Mitochondrial Respiration State Research Articles

Characterization of Mitochondrial Bioenergetics of Adipose‐Derived Stem Cells from Burned and Non‐burned Patients

Prolonged hypermetabolism is a hallmark of severe burn injury (over 20% of the total body surface area) that produces cachexia and hinders the wound healing process. Topical treatment with adipose‐derived stem cells (ASCs) have been proposed as an attractive solution to hasten the recovery of burn wounds. A potentially viable source of ASC is from the subcutaneous adipose tissue of severely burned patients, which would normally be discarded during surgical debridement. While previous studies found that severe burn injury alters the metabolic profile of subcutaneous adipocytes, the effect of severe burns on ASCs from burned victims is largely unknown. The purpose of this study is to compare the metabolic activity of ASCs from burned patients to those from non‐burned abdominoplasty patients by analyzing mitochondrial respiration and glycolysis. ASCs were isolated from adipose tissues from severely burned patients (BP, n=6) and abdominoplasty patients (AP, n=6). Cells were cultured to 80% confluence and harvested at at passage 2, 4, and 6 for analysis. Flow cytometry was used to determine ASC cell surface markers CD90, CD105, and CD73 and negative for CD34, CD11, CD19 and CD45. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively. JC‐10 Mitochondrial Membrane Potential Assays were used to determine mitochondrial membrane integrity. Mitochondrial respiration and glycolysis were analyzed in technical quadruplicates by a Seahorse XFe24 Analyzer. No significant differences were found in ASC surface markers, mitochondrial abundance or membrane potential among passages or between groups. A two‐way ANOVA showed that there was a significant effect (p<0.01) of passage number on mitochondrial ROS production. Mitochondrial respiratory states Basal, Leak (uncoupled), and Non‐mitochondrial respiration was significantly higher (p<0.05) at passage 6 than passage 2 for BP, but not AP. Additionally, Basal respiration was significantly higher at passage 6 in BP than AP (63.5±3.49 vs 45.7±4.26 pmol O2/min, p<0.001). Glycolysis significantly decreased (p<0.05) from passage 4 to 6 in AP but not BP ASCs. In summary, BP ASCs showed increased mitochondrial respiration with increasing passages and basal respiration was higher than AP ASCs patients at passage 6. While all ASCs retained their stem cell properties, they also showed increased ROS production through multiple passages which may cause oxidative stress that could limit the expansion of these cells ex vivo. The elevated mitochondrial activity from ASCs of burned patients may suggest these cells are more metabolically primed than those from abdominoplasty patients, but whether this thwarts their therapeutic potential requires additional investigation. Adipose‐derived stem cells from burned injured patients remain a potential viable source for burn wound therapies. While other studies have demonstrated efficacy in healing burn wounds, this study uncovers potential limitations in expansion of these cells ex vivo to use as coverage options for burned victims.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alterations in the Mitochondrial Physiology of Biomphalaria glabrata (Mollusca: Gastropoda) After Experimental Infection by Angiostrongylus cantonensis (Nematoda: Metastrongylidae).

Angiostrongylus cantonensis is a metastrongylid nematode that has a heteroxenous cycle, where snails act as intermediate hosts and the rodents Rattus rattus and Rattus novergicus are the definitive hosts. However, humans may act as accidental hosts presenting an atypical form of parasitism. This fact has motivated research to better understand systems of relationships involving A. cantonensis, targeting the control of species of gastropods that act as intermediary hosts. For this, six groups were formed: three control groups (uninfected) and three infected groups, exposed to approximately 1200 L1 larvae of A. cantonensis. At the end of each week (1, 2, and 3 weeks), snails were dissected without anesthesia and the gonad-digestive gland (DGG) complex was separated for determination of oxygen consumption through high-resolution titration-injection respirometer (Oroboros, Oxygraph; Innsbruck, Austria). The results indicate suppression of mitochondrial oxidative metabolism of the host and compromised in different mitochondrial respiratory states. This effect, mainly observed in the group exposed to 1 week of infection, showed a decrease of approximately 38% (2.78±0.37 pmol O2/mg of tissue; P<0.05), 41% (2.76±0.34 pmol O2/mg of tissue; P<0.05) e 46% (2.91±0.36 pmol O2/mg of tissue; P<0.05) in the basal oxygen consumption after sequential addition (P + M), succinate and (ADP) in the respiratory medium, differing significantly from the control group. The results presented indicate that the prepatent infection by this metastrongylid impairs the aerobic oxidative metabolism of its host, causing a reduction in basal oxygen consumption. This effect, observed at the start of development of the parasites, indicates that this stage is the most critical for the success of the infection, and can be explained by a reduction of the mitochondrial density of the tissue analyzed, or also by suppression of enzyme centers related to the oxidative reactions.

