Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/brandLab © 2011 Buck Institute, All Rights Reserved Oxidative phosphorylation is an important energyconserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton and electron leaks. In this chapter, methods are presented to measure proton and electron leak activities in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasised. http://www.ncbi.nlm.nih.gov/pubmed/22057567 Sat, 31 Dec 2011 00:00:00 -0800 Oscillations in plasma membrane potential play a central role in glucoseinduced insulin secretion from pancreatic cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (p) indicator in combination with indicators of cytoplasmic free Ca2 (Ca2c), mitochondrial membrane potential (m), matrix ATP concentration and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS1 832/13 cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca2 concentration over the same concentration range required for insulin release, although considerable celltocell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8mM glucose. Increased glucose and pyruvate each produced a concentrationdependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters p and Ca2c, p and NAD(P)H, matrix ATP and Ca2c, and m and Ca2c were investigated at single cell level. It is concluded that, in these cells, depolarizing oscillations in p are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded an autonomous p oscillatory mechanism is initiated. http://www.ncbi.nlm.nih.gov/pubmed/22418435 Sat, 31 Dec 2011 00:00:00 -0800 Mitochondrial membrane potential (M) is a central intermediate in oxidative energy metabolism. Although M is routinely measured qualitatively or semiquantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulkscale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bisoxonoltype indicator of plasma membrane potential (P) and the M probe tetramethylrhodamine methyl esther (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescencebased quantitative assay to measure absolute values of M in millivolts as they vary in time in individual cells in monolayer culture. The Pdependent distribution of the probes is modeled by Eyring rate theory. Solutions of the model are used to deconvolute P and M in time from the probe fluorescence intensities, taking into account their slow, Pdependent redistribution and Nernstian behavior. The calibration accounts for matrix:cell volume ratio, high and lowaffinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons M is 139 mV at rest, and is regulated between 108 mV and 158 mV by concerted increases in ATP demand and Ca2dependent metabolic activation. Sensitivity analysis showed that the standard error in the absolute calibrated values of resting M including all biological and systematic measurement errors introduced by the calibration parameters, is less than 11 mV. Between samples treated in different ways, the typical equivalent error is 5 mV. http://www.ncbi.nlm.nih.gov/pubmed/22495585 Sat, 31 Dec 2011 00:00:00 -0800 Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESCderived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77 of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisomeproliferatoractivated receptor coactivator (PGC)1, PGC1 and receptorinteracting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energydemanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology. http://www.ncbi.nlm.nih.gov/pubmed/21242311 Mon, 31 Jan 2011 00:00:00 -0800 Dopaminergic neurons of the substantia nigra pars compacta are defective in Parkinson's disease, but the specificity of this dysfunction is not understood. One hypothesis is that mitochondrial bioenergetic capacity is intrinsically lower in striatal dopaminergic presynaptic nerve varicosities, making them unusually susceptible to inhibition of electron transport by oxidative damage. To test this hypothesis, we separated isolated synaptosomes bearing dopamine transporters using immunomagnetic beads and compared their respiration with that of the residual nondopaminergic synaptosomes. As predicted, dopaminergic synaptosomes from striatum had lower respiratory rates. However, so did dopaminergic synaptosomes from cortex, indicating a lack of the predicted striatal specificity. We used fluorescent probes to analyze the bioenergetic competence of individual synaptosomes in the two fractions. The respiratory differences became nonsignificant when respiration rates were normalized to the number of respirationcompetent synaptosomes, suggesting that differences reflected the quality of the different fractions. To circumvent damage induced by synaptosomal separation, we monitored membrane potentials in whole unseparated single synaptosomes using fluorescent imaging, and then identified the dopaminergic subpopulation using a fluorescent dopamine transporter substrate (ASP() 4(4diethylaminostyryl)Nmethylpyridinium iodide). The capacity of dopaminergic and nondopaminergic synaptosomes to maintain plasma membrane and mitochondrial membrane potential under several stresses did not differ. In addition, this capacity did not decline in either subpopulation with age, a risk factor for Parkinson's disease. We conclude that the intrinsic bioenergetic capacities of dopaminergic and nondopaminergic presynaptic synaptosomes from mice do not differ. http://www.ncbi.nlm.nih.gov/pubmed/21430153 Fri, 31 Dec 2010 00:00:00 -0800 Despite decades of speculation, the proton pumping mechanism of complex I (NADHubiquinone oxidoreductase) is unknown and continues to be controversial. Recent descriptions of the architecture of the hydrophobic region of complex I have resolved one vital issue: this region appears to have multiple proton transporters that are mechanically interlinked. Thus transduction of conformational changes to drive the transmembrane transporters linked by a "connecting rod" during the reduction of ubiquinone (Q) can account for two or three of the four protons pumped per NADH oxidized. The remaining proton(s) must be pumped by direct coupling at the Qbinding site. Here, we present a mixed model based on a crucial constraint: the strong dependence on the pH gradient across the membrane (pH) of superoxide generation at the Qbinding site of complex I. This model combines direct and indirect coupling mechanisms to account for the pumping of the four protons. It explains the observed properties of the semiquinone in the Qbinding site the rapid superoxide production from this site during reverse electron transport its low superoxide production during forward electron transport except in the presence of inhibitory Qanalogues and high protonmotive force, and the strong dependence of both modes of superoxide production on pH. http://www.ncbi.nlm.nih.gov/pubmed/21454533 Fri, 31 Dec 2010 00:00:00 -0800 Complex I (NADH:Q oxidoreductase) can form superoxide during forward electron flow (NADH oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone(Q)pool (NAD() reducing). We designed an assay system to allow titration of the redox state of the superoxidegenerating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Qpool, and inhibition of complex III to prevent QH(2) oxidation via the Qcycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenonesensitive superoxide production by complex I. However, superoxide production rate was not uniquely related to the resultant NAD redox state. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Qpool redox state, and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a twosite model of complex I superoxide production one site in equilibrium with the NADpool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Qpool, presumably a semiquinone in the Qbinding site (site I(Q)). http://www.ncbi.nlm.nih.gov/pubmed/21659507 Fri, 31 Dec 2010 00:00:00 -0800 Mitochondria couple respiration to ATP synthesis through an electrochemical proton gradient. Proton leak across the inner membrane allows adjustment of the coupling efficiency. The aim of this review is threefold: 1) introduce the unfamiliar reader to proton leak and its physiological significance, 2) review the role and regulation of uncoupling proteins, and 3) outline the prospects of proton leak as an avenue to treat obesity, diabetes, and agerelated disease. http://www.ncbi.nlm.nih.gov/pubmed/21670165 Fri, 31 Dec 2010 00:00:00 -0800 Superoxide production from antimycininhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bellshaped relationship to the reduction state of cytochrome b566, suggesting that superoxide production peaks at intermediate Qreduction state because it comes from a semiquinone in the Qo site. Imposition of a membrane potential changed the relationships between superoxide production and b566 reduction, and between b562 and b566 redox states, suggesting that b562 reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Qcycle mechanism of complex III, we generated a kinetic model of the antimycininhibited Qo site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000fold and the rate constant for semiquinone oxidation by b566 to depend on b562 redox state, the model fit the energized and deenergized data well. In such fits, quinol oxidation was much slower than literature values, and slowed further when b566 was reduced, and reduction of b562 stabilized the semiquinone when b566 was oxidized. Thus superoxide production at Qo depends on the reduction states of b566 and b562, and fits the Qcycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and shortcircuiting of the Qcycle when electron transfer slows. http://www.ncbi.nlm.nih.gov/pubmed/21708945 Fri, 31 Dec 2010 00:00:00 -0800 Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review. http://www.ncbi.nlm.nih.gov/pubmed/21726199 Fri, 31 Dec 2010 00:00:00 -0800 Reactive oxygen species (ROS), byproducts of aerobic metabolism, cause oxidative damage to cells and tissue and not surprisingly many theories have arisen to link ROSinduced oxidative stress to aging and health. While studies clearly link ROS to a plethora of divergent diseases, their role in aging is still debatable. Genetic knockdown manipulations of antioxidants alter the levels of accrued oxidative damage, however, the resultant effect of increased oxidative stress on lifespan are equivocal. Similarly the impact of elevating antioxidant levels through transgenic manipulations yield inconsistent effects on longevity. Furthermore, comparative data from a wide range of endotherms with disparate longevity remain inconclusive. Many longliving species such as birds, bats and molerats exhibit highlevels of oxidative damage, evident already at young ages. Clearly, neither the amount of ROS per se nor the sensitivity in neutralizing ROS are as important as whether or not the accrued oxidative stress leads to oxidativedamagelinked ageassociated diseases. In this review we examine the literature on ROS, its relation to disease and the lessons gleaned from a comparative approach based upon species with widely divergent responses. We specifically focus on the longest lived rodent, the naked molerat, which maintains good health and provides novel insights into the paradox of maintaining both an extended healthspan and lifespan despite high oxidative stress from a young age. http://www.ncbi.nlm.nih.gov/pubmed/21736541 Fri, 31 Dec 2010 00:00:00 -0800 Recently developed technologies have enabled multiwell measurement of O(2) consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multiwell tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multiwell assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (110 g of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncouplerstimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex Idependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples. http://www.ncbi.nlm.nih.gov/pubmed/21799747 Fri, 31 Dec 2010 00:00:00 -0800 Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a halflife of 0.54h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation through this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 1020mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is deubiquitylated and degraded. http://www.ncbi.nlm.nih.gov/pubmed/21820402 Fri, 31 Dec 2010 00:00:00 -0800 Glucosestimulated insulin secretion (GSIS) by pancreatic cells is regulated by mitochondrial uncoupling protein2 (UCP2) but opposing phenotypes, GSIS improvement and impairment, have been reported for different Ucp2ablated mouse models. By measuring mitochondrial bioenergetics in attached INS1E insulinoma cellsUCP2, we show that UCP2 contributes to proton leak and attenuates glucoseinduced rises in both respiratory activity and the coupling efficiency of oxidative phosphorylation. Strikingly, the GSIS improvement seen upon UCP2 knockdown in INS1E cells is annulled completely by the cellpermeable antioxidant MnTMPyP. Consistent with this observation, UCP2 lowers mitochondrial reactive oxygen species at high glucose levels. We conclude that UCP2 plays both regulatory and protective roles in cells by acutely lowering GSIS and chronically preventing oxidative stress. Our findings thus provide a mechanistic explanation for the apparently discrepant findings in the field. http://www.ncbi.nlm.nih.gov/pubmed/21172424 Tue, 30 Nov 2010 00:00:00 -0800 The quest to understand why we age has given rise to numerous lines of investigation that have gradually converged to include metabolic control by mitochondrial activity as a major player. That is, the ideal balance between nutrient uptake, its transduction into usable energy, and the mitigation of damaging byproducts can be regulated by mitochondrial respiration and output (ATP, reactive oxygen species (ROS), and heat). Mitochondrial inefficiency through proton leak, which uncouples substrate oxidation from ADP phosphorylation, can comprise as much as 30 of the basal metabolic rate. This uncoupling is hypothesized to protect cells from conditions that favor ROS production. Uncoupling can also occur through pharmacological induction of proton leak and activity of the uncoupling proteins. Mitochondrial uncoupling is implicated in lifespan extension through its effects on metabolic rate and ROS production. However, evidence to date does not suggest a consistent role for uncoupling in lifespan. The purpose of this review is to discuss recent work examining how mitochondrial uncoupling impacts lifespan. http://www.ncbi.nlm.nih.gov/pubmed/20363244 Tue, 31 Aug 2010 00:00:00 -0700 Converting food to chemical energy (ATP) that is usable by cells is a principal requirement to sustain life. The rate of ATP production has to be sufficient for housekeeping functions, such as protein synthesis and maintaining membrane potentials, as well as for growth and locomotion. Energy metabolism is temperature sensitive, and animals respond to environmental variability at different temporal levels, from withinindividual to evolutionary timescales. Here we review principal molecular mechanisms that underlie control of oxidative ATP production in response to climate variability. Nuclear transcription factors and coactivators control expression of mitochondrial proteins and abundance of mitochondria. Fatty acid and phospholipid concentrations of membranes influence the activity of membranebound proteins as well as the passive leak of protons across the mitochondrial membrane. Passive proton leak as well as proteinmediated proton leak across the inner mitochondrial membrane determine the efficacy of ATP production but are also instrumental in endothermic heat production and as a defense against reactive oxygen species. Both transcriptional mechanisms and membrane composition interact with environmental temperature and diet, and this interaction between diet and temperature in determining mitochondrial function links the two major environmental variables that are affected by changing climates. The limits to metabolic plasticity could be set by the production of reactive oxygen species leading to cellular damage, limits to substrate availability in mitochondria, and a disproportionally large increase in proton leak over ATP production. http://www.ncbi.nlm.nih.gov/pubmed/20586603 Sat, 31 Jul 2010 00:00:00 -0700 Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of agingrelated damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of agingrelated damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of agingrelated damage. Using this approach, we assessed several commonly used biomarkers of agingrelated damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of agingrelated damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of agingrelated damage. Our approach provides a powerful method for identification of biomarkers of aging. http://www.ncbi.nlm.nih.gov/pubmed/20367621 Wed, 30 Jun 2010 00:00:00 -0700 Mitochondria are the major cellular producers of reactive oxygen species (ROS), and mitochondrial ROS production increases steeply with increased protonmotive force. The uncoupling proteins (UCP1, UCP2, and UCP3) and adenine nucleotide translocase induce proton leak in response to exogenously added fatty acids, superoxide, or lipid peroxidation products. "Mild uncoupling" by these proteins may provide a negative feedback loop to decrease protonmotive force and attenuate ROS production. Using wildtype and Ucp3(/) mice, we found that native UCP3 actively lowers the rate of ROS production in isolated energized skeletal muscle mitochondria, in the absence of exogenous activators. The estimated specific activity of UCP3 in lowering ROS production was 90 to 500 times higher than that of the adenine nucleotide translocase. The mild uncoupling hypothesis was tested by measuring whether the effect of UCP3 on ROS production could be mimicked by chemical uncoupling. A chemical uncoupler mimicked the effect of UCP3 at early time points after mitochondrial energization, in support of the mild uncoupling hypothesis. However, at later time points the uncoupler did not mimic UCP3, suggesting that UCP3 can also affect ROS production through a membrane potentialindependent mechanism. http://www.ncbi.nlm.nih.gov/pubmed/20493945 Wed, 30 Jun 2010 00:00:00 -0700 Mitochondrial superoxide production is an important source of reactive oxygen species in cells, and may cause or contribute to ageing and the diseases of ageing. Seven major sites of superoxide production in mammalian mitochondria are known and widely accepted. In descending order of maximum capacity they are the ubiquinonebinding sites in complex I (site IQ) and complex III (site IIIQo), glycerol 3phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid betaoxidation, and pyruvate and 2oxoglutarate dehydrogenases. None of these sites is fully characterized and for some we only have sketchy information. The topology of the sites is important because it determines whether or not a site will produce superoxide in the mitochondrial matrix and be able to damage mitochondrial DNA. All sites produce superoxide in the matrix site IIIQo and glycerol 3phosphate dehydrogenase also produce superoxide to the intermembrane space. The relative contribution of each site to mitochondrial reactive oxygen species generation in the absence of electron transport inhibitors is unknown in isolated mitochondria, in cells or in vivo, and may vary considerably with species, tissue, substrate, energy demand and oxygen tension. http://www.ncbi.nlm.nih.gov/pubmed/20064600 Mon, 31 May 2010 00:00:00 -0700 Uncoupling proteins (UCP1, UCP2 and UCP3) are important in regulating cellular fuel metabolism and as attenuators of reactive oxygen species production through strong or mild uncoupling. The generic function and broad tissue distribution of the uncoupling protein family means that they are increasingly implicated in a range of pathophysiological processes including obesity, insulin resistance and diabetes mellitus, neurodegeneration, cardiovascular disease, immunity and cancer. The significant recent progress describing the turnover of novel uncoupling proteins, as well as current views on the physiological roles and regulation of UCPs, is outlined. http://www.ncbi.nlm.nih.gov/pubmed/20211596 Mon, 31 May 2010 00:00:00 -0700 The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90 as a consequence of pretreatment with 1chloro2,4dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrixdirected superoxide by up to twofold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.22.5 nmol H(2)O(2) min(1) mg protein(1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNBpretreated rate = control rate 1.43 x (control rate)/(0.55 control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/20491900 Mon, 31 May 2010 00:00:00 -0700 Mitochondrial proton and electron leak have a major impact on mitochondrial coupling efficiency and production of reactive oxygen species. In the first part of this chapter, we address the molecular nature of the basal and inducible proton leak pathways, and their physiological importance. The basal leak is unregulated, and a major proportion can be attributed to mitochondrial anion carriers, whereas the proton leak through the lipid bilayer appears to be minor. The basal proton leak is celltype specific and correlates with metabolic rate. The inducible leak through the ANT (adenine nucleotide translocase) and UCPs (uncoupling proteins) can be activated by fatty acids, superoxide or lipid peroxidation products. The physiological role of inducible leak through UCP1 in mammalian brown adipose tissue is heat production, whereas the roles of nonmammalian UCP1 and its paralogous proteins, in particular UCP2 and UCP3, are not yet resolved. The second part of the chapter focuses on the electron leak that occurs in the mitochondrial electron transport chain. Exit of electrons prior to the reduction of oxygen to water at cytochrome c oxidase causes superoxide production. As the mechanisms of electron leak are crucial to understanding their physiological relevance, we summarize the mechanisms and topology of electron leak from complexes I and III in studies using isolated mitochondria. We also highlight recent progress and challenges of assessing electron leak in the living cell. Finally, we emphasize the importance of proton and electron leak as therapeutic targets in body mass regulation and insulin secretion. http://www.ncbi.nlm.nih.gov/pubmed/20533900 Mon, 31 May 2010 00:00:00 -0700 Mitochondrial uncoupling proteins disengage substrate oxidation from ADP phosphorylation by dissipating the proton electrochemical gradient that is required for ATP synthesis. In doing this, the archetypal uncoupling protein, UCP1, mediates adaptive thermogenesis. By contrast, its paralogues UCP2 and UCP3 are not thought to mediate whole body thermogenesis in mammals. Instead, they have been implicated in a variety of physiological and pathological processes, including protection from oxidative stress, negative regulation of glucose sensing systems and the adaptation of fatty acid oxidation capacity to starving. Although much work has been devoted to how these proteins are activated, little is known of the mechanisms that reverse this activation. http://www.ncbi.nlm.nih.gov/pubmed/20006514 Fri, 30 Apr 2010 00:00:00 -0700 Caged versions of the most common mitochondrial uncouplers (proton translocators) have been