Role of the Ca2+ cycle in uncoupling of oxidative phosphorylation in liver mitochondria of cold-acclimated rats

Cold acclimation of Wistar rats for 2-4 weeks at about 3 degrees C resulted in an increased respiration rate and a reduced ADP/O ratio in liver mitochondria. With increasing duration of acclimation up to 10-12 weeks, these parameters returned to a normal level. The increase in the respiration rate and the decline of the mitochondrial ADP/O ratio were associated with a significant activation of the electroneutral release of Ca2+. When the animals were acclimated for 10-12 weeks the rate of Ca2+ release reduced to control values. The addition of 1 microM ruthenium red resulted in a decrease in the rates of mitochondrial respiration in control and cold-acclimated rats to approximately equal values and in a partial restoration of the ADP/O ratio in liver mitochondria of rats kept in the cold for 2-4 weeks. The respiratory activity of mitochondria isolated in the presence of 1 mM EGTA unaffected by ruthenium red.     

Studies on mitochondria from dystrophic skeletal muscle of mice

Mitochondrial respiration and oxidative phosphorylation were compared in normal and dystrophic mouse skeletal muscles. To obtain the maximum respiration control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio from isolated muscle mitochondria, it is found that there is an advantage in having a low concentration of proteinase and EGTA present in the medium during preparation of mitochondria by centrifugation fractionation. Using pyruvate, acetylcarnitine, and palmitylcarnitine as substrates for oxidation, a highly significant reduction (40-60%) is shown in oxygen uptake by dystrophic muscle mitochondria as compared to normal muscle mitochondria. Studies of the integrity of the oxidative phosphorylation apparatus in these samples showed that there is a reduction of the RCR and ADP/O ratio in dystrophic muscle mitochondria as compared to normal muscle mitochondria.     

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles. Possible role in rescuing cellular energy homeostasis

Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK-/-), mitochondrial (Mi-CK-/-) and double knock-out (MiM-CK-/-), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2 x mg mitochondrial protein(-1) x min(-1)) and ADP affinity (K50ADP; microM) were determined by respirometry. State 3 Vmax and of M-CK-/- and MiM-CK-/- gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK-/-. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK-/- phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK-/- muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes.     

Proton leak induced by reactive oxygen species produced during in vitro anoxia/reoxygenation in rat skeletal muscle mitochondria

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenationk in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenationk. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenationk cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenationk. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenationk by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenationk and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenationk.     

‘Idealized’ State 4 and State 3 in Mitochondria vs. Rest and Work in Skeletal Muscle

A computer model of oxidative phosphorylation (OXPHOS) in skeletal muscle is used to compare state 3, intermediate state and state 4 in mitochondria with rest and work in skeletal muscle. ‘Idealized’ state 4 and 3 in relation to various ‘experimental’ states 4 and 3 are defined. Theoretical simulations show, in accordance with experimental data, that oxygen consumption (V’O₂), ADP and P_i are higher, while ATP/ADP and Δp are lower in rest than in state 4, because of the presence of basal ATP consuming reactions in the former. It is postulated that moderate and intensive work in skeletal muscle is very different from state 3 in isolated mitochondria. V’O₂, ATP/ADP, Δp and the control of ATP usage over V’O₂ are much higher, while ADP and Pi are much lower in the former. The slope of the phenomenological V’O₂-ADP relationship is much steeper during the rest-work transition than during the state 4-state 3 transition. The work state in intact muscle is much more similar to intermediate state than to state 3 in isolated mitochondria in terms of ADP, ATP/ADP, Δp and metabolic control pattern, but not in terms of V’O₂. The huge differences between intact muscle and isolated mitochondria are proposed to be caused by the presence of the each-step activation (ESA) mechanism of the regulation of OXPHOS in intact skeletal muscle. Generally, the present study suggests that isolated mitochondria (at least in the absence of Ca²⁺) cannot serve as a good model of OXPHOS regulation in intact skeletal muscle.     

