Increased respiration in skeletal muscle mitochondria from cold-acclimated ducklings: uncoupling effects of free fatty acids

Intermyofibrillar mitochondria from skeletal muscle (m. gastrocnemius) and liver mitochondria were isolated from cold-acclimated (4 degrees C) or control (30 degrees C) 4-week old ducklings. The respiratory rate of isolated mitochondria, with Na-succinate as substrate, was followed polarographically at 25 degrees C in order to determine the basal respiratory rate, the rate of respiration in the presence of free fatty acids (FFA) (Na-palmitate), and the fully uncoupled rate, after addition of FCCP. The basal respiration (which in liver mitochondria was unaffected by acclimation to cold) was higher (+53%) in intermyofibrillar mitochondria from cold-acclimated ducklings than from controls, and the maximal FCCP-stimulated respiration was also increased (+98%) by acclimation to cold. FFA-stimulated respiration increased as a function of FFA concentration in both types of mitochondria. The increase in respiration due to FFA was about double in intermyofibrillar mitochondria from cold-acclimated ducklings than that of controls, but in liver mitochondria there was no increase due to cold. The membrane potential was estimated by the dye safranine in the absence or in the presence of FFA in the incubation medium. There were no significant differences in the basal membrane potential in the two groups and the addition of FFA led to the same depolarization in both groups. The significance of these alterations for acclimation to cold is discussed.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.     

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.     

Dietary inorganic nitrate improves mitochondrial efficiency in humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.     

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.     

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens.     

Dexamethasone treatment specifically increases the basal proton conductance of rat liver mitochondria

We investigated the role that mitochondrial proton leak may play in the glucocorticoid-induced hypermetabolic state. Sprague-Dawley rats were injected with dexamethasone over a period of 5 days. Liver mitochondria and gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from dexamethasone-treated, pair-fed and control rats. Respiration and membrane potential were measured simultaneously using electrodes sensitive to oxygen and to the potential-dependent probe triphenylmethylphosphonium, respectively. Five days of dexamethasone injection resulted in a marked increase in the basal proton conductance of liver mitochondria, but not in the muscle mitochondrial populations. This effect would have a modest impact on energy expenditure in rats.     

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.     

Increased oxidative capacity in skeletal muscles from cold-acclimated ducklings: a comparison with rats

1. The effects of prolonged cold exposure on cytochrome oxidase activity were investigated in skeletal muscles, liver and adipose tissues from cold-acclimated (CA) and control (TN) ducklings and rats. 2. Cold acclimation increased the oxidative capacity of skeletal muscles (+33% in gastrocnemius and +195% in pectoral) and liver (+47%) from CA ducklings, but decreased the oxidative capacity of gastrocnemius muscle (-22%) from CA rats. On the other hand, in these CA rats it increased the oxidative capacity of liver by 88% and, above all, brown adipose tissue by 544%. 3. The significance of these changes due to acclimation to cold in ducklings and rats is discussed. Such an increase in oxidative capacity of CA duckling muscles may explain the non-shivering thermogenesis observed in these birds.     

Energization-dependent endogenous activation of proton conductance in skeletal muscle mitochondria

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.     

Functional characterisation of UCP1 in the common carp: uncoupling activity in liver mitochondria and cold-induced expression in the brain

Mammalian uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis in brown adipose tissue. We previously reported on the presence of a UCP1 orthologue in ectothermic fish and observed downregulation of UCP1 gene expression in the liver of the common carp. Neither the function of UCP1, nor the mode of UCP1 activation is known in carp liver mitochondria. Here, we compared the proton conductance at 25°C of liver mitochondria isolated from carp either maintained at 20°C (warm-acclimated, WA) or exposed to 8°C (cold-acclimated, CA) water temperature for 7–10 days. Liver mitochondria from WA carp had higher state four rates of oxygen consumption and greater proton conductance at high membrane potential. Liver mitochondria from WA, but not from CA, carp showed a strong increase in proton conductance when palmitate (or 4-hydroxy-trans-2-nonenal, HNE) was added, and this inducible proton conductance was prevented by addition of GDP. This fatty acid sensitive proton leak is likely due to the expression of UCP1 in the liver of WA carp. The observed biochemical properties of proton leak strongly suggest that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as mammalian UCP1. Significant UCP1 expression was also detected in our previous study in whole brain of the carp. We here observed a twofold increase of UCP1 mRNA in carp brain following cold exposure, suggesting a role of UCP1 in the thermal adaptation of brain metabolism. In situ hybridization located the UCP1 gene expression to the optic tectum responsible for visual system control, the descending trigeminal tract and the solitary tract. Taken together, this study characterises uncoupling protein activity in an ectotherm for the first time. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus)

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.     

Effects of acute leptin administration on the differences in proton leak rate in liver mitochondria from ob/ob mice compared to lean controls.

In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.     

Active proton leak in mitochondria: A new way to regulate substrate oxidation

The main function of mitochondria is energy transduction, from substrate oxidation to the free energy of ATP synthesis, through oxidative phosphorylation. For physiological reasons, the degree of coupling between these two processes must be modulated in order to adapt redox potential and ATP turnover to cellular needs. Such a modulation leads to energy wastage. To this day, two energy wastage mechanisms have been described: the membrane passive proton conductance (proton leak) and the decrease in the coupling efficiency between electrons transfer and proton extrusion at the proton pumps level (redox or proton slipping). In this paper, we describe a new energy wastage mechanism of interest. We show that in isolated yeast mitochondria, the membrane proton conductance is strictly dependent on the external dehydrogenases activity. An increase in their activity leads to an increase in the membrane proton conductance. This proton permeability is independent of the respiratory chain and ATP synthase proton pumps. We propose to name this new mechanism “active proton leak.” Such a mechanism could allow a wide modulation of substrate oxidation in response to cellular redox constraints.     

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.     

Thermal acclimation,mitochondrial capacities and organ metabolic profiles in a reptile (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

Reptiles thermoregulate behaviourally, but change their preferred temperature and the optimal temperature for performance seasonally. We evaluated whether the digestive and locomotor systems of the alligator show parallel metabolic adjustments during thermal acclimation. To this end, we allowed juvenile alligators to grow under thermal conditions typical of winter and summer, providing them with seasonally appropriate basking opportunities. Although mean body temperatures of alligators in these groups differed by approximately 10°C, their growth and final anatomic status was equivalent. While hepatic mitochondria isolated from cold-acclimated alligators had higher oxidative capacities at 30°C than those from warm-acclimated alligators, the capacities did not differ at 20°C. Cold acclimation decreased maximal oxidative capacities of muscle mitochondria. For mitochondria from both organs and acclimation groups, palmitate increased oligomycin-inhibited respiration. GDP addition reduced palmitate-uncoupled rates more in liver mitochondria from warm- than cold-acclimated alligators. In muscle mitochondria, carboxyatractyloside significantly reduced palmitate-uncoupled rates. This effect was not changed by thermal acclimation. The aerobic capacity of liver, skeletal muscle and duodenum, as estimated by activities of cytochrome c oxidase (COX), increased with cold acclimation. At acclimation temperatures, the activities of COX and citrate synthase (CS) in these organs were equivalent. By measuring COX and CS in isolated mitochondria and tissue extracts, we estimated that cold acclimation did not change the mitochondrial content in liver, but increased that of muscle. The thermal compensation of growth rates and of the aerobic capacity of the locomotor and digestive systems suggests that alligators optimised metabolic processes for the seasonally altered, preferred body temperature. The precision of this compensatory response exceeds that typically shown by aquatic ectotherms whose body temperatures are at the mercy of their habitat.     

Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle

This study deals with mitochondrial energy efficiency in liver and skeletal muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly increases the sensitivity to uncoupling by added palmitate of skeletal muscle but not liver mitochondria, while mitochondrial energy coupling in the absence of fatty acids is only slightly affected by cold in liver and skeletal muscle. In addition, uncoupling protein 3 content does not follow changes in skeletal muscle mitochondrial coupling. It is therefore concluded that skeletal muscle could play a direct thermogenic role based on fatty acid-induced mild uncoupling of mitochondrial oxidative phosphorylation.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: effect of cold acclimation

We report the first evidence of a mitochondrial NO synthase (mtNOS) in bird skeletal muscle. In vitro, mtNOS activity stimulated by l-arginine reduced intermyofibrillar mitochondrial oxygen uptake and ATP synthesis rates, stimulated endogenous H₂O₂ generation, but had no effect on oxidative phosphorylation efficiency. Arginine-induced effects were fully reversed by l-NAME, a known NOS inhibitor. When ducklings were cold exposed for 4 weeks, muscle mitochondria displayed an increased state 3 respiration, a reduced H₂O₂ generation but no significant alteration in mtNOS activity. We conclude that mtNOS is expressed in avian skeletal muscle.