392 Increased Mitochondrial Respiration and ROS Production From Adipose-Derived Stem Cells is Passage Dependent

Victims of severe burn injury (over 30% of the total body surface area) experience profound hypermetabolism which prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds. Moreover, the subcutaneous adipose tissue of severely burned patients normally discarded during surgical debridement is a viable source of ASCs. While previous studies found that severe burn injury alters the metabolic profile of subcutaneous adipocyte, the effect of severe burns on ASCs is largely unknown. The purpose of this study is to compare the metabolic activity of ASCs from burned patients to those from abdominoplasty patients by analyzing mitochondrial respiration and characteristics. Isolated ASCs from severely burned patients (BP, n=6) and abdominoplasty patients (AP, n=6) were grown to 80% confluency, trypsinized, and harvested for analysis at passage 2, 4, and 6. Flow cytometry was used to determine ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and ROS production were determined with MitoTracker Green and MitoSOX Red, respectively. JC-10 Mitochondrial Membrane Potential Assays were used to determine mitochondrial membrane integrity. Mitochondrial respiration and glycolysis were analyzed in technical quadruplicates by a Seahorse XFe24 Analyzer. No significant differences were found in ASC surface markers, mitochondrial abundance or membrane potential among passages or between groups. A two-way ANOVA showed that there was a significant effect (p<0.01) of passages on mitochondrial ROS production. Mitochondrial respiratory states Routine, Leak (uncoupled), and Non-mitochondrial respiration was significantly higher (p<0.05) at passage 6 than passage 2 for BP, but not AP. Glycolysis significantly decreased (p<0.05) from passage 4 to 2 in AP ASCs. Stem cell positive staining and mitochondrial activity were not different between cells from burned or non-burned humans. BP ASCs showed increased mitochondrial respiration with increasing passages. All ASCs showed increased ROS production through multiple passages. Adipose-derived stem cells from burned injured patients remain an exciting potential source for therapeutic purposes. However, increased ROS production and mitochondrial leak may cause oxidative stress that could limit the expansion of these cells ex vivo.

Mitochondrial complex activity in permeabilised cells of chronic fatigue syndrome patients using two cell types.

Abnormalities in mitochondrial function have previously been shown in chronic fatigue syndrome (CFS) patients, implying that mitochondrial dysfunction may contribute to the pathogenesis of disease. This study builds on previous work showing that mitochondrial respiratory parameters are impaired in whole cells from CFS patients by investigating the activity of individual mitochondrial respiratory chain complexes. Two different cell types were used in these studies in order to assess individual complex activity locally in the skeletal muscle (myotubes) (n = 6) and systemically (peripheral blood mononuclear cells (PBMCs)) (control n = 6; CFS n = 13). Complex I, II and IV activity and respiratory activitysupported by fatty acid oxidation and glutaminolysis were measured usingextracellular flux analysis. Cells were permeabilised and combinations of substrates and inhibitors were added throughout the assays to allow states of mitochondrial respiration to be calculated and the activity of specific aspects of respiratory activity to be measured. Results showed there to be no significant differences in individual mitochondrial complex activity or respiratory activity supported by fatty acid oxidation or glutaminolysis between healthy control and CFS cohorts in either skeletal muscle (p ≥ 0.190) or PBMCs (p ≥ 0.065). This is the first study to use extracellular flux analysisto investigate individual mitochondrial complex activity in permeabilised cells in the context of CFS. The lack of difference in complex activity in CFS PBMCs suggests that the previously observed mitochondrial dysfunction in whole PBMCs is due to causes upstream of the mitochondrial respiratory chain.

