Basal Mitochondrial Respiration Research Articles

Mitochondrial energetic metabolism perturbations in skeletal muscles and brain of zebrafish ( Danio rerio) exposed to low concentrations of waterborne uranium

Anthropogenic release of uranium (U), originating from the nuclear fuel cycle or military activities, may considerably increase U concentrations in terrestrial and aquatic ecosystems above the naturally occurring background levels found throughout the environment. With a projected increase in the world-wide use of nuclear power, it is important to improve our understanding of the possible effects of this metal on the aquatic fauna at concentrations commensurate with the provisional drinking water guideline value of the World Health Organization (15 μg U/L). The present study has examined the mitochondrial function in brain and skeletal muscles of the zebrafish, Danio rerio, exposed to 30 and 100 μg/L of waterborne U for 10 and 28 days. At the lower concentration, the basal mitochondrial respiration rate was increased in brain at day 10 and in muscles at day 28. This is due to an increase of the inner mitochondrial membrane permeability, resulting in a decrease of the respiratory control ratio. In addition, levels of cytochrome c oxidase subunit IV (COX-IV) increased in brain at day 10, and those of COX-I increased in muscles at day 28. Histological analyses performed by transmission electron microscopy revealed an alteration of myofibrils and a dilatation of endomysium in muscle cells. These effects were largest at the lowest concentration, following 28 days of exposure.

Abstract LB-108: Glutamine addition and glucose addiction - linking metabolic abnormalities to genetic alterations in glioblastoma SF188 and hereditary leiomyomatosis renal carcinoma UOK262

Abstract Metabolic transformations of malignant cells are essential for tumor development and progression. Oncogene Akt and c-myc stimulate glucose metabolism and glutamine metabolism, respectively, and render the cancers dependent on glucose or glutamine for growth and survival (otherwise known as glucose addiction and glutamine addiction). However, many other oncogenes, tumor suppressor genes and signaling molecules also regulate glucose and glutamine metabolism, which results in various metabolic abnormalities in different cancers. The question we want to ask is how one or more genetic mutations can lead to the ultimate metabolic abnormalities of a given cancer. Toward this end, we report here the metabolic abnormalities of two human cancer cell lines, glioblastoma SF188, which is c-myc-dependent and is addicted to glutamine, and hereditary leiomyomatosis renal carcinoma (HLRCC) UOK262, which is fumarate hydratase-deficient and is addicted to glucose. We performed our metabolic analysis using the XF24 Extracellular Flux Analyzer and pharmacological modulators of cellular metabolism. The XF24 Analyzer simultaneously monitors oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of mitochondrial respiration and aerobic glycolysis respectively. Oxygen consumption rate is also used as an indicator for the oxidation of glucose and glutamine when they are added to the cells after baseline measurement. We found that SF188 cells showed a small increase in acidification rate upon glucose addition, suggesting a relatively low basal glycolysis rate. We estimated the glycolytic capacity of the cells by determining the acidification rate after the addition of mitochondrial ATP syntase inhibitor oligomycin. The glycoltic capacity remained high although basal glycolysis rate was low. Glucose was not oxidized at all by these cells. Most importantly, we observed significant increases in oxygen consumption rate upon addition of glutamine indicating active uptake and oxidation of glutamine. Associated with active glutamine oxidation, SF188 cells exhibited high basal reparation rate and high respiratory capacity. Finally, SF188 cells grew and survived in medium containing glutamine but no glucose; however, they started to die in the medium containing glucose but no glutamine. In contrast with glioblastoma SF188, we found that HLRCC UOK262 cells exhibit high basal glycolysis rate, which is close to its maximal capacity. Interestingly, basal mitochondrial respiration and respiratory capacity are extremely low, which is associated with reported defective mitochondrial complex I. The growth and survival of UOK262 is dependent on a high concentration of glucose in the culture medium. Collectively, we established metabolic abnormalities of glioblastama SF188 and HLRCC UOK262, which contain genetic alterations in c-myc and fumarate hydratase, respectively, that makes them addicted to glutamine and glucose. The metabolic abnormalities we found include the preferential use of glucose and glutamine, the activation or inactivation of relevant enzymatic pathways that catabolize glucose and glutamine, and the modulation of mitochondrial bioenergetic activities in the two cancer cell lines. Most of cancer cell lines that we have studied showed metabolic abnormalities falling between the two extremes. Therefore, this type of metabolic analysis coupled with the analysis of genetic abnormalities in cancers will provides a rapid means to gain insight into the aberrant metabolism of each type of cancer and improve the efficacy of cancer therapy for a cancer with a particular set of metabolic abnormalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-108.

Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells.

BackgroundMitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate.Methodology/Principal FindingsMitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H2O2 level at the onset of differentiation.Conclusion/SignificanceA defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.

