Reversible inhibition of mitochondrial respiratory control by tetrabutylammonium bromide.

Influences of tetrabutylammonium bromide as a phosphorylation inhibitor and as an uncoupler of oxidative phosphorylation in rat liver mitochondria were reversed by washing the organelles. Uncoupling by 2,4-dinitrophenol was also reversible whereas gramicidin and the detergents, sodium dodecylsulfate and cetyltrimethylammonium bromide, were tightly bound uncouplers and they were not substantially removed by simple washing.     

Comparative effects of three 1,4-dihydropyridine derivatives [OSI-1210, OSI-1211 (etaftoron), and OSI-3802] on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers: relevance to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring

The 1,4-dihydropyridines OSI-1210, OSI-1211 (etaftoron), and OSI-3802 are compounds with similar chemical structure. They differ by the length of the alkoxyl chain in positions 3 and 5 of the dihydropyridine (DHP) ring and by their pharmacological action characteristics. However, as far as we know, a clear relationship between the effects of these compounds and the length of the alkoxyl chain in positions 3 and 5 of the DHP has not been established. The goal of this study was to compare the influence of OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers, correlating their actions with the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. Using either glutamate/malate or succinate as respiratory substrates, all the compounds, in concentrations of up to 500 microM, depressed state 3 and uncoupled respiration, respiratory control (RCR) and ADP/O ratios, and phosphorylation rate, whereas state 4 respiration was stimulated. However, the stimulatory effect on state 4 induced by OSI-3802, the compound with the longest chain in positions 3 and 5 of the DHP ring, as well as its inhibitory effects on RCR and ADP/O ratios and phosphorylation rate were more pronounced than that induced by OSI-1210 and OSI-1211 (etaftoron), the compounds with the shortest and intermediate chains, respectively. Moreover, OSI-3802 maximized state 4 stimulation and minimized RCR and ADP/O ratios, and phosphorylation rate at a concentration of 100 microM, whereas low graduate effects were detected with OSI-1210 and OSI-1211 (etaftoron) for concentrations of up to 500 microM. At low concentrations (< or =30 microM), OSI-3802, like its analogue OSI-1212 (cerebrocrast), reduced the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of dimyristoylphosphatidylcholine (DMPC) membrane bilayers. A good correlation was established between the effects of 200 microM OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on glutamate/malate- and succinate-dependent RCR of rat liver mitochondria and on the enthalpy change (Delta H) for the thermotropic profile of DMPC membrane bilayers at a 0.2 drug/DMPC molar ratio, indicating that the changes induced by these compounds on both mitochondrial membrane integrity and physical properties of DMPC membrane bilayers are strongly related to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. A putative relationship between membrane physical perturbation, bioenergetics impairment and the molecular characteristics of the compounds will be established as an approach to better understand their differentiated toxicological and pharmacological actions.     

Comparison of dietary mercury exposure in two sympatric top predator fishes, largemouth bass and northern pike: a bioenergetics modeling approach

Physical and ecologicalfactors, including lake temperature, fishphysiology, and diet, influence methylmercury(MeHg) exposure in fish. We employedbioenergetics modeling to compare dietary MeHgexposure in sympatric top predators, largemouthbass (Micropterus salmoides) and northernpike (Esox lucius). We comparedsimulations using field data to hypotheticalsimulations with (1) ± 25% change in meandaily lake temperature for juvenile and adultbass and pike; (2) ± 25% change inlong-term growth rate of pike; (3) adult bassdiet shift from generalist predator to strictpiscivore. Bass and pike MeHg exposures weresimilar in baseline simulations and reflectedpatterns in field tissue concentrations. Thisoccurred despite the fact that bass consumedhighly contaminated benthic invertebrates,while pike exclusively consumed lesscontaminated fish prey. Higher temperaturesincreased adult bass and pike MeHg exposures by35% and 27%, respectively. Shifting adultbass diets to 100% fish resulted in a 54%decrease in exposure, while increasing pikegrowth rates resulted in a 24% decrease. Bioenergetics modeling proved useful inunderstanding the influence of temperature,prey-base, and predator growth on differencesin Hg exposure across fish species.     

An application of a bioenergetics model to Eurasian perch (Perca fluviatilis L.)

A bioenergetics model was developed for Eurasian perch ( Perca fluviatilis L.) by revising an existing model for yellow perch and walleye. Data were gathered from field studies and the literature. Besides adjusting the original parameters of the model, effects of season on consumption and metabolic rates were added. The predictive capability of the revised model was high both concerning the levels of growth and its seasonal development in the Baltic coastal waters to which the model was applied. Perch young-of-the-year attained almost maximum consumption and growth except in the highest temperatures experienced. In larger fish, the model estimated consumption to be about 50 per cent of the maximum possible rate.     