Effects of thyroid hormones and cold acclimation on the energy metabolism of the striped hamster (Cricetulus barabensis).

To examine the effects of low ambient temperature and thyroid hormones on the energy metabolism of the striped hamster (Cricetulus barabensis), adult male striped hamsters were kept at 30°C, or acclimated to 5°C, for 4weeks. During this time, hamsters were treated with a synthetic thyroxine, levothyroxine sodium (LTS), the antithyroid drug methimazole, or saline solution (control). Hamster's food intake, basal metabolic rate (BMR), non-shivering thermogenesis (NST), thyroid hormones, body fat content, mitochondrial state-4 respiration, cytochrome c oxidase, and uncoupling protein 1 (UCP1) gene expression in brown adipose tissue (BAT), were measured. Both acclimation to 5°C and LTS increased serum levels of triiodothyronine, which was associated with increased food and energy intake and BMR. Interestingly, although acclimation to 5°C also increased NST and UCP1 gene expression in BAT, and decreased body fat content, these changes were not induced by LTS treatment. Finally, exposure to 5°C reduced the effects of LTS on energy intake and expenditure in specific metabolic markers and organs. Together, these data illustrate that ambient temperature and thyroid hormones can have both independent, and interactive, effects on the metabolic changes in striped hamsters induced by cold acclimation.

Determination of Polycation-Mediated Perturbation of Mitochondrial Respiration in Intact Cells by High-Resolution Respirometry (Oxygraph-2k, OROBOROS).

A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.

Food restriction decreases thermoregulation in the silky starling Sturnus sericeus (Aves: Passeriformes)

The capacity of small birds to sustain periods of food shortage largely depends on their ability to adaptively regulate expenditure in response to reduced food supply. To investigate changes in thermoregulation during periods of food restriction, we measured changes in the body mass, basal metabolic rate (BMR), organ mass, thyroid hormone levels, and several physiological and biochemical markers, of silky starlings (Sturnus sericeus) subject to 16 days food restriction. Birds in the control group were provided with food ad libitum whereas those in the food restriction (FR) group were provided with half of the usual quantity of food. Oxygen consumption was measured using an open–circuit respirometry system. Mitochondrial state-4 respiration and cytochrome c oxidase (COX) activity in the liver and pectoral muscle were measured with a Clark electrode. The concentrations of T3 and T4 in plasma were determined by radioimmunoassay. Starlings in the FR group had lower body mass and BMR compared to the control group. Starlings in the FR group also had lower state-4 respiration and COX activity in the liver and muscle, and lower T3/T4 ratio in plasma, relative to the control group. BMR was positively correlated with body mass, state-4 respiration and COX activity, in liver and muscle tissue. These results indicate that silky starlings can decrease their energy metabolism during periods of food shortage by making a suite of adjustments from the organismal to the biochemical level. These morphological, physiological and biochemical responses could be adaptations that allow starlings to adjust to seasonal changes in food abundance, such as the typically lower food abundance over winter.

Absence of effect of the antiretrovirals Duovir and Viraday on mitochondrial bioenergetics.

Most toxicity associated with antiretroviral drugs is thought to result from disruption of mitochondrial function. Unfortunately, there are no validated laboratory markers for clinically assessing the onset of mitochondrial toxicity associated with antiretroviral therapy. In a previous study on mitochondrial hepatocytes, the protease inhibitor lopimune was shown to induce mitochondrial toxicity by increasing reactive oxygen species (ROS) production and decreasing respiratory control ratio (RCR) reflecting compromised mitochondrial efficiency in adenosine triphosphate production. Mitochondrial dysfunction and ROS production were directly correlated with the expression of uncoupling protein 2 (UCP2). In the current study we aim to determine the toxicity of nucleoside or nucleotide and nonnucleoside reverse-transcriptase inhibitors, Duovir and Viraday on liver mitochondria isolated from treated mice by monitoring UCP2 expression. Our results showed that both Duovir and Viraday had no effect on mitochondrial respiration states 2, 3, 4, and on RCR. In addition, ROS generation and UCP2 expression were not affected. In conclusion, our results indicate the difference in the mechanism of action of distinct classes of antiretroviral drugs on mitochondrial functions and may associate UCP2 expression with subclinical mitochondrial damage as marker of cellular oxidative stress.