Breathing lessons: Tor tackles the mitochondria

Breathing lessons: Tor tackles the mitochondria

Mitochondrial respiration and membrane potential after low-flow ischemia are not affected by ischemic preconditioning

Mitochondrial function following prolonged ischemia and subsequent reperfusion is better preserved by ischemic preconditioning (IP). In the present study, we analyzed whether or not IP has an impact on mitochondrial function at the end of a sustained ischemic period. Göttinger minipigs were subjected to 90-min low-flow ischemia without ( n = 5) and with ( n = 5) a preconditioning cycle of 10-min ischemia and 15-min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall (AW) and the control posterior wall (PW) at the end of ischemia. Basal mitochondrial respiration was not different between AW and PW. The ADP-stimulated (state 3) respiration in AW mitochondria compared to PW mitochondria was equally decreased in non-preconditioned and preconditioned pigs. The uncoupled respiration as well as the membrane potential (rhodamine 123 fluorescence) were not significantly different between groups. However, the recovery of the membrane potential (Δ rhodamine 123 fluorescence/s) after the addition of ADP was delayed in mitochondria obtained from AW compared to PW, both in non-preconditioned and in preconditioned pig hearts. Neither the amount of marker proteins for complexes of the electron transport chain nor the level of reactive oxygen species were affected by ischemia without or with IP. State 3 respiration and recovery of membrane potential were impaired in pig mitochondria after 90 min of low-flow ischemia. IP did not improve mitochondrial function during ischemia. Therefore, the preservation of mitochondrial function by IP may occur during reperfusion rather than during the sustained ischemic period.

Involvement of SH-groups during interaction of diazoxide with the inner membrane of rat heart mitochondria

It is known that adenine nucleotide translocase plays an important role in the formation of calcium-dependent mitochondrial permeability transition pore in the inner mitochondrial membrane [1] and activation of basal mitochondrial respiration by diazoxide, an agonist of mitochondrial K ATP channels [2]. Recent findings suggest that mitochondrial permeability transition pore and mitochondrial K ATP channel, which are regulated by [Ca 2+ ] m and ∆Ψ , respectively, and play a key role in the initiation of apoptosis [3], have close structural interconnections. It was hypothesized that some molecules in the inner mitochondrial membrane (in particular, adenine nucleotide translocase) are involved in formation of both the calcium-dependent mitochondrial permeability transition pore and mitochondrial K ATP channel [4]. It was previously shown that diazoxide, which mimicked the effect of pharmacological preconditioning during ischemia/reperfusion of an isolated heart [5], induced the opening of calcium-dependent mitochondrial permeability transition pore in the low-conductance state in the presence of

Nuclear and Mitochondrial Interaction Involving mt-Nd2 Leads to Increased Mitochondrial Reactive Oxygen Species Production

NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2(a)) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2(a) affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mt(NOD)) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mt(ALR)). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mt(NOD) mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mt(ALR) or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2(a) does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production.

Tyk2 Tyrosine Kinase Expression Is Required for the Maintenance of Mitochondrial Respiration in Primary Pro-B Lymphocytes

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.

PKCε activation augments cardiac mitochondrial respiratory post-anoxic reserve—a putative mechanism in PKCε cardioprotection

Modest cardiac-overexpression of constitutively active PKCε (aPKCε) in transgenic mice evokes cardioprotection against ischemia. As aPKCε interacts with mitochondrial respiratory-chain proteins we hypothesized that aPKCε modulates respiration to induce cardioprotection. Using isolated cardiac mitochondria wild-type and aPKCε mice display similar basal mitochondrial respiration, rate of ATP synthesis and adenosine nucleotide translocase (ANT) functional content. Conversely, the aPKCε mitochondria exhibit modest hyperpolarization of their inner mitochondrial membrane potential (ΔΨ m) compared to wild-type mitochondrial by flow cytometry. To assess whether this hyperpolarization engenders resilience to simulated ischemia, anoxia-reoxygenation experiments were performed. Mitochondria were exposed to 45 min anoxia followed by reoxygenation. At reoxygenation, aPKCε mitochondria recovered ADP-dependent respiration to 44 ± 3% of baseline compared to 28 ± 2% in WT controls ( P = 0.03) in parallel with enhanced ATP synthesis. This preservation in oxidative phosphorylation is coupled to greater ANT functional content [42% > concentration of atractyloside for inhibition in the aPKCε mitochondria vs. WT control ( P < 0.0001)], retention of mitochondrial cytochrome c and conservation of ΔΨ m. These data demonstrate that mitochondria from PKCε activated mice are intrinsically resilient to anoxia-reoxygenation compared to WT controls. This resilience is in part due to enhanced recovery of oxidative phosphorylation coupled to maintained ANT activity. As maintenance of ATP is a prerequisite for cellular viability we conclude that PKCε activation augmented mitochondrial respiratory capacity in response to anoxia-reoxygenation may contribute to the PKCε cardioprotective program.

Catechol inhibits FADH2-linked respiration in rat liver mitochondrial fraction

The aim of this work was to investigate the hypothesis that catechol inhibits FADH -linked basal respiration in mitochondria isolated from rat liver homogenates. Moreover, catechol ability to induce peroxidation of biomolecules in liver nuclear fractions was also studied. Rat liver homogenates were incubated with 1mM 1,2-dihydroxybenzene (catechol) at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate. Nuclear fractions were incubated in the presence of 1 mM catechol for 17 hours at room temperature and the peroxidation of biomolecules was investigated by the reaction with thiobarbituric acid, which was determined spectrophotometrically at 535 nm. Catechol induced a time-dependent partial inhibition of FADH -linked basal mitochondrial respiration, however this substance was unable to induce a direct peroxidation of biomolecules in hepatic nuclear fractions. Catechol produced an inhibition of basal respiration associated to FADH2 in isolated liver mitochondria that could lead to cytotoxicity, ROS generation and cell death.

Cd 2+ versus Ca 2+-produced mitochondrial membrane permeabilization: a proposed direct participation of respiratory complexes I and III

A comparison of Cd 2+ and Ca 2+ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd 2+ and/or Ca 2+-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca 2+ and Cd 2+-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca 2+, catalase and dibucaine did not influence on main Cd 2+ effects. In NH 4NO 3 medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd 2+-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd 2+-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd 2+-produced swelling in NH 4NO 3 medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca 2+ and P i were simultaneously present in NH 4NO 3 medium, DTT reversed only partially Cd 2+-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca 2+ or P i were separately administered to the Cd 2+-treated mitochondria. Besides, DTT added following a low Cd 2+ pulse in KCl medium containing exogenous Ca 2+ induced a substantial enhancing of sustained Cd 2+ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd 2+] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd 2+ in the absence of added Ca 2+ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd 2+and/or Ca 2+-produced MMP was discussed.

Cyclosporin A treatment increases rat soleus muscle oxidative capacities.

Previous studies suggested that administration of cyclosporin A (CsA), an immunosuppressive agent, contributes to the increased fatigability of heart transplant recipients. The aim of this study was to investigate whether CsA itself, without vehicle, affects the function of mitochondria maintained in situ, in rats treated with CsA (25mg/kg/day) dissolved in ethanol and olive oil. Treatment with CsA induced a 16% decrease in slow myosin heavy chain (MHC) associated with a 225% increase in fast MHCIIa. The proportion of fibers expressing type IIa MHC increased as a result of CsA treatment. Soleus from the CsA-treated animals showed an increase in both basal (+85%) and maximal (+37%) mitochondrial respiration (P < 0.001), consistent with a 24% increase in citrate synthase activity, whereas the apparent Km for adenosine diphosphate was unchanged. By itself, CsA has no deleterious effects on muscle oxidative capacity but induces alterations in energy metabolism in accordance with the increased proportion of fast-twitch oxidative fibers.

Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle.

Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle.

Cyclosporin A-sensitive permeability transition pore is involved in Cd 2+-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd 2+-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca 2+ and P i, which is prevented by Sr 2+. Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P i, and Ca 2+) affect in a corresponding way Cd 2+-promoted membrane permeabilization in NH 4NO 3, KCl, and sucrose media. Maximal depression of Cd 2+-induced swelling is achieved by simultaneous addition of ADP, Mg 2+, and CsA that produces either synergistic (NH 4NO 3) or additive (KCl and sucrose media) action. Sustained activation by low [Cd 2+] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone + oligomycin) CsA inhibits completely Cd 2+ activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd 2+ effects are discussed in terms of comparison with those of Zn 2+ and PhAsO.

Oxygen consumption and biosynthetic function in perfused liver from rats at different stages of development.

Changes in mitochondrial function were studied in perfused liver from rats aged 24-365 days. Oxygen consumption together with the rates of gluconeogenesis, urea synthesis and ketogenesis were determined. Basal mitochondrial respiration as well as the ability of the liver to synthesize glucose, urea and ketone bodies declined from 24- to 365-day-old rats. On the other hand, on transition from 24 to 60 days the liver oxidation rate of hexanoate, sorbitol and glycerol is enhanced, but not of ketone bodies or palmitate. Our results show that the transition from weaning to middle age is accompanied by defined changes in hepatic substrate oxidation. From the observed time course of the decrease in basal and substrate-stimulated oxygen consumption, it is concluded that in rat liver cells a decline in respiratory chain function, long-chain fatty acid and ketone body metabolism, gluconeogenesis and ureogenesis occurs at a relatively early life stage.

Effect of constituents of burned skin and in vivo skin burning on the respiratory activity of rat liver mitochondria

The formation of toxic substances in the skin, due to the influence of thermal energy, is supposed to be one possible mechanism responsible for the high mortality rate after severe burns covering large areas of the body surface. The effects of low-molecular-weight volatile substances (dry distillates) generated after burning mouse skin were investigated. They led to reductions of the respiratory activity of rat liver mitochondria. Among other substances carbon disulphide, methylethylketone and pyrrole were isolated and characterized using gas chromatography. All of them had strong uncoupling effects on mitochondrial respiration. In a second study respiratory measurements were made on mitochondria from rats with 20–30 per cent TBSA full skin thickness burns. A decreased respiratory control rdtio and thus uncoupling of mitochondrial respiration was found on days 1, 2 and 5 postburn. The basal mitochondrial respiration was significantly decreased on day 5, which could be important for the mortality mentioned above. Finally after cross-transplantations rats having burned skin grafted showed a reduction of the RCR when compared with rats with the unburned graft. Thus it can be concluded that toxic substances must have been present in burned skin.