Laboratory evaluation of two bioenergetics models applied to yellow perch: identification of a major source of systematic error

Laboratory growth and food consumption data for two size classes of age 2 year yellow perch Perca flavescens , each fed on two distinct feeding schedules at 21° C, were used to evaluate the abilities of the Wisconsin (WI) and Karas–Thoresson (KT) bioenergetics models to predict fish growth and cumulative consumption. Neither model exhibited consistently better performance for predicting fish body masses across all four fish size and feeding regime combinations. Results indicated deficiencies in estimates of resting routine metabolism by both models. Both the WI and KT models exhibited errors for predicting growth rates, which were strongly correlated with food consumption rate. Consumption-dependent prediction errors may be common in bioenergetics models and are probably the result of deficiencies in parameter values or assumptions within the models for calculating energy costs of specific dynamic action, feeding activity metabolism or egestion and excretion. Inter-model differences in growth and consumption predictions were primarily the result of differences in egestion and excretion costs calculated by the two models. The results highlighted the potential importance of parameters describing egestion and excretion costs to the accuracy of bioenergetics model predictions, even though bioenergetics models are generally regarded as being insensitive to these parameters. The findings strongly emphasize the utility and necessity of performing laboratory evaluations of all bioenergetics models for assurance of model accuracy and for facilitation of model refinement.     

Development of a bioenergetics model for fish embryos and larvae during the yolk feeding period

A mechanistic model of an energy budget in fish embryos and yolk-sac larvae was developed using data for five freshwater fish species: rainbow trout Oncorhynchus mykiss , nase Chondrostoma nasus , carp Cyprinus carpio , tench Tinca tinca and African catfish Clarias gariepinus , based on the existing models for adult and juvenile fishes. The model simulates changes in the components of the budget under various conditions. Besides the effects of body mass and temperature on consumption and metabolic rate, the dependence of ration size on amount of available yolk and initial egg size was implemented in the model. The model parameters were found through optimization. A sensitivity analysis of the model was conducted by varying its parameters and observing changes in the output. A comparative analysis showed that the values generated by the model closely approximated independent empirical observations. Simulations of energy budgets demonstrated that the overall pattern of energy partitioning was the same for different species, irrespective of egg size and temperature preferences. Most energy was allocated to body growth. Clarias gariepinus showed the fastest growth and had the highest yolk conversion efficiency.     

To err and win a nobel prize: Paul Boyer,ATP synthase and the emergence of bioenergetics

Paul Boyer shared a Nobel Prize in1997 for his work on the mechanism of ATPsynthase. His earlier work, though (whichcontributed indirectly to his triumph),included major errors, both experimental andtheoretical. Two benchmark cases offer insightinto how scientists err and how they deal witherror. Boyer's work also parallels andillustrates the emergence of bioenergetics inthe second half of the twentieth century,rivaling achievements in evolution andmolecular biology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Juvenile chum salmon consumption of zooplankton in marine waters of southeastern Alaska: a bioenergetics approach to implications of hatchery stock interactions

Bioenergetics modeling was used to estimate zooplankton prey consumption of hatchery and unmarked stocks of juvenile chum salmon (Oncorhynchus keta) migrating seaward in littoral (nearshore) and neritic (epipelagic offshore) marine habitats of southeastern Alaska. A series of model runs were completed using biophysical data collected in Icy Strait, a regional salmon migration corridor, in May, June, July, August, and September of 2001. These data included a temperature (1-m surface versus surface to 20-m average), zooplankton standing crop (surface to 20-m depth versus entire water column), chum salmon diet (percent weight of prey type consumed), energy densities, and weight. Known numbers of hatchery releases were used in a cohort reconstruction model to estimate total abundance of hatchery and wild chum salmon in the northern region of southeastern Alaska, given average survival to adults and for two different (low and high) early marine littoral mortality rate assumptions. Total prey consumption was relatively insensitive to temperature differences associated with the depths potentially utilized by juvenile chum salmon. However, the magnitudes and temporal patterns of total prey consumed differed dramatically between the low and high mortality rate assumptions. Daily consumption rates from the bioenergetics model and CPUE abundance from sampling in Icy Strait were used to estimate amount and percentage of zooplankton standing crop consumed by mixed stocks of chum salmon. We estimated that only a small percentage of the available zooplankton was consumed by juvenile chum salmon, even during peak abundances of marked hatchery and unmarked mixed stocks in July. Total daily consumption of zooplankton by all stock groups of juvenile chum salmon was estimated to be between 330 and 1764 g/km²d¹ from June to September in the neritic habitat of Icy Strait. As with any modeling exercise, model outputs can be misleading if input parameters and underlying assumptions are not valid; therefore, additional studies are warranted, especially to determine physiological input parameters, and to improve abundance and mortality estimates specific to juvenile chum salmon. Future bioenergetics modeling is also needed to evaluate consumption by the highly abundant, vertically migrating planktivorous that co-occurred in our study; we suggest that these fishes have a greater impact on the zooplankton standing crop in Icy Strait than do hatchery stock groups of juvenile chum salmon.     

