Cellular Respiration

The physiological mechanisms limiting and adjusting cold and heat tolerance have regained interest in the light of global warming and associated shifts in the geographical distribution of ectothermic animals. Recent comparative studies, largely carried out on marine ectotherms, indicate that the processes and limits of thermal tolerance are linked with the adjustment of aerobic scope and capacity of the whole animal as a crucial step in thermal adaptation on top of parallel adjustments at the molecular or membrane level. In accordance with Shelford's law of tolerance decreasing whole animal aerobic scope characterises the onset of thermal limitation at low and high pejus thresholds (pejussgetting worse). The aerobic scope of an animal indicated by falling oxygen levels in the body fluids and or the progressively limited capacity of circulatory and ventilatory mechanisms. At high temperatures, excessive oxygen demand causes insufficient oxygen levels in the body fluids, whereas at low temperatures the aerobic capacity of mitochondria may become limiting for ventilation and circulation. Further cooling or warming beyond these limits leads to low or high critical threshold temperatures (T ) where aerobic scope disappears and transition to an anaerobic mode of mitochondrial metabolism and c progressive insufficiency of cellular energy levels occurs. The adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows. The finding of an oxygen limited thermal tolerance owing to loss of aerobic scope is in line with Taylor's and Weibel's concept of symmorphosis, which implies that excess capacity of any component of the oxygen delivery system is avoided. The present study suggests that the capacity of oxygen delivery is set to a level just sufficient to meet maximum oxygen demand between the average highs and lows of environmental temperatures. At more extreme temperatures only time limited passive survival is supported by anaerobic metabolism or the protection of molecular functions by heat shock proteins and antioxidative defence. As a corollary, the first line of thermal sensitivity is due to capacity limitations at a high level of organisational complexity, i.e. the integrated function of the oxygen delivery system, before individual, molecular or membrane functions become disturbed. These interpretations are in line with the more general consideration that, as a result of the high level of complexity of metazoan organisms compared with simple eukaryotes and then prokaryotes, thermal tolerance is reduced in metazoans. A similar sequence of sensitivities prevails within the metazoan organism, with the highest sensitivity at the organismic level and wider tolerance windows at lower levels of complexity. However, the situation is different in that loss in aerobic scope and progressive hypoxia at the organismic level define the onset of thermal limitation which then transfers to lower hierarchical levels and causes cellular and molecular disturbances. Oxygen limitation contributes to oxidative stress and, finally, denaturation or malfunction of molecular repair, e.g. during suspension of protein synthesis. The sequence of thermal tolerance limits turns into a hierarchy, ranging from systemic to cellular to molecular levels.

El estudiante identificará las biomoléculas, las rutas metabólicas y sus interacciones con los seres vivos, para comprender su relación con el medio ambiente. Competencia a la que contribuye la asignatura Evaluar la calidad de sistemas ambientales mediante la realización de muestreos y análisis físico-químicos y microbiológicos conforme a protocolos establecidos, para la verificación del cumplimiento del marco jurídico que aplique a las organizaciones.

Background-Organ failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver. Methods-C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid betaoxidation and anaplerotic contributions from amino acids. Conclusions-Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic

Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonemia occurs in advanced hepatic, cardiac, pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced co...

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The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

Isolated mitochondria of the bivalve Solemya reidi Bernard oxidize sulphide and couple this oxidation to ADP phosphorylation. The products of mitochondrial sulphide oxidation were analyzed by HPLC using monobromobimane derivatization. Concurrent measurements of respiration were made using sulphideinsensitive oxygen electrodes. 5. reidi mitochondria oxidized sulphide exclusively to thiosulphate. The reaction occurred in two steps. One sulphide molecule was first oxidized to sulphite. A second molecule of sulphide was then added oxidatively to form the free product thiosulphate. This oxidation was obligately linked to mitochondrial electron transport and could be inhibited by the cytochrome c oxidase inhibitor hydrogen cyanide, or by low oxygen concentration. The site II inhibitor antimycin A did not inhibit thiosulphate production, indicating that sulphide oxidation is linked through only one ATP coupling site (site III). A calculation of the respiratory potential for ATP synthesis b...

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.

Significance Bacterial aerobic respiration is a metabolic pathway that can adapt to changing environmental conditions. Most bacteria encode for two general types of terminal respiratory oxygen reductases that catalyze the reduction of molecular oxygen to water: heme-copper oxidases (HCO) and cytochrome bd- type oxidases. While HCO-type enzymes are conserved throughout all domains of life, cytochrome bd oxidases are present solely in prokaryotes. Due to their high oxygen affinity and insensitivity toward a wide range of naturally occurring HCO inhibitors, cytochrome bd oxidases confer bacteria with the ability to maintain aerobic respiration under hostile conditions, especially low-oxygen environments. The importance of cytochrome bd as a survival factor supports a key role for this enzyme as a drug target for combating infectious diseases.

In a previous study, Hatcher (1989: Mar. Biol. 102: 445-452) found that variations in COz and 02 respiration rates in individual marine invertebrates led to RQ (respiratory quotient) values which were variable and often outside theoretical limits. The present study was designed to examine the variability in several excretionbased metabolic ratios which are often used as alternatives for the RQ in qualitative predictions of catabolic substrates. The experimental organism was a solitary ascidian, Herdmania momus (Savigny), collected near Perth, Western Australia, between January and July 1984. Respiration and excretion rates of H. momus were examined as a function of a progressive nutritional stress, and covariation was examined. Nutritional stress accounted for more of the variation over time in respiration rates (40 to 50%) than in NH + excretion rates (20%). Significant net exchanges of dissolved organic compounds were measured. Qualitative predictions of catabolic substrates were based on a comparison of metabolic ratios with theoretical limits. The O:PO, 3-ratios were lower than the theoretical limits. The values of the O : NH + and NH + : PO~-ratios were not influenced by nutritional conditions but changed as a function of reproductive condition of the ascidians. Based on the results of this study, it was concluded that metabolic ratios measured on individual ascidians cannot be reliable predictors of catabolic substrates.

The Warburg effect, commonly depicted as an inherent metabolic trait of cancer in literature, is under intensive investigation to comprehend its origins. However, while the prolonged presence of excessive lactic acid production in tumors has been noted, it merely constitutes a fraction of the potential metabolic states cancer cells can adopt. This study aimed to elucidate the emergence of spatiotemporal diversity in tumor energy metabolism by expanding an existing model based on experimental facts. The resulting hybrid model integrates discrete formulations for individual cells and their processes, along with continuous elements for metabolism and the diffusion of crucial environmental substrates like oxygen, glucose, lactate, and the often underestimated acidity. This model enables simulation of a tumor spheroid, a standard experimental model, composed of numerous cells which can have distinct traits. By subjecting the spheroid to alterations of the environment such as cyclic hypox...

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser-(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r 2 ϭ 0.39, P Ͻ .001) and 532 nm, (r 2 ϭ 0.18, P ϭ .003); reflecting laser/fluorescence-induced, 442 nm (r 2 ϭ 0.20, P ϭ .002) and 532 nm (r 2 ϭ 0.004, P ϭ .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r 2 ϭ 0.43, P ϭ .0004), state 4 respiration (r 2 ϭ 0.03, P ϭ 0.38), RCR (r 2 ϭ 0.28, P ϭ .007), and membrane swelling (r 2 ϭ 0.02, P ϭ .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.

Previous studies have shown that increased oxygen delivery, via increased convection or arterial oxygen content, does not speed the dynamics of oxygen uptake, V̇o2m, in dog muscle electrically stimulated at a submaximal metabolic rate. However, the dynamics of transport and metabolic processes that occur within working muscle in situ is typically unavailable in this experimental setting. To investigate factors affecting V̇o2m dynamics at contraction onset, we combined dynamic experimental data across working muscle with a mechanistic model of oxygen transport and metabolism in muscle. The model is based on dynamic mass balances for O2, ATP, and PCr. Model equations account for changes in cellular ATPase, oxidative phosphorylation, and creatine kinase fluxes in skeletal muscle during exercise, and cellular respiration depends on [ADP] and [O2]. Model simulations were conducted at different levels of arterial oxygen content and blood flow to quantify the effects of convection and diff...

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.

Lactate, the redox-balanced end product of glycolysis, travels within and between cells to fulfill an array of physiologic functions. While evidence for the centrality of this lactate shuttling in mammalian metabolism continues to mount, its application to physical bioenergetics remains underexplored. Lactate represents a metabolic “cul-de-sac,” as it can only re-enter metabolism by first being converted back to pyruvate by lactate dehydrogenase (LDH). Given the differential distribution of lactate producing/consuming tissues during metabolic stresses (e.g., exercise), we hypothesize that lactate shuttling vis-à-vis the exchange of extracellular lactate between tissues serves a thermoregulatory function, i.e., an allostatic strategy to mitigate the consequences of elevated metabolic heat. To explore this idea, the rates of heat and respiratory oxygen consumption in saponin-permeabilized rat cortical brain samples fed lactate or pyruvate were measured. Heat and respiratory oxygen con...

SUMMARYCertain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation fromAngomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affectA. deaneirespiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2consumption. In theA. deaneigen...

Significance Bacterial aerobic respiration is a metabolic pathway that can adapt to changing environmental conditions. Most bacteria encode for two general types of terminal respiratory oxygen reductases that catalyze the reduction of molecular oxygen to water: heme-copper oxidases (HCO) and cytochrome bd- type oxidases. While HCO-type enzymes are conserved throughout all domains of life, cytochrome bd oxidases are present solely in prokaryotes. Due to their high oxygen affinity and insensitivity toward a wide range of naturally occurring HCO inhibitors, cytochrome bd oxidases confer bacteria with the ability to maintain aerobic respiration under hostile conditions, especially low-oxygen environments. The importance of cytochrome bd as a survival factor supports a key role for this enzyme as a drug target for combating infectious diseases.

To identify the most temperature-sensitive steps in the energy production pathways, we measured the thermal sensitivity of mitochondrial oxidative phosphorylation (OXPHOS), as well as that of the individual steps in this process in rat heart mitochondria. OXPHOS measured in the presence of pyruvate+ malate as substrates have an unusually high thermal sensitivity between 5 and 15 1C. Furthermore, the thermal sensitivity of OXPHOS correlates with the thermal sensitivity of pyruvate dehydrogenase between 5 and 35 1C. Pyruvate dehydrogenase is a potential control point for pyruvatesupported mitochondrial respiration below physiological temperature in rat heart.

Recently, marked oxygen dependence of respiration by isolated mitochondria after exposure to prolonged hypoxia has been described. Because mitochondrial oxygen-dependent respiration could significantly influence oxygen consumption during critical illness, we sought to confirm the oxygen-dependent behavior of mitochondria. We hypothesized that mitochondria isolated during sepsis would exhibit increased oxygen dependence. We isolated rat liver mitochondria 16 h after cecal ligation and puncture and found a 30–40% greater oxygen uptake compared with control rats under state 3 conditions. Mitochondria incubated in deoxygenated buffer were studied for oxygen dependence at 10-min intervals for 90 min. Mitochondrial respiration after reoxygenation was stable over a 60-min period of hypoxia for control rats and decreased slightly for septic rats (10–15%). State 3 respiration was 10% lower when mitochondria were reoxygenated at low (15–25 Torr) versus high (90–100 Torr) and low (10–15 Torr) ...

In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolites and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism ± NAD + or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K 0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K 0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel. (

In nonphotosynthetic organisms, mitochondria are the power plant of the cell, emphasizing their great potentiality for adenosine triphosphate (ATP) synthesis from the redox span between nutrients and oxygen. Also of great importance is their role in the maintenance of the cell redox balance. Even though crystallographic structures of respiratory complexes, ATP synthase, and ATP/adenosine diphosphate (ADP) carrier are now quite well known, the coupling between ATP synthesis and cell redox state remains a controversial issue. In this review, we will present some of the processes that allow a modular coupling between ATP synthesis and redox state. Furthermore, we will present some theoretical approaches of this highly integrated system.

Background. We have previously shown that chronic metabolic acidosis, induced in rats by NH 4 Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys. Since mitochondria are the main source of metabolic energy that drives ion transport in kidney tubules, we examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) in mitochondria isolated from rat kidney and liver at 48 h after metabolic acidosis induced by NH 4 Cl. Methods. Mitochondria isolated from the kidneys and liver of metabolic acidotic rats, induced by NH 4 Cl, was used to study of the oxygen consumption by Clarktype electrode, mitochondrial electrical transmembrane potential estimated by the safranine O method and the variations in free medium Ca 2+ concentrations examined by absorbance spectrum of Arsenazo III set at the 675-685 nm wavelength pair. Results. Whole kidney and liver mitochondria isolated from 48 h acidotic rats presented higher resting respiration, lower respiratory control and a lower ADP/O ratio than controls. These differences in mitochondrial coupling, between respiration and oxidative phosphorylation (ATP synthesis), were totally corrected when experiments were carried out in the presence of carboxyatractyloside, GDP and BSA, indicating that mitochondrial uncoupling proteins are more active in acidotic rat kidneys. Interestingly, determination of Ca 2þ transport demonstrated a faster rate of initial Ca 2þ uptake by acidotic kidney mitochondria, which resulted in a lower concentration of extra-mitochondrial Ca 2þ under steady-state conditions (Ca 2þ set point) when compared with control mitochondria. In contrast, there were no significant differences in the rates of Na þ or ruthenium red induced Ca 2þ efflux. Conclusions. We suggest that the mild uncoupling and higher Ca 2þ accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca 2þ , which are associated with a decreased efficiency of oxidative phosphorylation that may explain, at least in part, the striking natriuresis observed under chronic acidosis. Finally, there were no changes in Ca 2þ transport or coupling in liver mitochondria isolated from the acidotic rats.

To gain insight into the regulation of pancreatic β-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic β-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that β-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. α-Glycerophosphate (α-GP) oxidation was Ca2+-dependent with a half-maximal rate ...

The present study investigates energy metabolism by trout red blood cells. It is shown that they are able to use pyruvate at physiological concentrations as an aerobic source of energy. Moreover, microcalorimetric data suggest that trout erythrocytes are also able to use internal substrates, at least when maintained in vitro. Although the actual nature of these substrates has not been elucidated, glycogen appears to be the most probable. The relationship between heat dissipation and oxygen consumption suggests that most of the oxygen is used to oxidize substrates, and the inhibition of respiratory activity by antimycin A indicates that there is no substantial utilisation of non-respiratory oxygen. However, the oxygen uptake by these cells does not appear to be related to substrate utilisation, measured from transformation of labelled molecules (either pyruvate or glucose); this may be due to mixing of labelled compounds with non-labelled molecules in the intracellular pools, because...

Studies of metabolic regulation in yeasts have a long history. Yeasts have served as models for the regulation of fermentaive metabolism. Yeasts differ in their partitioning of metabolism between respiration and fermentation, that is, between their use of oxygen and organic compounds as terminal electron acceptors. The respirative route is assumed to yield more ATP. Many yeasts carry out metabolism solely by the respirative route. Other show a predominantly fermentaive metabolism, even when oxygen is freely available. Fermentaion under aeobic conditions, that is, when respiration should be possible, was believed to result from repression of respiration by fermentation. This phenomenon is known as the Crabtree effect. However, recent work has shown that aerobic fermentation results from an inherently-limited respiratory capacity of some yeasts, rather than from a specific repression of respiration, Nevertheless, considerable enzymatic evidence exists that suggests that a Crabtree effect does indeed operate in some yeasts, specifically Saccharomyces cerevisiae. Because multiple electron transport systems are known to exist in yeasts, repression of the normal ATP-producing system can be accompanied by the induction of an alternate pathway. No decrease in the overall rate of oxygen utilization would then be apparent. Repression would, however, affect the yield of ATP from oxidative metabolism. This effect should be detectable using a suitable analysis of growth energetics. To this end, a model has been developed and applied to a variety of yeasts in order to examine them for changes in respiratory efficiency indicative of a Crabtree effect. A Crabtree effect consistent with previous enzmatic findings was detected in S cerevisiae and S. uvarum, but not in Schizosaccharomyces prombe. New regulatory classifications based on model findings are proposed and methods for independently verifying these findings are outlined.

EM 49 (recently renamed octapeptin) is a membrane-active peptide antibiotic that has been reported to affect the structure of bacterial membranes (K. S. Rosenthal, P. E. Swanson, and D. R. Storm, Biochemistry 15: 5783–5792, 1976). In this study, it is shown that the effects of EM 49 on bacterial metabolism are similar to those of uncouplers of oxidative phosphorylation. EM 49 stimulated bacterial respiration within a narrow concentration range corresponding to minimum inhibitory concentrations and inhibited respiration at concentrations comparable to minimum biocidal concentrations. In addition, the peptide increased membrane proton permeability and lowered the adenosine 5′-triphosphate pool size. Parallel studies done with the related antibiotic polymyxin B demonstrated that the two peptides differed considerably in their effects on bacterial respiration. In contrast to EM 49, polymyxin B did not stimulate respiration at any concentration. It is proposed that the primary action of ...

Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by pier...

The energetic parameters of Escherichia coli were analyzed for the aerobic/anaerobic transition. The electrochemical proton potential (Δ p) across the cytoplasmic membrane was determined in the steady state of respiration with O2, nitrate, fumarate, dimethylsulfoxide (Me2SO), and for fermentation. With O2, a proton potential of −160 mV was obtained. For anaerobic respiration with nitrate, fumarate or Me2SO, Δ p decreased only slightly by about 20 mV in contrast to earlier assumptions, whereas Δ p dropped by approximately 40 mV during fermentation. Under all conditions, the membrane potential (Δ @KY) contributed the major portion to Δ p. The cellular ATP levels were highest for aerobic growth (about 13 μmol/g dry cells) and decreased to 3−6 μmol/g in anaerobic metabolism. Δ .cf2.G.cf2..cf1..es.cf1.′.cf5..rb.eiPhos.rb, however, was constant due to equivalent changes of the ADP contents. Transition to the stationary growth phase caused a massive drop in the ATP content. It is concluded...

Objective: To compare the effect of shifting anaerobic to aerobic metabolism on key regulators of oxidative stress, including extracellular superoxide dismutase (SOD3), inducible nitric oxide synthase (iNOS), and its product, nitric oxide (NO), as well as mitochondrial potential (Djm) and apoptosis in fibroblasts established from normal peritoneum and adhesion tissues. Design: Prospective, experimental study. Setting: University medical center. Patient(s): Fibroblasts established from normal peritoneum and adhesion tissues from the same patients. Intervention(s): Treatment with dichloroacetate (0, 20, 40, and 80 mg/mL, 24 hours). Main Outcome Measure(s): The expression of SOD3, iNOS, and NO levels were measured utilizing real-time reverse transcriptionpolymerase chain reaction and Greiss assay. The Djm was evaluated by the JC-1 Mitochondrial Membrane Potential Assay. Apoptosis was determined by caspase-3 activity and TUNEL assays. Data were analyzed using SPSS 19.0. Mixed model repeatedmeasures analysis of variance was used with a Bonferroni correction. Significant interactions were analyzed with independent sample t tests. Result(s): Dichloroacetate increased apoptosis, SOD3 messenger RNA, iNOS messenger RNA, and NO levels in fibroblasts from peritoneum and adhesions. There was enhanced Djm adhesion as compared with normal peritoneal fibroblasts. Creating oxidative stress by exposure by hypoxia markedly increased Djm in fibroblasts from normal peritoneum to levels observed in adhesions; dichloroacetate protected against the effects of hypoxia. Conclusion(s): Anaerobic metabolism and oxidative stress are associated with the development of the adhesion phenotype, which manifests decreased apoptosis. Dichloroacetate induces adhesion fibroblasts to undergo apoptosis via modulation of redox homeostasis. These findings may provide targets for therapeutic treatment for reduction of profibrotic disorders, including postoperative adhesions. (Fertil Steril Ò 2015;104:1022-9. Ó2015 by American Society for Reproductive Medicine.

The principal objective of the present study was to identify specific alterations in mitochondrial respiratory functions during the aging process. Respiration rates and the activities of electron transport chain complexes were measured at various ages in mitochondria isolated from thoraces of the fruit fly, Drosophila melanogaster, which consist primarily of flight muscles. The rates of state 3 respiration (ADP-stimulated), RCRs (respiratory control ratios) and uncoupled respiration rates decreased significantly as a function of age, using either NAD+- or FAD-linked substrates; however, there were no differences in state 4 respiration (ADP-depleted) rates. There was also a significant age-related decline in the activity of cytochrome c oxidase (complex IV), but not of the other mitochondrial oxidoreductases examined. Exposure of mitochondria isolated from young flies to low doses of KCN or NaAz (sodium azide), complex IV inhibitors, decreased cytochrome c oxidase activity and increa...

The "Warburg effect" refers to the situation wherein cellular energetics (ATP formation) use "aerobic glycolysis" (i.e., glucose use with the release of lactate (2 ATP per glucose)) even if oxygen present would authorize full oxidation with a much higher yield (34 ATP per glucose). The present article reviews possible reasons to explain this metabolic bias.

Endocrine functions of the gut are supported by a scattered population of cells, the enteroendocrine cells (EECs). EECs sense their environment to secrete hormones in a regulated manner. Distal EECs are in contact with various microbial compounds including hydrogen sulfide (H 2 S) which modulate cell respiration with potential consequences on EEC physiology. However, the effect of H 2 S on gut hormone secretion remains discussed and the importance of the modulation of cell metabolism on EEC functions remains to be deciphered. The aim of this project was to characterize the metabolic response of EECs to H 2 S and the consequences on GLP-1 secretion. We used cell line models of EECs to assess their capacity to metabolize H 2 S at low concentration and the associated modulation of cell respiration. We confirmed that like what is observed in colonocytes, colonic EEC model, NCI-h716 cell line rapidly metabolizes H 2 S at low concentrations, resulting in transient increased respiration. Higher concentrations of H 2 S inhibited this respiration, with the concentration threshold for inhibition depending on cell density. However, increased or inhibited oxidative respiration had little effect on acute GLP-1 secretion. Overall, we present here a first study showing the EEC capacity to detoxify low concentrations of H 2 S and used this model to acutely address the importance of cell respiration on secretory activity.

Understanding the strategies employed by plant species that live in extreme environments offers the possibility to discover stress tolerance mechanisms. We studied the physiological, antioxidant and metabolic responses to three temperature conditions (4, 15, and 23°C) of Colobanthus quitensis (CQ), one of the only two native vascular species in Antarctica. We also employed Dianthus chinensis (DC), to assess the effects of the treatments in a non-Antarctic species from the same family. Using fused LASSO modelling, we associated physiological and biochemical antioxidant responses with primary metabolism. This approach allowed us to highlight the metabolic pathways driving the response specific to CQ. Low temperature imposed dramatic reductions in photosynthesis (up to 88%) but not in respiration (sustaining rates of 3.0-4.2 lmol CO 2 m À2 s À1 ) in CQ, and no change in the physiological stress parameters was found. Its notable antioxidant capacity and mitochondrial cytochrome respiratory activity (20 and two times higher than DC, respectively), which ensure ATP production even at low temperature, was significantly associated with sulphur-containing metabolites and polyamines. Our findings potentially open new biotechnological opportunities regarding the role of antioxidant compounds and respiratory mechanisms associated with sulphur metabolism in stress tolerance strategies to low temperature. Antarctica is probably the most extreme continent for supporting life: it is the coldest, driest, and windiest continent on Earth. Climatic conditions are so hostile that, even during summer, plants frequently face subzero temperatures, strong winds, and very long photoperiods. Moreover, water availability is restricted to ice and glacier melting. In combination with strong dry winds (that impose elevated evaporation rates), this make life extremely

Although reactive oxygen species (ROS) are highly toxic substances that are produced during aerobic respiration and photosynthesis, many studies have demonstrated that ROS, such as superoxide anion radical (O2(-)) and hydrogen peroxide (H2O2), are produced in the plant cell wall in a highly regulated manner. These molecules are important signalling messengers playing key roles in controlling a broad range of physiological processes, such as cellular growth and development, as well as adaptation to environmental changes. Given the toxicity of ROS, especially of hydroxyl radical (OH), the enzymatic ROS production needs to be tightly regulated both spatially and temporally. Respiratory burst oxidase homologues (Rboh) have been identified as ROS-producing NADPH oxidases, which act as key signalling nodes integrating multiple signal transduction pathways in plants. Also other enzyme systems, such as class III peroxidases, amine oxidases, quinone reductases and oxalate oxidases contribute...

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O 2 consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O 2 consumption. As a first evaluation of the technique, we compared whole slice bioenergetics to previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O 2 consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O 2 consumption that was well-coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared to previous measurements in cells or synaptosomes, although enhanced slightly (to ~150% of basal respiration) by the acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O 2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to efficiently measure O 2 consumption from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

Nitric oxide (NO) plays a key role in many physiological and pathophysiological events, including the control of cell respiration. Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of NO by cells. We have exposed the murine macrophage cell line J774 to high concentrations of NO, such as are generated in some pathological conditions, and determined their effect on oxygen consumption. We observed a persistent inhibition of respiration which was due to a redox-dependent, progressive inhibition of complex I activity. No other enzyme of the respiratory chain was inhibited in this way. At the same time, we detected a paradoxical removal of oxygen by the extracellular medium. This removal

Bacteria capahle of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Comparative spectroscopic analyses revealed that phylogenetically distinct organisms expressed copious quantities of spectrally distinct redox-active biomolecules during autotrophic growth on soluble iron. Thiobaeillus J~,rmoaidans, L~Tm~spirillum [~'rrooxidans, Su![~bacil[us thennosu!f)'doo.tidans, and Metallosphaera sedula possessed iron respiratory chains dominated by a blue copper protein, a novel red cytochrome, a novel yellow protein, and a novel yellow eytochrome, respectively. Further investigation of each type of respiratory chain will be necessary to deduce the advantages and disadvantages of each.

Out of all the complex systems in science education curricula, cellular respiration is considered to be one of the most complex and abstract processes. Students are known to have low interest and difficulties in conceptual understanding of cellular respiration which provides a challenge for teaching and learning. In this study, we took literature about modelling and teaching and learning cellular respiration as a starting point for the design of a concrete dynamic model in which students (n = 126) use Lego® to simulate the process of cellular respiration. Students used the simulation embedded in the context of finding the efficiency of a sediment battery as a future source of green energy and we tested the effects on conceptual learning and situational interest in an experimental study. Results on conceptual learning show that both experimental and control groups had comparable results in the test. The questions that students in the experimental group asked during enactment, however...

Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respira...

Background: Mitochondria are both the cellular powerhouse and the major source of reactive oxygen species. Coenzyme Q 10 plays a key role in mitochondrial energy production and is recognized as a powerful antioxidant. For these reasons it can be argued that higher mitochondrial ubiquinone levels may enhance the energy state and protect from oxidative stress. Despite the large number of clinical studies on the effect of CoQ 10 supplementation, there are very few experimental data about the mitochondrial ubiquinone content and the cellular bioenergetic state after supplementation. Controversial clinical and in vitro results are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability.

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20Z R were investigated. We found that the bhr knockout (20Z R bhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20Z R bhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery.

Coke strength is mainly determined by pores and cracks that cause fracture of coke. In this study, connected pores that were considered to cause fracture of coke were investigated. In order to evaluate connected pores quantitatively, low roundness pores (whose roundness were below 0.2) were measured by image analysis technique of microscopic photographs of cokes. The relationship between the amount of low roundness pores and coke strength (DI 150 6) showed a good correlation. It is thought that the amount of low roundness pores is one of the factors determining coke strength.

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca 2+ -dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial bioenergetics have mainly been performed in isolated PACs using confocal microscopy, with assessment of mitochondrial membrane potential, NADH/FAD + and ATP levels, coupled with patch-clamp electrophysiology. These studies are technically demanding and time-consuming. Application of Seahorse flux analysis now allows detailed investigations of bioenergetics changes to be performed in cell populations using a multi-well platereader format; rates of oxygen consumption (OCR) and extracellular acidification (ECAR) provide important information about cellular respiration and glycolysis, respectively. Parameters such as maximal respiration, ATP-linked capacity and proton leak can be derived from application of a respiratory function "stress" test that involves pharmacological manipulation of the electron transport chain. The use of Seahorse Flux analysis therefore provides a quick, and convenient means to measure detailed cellular bioenergetics and allows results to be coupled with other plate-reader based assays, providing a fuller understanding of the pathophysiological consequences of mitochondrial bioenergetics alterations.