Optimal conditions for studies of amino acid incorporation in vitro by isolated skeletal muscle mitochondria

Optimal conditions for amino acid incorporation into protein in vitro by isolated skeletal muscle mitochondria were established. Maximum incorporation rates were obtained when atractylate and glutamate were added to the incubation medium in the absence of any exogenous adenine nucleotides. Under these conditions, the rate of amino acid incorporation was more than 5-fold greater than that observed with glutamate and ADP and nearly 12-fold greater than that observed with ATP and an ATP-regenerating system consisting of phosphoenolpyruvate and pyruvate kinase. The optimal concentrations of adenine nucleotides, glutamate, cofactors and the substrate leucine were determined for all three energy-providing systems. The inhibitors of protein synthesis, puromycin and chloramphenicol, completely blocked amino acid incorporation by isolated skeletal muscle in mitochondria, while cycloheximide had no effect. Analysis of the labeled mitochondrial proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis revealed five labeled bands of molecular weights ranging from 38,000 to 10,000.Amino acid incorporation by skeletal muscle mitochondria isolated from diabetic rats was decreased over 60% as compared to mitochondria from controls when measured in the presence of glutamate and atractylate, ADP and glutamate or the ATP regenerating system. By contrast, amino acid incorporation by liver mitochondria isolated from diabetic rats did not differ significantly from control values when measured with four different energy sources.     

Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus)

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b(562)). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.     

Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.     

Acclimation temperature affects the metabolic response of amphibian skeletal muscle to insulin

Frog skeletal muscle mainly utilizes the substrates glucose and lactate for energy metabolism. The goal of this study was to determine the effect of insulin on the uptake and metabolic fate of lactate and glucose at rest in skeletal muscle of the American bullfrog, Lithobates catesbeiana, under varying temperature regimens. We hypothesize that lactate and glucose metabolic pathways will respond differently to the presence of insulin in cold versus warm acclimated frog tissues, suggesting an interaction between temperature and metabolism under varying environmental conditions. We employed radiolabeled tracer techniques to measure in vitro uptake, oxidation, and incorporation of glucose and lactate into glycogen by isolated muscles from bullfrogs acclimated to 5 °C (cold) or 25 °C (warm). Isolated bundles from Sartorius muscles were incubated at 5 °C, 15 °C, or 25 °C, and in the presence and absence of 0.05 IU/mL bovine insulin. Insulin treatment in the warm acclimated and incubated frogs resulted in an increase in glucose incorporation into glycogen, and an increase in intracellular [glucose] of 0.5 μmol/g (P<0.05). Under the same conditions lactate incorporation into glycogen was reduced (P<0.05) in insulin-treated muscle. When compared to the warm treatment group, cold acclimation and incubation resulted in increased rates of glucose oxidation and glycogen synthesis, and a reduction in free intracellular glucose levels (P<0.05). When muscles from either acclimation group were incubated at an intermediate temperature of 15 °C, insulin's effect on substrate metabolism was attenuated or even reversed. Therefore, a significant interaction between insulin and acclimation condition in controlling skeletal muscle metabolism appears to exist. Our findings further suggest that one of insulin's actions in frog muscle is to increase glucose incorporation into glycogen, and to reduce reliance on lactate as the primary metabolic fuel.     

Identification and characterization of uncoupling protein in heart and muscle mitochondria of canary birds

An uncoupling protein (cUCP) was identified in heart and skeletal muscle mitochondria of canary birds. cUCP was immunodetected using polyclonal antibodies raised against murine UCP2. Its molecular mass was similar to those of mammalian UCPs (32 kDa). The activity of cUCP was stimulated by palmitic acid (PA) and inhibited by GTP mainly in state 3 respiration. Additions of PA augmented state 4 respiration and lowered the ADP/O ratio. Thus, the activity of cUCP diverted energy from oxidative phosphorylation in state 3 respiration. cUCP in heart and skeletal muscles of canary birds might have implications in thermogenesis as well as protection against free radical production.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Temperature acclimation in crucian carp, Carassius carassius L., morphometric analyses of muscle fibre ultrastructure

Carp show a partial compensation in metabolic rate and activity following temperature acclimation. In the present study crucian carp, Carassius carassius , were acclimated for eight weeks to either 2deg; C or 28deg; C. The effects of temperature acclimation on muscle fibre ultrastructure has been investigated. The fractional volume (%) of each fibre type occupied by mitochondria and myofibrils was determined using a point counting morphometric method. Mitochondrial density was found to be higher in the muscles of cold (red fibres 25%; pink fibres 20% and white fibres 4%) than in those of warm acclimated fish (red fibres 14%, pink fibres 11%, white fibres 1%). The proportion of subsarcolemmal to intra-myofibrillar mitochondria was significantly lower in the red fibres of cold acclimated fish. Metabolic compensation to low temperatures are therefore associated with an increase in the number of mitochondria per cell. In contrast, the fractional volume occupied by myofibrils actually decreased following cold acclimation. Evidence is reviewed that temperature compensation of contractile activity results from qualitative rather than quantitative changes in myofibrillar proteins.     

Temperature- and developmentally-induced variation in the histochemical profile of myofibrillar ATPase activity in carp

The histochemical profile of calcium activated acid stable myofibrillar ATPase (mATPase) activity in developing larval and juvenile carp was investigated. In the larval fish, differentiation of pink muscle fibres occurred after metamorphosis which was delayed by a week at 17° C compared to larvae grown at 27° C. After metamorphosis the 27° C group exhibited some small myofibres with acid stable mATPase activity in the deep white muscle. This was similar for the juvenile carp which were acclimated for more than a month at 25° C. In contrast, the cold (12° C) acclimated juvenile fish, contained very few small white muscle fibres with acid stable mATPase activity. It was also noted that the cold acclimated fish had lower background acid stable mATPase activity than the warm acclimated fish. Results indicate that after metamorphosis and more evidently in juveniles, temperature can influence the rate of myofibre hyperplasia.     

Formation of hydrogen peroxide in the mitochondria of skeletal muscles

The generation of H2O2 in skeletal muscle mitochondria during the oxidation of NAD-dependent substrates and succinate is initiated by antimycin A but not by rotenone, which points to H2O2 formation at the respiratory chain site between the rotenone and antimycin blocks. The O2-/H2O2 ratio for alpha-ketoglutarate and succinate oxidation is approximately 1.4, which suggests that in skeletal muscle mitochondria H2O2 is predominantly formed via the superoxide radical generation. Heart and skeletal muscle mitochondria appeared to have the similar values of Vmax for H2O2 production; the catalase activity in skeletal muscle mitochondria is much lower.     

Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1

The recruitment process induced by acclimation of mammals to cold includes a marked alteration in the acyl composition of the phospholipids of mitochondria from brown adipose tissue: increases in 18:0, 18:2(n-6), and 20:4(n-6) and decreases in 16:0, 16:1, 18:1, and 22:6(n-3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein-1 (UCP1) or the thermogenesis it mediates-implying that they are secondary effects-or whether they are an integrated, independent part of the recruitment process. This question was addressed here using wild-type and UCP1-ablated C57BL/6 mice acclimated to 24 degrees C or 4 degrees C. In wild-type mice, the phospholipid fatty acyl composition of mitochondria from brown adipose tissue showed the changes in response to cold that were expected from observations in other species and strains. The changes were specific, as different changes occurred in skeletal muscle mitochondria. In UCP1-ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wild-type mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wild-type mice had little effect on phospholipid acyl composition in muscle mitochondria, but cold-acclimation in UCP1-ablated mice caused significant alterations, probably due to sustained shivering. Thus, the alterations in brown adipose tissue phospholipid acyl composition are revealed to be an independent part of the recruitment process, and their functional significance for thermogenesis should be elucidated.     

Mitochondrial oxidation of p-phenylenediamine derivatives in vitro: structure-activity relationships and correlation with myotoxic activity in vivo.

A number of p-phenylenediamine derivatives are known to cause necrosis of skeletal and/or cardiac muscle when administered to experimental animals. Compounds of this type are oxidized to semiquinonedi-imines or quinonedi-imines by mitochondria in vitro, establishing alternative pathways for electron transport in the respiratory chain with concomitant decreases in respiratory control and ADP:O ratios. Muscle mitochondria were found to be particularly effective in promoting p-phenylenediamine oxidation in vitro and the magnitude of the mitochondrial effects of the various compounds tested correlated well with their ability to cause muscle necrosis in vivo. It is suggested that mitochondrial oxidation may be involved in the initiation of the myotoxic effects of these compounds and account for their target-site specificity.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.