Molecular Wiring of a Mitochondrial Translational Feedback Loop

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Muscle ATP synthesis and utilisation,balanced during flow-induced increase of respiration

To investigate the control of cell energetic metabolism, creatine charge, ATP/ADP ratio and oxygen consumption (as indicators of an energetic status, the balance between ATP synthesis and degradation and the aerobic ATP turnover, respectively) were evaluated in the rat gracilis muscle, perfused-superfused in vitro. During the perfusion rate of 70 l/min the ATP/ADP ratio, as well as the creatine charge are kept at the in vivo level. With the decrease of the rate toward 54 l/min (of an abundant oxygen delivery), the values of both parameters are lower than levels in vivo. With the increase of the rate up to 100 l/min, both parameters are kept at the in vivo level, when respiration increases by 125%. The data demonstrate the 'unmatched' control of ATP utilisation and synthesis steady rates during the low perfusion rate; during the increasing steady ATP turnover following the increased perfusion rate, the two fluxes are strikingly 'matched', i.e. precisely balanced.     

Synthesis of Mitochondrial DNA Precursors during Myogenesis,an Analysis in Purified C2C12 Myotubes

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.     

Properties of liver mitochondria from iron-loaded rats.

The increased iron content in livers from iron-loaded rats is almost exclusively confined to the mitochondria. The ten- to twenty-fold higher level of nonheme iron in such mitochondria decreases the respiratory control with pyruvate-malate, but not with 3-hydroxybutyrate or succinate as substrates, and has no effect on the capacity for phosphorylation and substrate oxidation. Iron-loaded mitochondria have a malondialdehyde level which is about three times higher than that of control mitochondria, even after repeated washings with bovine serum albumin and EDTA. This is suggestive of an on-going process of lipid oxidation presumably catalyzed by the accumulated iron. Differences between the present in vivo data and in vitro results obtained by others are discussed.     

Application of a bioenergetics model for analysis of growth and food consumption of subarctic whitefish Coregonus lavaretus (L.) in Lake Kilpisjärvi, Finnish Lapland

A bioenergetics model was used to estimate daily food consumption and growth of benthic whitefish Coregonus lavaretus (L.) of age groups 1+ to 10+, in oligotrophic Lake Kilpisjärvi, northern Finnish Lapland. Fish and zooplankton samples and water temperature data were collected twice per month from February to December 1993. Simulation results indicated wide seasonal variations in consumption and growth rates. Growth was in general slow; especially in age-group 6 the net increase in weight was slight. The fit of the model to the weight data was good, and the model was able to follow even rapid seasonal variations in the weight. There were clear changes in the diet during the year. In March, when the food intake was reduced, copepods, mainly adult calanoids, formed the bulk of the food consumed. The most intensive consumption period lasted from June to late September. Chironomid pupae and planktonic cladocerans were the major summer food items. The largest individuals fed mainly on molluscs during summer. Zooplankton survey and consumption estimates did not show directly that the population density of copepodids and adults of Eudiaptomus graciloides were affected by whitefish predation although their population density decreased in spring. Nauplii, showing the maximum population density in April, were not consumed. The role of copepod consumption in the timing of Diphyllobothrium ditremum plerocercoid transmission to whitefish was also discussed. The increased copepod consumption rate during winter results in high energy intake but also an increased risk of food transmitted parasite infection.     

Myc-dependent Mitochondrial Generation of Acetyl-CoA Contributes to Fatty Acid Biosynthesis and Histone Acetylation during Cell Cycle Entry

Cell reprogramming from a quiescent to proliferative state requires coordinate activation of multiple -omic networks. These networks activate histones, increase cellular bioenergetics and the synthesis of macromolecules required for cell proliferation. However, mechanisms that coordinate the regulation of these interconnected networks are not fully understood. The oncogene c-Myc (Myc) activates cellular metabolism and global chromatin remodeling. Here we tested for an interconnection between Myc regulation of metabolism and acetylation of histones. Using [¹³C₆]glucose and a combination of GC/MS and LC/ESI tandem mass spectrometry, we determined the fractional incorporation of ¹³C-labeled 2-carbon fragments into the fatty acid palmitate, and acetyl-lysines at the N-terminal tail of histone H4 in myc^−/− and myc^+/+ Rat1A fibroblasts. Our data demonstrate that Myc increases mitochondrial synthesis of acetyl-CoA, as the de novo synthesis of ¹³C-labeled palmitate was increased 2-fold in Myc-expressing cells. Additionally, Myc induced a forty percent increase in ¹³C-labeled acetyl-CoA on H4-K16. This is linked to the capacity of Myc to increase mitochondrial production of acetyl-CoA, as we show that mitochondria provide 50% of the acetyl groups on H4-K16. These data point to a key role for Myc in directing the interconnection of -omic networks, and in particular, epigenetic modification of proteins in response to proliferative signals.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability.