu (with the stronger effects observed for ADP-stimulated compared to the resting respiration) while the mitochondrial membrane potential was unaffected. pH modulated the effects of Cu and Cd on mitochondrial respiration of the bivalves. In oysters, Cu suppressed ADP-stimulated mitochondrial respiration at high and low pH values (6.6 and 7.8, respectively), but had no effect in the intermediate pH range (7.0–7.4). In clams, the negative effect of Cu on ADP-stimulated respiration was only observed at extremely high pH (7.8). A decrease in p

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation

DEFF Research Database (Denmark)

Larsen, Filip J; Schiffer, Tomas A; Ørtenblad, Niels

2016-01-01

. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6-fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response...

Increased mitochondrial substrate sensitivity in skeletal muscle of patients with type 2 diabetes

DEFF Research Database (Denmark)

Larsen, S; Stride, N; Hey-Mogensen, Martin

2011-01-01

AIMS/HYPOTHESIS: Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants. METHODS: Mitochondrial respiration was measured.......4). Substrate sensitivity for octanoyl-carnitine did not differ between groups. CONCLUSIONS/INTERPRETATION: Increased mitochondrial substrate sensitivity is seen in skeletal muscle from type 2 diabetic patients and is confined to non-lipid substrates. Respiratory capacity per mitochondrion is not decreased...... and maximal oxygen uptake (VO2) were also determined. Insulin sensitivity was determined with the isoglycaemic-hyperinsulinaemic clamp technique. RESULTS: Insulin sensitivity was different (p

Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

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Zhang, Tiantian; Bu, Pengli; Zeng, Joey; Vancura, Ales

2017-10-13

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4 , the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1 , which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

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Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

2010-10-01

The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects

Science.gov (United States)

Huang, Zih-Ning; Chung, Her Min; Fang, Su-Chiung; Her, Lu-Shiun

2017-01-01

Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction. PMID:29209146

An Unusual Prohibitin Regulates Malaria Parasite Mitochondrial Membrane Potential

Directory of Open Access Journals (Sweden)

Joachim Michael Matz

2018-04-01

Full Text Available Summary: Proteins of the stomatin/prohibitin/flotillin/HfIK/C (SPFH family are membrane-anchored and perform diverse cellular functions in different organelles. Here, we investigate the SPFH proteins of the murine malaria model parasite Plasmodium berghei, the conserved prohibitin 1, prohibitin 2, and stomatin-like protein and an unusual prohibitin-like protein (PHBL. The SPFH proteins localize to the parasite mitochondrion. While the conserved family members could not be deleted from the Plasmodium genome, PHBL was successfully ablated, resulting in impaired parasite fitness and attenuated virulence in the mammalian host. Strikingly, PHBL-deficient parasites fail to colonize the Anopheles vector because of complete arrest during ookinete development in vivo. We show that this arrest correlates with depolarization of the mitochondrial membrane potential (ΔΨmt. Our results underline the importance of SPFH proteins in the regulation of core mitochondrial functions and suggest that fine-tuning of ΔΨmt in malarial parasites is critical for colonization of the definitive host. : Matz et al. present an experimental genetics study of an unusual prohibitin-like protein in the malaria parasite and find that it regulates mitochondrial membrane polarity. Ablation of this protein causes almost complete mitochondrial depolarization in the mosquito vector, which, in turn, leads to a block in malaria parasite transmission. Keywords: Plasmodium berghei, malaria, SPFH, prohibitin, stomatin-like protein, mitochondrion, membrane potential, ookinete, transmission

Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

Science.gov (United States)

Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Sloan, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth W; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

2018-01-05

Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

Science.gov (United States)

Lyra-Leite, Davi M; Andres, Allen M; Petersen, Andrew P; Ariyasinghe, Nethika R; Cho, Nathan; Lee, Jezell A; Gottlieb, Roberta A; McCain, Megan L

2017-10-01

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O 2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix

Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2

Science.gov (United States)

Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong

2011-01-01

Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114

The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells

International Nuclear Information System (INIS)

Xiao, Anqi; Gan, Xueqi; Chen, Ruiqi; Ren, Yanming; Yu, Haiyang; You, Chao

2017-01-01

Oxidative stress plays a central role in the pathogenesis of various neurodegenerative diseases. Increasing evidences have demonstrated that structural abnormalities in mitochondria are involved in oxidative stress related nerve cell damage. And Drp1 plays a critical role in mitochondrial dynamic imbalance insulted by oxidative stress-derived mitochondria. However, the status of mitochondrial fusion and fission pathway and its relationship with mitochondrial properties such as mitochondrial membrane permeability transition pore (mPTP) have not been fully elucidated. Here, we demonstrated for the first time the role of Cyclophilin D (CypD), a crucial component for mPTP formation, in the regulation of mitochondrial dynamics in oxidative stress treated nerve cell. We observed that CypD-mediated phosphorylation of Drp1 and subsequently augmented Drp1 recruitment to mitochondria and shifts mitochondrial dynamics toward excessive fission, which contributes to the mitochondrial structural and functional dysfunctions in oxidative stress-treated nerve cells. CypD depletion or over expression accompanies mitochondrial dynamics/functions recovery or aggravation separately. We also demonstrated first time the link between the CypD to mitochondrial dynamics. Our data offer new insights into the mechanism of mitochondrial dynamics which contribute to the mitochondrial dysfunctions, specifically the role of CypD in Drp1-mediated mitochondrial fission. The protective effect of CsA, or other molecules affecting the function of CypD hold promise as a potential novel therapeutic strategy for governing oxidative stress pathology via mitochondrial pathways. - Highlights: • Demonstrated first time the link between the mPTP to mitochondrial dynamics. • The role of Cyclophilin D in the regulation of Drp1-mediated mitochondrial fission. • CsA as a potential target for governing oxidative stress related neuropathology.

Application of the principles of systems biology and Wiener's cybernetics for analysis of regulation of energy fluxes in muscle cells in vivo.

Science.gov (United States)

Guzun, Rita; Saks, Valdur

2010-03-08

The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener's cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener's cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures - intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations of

Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. I. Direct effects of in vitro mercuric chloride on renal cortical mitochondrial function

Energy Technology Data Exchange (ETDEWEB)

Weinberg, J.M. (Veterans Administration Medical Center, Ann Arbor, MI); Harding, P.G.; Humes, H.D.

1982-01-01

Increasing data suggest that mitochondrial dysfunction may be an important early component of nephrotoxin-induced changes in renal cell function and viability. This study was designed to obtain more detailed information about the effects on several basic bioenergetic parameters of the direct interaction of Hg/sup 2 +/ with renal cortical mitochondria in vitro as a necessary prelude to studies of mitochondrial functional changes after treatment with mercuric chloride in vivo. Beginning at a threshhold level of 2 nmol of Hg/sup 2 +//mg of mitochondrial protein Hg/sup 2 +/ induced marked stimulation of State 4 respiration, mild inhibition of State 3 respiration, and 2,4-dinitrophenol uncoupled respiration, a striking increase in atractyloside-insensitive ADP uptake and stimulation of both basal- and Mg/sup 2 +/-activated oligomycin-sensitive mitochondrial ATPase activity. These effects of Hg/sup 2 +/ could be prevented and reversed by the sulfhydryl reagent dithioerythritol and by albumin but were not affected by Mg/sup 2 +/. Detailed studies on the addition of HgCl/sub 2/ to the preparation at different stages of the mitochondrial isolation procedure demonstrated that the presence of other proteins decreased mitochondrial Hg/sup 2 +/ binding, that the Hg/sup 2 +/ was not readily washed off the mitochondria by nonprotein-containing solutions, and that prolonged exposure of mitochondria to Hg/sup 2 +/ during the isolation procedure did not markedly alter its functional effects on their reversibility as assessed on the final mitochondrial preparation. These data provide an important basis for critically assessing the changes in function of mitochondria isolated after in vivo treatment with mercuric chloride.

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Energy Technology Data Exchange (ETDEWEB)

Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko, E-mail: amasaki@mail.ecc.u-tokyo.ac.jp

2014-08-15

Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

Respiration of midges (Diptera; Chironomidae) in British Columbian lakes: oxy-regulation, temperature and their role as palaeo-indicators

DEFF Research Database (Denmark)

Brodersen, Klaus Peter; Pedersen, Ole; Walker, Ian R.

2008-01-01

1. The specific respiration rate of 13 chironomid taxa and Chaoborus were measured to test the hypothesis of the relation between a species' ability to regulate their oxygen uptake and their distributional patterns among nine study lakes in British Columbia, Canada. 2. Respiration patterns of ind...

Reduced skeletal muscle mitochondrial respiration and improved glucose metabolism in nondiabetic obese women during a very low calorie dietary intervention leading to rapid weight loss

DEFF Research Database (Denmark)

Rabøl, Rasmus; Svendsen, Pernille F; Skovbro, Mette

2009-01-01

Reduced oxidative capacity of skeletal muscle has been proposed to lead to accumulation of intramyocellular triglyceride (IMTG) and insulin resistance. We have measured mitochondrial respiration before and after a 10% low-calorie-induced weight loss in young obese women to examine the relationship...... between mitochondrial function, IMTG, and insulin resistance. Nine obese women (age, 32.3 years [SD, 3.0]; body mass index, 33.4 kg/m(2) [SD, 2.6]) completed a 53-day (SE, 3.8) very low calorie diet (VLCD) of 500 to 600 kcal/d without altering physical activity. The target of the intervention was a 10...... resistance in young obese women and do not support a direct relationship between IMTG and insulin sensitivity in young obese women during weight loss....

Caloric Restriction-Induced Extension of Chronological Lifespan Requires Intact Respiration in Budding Yeast.

Science.gov (United States)

Kwon, Young-Yon; Lee, Sung-Keun; Lee, Cheol-Koo

2017-04-01

Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Science.gov (United States)

Martínez-Reyes, Inmaculada; Diebold, Lauren P; Kong, Hyewon; Schieber, Michael; Huang, He; Hensley, Christopher T; Mehta, Manan M; Wang, Tianyuan; Santos, Janine H; Woychik, Richard; Dufour, Eric; Spelbrink, Johannes N; Weinberg, Samuel E; Zhao, Yingming; DeBerardinis, Ralph J; Chandel, Navdeep S

2016-01-21

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation. Copyright © 2016 Elsevier Inc. All rights reserved.

Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum.

Science.gov (United States)

Bönnighausen, Jakob; Gebhard, Daniel; Kröger, Cathrin; Hadeler, Birgit; Tumforde, Thomas; Lieberei, Reinhard; Bergemann, Jörg; Schäfer, Wilhelm; Bormann, Jörg

2015-12-01

The cereal pathogen Fusarium graminearum threatens food and feed production worldwide. It reduces the yield and poisons the remaining kernels with mycotoxins, notably deoxynivalenol (DON). We analyzed the importance of gamma-aminobutanoic acid (GABA) metabolism for the life cycle of this fungal pathogen. GABA metabolism in F. graminearum is partially regulated by the global nitrogen regulator AreA. Genetic disruption of the GABA shunt by deletion of two GABA transaminases renders the pathogen unable to utilize the plant stress metabolites GABA and putrescine. The mutants showed increased sensitivity against oxidative stress, GABA accumulation in the mycelium, downregulation of two key enzymes of the TCA cycle, disturbed potential gradient in the mitochondrial membrane and lower mitochondrial oxygen consumption. In contrast, addition of GABA to the wild type resulted in its rapid turnover and increased mitochondrial steady state oxygen consumption. GABA concentrations are highly upregulated in infected wheat tissues. We conclude that GABA is metabolized by the pathogen during infection increasing its energy production, whereas the mutants accumulate GABA intracellularly resulting in decreased energy production. Consequently, the GABA mutants are strongly reduced in virulence but, because of their DON production, are able to cross the rachis node. © 2015 John Wiley & Sons Ltd.

Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells.