Mitochondrial respiratory states and rates: building blocks of mitochondrial physiology

Mitochondrial respiratory states and rates: building blocks of mitochondrial physiology

Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology (Part 1)

Supporting co-authors: Bakker BM, Bernardi P, Boetker HE, Borsheim E, Borutaitė V, Bouitbir J, Calbet JA, Calzia E, Chaurasia B, Clementi E, Coker RH, Collin A, Das AM, De Palma C, Dubouchaud H, Durham WJ, Dyrstad SE, Engin AB, Fornaro M, Gan Z, Garlid KD, Garten A, Gourlay CW, Granata C, Haas CB, Haavik J, Haendeler J, Hand SC, Hepple RT, Hickey AJ, Hoel F, Jang DH, Kainulainen H, Khamoui AV, Klingenspor M, Koopman WJH, Kowaltowski AJ, Krajcova A, Lane N, Lenaz G, Malik A, Markova M, Mazat JP, Menze MA, Methner A, Neuzil J, Oliveira MT, Pallotta ML, Parajuli N, Pettersen IKN, Porter C, Pulinilkunnil T, Ropelle ER, Salin K, Sandi C, Sazanov LA, Silber AM, Skolik R, Smenes BT, Soares FAA, Sokolova I, Sonkar VK, Swerdlow RH, Szabo I, Trifunovic A, Thyfault JP, Valentine JM, Vieyra A, Votion DM, Williams C, Zischka H

Indirubin and NAD+ prevent mitochondrial ischaemia/reperfusion damage in fatty livers.

Fatty livers are considerably more susceptible to acute stressors, such as ischaemia/reperfusion (I/R). As the incidence of I/R is high due to surgical events and some pathologies, there is an urgent need to find strategies against I/R injury (I/RI) in fatty livers. We postulate that an acute pretreatment with indirubin-3'-oxime (Ind) or NAD+ prevents mitochondrial dysfunction associated with warm I/RI in fatty livers. Zucker fatty rats were subjected to warm ischaemia and 12 hours of reperfusion. Ind or NAD+ was administered in the hepatic artery 30 minutes before ischaemia. Hepatic mitochondrial isolation was performed, and functional assays as well as molecular analysis were performed. Pretreatment decreased markers of liver injury while preserving mitochondrial cytochrome c content, which is related to the prevention of calcium-induced mitochondrial permeability transition (mPT), the decline in mitochondrial respiratory state 3 and ATP content. The generation of reactive oxygen species (ROS) was also diminished. Inhibition of GSK-3ß by Ind resulted in the prevention of cyclophilin-D (CypD) phosphorylation, unabling it to bind to the adenine nucleotide translocator (ANT), thus, preventing mPT induction. Furthermore, deacetylation of CypD at Lys residue by sirtuin 3 (SIRT3) caused its dissociation from ANT, contributing to an increase in mPT threshold in NAD+ -pretreated animals. Pretreatment with Ind or NAD+ protects fatty livers by maintaining mitochondrial calcium homoeostasis, thus, preserving mitochondrial function and energetic balance. As such, CypD might be a new protective target against I/RI in fatty livers.

2,4 Dinitrophenol Attenuates Mitochondrial Dysfunction and Improves Neurobehavioral Outcomes Postanoxia in Neonatal Rats.

Following anoxia, a rapid and marked mitochondrial-linked cell death occurs in the cerebral cortex of newborn rats which leads to insult advancement within a couple of days and causes lifelong neurobehavioral abnormalities. The present study investigated the role of 2,4 dinitrophenol (2,4 DNP) in three doses, i.e.,1, 2.5, and 5mg/kg on anoxia-induced time-dependent mitochondrial dysfunction and associated neurobehavioral outcome using a well-established global model of anoxia. Briefly, rat pups of 30-h age (P2) were subjected to two episodes of anoxia (10min each) at 24h of the time interval in an enclosed chamber supplied with 100% N2 and immersed in a water bath (35-37°C) to avoid hypothermia. Results demonstrated that the uncoupler 2,4 DNP, in the dose 2.5 and 5mg/kg injected i.p. within 5min after second anoxic episode significantly (P < 0.05) preserved mitochondrial function on day 7 preferentially by maintaining mitochondrial membrane potential (MMP) and inhibiting mitochondrial permeability transition (MPT) pore. Further, 2,4 DNP preserved mitochondrial function by improving different states of mitochondrial respiration (s2, s3, s4, s5), respiratory control ratio (RCR), antioxidant enzyme system like superoxide dismutase (SOD) and catalase (CAT), and mitochondrial complex enzymes (I, II, IV, V) after anoxia. Furthermore, a marked decrease in the levels of expression of cytochrome C (cyt C) and pro-apoptotic (Bcl-2 family) and apoptotic (caspase-9/3) proteins was observed on day 7 indicating that the treatment with 2,4 DNP prevented mitochondrial dysfunction and further insult progression (day 1 to day 7). Moreover, 2,4 DNP decreased the apoptotic cell death on day 7 and overall improved the neurobehavioral outcomes like reflex latency and hanging latency which suggests its role in treating neonatal anoxia.

The Effect of Mitochondrial Complex I-Linked Respiration by Isoflurane Is Independent of Mitochondrial Nitric Oxide Production

Background: Anesthetic preconditioning (APC) of the myocardium is mediated in part by reversible alteration of mitochondrial function. Nitric oxide (NO) inhibits mitochondrial respiration and may mediate APC-induced cardioprotection. In this study, the effects of isoflurane on different states of mitochondrial respiration during the oxidation of complex I-linked substrates and the role of NO were investigated. Methods: Mitochondria were isolated from Sprague-Dawley rat hearts. Respiration rates were measured polarographically at 28ºC with a computer-controlled Clark-type O₂ electrode in the mitochondria (0.5 mg/mL) with complex I substrates glutamate/malate (5 mM). Isoflurane (0.25 mM) was administered before or after adenosine diphosphate (ADP)-initiated state 3 respiration. The NO synthase (NOS) inhibitor L-N⁵-(1-iminoethyl)-ornithine (L-NIO, 10 μM) and the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1 μM) were added before or after the addition of ADP. Results: Isoflurane administered in state 2 increased state 2 respiration and decreased state 3 respiration. This attenuation of state 3 respiration by isoflurane was similar when it was given during state 3. L-NIO did not alter mitochondrial respiration or the effect of isoflurane. SNAP only, added in state 3, decreased state 3 respiration and enhanced the isoflurane-induced attenuation of state 3 respiration. Conclusion: Isoflurane has clearly distinguishable effects on different states of mitochondrial respiration during the oxidation of complex I substrates. The uncoupling effect during state 2 respiration and the attenuation of state 3 respiration may contribute to the mechanism of APC-induced cardioprotection. These effects of isoflurane do not depend on endogenous mitochondrial NO, as the NOS inhibitor L-NIO did not alter the effects of isoflurane on mitochondrial respiration.

Towards engineering distinct multi-lamellated outer and inner annulus fibrosus tissues.

The annulus fibrosus (AF) of the intervertebral disc (IVD) has a zonal distribution of phenotypically distinct cells. The outer AF (OAF) cells produce an extracellular matrix (ECM) rich in type I collagen with little proteoglycans, whereas the ECM of the inner AF (IAF) has abundant type II collagen and proteoglycans. The inhomogeneous distribution of the ECM in the AF may reflect the complex mechanical forces that the IVD experiences. A bioengineered AF tissue should recapitulate both the inner and outer zones in order to have proper functionality. The aim of this study is to generate multi-lamellated OAF and IAF tissues with ECM compositions that resemble their zonal origin using polycarbonate urethane (PU) scaffolds. It was observed that supplementation of the media with insulin-transferrin-selenium (ITS) and proline yielded tissues with good cellularity. However, IAF cells accumulated only type I collagen, similar to OAF cells. Addition of dexamethasone and sodium pyruvate induced the accumulation of IAF tissues rich in type II collagen and aggrecan, without altering the accumulation of type I collagen in OAF tissues. Dexamethasone stimulated mitochondrial membrane potential in both tissues in the presence of sodium pyruvate, suggesting a relationship between the mitochondrial aerobic respiratory state and dexamethasone signalling during the in vitro-tissue formation by OAF and IAF cells. Inhibition of the glucocorticoid receptor blocked the stimulation of mitochondrial membrane potentials and type II collagen accumulation. In summary, biologically distinct multi-lamellated OAF and IAF tissues can be generated, which will facilitate advancement towards the goal of engineering a biological IVD replacement. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1346-1355, 2018.

Identification of a previously undetected metabolic defect in the Complex II Caenorhabditis elegans mev-1 mutant strain using respiratory control analysis.

Hypometabolism may play an important role in the pathogenesis of ageing and ageing-related diseases. The nematode Caenorhabditis elegans offers the opportunity to study "living mitochondria" in a small (~1mm) animal replete with a highly stereotypical, yet complex, anatomy and physiology. Basal oxygen consumption rate is often employed as a proxy for energy metabolism in this context. This parameter is traditionally measured using single-chamber Clark electrodes without the addition of metabolic modulators. Recently, multi-well oxygen electrodes, facilitating addition of metabolic modulators and hence study of respiratory control during different mitochondrial respiration states, have been developed. However, only limited official protocols exist for C. elegans, and key limitations of these techniques are therefore unclear. Following modification and testing of some of the existing protocols, we used these methods to explore mitochondrial bioenergetics in live nematodes of an electron transfer chain Complex II mutant strain, mev-1, and identified a previously undetected metabolic defect. We find that mev-1 mutants cannot respond adequately to increased energy demands, suggesting that oxidative phosphorylation is more severely impaired in these animals than has previously been appreciated.

Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies

The research on mitochondrial functions in adipocytes has increasingly evidenced that mitochondria plays an important role in the onset and/or progression of obesity and related pathologies. Mitochondrial function in brown adipose tissue (BAT) has been classically assessed by measuring either the levels/activity of mitochondrial enzymes, or the respiration in isolated mitochondria. Isolation of mitochondria is not advantageous because it demands significant time and amount of tissue and, as tissue homogenates, disrupts biochemical and physical connections of mitochondria within the cell. Here, we described a new and efficient protocol to analyze the mitochondrial respiratory states in BAT biopsies that relies on intracellular triglyceride depletion followed by tissue permeabilization. In addition to minimizing tissue requirements to ∼17 mg wet weight, the proposed protocol enabled analysis of all mitochondrial respiratory states, including phosphorylation (OXPHOS), no-phosphorylation (LEAK), and uncoupled (ETS) states, as well as the use of substrates for complex I, complex II, and cytochrome c; together, these features demonstrated mitochondrial integrity and validated the preparation efficacy. Therefore, the protocol described here increases the possibilities of answering physiological questions related to small BAT regions of human and animal models, which shall help to unravel the mechanisms that regulate mitochondrial function in health and disease.

Mitochondrial dysfunction in peripheral blood mononuclear cells in early experimental and clinical acute pancreatitis

Background/objectivesMitochondrial dysfunction occurs in vital organs in experimental acute pancreatitis (AP) and may play an important role in determining severity of AP. However, obtaining vital organ biopsies to measure mitochondrial function (MtF) in patients with AP poses considerable risk of harm. Being able to measure MtF from peripheral blood will bypass this problem. Furthermore, whether mitochondrial dysfunction is detectable in peripheral blood in mild AP is unknown. Therefore, the objective was to evaluate peripheral blood MtF in experimental and clinical AP. MethodMitochondrial respiration was measured using high resolution oxygraphy in an experimental study in caerulein induced AP and in a separate study, in patients with mild AP. Superoxide, cytochrome c, mitochondrial membrane potential (ΔΨ) and adenine triphosphate (ATP) were also measured as other markers of MtF. ResultsEven though some states of mitochondrial respiration were increased in both experimental and clinical AP, this did not lead to an increase in net ATP in patients with AP. The increased leak respiration in both studies was further proof of dyscoupled mitochondria. In the clinical study there were also features of mitochondrial dysfunction with increased leak flux control ratio, superoxide, ΔΨ and decreased cytochrome c. ConclusionThere is evidence of mitochondrial dysfunction with dyscoupled mitochondria, increased superoxide and decreased cytochrome c in patients with mild acute pancreatitis. Further studies should now determine whether mitochondrial function alters with severity in AP and whether mitochondrial dysfunction responds to treatments.

Relationships between interspecific differences in the mass of internal organs, biochemical markers of metabolic activity, and the thermogenic properties of three small passerines

The capacity for thermogenesis is considered part of an animal’s adaptive strategy for survival, and basal metabolic rate (BMR) is one of the fundamental physiological standards for assessing the energy cost of thermoregulation in endotherms. BMR has been shown to be a highly flexible phenotypic trait both between, and within, species, but the metabolic mechanisms involved in the regulation of BMR, which range from variation in organ mass to biochemical adjustments, remain unclear. In this study, we investigated the relationship between organ mass, biochemical markers of metabolic tissue activity, and thermogenesis, in three species of small passerines: wild Bramblings (Fringilla montifringilla), Little Buntings (Emberiza pusilla) and Eurasian Tree Sparrows (Passer montanus), caught in Wenzhou, southeastern China. Oxygen consumption was measured using an open-circuit respirometry system. Mitochondrial state-4 respiration and cytochrome c oxidase (COX) activity in liver and pectoral muscle were measured with a Clark electrode. Our results show that Eurasian Tree Sparrows had significantly higher BMR, digestive organ mass, mitochondrial state-4 respiration capacity and COX activity in liver and muscle, than Bramblings and Little Buntings. Furthermore, interspecific differences in BMR were strongly correlated with those indigestive tract mass, state-4 respiration and COX activity. Our findings suggest that the digestive organ mass, state-4 respiration and COX activity play an important role in determining interspecific differences in BMR.

Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse

Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with H2O2 generation in freshly isolated synaptosomes using high resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that although synaptic mitochondria exhibit substantially higher respiratory activities (8–82-fold greater than NOX oxygen consumption depending on mitochondrial respiratory state), NADPH-dependent oxygen consumption is associated with greater H2O2 production (6-7-fold higher NOX-H2O2). We also show that, in terms of the consumed oxygen, while synaptic mitochondria “leaked” 0.71% ± 0.12 H2O2 during NAD+-linked resting, 0.21% ± 0.04 during NAD+-linked active respiration, and 0.07% ± 0.02 during FAD+-linked active respiration, NOX converted 38% ± 13 of O2 into H2O2. Our results indicate that NOX rather than mitochondria is the major source of synaptic H2O2. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons.

Impairment of mitochondrial energy metabolism of two marine fish by in vitro mercuric chloride exposure

The goal of this work was to understand the extent of mercury toxic effects in liver metabolism under an episode of acute contamination. Hence, the effects of in vitro mercuric chloride in liver mitochondria were assessed in two commercial marine fish: Senegalese sole (Solea senegalensis) and gilthead seabream (Sparus aurata). Liver mitochondria were exposed to 0.2mgL−1 of mercury, the average concentration found in fish inhabiting contaminated areas. Mercuric chloride depressed mitochondrial respiration state 3 and the maximal oxygen consumption in the presence of FCCP indicating inhibitory effects on the oxidative phosphorylation and on the electron transport chain, respectively. The inhibition of F1Fo-ATPase and succinate-dehydrogenase activities also corroborated the ability of mercury to inhibit ADP phosphorylation and the electron transport chain. This study brings new understanding on the mercury levels able to impair fish mitochondrial function, reinforcing the need for further assessing bioenergetics as a proxy for fish health status.