Science.gov (United States)

Granger, D L; Lehninger, A L

1982-11-01

Previous work has shown that injury of neoplastic cells by cytotoxic macrophages (CM) in cell culture is accompanied by inhibition of mitochondrial respiration. We have investigated the nature of this inhibition by studying mitochondrial respiration in CM-injured leukemia L1210 cells permeabilized with digitonin. CM-induced injury affects the mitochondrial respiratory chain proper. Complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) are markedly inhibited. In addition a minor inhibition of cytochrome oxidase was found. Electron transport from alpha-glycerophosphate through the respiratory chain to oxygen is unaffected and permeabilized CM-injured L1210 cells oxidizing this substrate exhibit acceptor control. However, glycerophosphate shuttle activity was found not to occur within CM-injured or uninjured L1210 cells in culture hence, alpha-glycerophosphate is apparently unavailable for mitochondrial oxidation in the intact cell. It is concluded that the failure of respiration of intact neoplastic cells injured by CM is caused by the nearly complete inhibition of complexes I and II of the mitochondrial electron transport chain. The time courses of CM-induced electron transport inhibition and arrest of L1210 cell division are examined and the possible relationship between these phenomena is discussed.

Application of the Principles of Systems Biology and Wiener’s Cybernetics for Analysis of Regulation of Energy Fluxes in Muscle Cells in Vivo

Science.gov (United States)

Guzun, Rita; Saks, Valdur

2010-01-01

The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener’s cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener’s cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures – intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations

Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

Science.gov (United States)

Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

2014-01-01

Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

Science.gov (United States)

Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S; Dringen, Ralf

2017-11-01

Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U- 13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Science.gov (United States)

Woodman, Andrew G; Mah, Richard; Keddie, Danae; Noble, Ronan M N; Panahi, Sareh; Gragasin, Ferrante S; Lemieux, Hélène; Bourque, Stephane L

2018-06-01

Prenatal iron deficiency alters fetal developmental trajectories, which results in persistent changes in organ function. Here, we studied the effects of prenatal iron deficiency on fetal kidney and liver mitochondrial function. Pregnant Sprague-Dawley rats were fed partially or fully iron-restricted diets to induce a state of moderate or severe iron deficiency alongside iron-replete control rats. We assessed mitochondrial function via high-resolution respirometry and reactive oxygen species generation via fluorescence microscopy on gestational d 21. Hemoglobin levels were reduced in dams in the moderate (-31%) and severe groups (-54%) compared with controls, which was accompanied by 55% reductions in fetal hemoglobin levels in both moderate and severe groups versus controls. Male iron-deficient kidneys exhibited globally reduced mitochondrial content and respiration, as well as increased cytosolic superoxide and decreased NO. Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress. Male iron-deficient livers exhibited reduced complex IV respiration and increased cytosolic superoxide, whereas female liver tissues exhibited no alteration in oxidant levels or mitochondrial function. These findings indicate that prenatal iron deficiency causes changes in mitochondrial content and function as well as oxidant status in a sex- and organ-dependent manner, which may be an important mechanism that underlies the programming of cardiovascular disease.-Woodman, A. G., Mah, R., Keddie, D., Noble, R. M. N., Panahi, S., Gragasin, F. S., Lemieux, H., Bourque, S. L. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Use the Protonmotive Force: Mitochondrial Uncoupling and Reactive Oxygen Species.

Science.gov (United States)

Berry, Brandon J; Trewin, Adam J; Amitrano, Andrea M; Kim, Minsoo; Wojtovich, Andrew P

2018-04-04

Mitochondrial respiration results in an electrochemical proton gradient, or protonmotive force (pmf), across the mitochondrial inner membrane. The pmf is a form of potential energy consisting of charge (∆ψ m ) and chemical (∆pH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Although capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review, we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models-mitochondria and cells-only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo. Copyright © 2018 Elsevier Ltd. All rights reserved.

What controls respiration rate in stored sugarbeet roots

Science.gov (United States)

Although respiration is estimated to be responsible for 60 to 80% of the sucrose lost during storage, the mechanisms by which sugarbeet roots regulate their respiration rate are unknown. In plants, respiration rate is regulated by (1) available respiratory capacity, (2) cellular energy status, (3) ...

Structures and mechanisms of antitumor agents: xestoquinones uncouple cellular respiration and disrupt HIF signaling in human breast tumor cells.

Science.gov (United States)

Du, Lin; Mahdi, Fakhri; Datta, Sandipan; Jekabsons, Mika B; Zhou, Yu-Dong; Nagle, Dale G

2012-09-28

The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 μM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

Science.gov (United States)

Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

2014-08-01

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

Separating rhizosphere respiration from total soil respiration in two larch plantations in northeastern China.

Science.gov (United States)

Jiang, Lifen; Shi, Fuchen; Li, Bo; Luo, Yiqi; Chen, Jiquan; Chen, Jiakuan

2005-09-01

The potential capacity of soil to sequester carbon in response to global warming is strongly regulated by the ratio of rhizosphere respiration to respiration by soil microbial decomposers, because of their different temperature sensitivities. To quantify relative contributions of rhizosphere respiration to total soil respiration as influenced by forest stand development, we conducted a trenching study in two larch (Larix gmelini (Rupr.) Rupr.) plantations, aged 17 and 31 years, in northeastern China. Four plots in each plantation were randomly selected and trenched in early May 2001. Soil surface CO2 effluxes both inside and outside the plots were measured from May 2001 to August 2002. Soil respiration (i.e., the CO2 effluxes outside the trenched plots) varied similarly in the two plantations from 0.8 micromol m(-2) s(-1) in winter to 6.0 micromol m(-2) s(-1) in summer. Rhizosphere respiration (i.e., CO2 efflux outside the trenched plots minus that inside the plots) varied from 0.2 to 2.0 micromol m(-2) s(-1) in the old forest and from 0.3 to 4.0 micromol m(-2) s(-1) in the young forest over the seasons. Rhizosphere respiration, on average, accounted for 25% of soil respiration in the old forest and 65% in the young forest. Rhizosphere and soil respiration were significantly correlated with soil temperature but not with soil water content. We conclude that the role forests play in regulating climate change may depend on their age.

FAM49B, a novel regulator of mitochondrial function and integrity that suppresses tumor metastasis.

Science.gov (United States)

Chattaragada, M S; Riganti, C; Sassoe, M; Principe, M; Santamorena, M M; Roux, C; Curcio, C; Evangelista, A; Allavena, P; Salvia, R; Rusev, B; Scarpa, A; Cappello, P; Novelli, F

2018-02-08

Mitochondrial dysregulation plays a central role in cancers and drives reactive oxygen species (ROS)-dependent tumor progression. We investigated the pro-tumoral roles of mitochondrial dynamics and altered intracellular ROS levels in pancreatic ductal adenocarcinoma (PDAC). We identified 'family with sequence similarity 49 member B' (FAM49B) as a mitochondria-localized protein that regulates mitochondrial fission and cancer progression. Silencing FAM49B in PDAC cells resulted in increased fission and mitochondrial ROS generation, which enhanced PDAC cell proliferation and invasion. Notably, FAM49B expression levels in PDAC cells were downregulated by the tumor microenvironment. Overall, the results of this study show that FAM49B acts as a suppressor of cancer cell proliferation and invasion in PDAC by regulating tumor mitochondrial redox reactions and metabolism.

Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana.

Science.gov (United States)

Barros, Jessica A S; Cavalcanti, João Henrique F; Medeiros, David B; Nunes-Nesi, Adriano; Avin-Wittenberg, Tamar; Fernie, Alisdair R; Araújo, Wagner L

2017-09-01

Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis ( Arabidopsis thaliana ) mutants with disruption of autophagy ( atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cdkal1, a type 2 diabetes susceptibility gene, regulates mitochondrial function in adipose tissue

Directory of Open Access Journals (Sweden)

Colin J. Palmer

2017-10-01

Conclusions: Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

Science.gov (United States)

Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

2017-08-15

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

DEFF Research Database (Denmark)

Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

2002-01-01

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...... procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit approximately 0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated...... as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore...

Impaired cardiac mitochondrial oxidative phosphorylation and enhanced mitochondrial oxidative stress in feline hypertrophic cardiomyopathy

DEFF Research Database (Denmark)

Christiansen, Liselotte Bruun; Dela, Flemming; Koch, Jørgen

2015-01-01

respiration with complex I-linked nonfatty acid substrates and with fatty acid substrates, respectively, was significantly lower in the hearts of HCM cats compared with control cats. Mitochondrial ROS release during state 3 with complex I-linked substrates and thiobarbituric acid-reactive substances...

Age-and Brain Region-Specific Differences in Mitochondrial ...

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Mitochondria are central regulators of energy homeostasis and play a pivotal role in mechanisms of cellular senescence. The objective of the present study was to evaluate mitochondrial bio­-energetic parameters in five brain regions [brainstem (BS), frontal cortex (FC), cerebellum (CER), striatum (STR), hippocampus (HIP)] of four diverse age groups [1 Month (young), 4 Month (adult), 12 Month (middle-aged), 24 Month (old age)] to understand age-related differences in selected brain regions and their contribution to age-related chemical sensitivity. Mitochondrial bioenergetics parameters and enzyme activity were measured under identical conditions across multiple age groups and brain regions in Brown Norway rats (n = 5). The results indicate age- and brain region-specific patterns in mitochondrial functional endpoints. For example, an age-specific decline in ATP synthesis (State 111 respiration) was observed in BS and HIP. Similarly, the maximal respiratory capacities (State V1 and V2) showed age-specific declines in all brain regions examined (young > adult > middle-aged > old age). Amongst all regions, HIP had the greatest change in mitochondrial bioenergetics, showing declines in the 4, 12 and 24 Month age groups. Activities of mitochondrial pyruvate dehydrogenase complex (PDHC) and electron transport chain (ETC) complexes I, II, and IV enzymes were also age- and brain-region specific. In general changes associated with age were more pronounced, with

Seeking homeostasis: Temporal trends in respiration, oxidation, and calcium in SOD1 G93A Amyotrophic Lateral Sclerosis mice

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Cameron W Irvin

2015-07-01

Full Text Available Impairments in mitochondria, oxidative regulation, and calcium homeostasis have been well documented in numerous amyotrophic lateral sclerosis (ALS experimental models, especially in the superoxide dismutase 1 glycine 93 to alanine (SOD1 G93A transgenic mouse. However, the timing of these deficiencies has been debatable. In a systematic review of 45 articles, we examine experimental measurements of cellular respiration, mitochondrial mechanisms, oxidative markers, and calcium regulation. We evaluate the quantitative magnitude and statistical temporal trend of these aggregated assessments in high transgene copy SOD1 G93A mice compared to wild type mice. Analysis of overall trends reveals cellular respiration, intracellular ATP, and corresponding mitochondrial elements (Cox, cytochrome c, complex I, enzyme activity are depressed for the entire lifespan of the SOD1 G93A mouse. Oxidant markers (H2O2, 8OH2’dG, MDA are initially similar to wild type but are double that of wild type by the time of symptom onset despite early post-natal elevation of protective heat shock proteins. All aspects of calcium regulation show early disturbances, although a notable and likely compensatory convergence to near wild type levels appears to occur between 40-80 days (pre-onset, followed by a post-onset elevation in intracellular calcium. The identified temporal trends and compensatory fluctuations provide evidence that the cause of ALS may lay within failed homeostatic regulation, itself, rather than any one particular perturbing event or cellular mechanism. We discuss the vulnerabilities of motoneurons to regulatory instability and possible hypotheses regarding failed regulation and its potential treatment in ALS.

DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

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LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

2012-01-01

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

Carbohydrate regulation of photosynthesis and respiration from branch girdling in four species of wet tropical rain forest trees.

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Asao, Shinichi; Ryan, Michael G

2015-06-01

How trees sense source-sink carbon balance remains unclear. One potential mechanism is a feedback from non-structural carbohydrates regulating photosynthesis and removing excess as waste respiration when the balance of photosynthesis against growth and metabolic activity changes. We tested this carbohydrate regulation of photosynthesis and respiration using branch girdling in four tree species in a wet tropical rainforest in Costa Rica. Because girdling severs phloem to stop carbohydrate export while leaving xylem intact to allow photosynthesis, we expected carbohydrates to accumulate in leaves to simulate a carbon imbalance. We varied girdling intensity by removing phloem in increments of one-quarter of the circumference (zero, one--quarter, half, three-quarters, full) and surrounded a target branch with fully girdled ones to create a gradient in leaf carbohydrate content. Light saturated photosynthesis rate was measured in situ, and foliar respiration rate and leaf carbohydrate content were measured after destructive harvest at the end of the treatment. Girdling intensity created no consistent or strong responses in leaf carbohydrates. Glucose and fructose slightly increased in all species by 3.4% per one-quarter girdle, total carbon content and leaf mass per area increased only in one species by 5.4 and 5.5% per one-quarter girdle, and starch did not change. Only full girdling lowered photosynthesis in three of four species by 59-69%, but the decrease in photosynthesis was unrelated to the increase in glucose and fructose content. Girdling did not affect respiration. The results suggest that leaf carbohydrate content remains relatively constant under carbon imbalance, and any changes are unlikely to regulate photosynthesis or respiration. Because girdling also stops the export of hormones and reactive oxygen species, girdling may induce physiological changes unrelated to carbohydrate accumulation and may not be an effective method to study carbohydrate feedback

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

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Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-07-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

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Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

2015-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

A nuclear mutation defective in mitochondrial recombination in yeast.

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Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

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Patterson, Jessica N; Cousteils, Katelyn; Lou, Jennifer W; Manning Fox, Jocelyn E; MacDonald, Patrick E; Joseph, Jamie W

2014-05-09

It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

The switch from fermentation to respiration in Saccharomyces cerevisiae is regulated by the Ert1 transcriptional activator/repressor.

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Gasmi, Najla; Jacques, Pierre-Etienne; Klimova, Natalia; Guo, Xiao; Ricciardi, Alessandra; Robert, François; Turcotte, Bernard

2014-10-01

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene. Copyright © 2014 by the Genetics Society of America.

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

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Sappal, Ravinder; MacDonald, Nicole; Fast, Mark; Stevens, Don; Kibenge, Fred; Siah, Ahmed; Kamunde, Collins

2014-01-01

Highlights: • Interacting effects of Cu and temperature were investigated in rainbow trout liver mitochondria. • Mitochondrial functional indices are highly sensitive to temperature change. • High and low temperatures sensitize mitochondria to adverse effects of Cu. • Cu induces a highly temperature-sensitive mitochondrial permeability transition pore. • Cu-imposed mitochondrial membrane potential dissipation is mediated by reactive oxygen species. - Abstract: Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q 10 effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control, Cu

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

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Sappal, Ravinder [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); MacDonald, Nicole [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Fast, Mark [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Stevens, Don [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Kibenge, Fred [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Siah, Ahmed [British Columbia Centre for Aquatic Health Sciences, 871A Island Highway, Campbell River, BC V9W 2C2 (Canada); Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada)

2014-12-15

Highlights: • Interacting effects of Cu and temperature were investigated in rainbow trout liver mitochondria. • Mitochondrial functional indices are highly sensitive to temperature change. • High and low temperatures sensitize mitochondria to adverse effects of Cu. • Cu induces a highly temperature-sensitive mitochondrial permeability transition pore. • Cu-imposed mitochondrial membrane potential dissipation is mediated by reactive oxygen species. - Abstract: Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q{sub 10} effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control

Obesity-induced down-regulation of the mitochondrial translocator protein (TSPO) impairs placental steroid production.

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Lassance, Luciana; Haghiac, Maricela; Minium, Judi; Catalano, Patrick; Hauguel-de Mouzon, Sylvie

2015-01-01

Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. One hundred forty-four obese (body mass index 30-35 kg/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.

Non-electron transfer chain mitochondrial defects differently regulate HIF-1α degradation and transcription

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Antonina N. Shvetsova

2017-08-01

Full Text Available Mitochondria are the main consumers of molecular O2 in a cell as well as an abundant source of reactive oxygen species (ROS. Both, molecular oxygen and ROS are powerful regulators of the hypoxia-inducible factor-1α-subunit (HIF-α. While a number of mechanisms in the oxygen-dependent HIF-α regulation are quite well known, the view with respect to mitochondria is less clear. Several approaches using pharmacological or genetic tools targeting the mitochondrial electron transport chain (ETC indicated that ROS, mainly formed at the Rieske cluster of complex III of the ETC, are drivers of HIF-1α activation. However, studies investigating non-ETC located mitochondrial defects and their effects on HIF-1α regulation are scarce, if at all existing. Thus, in the present study we examined three cell lines with non-ETC mitochondrial defects and focused on HIF-1α degradation and transcription, target gene expression, as well as ROS levels. We found that cells lacking the key enzyme 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (MECR, and cells lacking manganese superoxide dismutase (MnSOD showed a reduced induction of HIF-1α under long-term (20 h hypoxia. By contrast, cells lacking the mitochondrial DNA depletion syndrome channel protein Mpv17 displayed enhanced levels of HIF-1α already under normoxic conditions. Further, we show that ROS do not exert a uniform pattern when mediating their effects on HIF-1α, although all mitochondrial defects in the used cell types increased ROS formation. Moreover, all defects caused a different HIF-1α regulation via promoting HIF-1α degradation as well as via changes in HIF-1α transcription. Thereby, MECR- and MnSOD-deficient cells showed a reduction in HIF-1α mRNA levels whereas the Mpv17 lacking cells displayed enhanced HIF-1α mRNA levels under normoxia and hypoxia. Altogether, our study shows for the first time that mitochondrial defects which are not related to the ETC and Krebs cycle

AKIP1 expression modulates mitochondrial function in rat neonatal cardiomyocytes.

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Hongjuan Yu

Full Text Available A kinase interacting protein 1 (AKIP1 is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.

The mTOR inhibitor sirolimus suppresses renal, hepatic, and cardiac tissue cellular respiration.

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Albawardi, Alia; Almarzooqi, Saeeda; Saraswathiamma, Dhanya; Abdul-Kader, Hidaya Mohammed; Souid, Abdul-Kader; Alfazari, Ali S

2015-01-01

The purpose of this in vitro study was to develop a useful biomarker (e.g., cellular respiration, or mitochondrial O2 consumption) for measuring activities of mTOR inhibitors. It measured the effects of commonly used immunosuppressants (sirolimus-rapamycin, tacrolimus, and cyclosporine) on cellular respiration in target tissues (kidney, liver, and heart) from C57BL/6 mice. The mammalian target of rapamycin (mTOR), a serine/ threonine kinase that supports nutrient-dependent cell growth and survival, is known to control energy conversion processes within the mitochondria. Consistently, inhibitors of mTOR (e.g., rapamycin, also known as sirolimus or Rapamune®) have been shown to impair mitochondrial function. Inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin (e.g., tacrolimus and cyclosporine), on the other hand, strictly prevent lymphokine production leading to a reduced T-cell function. Sirolimus (10 μM) inhibited renal (22%, P=0.002), hepatic (39%, Prespiration. Tacrolimus and cyclosporine had no or minimum effects on cellular respiration in these tissues. Thus, these results clearly demonstrate that impaired cellular respiration (bioenergetics) is a sensitive biomarker of the immunosuppressants that target mTOR.

Nitric Oxide Regulates Seedling Growth and Mitochondrial Responses in Aged Oat Seeds

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Chunli Mao

2018-04-01

Full Text Available Mitochondria are the source of reactive oxygen species (ROS in plant cells and play a central role in the mitochondrial electron transport chain (ETC and tricarboxylic acid cycle (TCA cycles; however, ROS production and regulation for seed germination, seedling growth, as well as mitochondrial responses to abiotic stress, are not clear. This study was conducted to obtain basic information on seed germination, embryo mitochondrial antioxidant responses, and protein profile changes in artificial aging in oat seeds (Avena sativa L. exposed to exogenous nitric oxide (NO treatment. The results showed that the accumulation of H2O2 in mitochondria increased significantly in aged seeds. Artificial aging can lead to a loss of seed vigor, which was shown by a decline in seed germination and the extension of mean germination time (MGT. Seedling growth was also inhibited. Some enzymes, including catalase (CAT, glutathione reductase (GR, dehydroascorbate reductase (DHAR, and monodehydroascorbate reductase (MDHAR, maintained a lower level in the ascorbate-glutathione (AsA-GSH scavenging system. Proteomic analysis revealed that the expression of some proteins related to the TCA cycle were down-regulated and several enzymes related to mitochondrial ETC were up-regulated. With the application of 0.05 mM NO in aged oat seeds, a protective effect was observed, demonstrated by an improvement in seed vigor and increased H2O2 scavenging ability in mitochondria. There were also higher activities of CAT, GR, MDHAR, and DHAR in the AsA-GSH scavenging system, enhanced TCA cycle-related enzymes (malate dehydrogenase, succinate-CoA ligase, fumarate hydratase, and activated alternative pathways, as the cytochrome pathway was inhibited. Therefore, our results indicated that seedling growth and seed germinability could retain a certain level in aged oat seeds, predominantly depending on the lower NO regulation of the TCA cycle and AsA-GSH. Thus, it could be concluded that the

Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

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Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

2015-07-30

Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.

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Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R; Scott, Melanie J

2013-05-31

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.

Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress*

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Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R.; Scott, Melanie J.

2013-01-01

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed. PMID:23589298

Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?

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Philippe eGiegé

2012-09-01

Full Text Available Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and / or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.

Coordinated Evolution of Transcriptional and Post-Transcriptional Regulation for Mitochondrial Functions in Yeast Strains.

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Xuepeng Sun

Full Text Available Evolution of gene regulation has been proposed to play an important role in environmental adaptation. Exploring mechanisms underlying coordinated evolutionary changes at various levels of gene regulation could shed new light on how organism adapt in nature. In this study, we focused on regulatory differences between a laboratory Saccharomyces cerevisiae strain BY4742 and a pathogenic S. cerevisiae strain, YJM789. The two strains diverge in many features, including growth rate, morphology, high temperature tolerance, and pathogenicity. Our RNA-Seq and ribosomal footprint profiling data showed that gene expression differences are pervasive, and genes functioning in mitochondria are mostly divergent between the two strains at both transcriptional and translational levels. Combining functional genomics data from other yeast strains, we further demonstrated that significant divergence of expression for genes functioning in the electron transport chain (ETC was likely caused by differential expression of a transcriptional factor, HAP4, and that post-transcriptional regulation mediated by an RNA-binding protein, PUF3, likely led to expression divergence for genes involved in mitochondrial translation. We also explored mito-nuclear interactions via mitochondrial DNA replacement between strains. Although the two mitochondrial genomes harbor substantial sequence divergence, neither growth nor gene expression were affected by mitochondrial DNA replacement in both fermentative and respiratory growth media, indicating compatible mitochondrial and nuclear genomes between these two strains in the tested conditions. Collectively, we used mitochondrial functions as an example to demonstrate for the first time that evolution at both transcriptional and post-transcriptional levels could lead to coordinated regulatory changes underlying strain specific functional variations.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

Science.gov (United States)

Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

2017-07-14

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

Directory of Open Access Journals (Sweden)

Graham P. Holloway

2018-03-01

Full Text Available Summary: It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress. : Holloway et al. show that an inability of ADP to decrease mitochondrial reactive oxygen species emission contributes to redox stress in skeletal muscle tissue of older individuals and that this process is not recovered following prolonged resistance-type exercise training, despite the general benefits of resistance training for muscle health. Keywords: mitochondria, aging, muscle, ROS, H2O2, ADP, respiration, bioenergetics, exercise, resistance training

Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

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Enríquez, José A.; Fernández-Silva, Patricio; Garrido-Pérez, Nuria; López-Pérez, Manuel J.; Pérez-Martos, Acisclo; Montoya, Julio

1999-01-01

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria. PMID:9858589

Respirators: Supervisors Self-Study #43442

Energy Technology Data Exchange (ETDEWEB)

Chochoms, Michael [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2016-04-20

This course, Respirators: Supervisors Self-Study (#43442), addresses training requirements for supervisors of respirator wearers as specified by the American National Standard Institute (ANSI) Standard for Respiratory Protection, ANSI Z88.2, and as incorporated by reference in the Department of Energy (DOE) Worker Health and Safety Rule, 10 Code of Federal Regulations (CFR) 851. This course also presents the responsibilities of supervisors of respirator wearers at Los Alamos National Laboratory (LANL).

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss)

International Nuclear Information System (INIS)

Onukwufor, John O.; Kibenge, Fred; Stevens, Don; Kamunde, Collins

2015-01-01

Highlights: • Interactions of Cd and temperature exacerbate mitochondrial dysfunction and enhance Cd accumulation. • Cd uptake by mitochondria occurs through the Ca uniporter. • Temperature exacerbates Cd-induced mitochondrial volume changes. • Low concentrations of Cd inhibit mitochondrial swelling. - Abstract: We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0–100 μM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q 10 values for state 3 respiration increased at low temperature (5–13 °C) while those for state 4 increased at high temperature (13–25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss)

Energy Technology Data Exchange (ETDEWEB)

Onukwufor, John O. [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3 (Canada); Kibenge, Fred [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3 (Canada); Stevens, Don [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3 (Canada); Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3 (Canada)

2015-01-15

Highlights: • Interactions of Cd and temperature exacerbate mitochondrial dysfunction and enhance Cd accumulation. • Cd uptake by mitochondria occurs through the Ca uniporter. • Temperature exacerbates Cd-induced mitochondrial volume changes. • Low concentrations of Cd inhibit mitochondrial swelling. - Abstract: We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0–100 μM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q{sub 10} values for state 3 respiration increased at low temperature (5–13 °C) while those for state 4 increased at high temperature (13–25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and

Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1

Directory of Open Access Journals (Sweden)

Elena A. Matveeva

2015-10-01

Full Text Available In this study we show that binding of mitochondria to vimentin intermediate filaments (VIF is regulated by GTPase Rac1. The activation of Rac1 leads to a redoubling of mitochondrial motility in murine fibroblasts. Using double-mutants Rac1(G12V, F37L and Rac1(G12V, Y40H that are capable to activate different effectors of Rac1, we show that mitochondrial movements are regulated through PAK1 kinase. The involvement of PAK1 kinase is also confirmed by the fact that expression of its auto inhibitory domain (PID blocks the effect of activated Rac1 on mitochondrial motility. The observed effect of Rac1 and PAK1 kinase on mitochondria depends on phosphorylation of the Ser-55 of vimentin. Besides the effect on motility Rac1 activation also decreases the mitochondrial membrane potential (MMP which is detected by ∼20% drop of the fluorescence intensity of mitochondria stained with the potential sensitive dye TMRM. One of important consequences of the discovered regulation of MMP by Rac1 and PAK1 is a spatial differentiation of mitochondria in polarized fibroblasts: at the front of the cell they are less energized (by ∼25% than at the rear part.

Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions.

Science.gov (United States)

Mailloux, Ryan J; Jin, Xiaolei; Willmore, William G

2014-01-01

Mitochondria have a myriad of essential functions including metabolism and apoptosis. These chief functions are reliant on electron transfer reactions and the production of ATP and reactive oxygen species (ROS). The production of ATP and ROS are intimately linked to the electron transport chain (ETC). Electrons from nutrients are passed through the ETC via a series of acceptor and donor molecules to the terminal electron acceptor molecular oxygen (O2) which ultimately drives the synthesis of ATP. Electron transfer through the respiratory chain and nutrient oxidation also produces ROS. At high enough concentrations ROS can activate mitochondrial apoptotic machinery which ultimately leads to cell death. However, if maintained at low enough concentrations ROS can serve as important signaling molecules. Various regulatory mechanisms converge upon mitochondria to modulate ATP synthesis and ROS production. Given that mitochondrial function depends on redox reactions, it is important to consider how redox signals modulate mitochondrial processes. Here, we provide the first comprehensive review on how redox signals mediated through cysteine oxidation, namely S-oxidation (sulfenylation, sulfinylation), S-glutathionylation, and S-nitrosylation, regulate key mitochondrial functions including nutrient oxidation, oxidative phosphorylation, ROS production, mitochondrial permeability transition (MPT), apoptosis, and mitochondrial fission and fusion. We also consider the chemistry behind these reactions and how they are modulated in mitochondria. In addition, we also discuss emerging knowledge on disorders and disease states that are associated with deregulated redox signaling in mitochondria and how mitochondria-targeted medicines can be utilized to restore mitochondrial redox signaling.

Peroxidative damage of mitochondrial respiration is substrate-dependent

Czech Academy of Sciences Publication Activity Database

Endlicher, R.; Křiváková, P.; Rauchová, Hana; Nůsková, Hana; Červinková, Z.; Drahota, Zdeněk

2009-01-01

Roč. 58, č. 5 (2009), s. 685-692 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA303/06/1261 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondrial enzymes * peroxidative damage * tert-butyl hydroperoxide Subject RIV: CE - Biochemistry Impact factor: 1.430, year: 2009

Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

Energy Technology Data Exchange (ETDEWEB)

Sirvent, P., E-mail: pascal.sirvent@univ-bpclermont.fr [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France); Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l' Exercice en conditions Physiologiques et Pathologiques (AME2P), BP 80026, F-63171 Aubière cedex (France); Fabre, O.; Bordenave, S. [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France); Hillaire-Buys, D. [CHRU Montpellier, 34295 Montpellier (France); Raynaud De Mauverger, E.; Lacampagne, A.; Mercier, J. [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France)

2012-03-01

The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.

Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

International Nuclear Information System (INIS)

Sirvent, P.; Fabre, O.; Bordenave, S.; Hillaire-Buys, D.; Raynaud De Mauverger, E.; Lacampagne, A.; Mercier, J.

2012-01-01

The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

Science.gov (United States)

Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

2015-12-08

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. © 2016 Authors.

Nutritional Ketosis and Mitohormesis: Potential Implications for Mitochondrial Function and Human Health

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Villamena, Frederick A.

2018-01-01

Impaired mitochondrial function often results in excessive production of reactive oxygen species (ROS) and is involved in the etiology of many chronic diseases, including cardiovascular disease, diabetes, neurodegenerative disorders, and cancer. Moderate levels of mitochondrial ROS, however, can protect against chronic disease by inducing upregulation of mitochondrial capacity and endogenous antioxidant defense. This phenomenon, referred to as mitohormesis, is induced through increased reliance on mitochondrial respiration, which can occur through diet or exercise. Nutritional ketosis is a safe and physiological metabolic state induced through a ketogenic diet low in carbohydrate and moderate in protein. Such a diet increases reliance on mitochondrial respiration and may, therefore, induce mitohormesis. Furthermore, the ketone β-hydroxybutyrate (BHB), which is elevated during nutritional ketosis to levels no greater than those resulting from fasting, acts as a signaling molecule in addition to its traditionally known role as an energy substrate. BHB signaling induces adaptations similar to mitohormesis, thereby expanding the potential benefit of nutritional ketosis beyond carbohydrate restriction. This review describes the evidence supporting enhancement of mitochondrial function and endogenous antioxidant defense in response to nutritional ketosis, as well as the potential mechanisms leading to these adaptations. PMID:29607218

Lipophilic triphenylphosphonium cations inhibit mitochondrial electron transport chain and induce mitochondrial proton leak.

Directory of Open Access Journals (Sweden)

Jan Trnka

Full Text Available The lipophilic positively charged moiety of triphenylphosphonium (TPP+ has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism.We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP+ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP+ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity.More hydrophobic TPP+ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect.

Exercise improves mitochondrial and redox-regulated stress responses in the elderly: better late than never!

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Cobley, James N; Moult, Peter R; Burniston, Jatin G; Morton, James P; Close, Graeme L

2015-04-01

Ageing is associated with several physiological declines to both the cardiovascular (e.g. reduced aerobic capacity) and musculoskeletal system (muscle function and mass). Ageing may also impair the adaptive response of skeletal muscle mitochondria and redox-regulated stress responses to an acute exercise bout, at least in mice and rodents. This is a functionally important phenomenon, since (1) aberrant mitochondrial and redox homeostasis are implicated in the pathophysiology of musculoskeletal ageing and (2) the response to repeated exercise bouts promotes exercise adaptations and some of these adaptations (e.g. improved aerobic capacity and exercise-induced mitochondrial remodelling) offset age-related physiological decline. Exercise-induced mitochondrial remodelling is mediated by upstream signalling events that converge on downstream transcriptional co-factors and factors that orchestrate a co-ordinated nuclear and mitochondrial transcriptional response associated with mitochondrial remodelling. Recent translational human investigations have demonstrated similar exercise-induced mitochondrial signalling responses in older compared with younger skeletal muscle, regardless of training status. This is consistent with data indicating normative mitochondrial remodelling responses to long-term exercise training in the elderly. Thus, human ageing is not accompanied by diminished mitochondrial plasticity to acute and chronic exercise stimuli, at least for the signalling pathways measured to date. Exercise-induced increases in reactive oxygen and nitrogen species promote an acute redox-regulated stress response that manifests as increased heat shock protein and antioxidant enzyme content. In accordance with previous reports in rodents and mice, it appears that sedentary ageing is associated with a severely attenuated exercise-induced redox stress response that might be related to an absent redox signal. In this regard, regular exercise training affords some protection

Plant Respiration and Climate Change Effects

International Nuclear Information System (INIS)

Bruhn, D.

2002-04-01

Plant respiration is one of the key processes in terms of an understanding of plant growth and functioning in a future climate. Short- and long-term effects of temperature and CO 2 on plant respiration were investigated in a number of plant species. The experiments tested effects of either temperature and/or CO 2 from the level of individual respiratory enzymes, isolated mitochondria, whole-tissue, and up to the whole canopy level. The short-term effects of elevated atmospheric CO 2 on plant respiration appeared to be less than suggested so far in the literature. This was true both at the tissue level and for intact mitochondria. Respiratory enzymes can, however, be affected already at low CO 2 . These effects did not manifest itself at the tissue level, though, due to low degrees of control on the whole respiratory process exerted by the particular enzymes. Plant respiration on the other hand was affected by long-term growth at elevated atmospheric CO 2 . The findings of the reduced plant respiration at the leaf level were consistent with the literature and potential causes are discussed. Short-term effects of temperature on plant respiration were demonstrated to be dependent on the actual measurement temperature. Further, it is shown that mitochondrial leaf respiration in darkness and light differ substantially in the temperature sensitivity with the former being the far most sensitive. This has implications for modelling CO 2 exchange between vegetation and atmosphere as demonstrated here, since this has so far been neglected. Long-term effects of temperature resulted in respiratory acclimation in a number of species. Respiratory acclimation appeared not to occur to any one single type of growth temperature. The implications of this finding in combination with the timing of acclimation are discussed for modelling respiratory CO 2 release. (au)

Plant Respiration and Climate Change Effects

Energy Technology Data Exchange (ETDEWEB)

Bruhn, D

2002-04-01

Plant respiration is one of the key processes in terms of an understanding of plant growth and functioning in a future climate. Short- and long-term effects of temperature and CO{sub 2} on plant respiration were investigated in a number of plant species. The experiments tested effects of either temperature and/or CO{sub 2} from the level of individual respiratory enzymes, isolated mitochondria, whole-tissue, and up to the whole canopy level. The short-term effects of elevated atmospheric CO{sub 2} on plant respiration appeared to be less than suggested so far in the literature. This was true both at the tissue level and for intact mitochondria. Respiratory enzymes can, however, be affected already at low CO{sub 2}. These effects did not manifest itself at the tissue level, though, due to low degrees of control on the whole respiratory process exerted by the particular enzymes. Plant respiration on the other hand was affected by long-term growth at elevated atmospheric CO{sub 2}. The findings of the reduced plant respiration at the leaf level were consistent with the literature and potential causes are discussed. Short-term effects of temperature on plant respiration were demonstrated to be dependent on the actual measurement temperature. Further, it is shown that mitochondrial leaf respiration in darkness and light differ substantially in the temperature sensitivity with the former being the far most sensitive. This has implications for modelling CO{sub 2} exchange between vegetation and atmosphere as demonstrated here, since this has so far been neglected. Long-term effects of temperature resulted in respiratory acclimation in a number of species. Respiratory acclimation appeared not to occur to any one single type of growth temperature. The implications of this finding in combination with the timing of acclimation are discussed for modelling respiratory CO{sub 2} release. (au)

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

Science.gov (United States)

Raza Shaikh, Saame; Brown, David A

2013-01-01

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Understanding D-Ribose and Mitochondrial Function

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Diane E. Mahoney

2018-02-01

Full Text Available Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production.  With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

Science.gov (United States)

Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

2009-07-01

Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

Endogenous ovarian hormones affect mitochondrial efficiency in cerebral endothelium via distinct regulation of PGC-1 isoforms.

Science.gov (United States)

Kemper, Martin F; Zhao, Yuanzi; Duckles, Sue P; Krause, Diana N

2013-01-01

Mitochondria support the energy-intensive functions of brain endothelium but also produce damaging-free radicals that lead to disease. Previously, we found that estrogen treatment protects cerebrovascular mitochondria, increasing capacity for ATP production while decreasing reactive oxygen species (ROS). To determine whether these effects occur specifically in endothelium in vivo and also explore underlying transcriptional mechanisms, we studied freshly isolated brain endothelial preparations from intact and ovariectomized female mice. This preparation reflects physiologic influences of circulating hormones, hemodynamic forces, and cell-cell interactions of the neurovascular unit. Loss of ovarian hormones affected endothelial expression of the key mitochondrial regulator family, peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), but in a unique way. Ovariectomy increased endothelial PGC-1α mRNA but decreased PGC-1β mRNA. The change in PGC-1β correlated with decreased mRNA for crucial downstream mitochondrial regulators, nuclear respiratory factor 1 and mitochondrial transcription factor A, as well as for ATP synthase and ROS protection enzymes, glutamate-cysteine ligase and manganese superoxide dismutase. Ovariectomy also decreased mitochondrial biogenesis (mitochondrial/nuclear DNA ratio). These results indicate ovarian hormones normally act through a distinctive regulatory pathway involving PGC-1β to support cerebral endothelial mitochondrial content and guide mitochondrial function to favor ATP coupling and ROS protection.

Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions☆

Science.gov (United States)

Mailloux, Ryan J.; Jin, Xiaolei; Willmore, William G.

2013-01-01

Mitochondria have a myriad of essential functions including metabolism and apoptosis. These chief functions are reliant on electron transfer reactions and the production of ATP and reactive oxygen species (ROS). The production of ATP and ROS are intimately linked to the electron transport chain (ETC). Electrons from nutrients are passed through the ETC via a series of acceptor and donor molecules to the terminal electron acceptor molecular oxygen (O2) which ultimately drives the synthesis of ATP. Electron transfer through the respiratory chain and nutrient oxidation also produces ROS. At high enough concentrations ROS can activate mitochondrial apoptotic machinery which ultimately leads to cell death. However, if maintained at low enough concentrations ROS can serve as important signaling molecules. Various regulatory mechanisms converge upon mitochondria to modulate ATP synthesis and ROS production. Given that mitochondrial function depends on redox reactions, it is important to consider how redox signals modulate mitochondrial processes. Here, we provide the first comprehensive review on how redox signals mediated through cysteine oxidation, namely S-oxidation (sulfenylation, sulfinylation), S-glutathionylation, and S-nitrosylation, regulate key mitochondrial functions including nutrient oxidation, oxidative phosphorylation, ROS production, mitochondrial permeability transition (MPT), apoptosis, and mitochondrial fission and fusion. We also consider the chemistry behind these reactions and how they are modulated in mitochondria. In addition, we also discuss emerging knowledge on disorders and disease states that are associated with deregulated redox signaling in mitochondria and how mitochondria-targeted medicines can be utilized to restore mitochondrial redox signaling. PMID:24455476

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

International Nuclear Information System (INIS)

Liu, Cong; Sekine, Shuichi; Ito, Kousei

2016-01-01

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Liu, Cong; Sekine, Shuichi, E-mail: ssekine@faculty.chiba-u.jp; Ito, Kousei

2016-07-01

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

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Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

2005-06-01

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Changes in Respiratory Mitochondrial Machinery and Cytochrome and Alternative Pathway Activities in Response to Energy Demand Underlie the Acclimation of Respiration to Elevated CO2 in the Invasive Opuntia ficus-indica1[OA

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Gomez-Casanovas, Nuria; Blanc-Betes, Elena; Gonzalez-Meler, Miquel A.; Azcon-Bieto, Joaquim

2007-01-01

Studies on long-term effects of plants grown at elevated CO2 are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO2, the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO2 concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO2 during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO2 also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO2, the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO2. Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO2, the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO2 suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO2. However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO2, total mitochondrial ATP production was decreased by plant growth at elevated CO2 when compared to ambient-grown plants. Because plant growth at elevated CO2 increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O2 consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO2 results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested. PMID:17660349

Effects of acute and chronic endurance exercise on mitochondrial uncoupling in human skeletal muscle

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Fernström, Maria; Tonkonogi, Michail; Sahlin, Kent

2004-01-01

Mitochondrial proteins such as uncoupling protein 3 (UCP3) and adenine nucleotide translocase (ANT) may mediate back-leakage of protons and serve as uncouplers of oxidative phosphorylation. We hypothesized that UCP3 and ANT increase after prolonged exercise and/or endurance training, resulting...... respiration or state 3). Protein expression of UCP3 and ANT was measured with Western blotting. After endurance training, .VO2peak, citrate synthase activity (CS), state 3 respiration and ANT increased by 24, 47, 40 and 95%, respectively (all P ... mitochondrial resistance to Ca2+ overload but does not influence UCR or protein expression of UCP3 and ANT. The increased Ca2+ resistance may prevent mitochondrial degradation and the mechanism needs to be further explored....

Impaired Insulin Signaling is Associated with Hepatic Mitochondrial Dysfunction in IR+/−-IRS-1+/− Double Heterozygous (IR-IRS1dh Mice

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Andras Franko

2017-05-01

Full Text Available Mitochondria play a pivotal role in energy metabolism, but whether insulin signaling per se could regulate mitochondrial function has not been identified yet. To investigate whether mitochondrial function is regulated by insulin signaling, we analyzed muscle and liver of insulin receptor (IR+/−-insulin receptor substrate-1 (IRS-1+/− double heterozygous (IR-IRS1dh mice, a well described model for insulin resistance. IR-IRS1dh mice were studied at the age of 6 and 12 months and glucose metabolism was determined by glucose and insulin tolerance tests. Mitochondrial enzyme activities, oxygen consumption, and membrane potential were assessed using spectrophotometric, respirometric, and proton motive force analysis, respectively. IR-IRS1dh mice showed elevated serum insulin levels. Hepatic mitochondrial oxygen consumption was reduced in IR-IRS1dh animals at 12 months of age. Furthermore, 6-month-old IR-IRS1dh mice demonstrated enhanced mitochondrial respiration in skeletal muscle, but a tendency of impaired glucose tolerance. On the other hand, 12-month-old IR-IRS1dh mice showed improved glucose tolerance, but normal muscle mitochondrial function. Our data revealed that deficiency in IR/IRS-1 resulted in normal or even elevated skeletal muscle, but impaired hepatic mitochondrial function, suggesting a direct cross-talk between insulin signaling and mitochondria in the liver.

Lack of mitochondrial MutS homolog 1 in Toxoplasma gondii disrupts maintenance and fidelity of mitochondrial DNA and reveals metabolic plasticity.

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Tamila Garbuz

Full Text Available The importance of maintaining the fidelity of the mitochondrial genome is underscored by the presence of various repair pathways within this organelle. Presumably, the repair of mitochondrial DNA would be of particular importance in organisms that possess only a single mitochondrion, like the human pathogens Plasmodium falciparum and Toxoplasma gondii. Understanding the machinery that maintains mitochondrial DNA in these parasites is of particular relevance, as mitochondrial function is a validated and effective target for anti-parasitic drugs. We previously determined that the Toxoplasma MutS homolog TgMSH1 localizes to the mitochondrion. MutS homologs are key components of the nuclear mismatch repair system in mammalian cells, and both yeast and plants possess MutS homologs that localize to the mitochondria where they regulate DNA stability. Here we show that the lack of TgMSH1 results in accumulation of single nucleotide variations in mitochondrial DNA and a reduction in mitochondrial DNA content. Additionally, parasites lacking TgMSH1 function can survive treatment with the cytochrome b inhibitor atovaquone. While the Tgmsh1 knockout strain has several missense mutations in cytochrome b, none affect amino acids known to be determinants of atovaquone sensitivity and atovaquone is still able to inhibit electron transport in the Tgmsh1 mutants. Furthermore, culture of Tgmsh1 mutant in the presence atovaquone leads to parasites with enhanced atovaquone resistance and complete shutdown of respiration. Thus, parasites lacking TgMSH1 overcome the disruption of mitochondrial DNA by adapting their physiology allowing them to forgo the need for oxidative phosphorylation. Consistent with this idea, the Tgmsh1 mutant is resistant to mitochondrial inhibitors with diverse targets and exhibits reduced ability to grow in the absence of glucose. This work shows TgMSH1 as critical for the maintenance and fidelity of the mitochondrial DNA in Toxoplasma

Circadian rhythms in mitochondrial respiration

NARCIS (Netherlands)

de Goede, Paul; Wefers, Jakob; Brombacher, Eline Constance; Schrauwen, P; Kalsbeek, A.

2018-01-01

Many physiological processes are regulated with a 24h periodicity to anticipate the environmental changes of day to nighttime and vice versa. These 24h regulations, commonly termed circadian rhythms, amongst others control the sleep-wake cycle, locomotor activity and preparation for food

Genetically Determined Insulin Resistance is Characterized by Down-Regulation of Mitochondrial Oxidative Metabolism in Human Skeletal Muscle

DEFF Research Database (Denmark)

Kristensen, Jonas M; Skov, Vibe; Wojtaszewski, Jørgen

2010-01-01

Transcriptional profiling of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated a co-ordinated down-regulation of oxidative phosphorylation (OxPhos) genes, suggesting a link between insulin resistance and mitochondrial dysfunction. However, whether...... mitochondrial dysfunction is a cause or consequence of insulin resistance remains to be clarified. In the present study, we tested the hypothesis that mitochondrial oxidative metabolism was down-regulated in skeletal muscle of patients with genetically determined insulin resistance. Skeletal muscle biopsies.......02), and complex V (ATP5B; p=0.005). Our data demonstrate that genetically determined insulin resistance is associated with a co-ordinated down-regulation of OxPhos components both at the transcriptional and translational level. These findings suggest that an impaired biological response to insulin in skeletal...

Resveratrol Co-Treatment Attenuates the Effects of HIV Protease Inhibitors on Rat Body Weight and Enhances Cardiac Mitochondrial Respiration.

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Burger Symington

Full Text Available Since the early 1990s human immunodeficiency virus (HIV/acquired immunodeficiency syndrome (AIDS emerged as a global health pandemic, with sub-Saharan Africa the hardest hit. While the successful roll-out of antiretroviral (ARV therapy provided significant relief to HIV-positive individuals, such treatment can also elicit damaging side-effects. Here especially HIV protease inhibitors (PIs are implicated in the onset of cardio-metabolic complications such as type-2 diabetes and coronary heart disease. As there is a paucity of data regarding suitable co-treatments within this context, this preclinical study investigated whether resveratrol (RSV, aspirin (ASP or vitamin C (VitC co-treatment is able to blunt side-effects in a rat model of chronic PI exposure (Lopinavir/Ritonavir treatment for 4 months. Body weights and weight gain, blood metabolite levels (total cholesterol, HDL, LDL, triglycerides, echocardiography and cardiac mitochondrial respiration were assessed in PI-treated rats ± various co-treatments. Our data reveal that PI treatment significantly lowered body weight and cardiac respiratory function while no significant changes were found for heart function and blood metabolite levels. Moreover, all co-treatments ameliorated the PI-induced decrease in body weight after 4 months of PI treatment, while RSV co-treatment enhanced cardiac mitochondrial respiratory capacity in PI-treated rats. This pilot study therefore provides novel hypotheses regarding RSV co-treatment that should be further assessed in greater detail.

Cryopreservation of human skeletal muscle impairs mitochondrial function

DEFF Research Database (Denmark)

Larsen, Steen; Wright-Paradis, C; Gnaiger, E

2012-01-01

functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity...

Gene set of nuclear-encoded mitochondrial regulators is enriched for common inherited variation in obesity.

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Nadja Knoll

Full Text Available There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1 16 nuclear regulators of mitochondrial genes, (2 91 genes for oxidative phosphorylation and (3 966 nuclear-encoded mitochondrial genes. Gene set enrichment analysis (GSEA showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents and a population-based GWAS sample (KORA F4, n = 1,743. A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50(th and 95(th percentile of the set of all gene-wise corrected p-values as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50(th percentile for the set of the 16 nuclear regulators of mitochondrial genes (p(GSEA,50 = 0.0103. This finding was not confirmed in the trios (p(GSEA,50 = 0.5991, but in KORA (p(GSEA,50 = 0.0398. The meta-analysis again indicated a trend for enrichment (p(MAGENTA,50 = 0.1052, p(MAGENTA,75 = 0.0251. The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.

The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration.

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Szarka, András; Bánhegyi, Gábor; Asard, Han

2013-09-20

Ascorbate, this multifaceted small molecular weight carbohydrate derivative, plays important roles in a range of cellular processes in plant cells, from the regulation of cell cycle, through cell expansion and senescence. Beyond these physiological functions, ascorbate has a critical role in responses to abiotic stresses, such as high light, high salinity, or drought. The biosynthesis, recycling, and intracellular transport are important elements of the balancing of ascorbate level to the always-changing conditions and demands. A bidirectional tight relationship was described between ascorbate biosynthesis and the mitochondrial electron transfer chain (mETC), since L-galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate step of ascorbate biosynthesis, uses oxidized cytochrome c as the only electron acceptor and has a role in the assembly of Complex I. A similar bidirectional relationship was revealed between the photosynthetic apparatus and ascorbate biosynthesis since the electron flux through the photosynthetic ETC affects the biosynthesis of ascorbate and the level of ascorbate could affect photosynthesis. The details of this regulatory network of photosynthetic electron transfer, respiratory electron transfer, and ascorbate biosynthesis are still not clear, as are the potential regulatory role and the regulation of intracellular ascorbate transport and fluxes. The elucidation of the role of ascorbate as an important element of the network of photosynthetic, respiratory ETC and tricarboxylic acid cycle will contribute to understanding plant cell responses to different stress conditions.

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

DEFF Research Database (Denmark)

Larsen, Steen; Nielsen, Joachim; Hansen, Christina Neigaard

2012-01-01

Key points  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects.  The most commonly used...... markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein, and complex I-IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres......).  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.  mtDNA was found to be a poor biomarker of mitochondrial content.  Complex IV activity was closely associated with mitochondrial oxidative...

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

International Nuclear Information System (INIS)

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

2014-01-01

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations

Improved glycaemic control decreases inner mitochondrial membrane leak in type 2 diabetes

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Rabøl, R; Højberg, P M V; Almdal, T

2009-01-01

AIM: Several mechanisms have been targeted as culprits of weight gain during antihyperglycaemic treatment in type 2 diabetes (T2DM). These include reductions in glucosuria, increased food intake from fear of hypoglycaemia, the anabolic effect of insulin, decreased metabolic rate and increased eff...... to reductions in inner mitochondrial membrane leak and increased efficiency of mitochondria. This change in mitochondrial physiology could contribute to the weight gain seen with antihyperglycaemic treatment....... efficiency in fuel usage. The purpose of the study was to test the hypothesis that mitochondrial efficiency increases as a result of insulin treatment in patients with type 2 diabetes. METHODS: We included ten patients with T2DM (eight males) on oral antidiabetic treatment, median age: 51.5 years (range: 39......-67) and body mass index (BMI): 30.1 +/- 1.2 kg/m2 (mean +/- s.e.). Muscle biopsies from m. vastus lateralis and m. deltoideus were obtained before and after seven weeks of intensive insulin treatment, and mitochondrial respiration was measured using high-resolution respirometry. State 3 respiration...

The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

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Charizanis, C; Juhnke, H; Krems, B; Entian, K D

1999-10-01

In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

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Miyata, Non; Watanabe, Yasunori; Tamura, Yasushi; Endo, Toshiya; Kuge, Osamu

2016-07-04

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state. © 2016 Miyata et al.

The mitochondrial genome impacts respiration but not fermentation in interspecific Saccharomyces hybrids.

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Warren Albertin

Full Text Available In eukaryotes, mitochondrial DNA (mtDNA has high rate of nucleotide substitution leading to different mitochondrial haplotypes called mitotypes. However, the impact of mitochondrial genetic variant on phenotypic variation has been poorly considered in microorganisms because mtDNA encodes very few genes compared to nuclear DNA, and also because mitochondrial inheritance is not uniparental. Here we propose original material to unravel mitotype impact on phenotype: we produced interspecific hybrids between S. cerevisiae and S. uvarum species, using fully homozygous diploid parental strains. For two different interspecific crosses involving different parental strains, we recovered 10 independent hybrids per cross, and allowed mtDNA fixation after around 80 generations. We developed PCR-based markers for the rapid discrimination of S. cerevisiae and S. uvarum mitochondrial DNA. For both crosses, we were able to isolate fully isogenic hybrids at the nuclear level, yet possessing either S. cerevisiae mtDNA (Sc-mtDNA or S. uvarum mtDNA (Su-mtDNA. Under fermentative conditions, the mitotype has no phenotypic impact on fermentation kinetics and products, which was expected since mtDNA are not necessary for fermentative metabolism. Alternatively, under respiratory conditions, hybrids with Sc-mtDNA have higher population growth performance, associated with higher respiratory rate. Indeed, far from the hypothesis that mtDNA variation is neutral, our work shows that mitochondrial polymorphism can have a strong impact on fitness components and hence on the evolutionary fate of the yeast populations. We hypothesize that under fermentative conditions, hybrids may fix stochastically one or the other mt-DNA, while respiratory environments may increase the probability to fix Sc-mtDNA.

Mitochondrial uncoupling proteins in unicellular eukaryotes.

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Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

2010-01-01

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

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Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

2014-11-01

Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression

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Francesco Bellanti

2018-05-01

Full Text Available The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury.

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

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Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

2011-03-01

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Acceptable respiratory protection program and LASL respirator research

International Nuclear Information System (INIS)

Skaggs, B.J.

1979-01-01

A short history is presented on the LASL Respiratory Protection Training Programs. Then a discussion is given on the major points of an acceptable respiratory protection program utilizing the points required by the Occupational, Safety, and Health Administration (OSHA) Regulation 29 CFR 1910.134. Contributions to respirator research are reviewed. Discussion is presented under the following section headings: program administration; respirator selection; respirator use; fitting and training; respirator maintenance; medical clearance and surveillance; special problems; program evaluation; and documentation

SIRT3/SOD2 maintains osteoblast differentiation and bone formation by regulating mitochondrial stress

OpenAIRE

Gao, Jing; Feng, Zhihui; Wang, Xueqiang; Zeng, Mengqi; Liu, Jing; Han, Shujun; Xu, Jie; Chen, Lei; Cao, Ke; Long, Jiangang; Li, Zongfang; Shen, Weili; Liu, Jiankang

2017-01-01

Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by in...

m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

Science.gov (United States)

Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

2018-03-01

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.

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Piotr Bednarczyk

Full Text Available Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+-regulated potassium channel (mitoBKCa channel was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

Cytokine and nitric oxide levels in patients with sepsis--temporal evolvement and relation to platelet mitochondrial respiratory function

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Sjövall, Fredrik; Morota, Saori; Frostner, Eleonor Åsander

2014-01-01

the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis. METHODS AND RESULTS: Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon......-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content....... All mitochondrial respiratory states increased over the week analyzed (prespiration on day 6-7 (p = 0.02, r2 = 0.22) and was also higher in non-survivors compared to survivors on day 3-4 and day 6-7 (p = 0.03 respectively). Neither NO nor any...

The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

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Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

2015-05-19

The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels

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Bin Li

2011-01-01

Full Text Available Calorie restriction (CR induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX. Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators

International Nuclear Information System (INIS)

Claveria, Cristina; Torres, Miguel

2003-01-01

Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway

Lymphocyte respiration in children with Trisomy 21

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Aburawi Elhadi H

2012-12-01

Full Text Available Abstract Background This study measured lymphocyte mitochondrial O2 consumption (cellular respiration in children with trisomy 21. Methods Peripheral blood mononuclear cells were isolated from whole blood of trisomy 21 and control children and these cells were immediately used to measure cellular respiration rate. [O2] was determined as a function of time from the phosphorescence decay rates (1/τ of Pd (II-meso-tetra-(4-sulfonatophenyl-tetrabenzoporphyrin. In sealed vials containing lymphocytes and glucose as a respiratory substrate, [O2] declined linearly with time, confirming the zero-order kinetics of O2 conversion to H2O by cytochrome oxidase. The rate of respiration (k, in μM O2 min-1, thus, was the negative of the slope of [O2] vs. time. Cyanide inhibited O2 consumption, confirming that oxidation occurred in the mitochondrial respiratory chain. Results For control children (age = 8.8 ± 5.6 years, n = 26, the mean (± SD value of kc (in μM O2 per min per 107 cells was 1.36 ± 0.79 (coefficient of variation, Cv = 58%; median = 1.17; range = 0.60 to 3.12; -2SD = 0.61. For children with trisomy 21 (age = 7.2 ± 4.6 years, n = 26, the values of kc were 0.82 ± 0.62 (Cv = 76%; median = 0.60; range = 0.20 to 2.80, pp6.1 mU/L. Fourteen of 26 (54% children with trisomy 21 had kc values of 0.20 to 0.60 (i.e., kc positively correlated with body-mass index (BMI, R >0.302, serum creatinine (R >0.507, blood urea nitrogen (BUN, R >0.535 and albumin (R >0.446. Conclusions Children with trisomy 21 in this study have reduced lymphocyte bioenergetics. The clinical importance of this finding requires further studies.

Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

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Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

2014-11-15

Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

Direct Effects of (−-Epicatechin and Procyanidin B2 on the Respiration of Rat Heart Mitochondria

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Dalia M. Kopustinskiene

2015-01-01

Full Text Available Flavonol (−-epicatechin and its derived dimer procyanidin B2, present in high amounts in cocoa products, have been shown to exert beneficial effects on the heart and cardiovascular system; however, their mechanism of action has not been fully elucidated. We studied effects of (−-epicatechin and procyanidin B2 on the oxidative phosphorylation of isolated rat heart mitochondria. (−-Epicatechin and procyanidin B2 had stimulating effect (up to 30% compared to control on substrate-driven (State 2 mitochondrial respiration. Their effect was dependent on the respiratory substrates used. (−-Epicatechin at higher concentrations (from 0.27 µg/mL significantly decreased (up to 15% substrate- and ADP-driven (State 3 mitochondrial respiration in case of pyruvate and malate oxidation only. Procyanidin B2 (0.7–17.9 ng/mL inhibited State 3 respiration rate up to 19%, the most profound effect being expressed with succinate as the substrate. (−-Epicatechin at concentrations of 0.23 µg/mL and 0.46 µg/mL prevented loss of the cytochrome c from mitochondria when substrate was succinate, supporting the evidence of membrane stabilizing properties of this flavonol. Thus, both (−-epicatechin and procyanidin B2 directly influenced mitochondrial functions and the observed effects could help to explain cardiometabolic risk reduction ascribed to the consumption of modest amounts of cocoa products.

Mitochondrial Dynamics: Coupling Mitochondrial Fitness with Healthy Aging.

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Sebastián, David; Palacín, Manuel; Zorzano, Antonio

2017-03-01

Aging is associated with a decline in mitochondrial function and the accumulation of abnormal mitochondria. However, the precise mechanisms by which aging promotes these mitochondrial alterations and the role of the latter in aging are still not fully understood. Mitochondrial dynamics is a key process regulating mitochondrial function and quality. Altered expression of some mitochondrial dynamics proteins has been recently associated with aging and with age-related alterations in yeast, Caenorhabditis elegans, mice, and humans. Here, we review the link between alterations in mitochondrial dynamics, aging, and age-related impairment. We propose that the dysregulation of mitochondrial dynamics leads to age-induced accumulation of unhealthy mitochondria and contributes to alterations linked to aging, such as diabetes and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondrial-nuclear genome interactions in nonalcoholic fatty liver disease in mice

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Betancourt, Angela M.; King, Adrienne L.; Fetterman, Jessica L.; Millender-Swain, Telisha; Finley, Rachel D.; Oliva, Claudia R.; Crowe, David Ralph; Ballinger, Scott W.; Bailey, Shannon M.

2014-01-01

Nonalcoholic fatty liver disease (NAFLD) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation, and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. Herein, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, Mitochondrial-Nuclear eXchange (MNX) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared to wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation, and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD. PMID:24758559

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

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Almeida, Luciana O.; Goto, Renata N. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Neto, Marinaldo P.C. [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Sousa, Lucas O. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Curti, Carlos [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Leopoldino, Andréia M., E-mail: andreiaml@usp.br [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

2015-03-06

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

Long noncoding RNA Tug1 regulates mitochondrial bioenergetics in diabetic nephropathy.

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Long, Jianyin; Badal, Shawn S; Ye, Zengchun; Wang, Yin; Ayanga, Bernard A; Galvan, Daniel L; Green, Nathanael H; Chang, Benny H; Overbeek, Paul A; Danesh, Farhad R

2016-11-01

The regulatory roles of long noncoding RNAs (lncRNAs) in transcriptional coactivators are still largely unknown. Here, we have shown that the peroxisome proliferator-activated receptor γ (PPARγ) coactivator α (PGC-1α, encoded by Ppargc1a) is functionally regulated by the lncRNA taurine-upregulated gene 1 (Tug1). Further, we have described a role for Tug1 in the regulation of mitochondrial function in podocytes. Using a murine model of diabetic nephropathy (DN), we performed an unbiased RNA-sequencing (RNA-seq) analysis of kidney glomeruli and identified Tug1 as a differentially expressed lncRNA in the diabetic milieu. Podocyte-specific overexpression (OE) of Tug1 in diabetic mice improved the biochemical and histological features associated with DN. Unexpectedly, we found that Tug1 OE rescued the expression of PGC-1α and its transcriptional targets. Tug1 OE was also associated with improvements in mitochondrial bioenergetics in the podocytes of diabetic mice. Mechanistically, we found that the interaction between Tug1 and PGC-1α promotes the binding of PGC-1α to its own promoter. We identified a Tug1-binding element (TBE) upstream of the Ppargc1a gene and showed that Tug1 binds with the TBE to enhance Ppargc1a promoter activity. These findings indicate that a direct interaction between PGC-1α and Tug1 modulates mitochondrial bioenergetics in podocytes in the diabetic milieu.

Intrauterine growth retardation increases the susceptibility of pigs to high-fat diet-induced mitochondrial dysfunction in skeletal muscle.

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Jingbo Liu

Full Text Available It has been recognized that there is a relationship between prenatal growth restriction and the development of metabolic-related diseases in later life, a process involved in mitochondrial dysfunction. In addition, intrauterine growth retardation (IUGR increases the susceptibility of offspring to high-fat (HF diet-induced metabolic syndrome. Recent findings suggested that HF feeding decreased mitochondrial oxidative capacity and impaired mitochondrial function in skeletal muscle. Therefore, we hypothesized that the long-term consequences of IUGR on mitochondrial biogenesis and function make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. Normal birth weight (NBW, and IUGR pigs were allotted to control or HF diet in a completely randomized design, individually. After 4 weeks of feeding, growth performance and molecular pathways related to mitochondrial function were determined. The results showed that IUGR decreased growth performance and plasma insulin concentrations. In offspring fed a HF diet, IUGR was associated with enhanced plasma leptin levels, increased concentrations of triglyceride and malondialdehyde (MDA, and reduced glycogen and ATP contents in skeletal muscle. High fat diet-fed IUGR offspring exhibited decreased activities of lactate dehydrogenase (LDH and glucose-6-phosphate dehydrogenase (G6PD. These alterations in metabolic traits of IUGR pigs were accompanied by impaired mitochondrial respiration function, reduced mitochondrial DNA (mtDNA contents, and down-regulated mRNA expression levels of genes responsible for mitochondrial biogenesis and function. In conclusion, our results suggest that IUGR make the offspring more susceptible to HF diet-induced mitochondrial dysfunction.

Intrauterine Growth Retardation Increases the Susceptibility of Pigs to High-Fat Diet-Induced Mitochondrial Dysfunction in Skeletal Muscle

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Liu, Jingbo; Chen, Daiwen; Yao, Ying; Yu, Bing; Mao, Xiangbing; He, Jun; Huang, Zhiqing; Zheng, Ping

2012-01-01

It has been recognized that there is a relationship between prenatal growth restriction and the development of metabolic-related diseases in later life, a process involved in mitochondrial dysfunction. In addition, intrauterine growth retardation (IUGR) increases the susceptibility of offspring to high-fat (HF) diet-induced metabolic syndrome. Recent findings suggested that HF feeding decreased mitochondrial oxidative capacity and impaired mitochondrial function in skeletal muscle. Therefore, we hypothesized that the long-term consequences of IUGR on mitochondrial biogenesis and function make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. Normal birth weight (NBW), and IUGR pigs were allotted to control or HF diet in a completely randomized design, individually. After 4 weeks of feeding, growth performance and molecular pathways related to mitochondrial function were determined. The results showed that IUGR decreased growth performance and plasma insulin concentrations. In offspring fed a HF diet, IUGR was associated with enhanced plasma leptin levels, increased concentrations of triglyceride and malondialdehyde (MDA), and reduced glycogen and ATP contents in skeletal muscle. High fat diet-fed IUGR offspring exhibited decreased activities of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD). These alterations in metabolic traits of IUGR pigs were accompanied by impaired mitochondrial respiration function, reduced mitochondrial DNA (mtDNA) contents, and down-regulated mRNA expression levels of genes responsible for mitochondrial biogenesis and function. In conclusion, our results suggest that IUGR make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. PMID:22523560

p32, a novel binding partner of Mcl-1, positively regulates mitochondrial Ca{sup 2+} uptake and apoptosis

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Xiao, Kang [Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Wang, Yinyin; Chang, Zhijie [School of Medicine, Tsinghua University, Beijing (China); Lao, Yuanzhi, E-mail: laurence_ylao@163.com [School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai (China); Chang, Donald C., E-mail: bochang@ust.hk [Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)

2014-08-22

Highlights: • p32 binds to Mcl-1. • p32 affects apoptosis. • p32 and Mcl-1 regulate mitochondrial Ca{sup 2+}. - Abstract: Mcl-1 is a major anti-apoptotic Bcl-2 family protein. It is well known that Mcl-1 can interact with certain pro-apoptotic Bcl-2 family proteins in normal cells to neutralize their pro-apoptotic functions, thus prevent apoptosis. In addition, it was recently found that Mcl-1 can also inhibit mitochondrial calcium uptake. The detailed mechanism, however, is still not clear. Based on Yeast Two-Hybrid screening and co-immunoprecipitation, we identified a mitochondrial protein p32 (C1qbp) as a novel binding partner of Mcl-1. We found that p32 had a number of interesting properties: (1) p32 can positively regulate UV-induced apoptosis in HeLa cells. (2) Over-expressing p32 could significantly promote mitochondrial calcium uptake, while silencing p32 by siRNA suppressed it. (3) In p32 knockdown cells, Ruthenium Red treatment (an inhibitor of mitochondrial calcium uniporter) showed no further suppressive effect on mitochondrial calcium uptake. In addition, in Ruthenium Red treated cells, Mcl-1 also failed to suppress mitochondrial calcium uptake. Taken together, our findings suggest that p32 is part of the putative mitochondrial uniporter that facilitates mitochondrial calcium uptake. By binding to p32, Mcl-1 can interfere with the uniporter function, thus inhibit the mitochondrial Ca{sup 2+} uploading. This may provide a novel mechanism to explain the anti-apoptotic function of Mcl-1.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

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Wills, Lauren P. [MitoHealth Inc., Charleston, SC 29403 (United States); Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Beeson, Craig C. [MitoHealth Inc., Charleston, SC 29403 (United States); Peterson, Yuri K. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

2013-10-15

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

International Nuclear Information System (INIS)

Wills, Lauren P.; Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C.; Beeson, Craig C.; Peterson, Yuri K.; Schnellmann, Rick G.

2013-01-01

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants

Mitochondrial oxidative function and type 2 diabetes

DEFF Research Database (Denmark)

Rabøl, Rasmus; Boushel, Robert; Dela, Flemming

2006-01-01

The cause of insulin resistance and type 2 diabetes is unknown. The major part of insulin-mediated glucose disposal takes place in the skeletal muscle, and increased amounts of intramyocellular lipid has been associated with insulin resistance and linked to decreased activity of mitochondrial...... oxidative phosphorylation. This review will cover the present knowledge and literature on the topics of the activity of oxidative enzymes and the electron transport chain (ETC) in skeletal muscle of patients with type 2 diabetes. Different methods of studying mitochondrial function are described, including...... biochemical measurements of oxidative enzyme and electron transport activity, isolation of mitochondria for measurements of respiration, and ATP production and indirect measurements of ATP production using nuclear magnetic resonance (NMR) - spectroscopy. Biochemical markers of mitochondrial content are also...

Combined effects of cadmium, temperature and hypoxia-reoxygenation on mitochondrial function in rainbow trout (Oncorhynchus mykiss)

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Onukwufor, John O.; Stevens, Don; Kamunde, Collins, E-mail: ckamunde@upei.ca

2017-01-15

Highlights: • Ternary interactions of Cd, temperature and H-R alter their individual and binary effects on mitochondrial bioenergetics. • Oxidative stress explains many effects of Cd, H-R and temperature on mitochondria. • Cd accumulation does not explain increased sensitivity of mitochondria to multiple stressors. • Cd induces hormetic responses during H-R stress. • Cd at low dose blunts conversion of complex I active (A)- to deactive (D)-form after H-R. - Abstract: Although aquatic organisms face multiple environmental stressors that may interact to alter adverse outcomes, our knowledge of stressor–stressor interaction on cellular function is limited. We investigated the combined effects of cadmium (Cd), hypoxia-reoxygenation (H-R) and temperature on mitochondrial function. Liver mitochondria from juvenile rainbow trout were exposed to Cd (0–20 μM) and H-R (0 and 5 min) at 5, 13 and 25 °C followed by measurements of mitochondrial Cd load, volume, complex I active (A) ↔ deactive (D) transition, membrane potential, ROS release and ultrastructural changes. At high temperature Cd exacerbated H-R-imposed reduction of maximal complex I (CI) respiration whereas at low temperature 5 and 10 μM stimulated maximal CI respiration post H-R. The basal respiration showed a biphasic response at high temperatures with low Cd concentrations reducing the stimulatory effect of H-R and high concentrations enhancing this effect. At low temperature Cd monotonically enhanced H-R-induced stimulation of basal respiration. Cd and H-R reduced both the P/O ratio and the RCR at all 3 temperatures. Temperature rise alone increased mitochondrial Cd load and toxicity, but combined H-R and temperature exposure reduced mitochondrial Cd load but surprisingly exacerbated the mitochondrial dysfunction. Mitochondrial dysfunction induced by H-R was associated with swelling of the organelle and blocking of conversion of CI D to A form. However, low amounts of Cd protected against H

Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

Science.gov (United States)

Madsen, Christian T.; Sylvestersen, Kathrine B.; Young, Clifford; Larsen, Sara C.; Poulsen, Jon W.; Andersen, Marianne A.; Palmqvist, Eva A.; Hey-Mogensen, Martin; Jensen, Per B.; Treebak, Jonas T.; Lisby, Michael; Nielsen, Michael L.

2015-01-01

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells. PMID:26158509

Mitochondrial PKA mediates sperm motility.

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Mizrahi, Rashel; Breitbart, Haim

2014-12-01

Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics. Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy. Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria. Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60kDa proteins does not occur in the absence of bicarbonate. Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Mitochondrial role in cell aging

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Miquel, J.; Fleming, J.; Economos, A. C.; Johnson, J. E., Jr.

1980-01-01

The experimental studies on the mitochondria of insect and mammalian cells are examined with a view to an analysis of intrinsic mitochondrial senescence, and its relation to the age-related changes in other cell organelles. The fine structural and biochemical data support the concept that the mitochondria of fixed postmitotic cells may be the site of intrinsic aging because of the attack by free radicals and lipid peroxides originating in the organelles as a by-product of oxygen reduction during respiration. Although the cells have numerous mechanisms for counteracting lipid peroxidation injury, there is a slippage in the antioxidant protection. Intrinsic mitochondrial aging could thus be considered as a specific manifestation of oxygen toxicity. It is proposed that free radical injury renders an increasing number of the mitochondria unable to divide, probably because of damage to the lipids of the inner membrane and to mitochondrial DNA.

[New theory of holistic integrative physiology and medicine. III: New insight of neurohumoral mechanism and pattern of control and regulation for core axe of respiration, circulation and metabolism].

Science.gov (United States)

Sun, Xing-guo

2015-07-01

Systemic mechanism of neurohumoral control and regulation for human is limited. We used the new theory of holistic integrative physiology and medicine to approach the mechanism and pattern of neurohumoral control and regulation for life. As the core of human life, there are two core axes of functions. The first one is the common goal of respiration and circulation to transport oxygen and carbon dioxide for cells, and the second one is the goal of gastrointestinal tract and circulation to transport energy material and metabolic product for cells. These two core axes maintain the metabolism. The neurohumoral regulation is holistically integrated and unified for all functions in human body. We simplified explain the mechanism of neurohumoral control and regulation life (respiration and circulation) as the example pattern of sound system. Based upon integrated regulation of life, we described the neurohumoral pattern to control respiration and circulation.

Metformin-treated patients with type 2 diabetes have normal mitochondrial complex I respiration

DEFF Research Database (Denmark)

Larsen, Steen; Rabøl, R; Hansen, C N

2012-01-01

The glucose-lowering drug metformin has been shown to inhibit complex I of the mitochondrial electron transport chain in skeletal muscle. To investigate this effect in vivo we studied skeletal muscle mitochondrial respiratory capacity and content from patients with type 2 diabetes treated...

AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

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Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

2014-01-01

Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

Mitochondrial functions of THP-1 monocytes following the exposure to selected natural compounds.

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Schultze, Nadin; Wanka, Heike; Zwicker, Paula; Lindequist, Ulrike; Haertel, Beate

2017-02-15

The immune system is an important target of various xenobiotics, which may lead to severe adverse effects including immunosuppression or inappropriate immunostimulation. Mitochondrial toxicity is one possibility by which xenobiotics exert their toxic effects in cells or organs. In this study, we investigated the impact of three natural compounds, cyclosporine A (CsA), deoxynivalenol (DON) and cannabidiol (CBD) on mitochondrial functions in the THP-1 monocytic cell line. The cells were exposed for 24h to two different concentrations (IC 10 and IC 50 determined by MTT) of each compound. The cells showed concentration-dependent elevated intracellular reactive oxygen species (iROS) and induction of apoptosis (except DON) in response to the three test compounds. Mitochondrial functions were characterized by using bioenergetics profiling experiments. In THP-1 monocytes, the IC 50 of CsA decreased basal and maximal respiration as well as ATP production with an impact on spare capacity indicating a mitochondrial dysfunction. Similar reaction patterns were observed following CBD exposure. The basal respiration level and ATP-production decreased in the THP-1 cells exposed to the IC 50 of DON with no major impact on mitochondrial function. In conclusion, impaired mitochondrial function was accompanied by elevated iROS and apoptosis level in a monocytic cell line exposed to CsA and CBD. Mitochondrial dysfunction may be one explanation for the cytotoxicity of CBD and CsA also in other in immune cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

MicroRNA-761 regulates mitochondrial biogenesis in mouse skeletal muscle in response to exercise

Energy Technology Data Exchange (ETDEWEB)

Xu, Yanli [Affiliated Hospital of Hebei Engineering University, Handan, 056002, Hebei (China); Zhao, Chaoxian; Sun, Xuewen [Medical College of Hebei Engineering University, Handan, 056002, Hebei (China); Liu, Zhijun, E-mail: liuzhij1207@163.com [Affiliated Hospital of Hebei Engineering University, Handan, 056002, Hebei (China); Zhang, Jianzhong, E-mail: zhangjianzhong@icdc.cn [National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC), Beijing, 102206 (China)

2015-11-06

MicroRNAs (miRNAs) have been suggested to play critical roles in skeletal muscle in response to exercise. Previous study has shown that miR-761 was involved in a novel model regulating the mitochondrial network. However, its role in mitochondrial biogenesis remains poorly understood. Therefore, the current study was aimed to examine the effect of miR-761 on mitochondrial biogenesis in skeletal muscle. Real-time quantitative PCR analysis demonstrated that aberrantly expressed miR-761 is involved in exercise activity and miR-761 is decreased by exercise training compared with the sedentary control mice. miR-761 suppresses mitochondrial biogenesis of C{sub 2}C{sub 12} myocytes by targeting the 3′-UTR of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1α). Overexpression of miR-761 was capable of inhibiting the protein expression levels of PGC-1α. Moreover, miR-761 overexpression suppressed the p38 MAPK signaling pathway and down-regulated the expression of phosphorylated MAPK-activated protein kinase-2 (P-MK2), a downstream kinase of p38 MAPK. The phosphorylation of activating transcription factors 2 (ATF2) that plays a functional role in linking the activation of the p38 MAPK pathway to enhanced transcription of the PGC-1α was also inhibited by the overexpression of miR-761. These findings revealed a novel regulation mechanism for miR-761 in skeletal myocytes, and contributed to a better understanding of the modulation of skeletal muscle in response to exercise. - Highlights: • Endurance exercise decreases miR-761 expression in skeletal muscle. • MiR-761 suppresses mitochondrial biogenesis in C{sub 2}C{sub 12} myocytes. • MiR-761 directly targeted PGC-1α expression. • MiR-761 suppresses p38 MAPK signaling pathways in C{sub 2}C{sub 12} myocytes. • A novel mechanism for miR-761 in skeletal myocytes is demonstrated.

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

Science.gov (United States)

Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

2013-01-01

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and

The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Directory of Open Access Journals (Sweden)

Romain Rivalin

Full Text Available Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice.

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Betancourt, Angela M; King, Adrienne L; Fetterman, Jessica L; Millender-Swain, Telisha; Finley, Rachel D; Oliva, Claudia R; Crowe, David R; Ballinger, Scott W; Bailey, Shannon M

2014-07-15

NAFLD (non-alcoholic fatty liver disease) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. In the present study, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, MNX (mitochondrial-nuclear exchange) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared with wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR (respiratory control ratio) when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD.

/sup 31/P-NMR studies of respiratory regulation in the intact myocardium

Energy Technology Data Exchange (ETDEWEB)

From, A H.L.; Petein, M A; Zimmer, S W; Michurski, S P; Ugurbil, K

1986-10-06

The mechanism by which mitochondrial respiration is coupled to ATP consumption in intact tissues is unclear. The authors determined the relationship between high-energy phosphate levels and oxygen consumption rate in rat hearts operating over a range of workloads and perfused with different substrates. With pyruvate + glucose perfusion, ADP levels were in general very low, and varied with MVO/sub 2/ yielding an apparent K/sub m/ of 25 +- 5 ..mu..M, suggesting regulation of oxidative phosphorylation through availability of ADP. In contrast, with glucose perfusion in the presence or absence of insulin, ADP levels, ADP/ATP ratio or the phosphate potential were relatively constant over the workload range examined and generally not correlated with alterations in MVO/sub 2/; it is suggested that under these conditions, carbon substrate delivery to the mitochondria may control mitochondrial respiration. The common feature of both of the suggested regulatory mechanisms is substrate limitation which, however, is exercised at different metabolic points depending on the carbon substrate available to the myocardium. 19 refs.; 2 figs.; 1 table.

Toward an adverse outcome pathway for impaired growth: Mitochondrial dysfunction impairs growth in early life stages of the fathead minnow (Pimephales promelas).

Science.gov (United States)

Bolser, Derek G; Dreier, David A; Li, Erchao; Kroll, Kevin J; Martyniuk, Christopher J; Denslow, Nancy D

2018-07-01

Chemical contaminants present in the environment can affect mitochondrial bioenergetics in aquatic organisms and can have substantial effects on individual fitness. As early life stages of fish are particularly vulnerable to environmental contaminants, they are ideal models for examining the relationship between impaired mitochondrial bioenergetics (ATP-dependent respiration, basal oxidative respiration) and apical endpoints such as growth. Here, early life stages of the fathead minnow (Pimephales promelas), an ecologically relevant North American species, were used to investigate the relationship between mitochondrial bioenergetics and growth following perturbation with model mitochondrial toxicants 2,4-dinitrophenol and octylamine. Fathead minnows were exposed to 2,4-dinitrophenol and octylamine at 3 concentrations for 24 h and endpoints related to mitochondrial bioenergetics were measured with the Agilent Seahorse XFe24 Bioanalyzer. In order to link changes in mitochondrial bioenergetics to growth, fathead minnows were exposed to the same chemical contaminants for 7-14 days and growth was measured by measuring total length on a weekly basis. There was a significant correlation between decrease in average length at 14 days and basal respiration (r = 0.997, p = 0.050, n = 3), as well as maximal respiration (r = 0.998, p-value = 0.043, n = 3) for embryos exposed to 2,4 dinitrophenol. For octylamine, ATP production was highly correlated with average length at 7 days (p-value = 0.1) and spare respiratory capacity and average length at 14 days were highly correlated (p-value = 0.1). These data improve understanding of how mitochondrial toxicants impair growth in fish larvae and may be useful for developing an adverse outcome pathway for growth. Copyright © 2018 Elsevier Inc. All rights reserved.

Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

International Nuclear Information System (INIS)

Montaigne, David; Marechal, Xavier; Baccouch, Riadh; Modine, Thomas; Preau, Sebastien; Zannis, Konstantinos; Marchetti, Philippe; Lancel, Steve; Neviere, Remi

2010-01-01

The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 μM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt max of 105 ± 8 mN/s in control hearts vs. 49 ± 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 ± 0.2 in control hearts vs. 2.2 ± 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 ± 1 μM cytochrome c/min/mg in control hearts vs. 14 ± 3 μM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

The Mitochondrial GTPase Gem1 Contributes to the Cell Wall Stress Response and Invasive Growth of Candida albicans

Directory of Open Access Journals (Sweden)

Barbara Koch

2017-12-01

Full Text Available The interactions of mitochondria with the endoplasmic reticulum (ER are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure. In the model yeast Saccharomyces cerevisiae, the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in Candida albicans Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of GEM1 in C. albicans. There are two major virulence-related consequences of disrupting mitochondrial morphology by GEM1 inactivation: C. albicans becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the gem1Δ/Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the gem1Δ/Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP kinase Cek1 is reduced in the gem1Δ/Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in C. albicans. We propose that ER-mitochondria interactions and the ER

The Mitochondrial GTPase Gem1 Contributes to the Cell Wall Stress Response and Invasive Growth of Candida albicans.

Science.gov (United States)

Koch, Barbara; Tucey, Timothy M; Lo, Tricia L; Novakovic, Stevan; Boag, Peter; Traven, Ana

2017-01-01

The interactions of mitochondria with the endoplasmic reticulum (ER) are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure). In the model yeast Saccharomyces cerevisiae , the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in Candida albicans Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of GEM1 in C. albicans . There are two major virulence-related consequences of disrupting mitochondrial morphology by GEM1 inactivation: C. albicans becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the gem1 Δ / Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the gem1 Δ / Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP) kinase Cek1 is reduced in the gem1 Δ / Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in C. albicans . We propose that ER-mitochondria interactions and the ER-Mitochondria Organizing