prepared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4dinitrophenol (DNP) and carbonylcyanide ptrifluoromethoxyphenylhydrazone (FCCP) from caged states with second order rate constants of 10 (/0.8) M(1) s(1) and 64.8 (/0.6) M(1) s(1), respectively. The trigger mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen peroxideactivated uncouplers may be useful for studying the biological process of ageing. http://www.ncbi.nlm.nih.gov/pubmed/20418941 Wed, 31 Mar 2010 00:00:00 -0700 Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual O(2) gradient. We describe a compartment modelbased correction algorithm to deconvolute the biological oxygen consumption rate from the measured O(2). We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems. http://www.ncbi.nlm.nih.gov/pubmed/19555051 Sun, 28 Feb 2010 00:00:00 -0800 Mitochondrial uncoupling protein 2 (UCP2) is implicated in a wide range of pathophysiological processes, including immunity and diabetes mellitus, but its rapid degradation remains uncharacterized. Using pharmacological proteasome inhibitors, immunoprecipitation, dominant negative ubiqbiquitiuitin mutants, cellular fractionation and siRNA techniques, we demonstrate the involvement of the ubiquitinproteasome system in the rapid degradation of UCP2. Importantly, we resolve the issue of whether intramitochondrial proteins can be degraded by the cytosolic proteasome by reconstituting a cellfree system that shows rapid proteasomeinhibitorsensitive UCP2 degradation in isolated, energised mitochondria presented with an ATP regenerating system, ubiquitin and 26S proteasome fractions. These observations provide the first demonstration that a mitochondrial inner membrane protein is degraded by the cytosolic ubiquitinproteasome system. http://www.ncbi.nlm.nih.gov/pubmed/20103532 Sun, 31 Jan 2010 00:00:00 -0800 UCP3 (uncoupling protein 3) and its homologues UCP2 and UCP1 are regulators of mitochondrial function. UCP2 is known to have a short halflife of approx. 1 h, owing to its rapid degradation by the cytosolic 26S proteasome, whereas UCP1 is turned over much more slowly by mitochondrial autophagy. In the present study we investigate whether UCP3 also has a short halflife, and whether the proteasome is involved in UCP3 degradation. UCP3 halflife was examined in the mouse C2C12 myoblast cell line by inhibiting protein synthesis with cycloheximide and monitoring UCP3 protein levels by immunoblot analysis. We show that UCP3 has a short halflife of 0.54 h. Rapid degradation was prevented by a cocktail of proteasome inhibitors, supporting a proteasomal mechanism for turnover. In addition, this phenotype is recapitulated in vitro: UCP3 was degraded in mitochondria isolated from rat skeletal muscle or brown adipose tissue with a halflife of 0.54 h, but only in the presence of a purified 26S proteasomal fraction. This in vitro proteolysis was also sensitive to proteasome inhibition. This phenotype is in direct contrast with the related proteins UCP1 and the adenine nucleotide translocase, which have long halflives. Therefore UCP3 is turned over rapidly in multiple cell types in a proteasomedependent manner. http://www.ncbi.nlm.nih.gov/pubmed/19954423 Thu, 31 Dec 2009 00:00:00 -0800 Across a range of vertebrate species, it is known that there is a negative association between maximum lifespan and mitochondrial hydrogen peroxide production. In this report, we investigate the underlying biochemical basis of the low hydrogen peroxide production rate of heart mitochondria from a longlived species (pigeon) compared with a shortlived species with similar body mass (rat). The difference in hydrogen peroxide efflux rate was not explained by differences in either superoxide dismutase activity or hydrogen peroxide removal capacity. During succinate oxidation, the difference in hydrogen peroxide production rate between the species was localized to the DeltapHsensitive superoxide producing site within complex I. Mitochondrial DeltapH was significantly lower in pigeon mitochondria compared with rat, but this difference in DeltapH was not great enough to explain the lower hydrogen peroxide production rate. As judged by mitochondrial flavin mononucleotide content and blue native polyacrylamide gel electrophoresis, pigeon mitochondria contained less complex I than rat mitochondria. Recalculation revealed that the rates of hydrogen peroxide production per molecule of complex I were the same in rat and pigeon. We conclude that mitochondria from the longlived pigeon display low rates of hydrogen peroxide production because they have low levels of complex I. http://www.ncbi.nlm.nih.gov/pubmed/19968628 Thu, 31 Dec 2009 00:00:00 -0800 The mPTP (mitochondrial permeability transition pore) is a nonspecific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltagedependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in deenergized mitochondria from wildtype and Ucp1KO (Ucp1knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calciuminduced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50 inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80 inhibited, compared with approximately 60 in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin Asensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT. http://www.ncbi.nlm.nih.gov/pubmed/19622065 Mon, 30 Nov 2009 00:00:00 -0800 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 wildtype. 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. http://www.ncbi.nlm.nih.gov/pubmed/19705265 Wed, 30 Sep 2009 00:00:00 -0700 Glucose intolerance in C57Bl/6 mice has been associated with mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene. It has been proposed that the absence of NNT from mitochondria leads to increased mitochondrial reactive oxygen species production and subsequent activation of uncoupling protein 2 (UCP2). Activation of UCP2 has been suggested to uncouple electron transport from ATP synthesis in pancreatic beta cell mitochondria thereby decreasing glucose tolerance due to decreased insulin secretion through lower ATP/ADP ratios. The hypothesis tested in this paper is that UCP2 function is required for the dysregulation of glucose homeostasis observed in NNT ablated mice. Single and double Nnt and Ucp2 knockout mouse lines were used to measure glucose tolerance, whole animal energy balance and biochemical characteristics of mitochondrial uncoupling. As expected, glucose tolerance was diminished in mice lacking NNT. This was independent of UCP2 as it was observed either in the presence or absence of UCP2. The range of metabolic parameters examined in the mice and the proton conductance of isolated mitochondria remained unaltered in this double NNT and UCP2 knockout model. Ablation of UCP2 did not itself affect glucose tolerance and therefore previous observations of increased glucose tolerance of mice lacking UCP2 were not confirmed. We conclude that the decreased glucose tolerance in Nnt knockout mice observed in our experiments does not require UCP2. http://www.ncbi.nlm.nih.gov/pubmed/19539600 Mon, 31 Aug 2009 00:00:00 -0700 Leptin reduces body weight in ob/ob mice by decreasing food intake and increasing energy expenditure however, the mechanisms by which it does the latter are not known. Here we report that 30 of the weight loss induced by leptin treatment of ob/ob mice is due to changes in energy expenditure. In assessing leptin's effects on specific tissues, we found that hepatic basal metabolic rate was paradoxically decreased 1.7fold with leptin treatment, which was the result of a 1.6fold reduction in mitochondrial volume density and altered substrate oxidation kinetics. The altered kinetics were associated with a decrease in protein levels of 2 mitochondrial respiratory chain componentscytochrome c oxidase subunit VIa and cytochrome c oxidase subunit IV. In addition to reduced hepatic metabolism, there was reduced long chain fatty acid production and a 2.5fold increase in hepatic lipid export, both of which explain the reduced steatosis in leptintreated animals. These data help clarify the role of the liver in leptinmediated weight loss and define the mechanisms by which leptin alters hepatic metabolism and corrects steatosis. http://www.ncbi.nlm.nih.gov/pubmed/19622746 Fri, 31 Jul 2009 00:00:00 -0700 Studies were conducted to assess proton leak kinetics (proton conductance) in breast muscle mitochondria isolated from broiler breeder males within a single genetic line exhibiting either high (HFE) or low (LFE) feed efficiency. Proton leak kinetics were determined by simultaneously measuring mitochondrial membrane potential and state 2 (resting) respiration rate in breast muscle mitochondria as succinate oxidation was progressively decreased by malonate. Control proton conductance was similar in HFE and LFE mitochondria and decreased to a similar extent in both groups in response to BSA. Although treatment of mitochondria with Glu or guanosine diphosphate had no effect, retinal increased and carboxyatractylate alone or in combination with Glu decreased proton conductance relative to control proton conductance in both HFE and LFE mitochondria. After treatment with either guanosine diphosphate or carboxyatractylate alone, proton conductance was lower in HFE compared with LFE mitochondria. With the exception of BSA, proton conductance in HFE mitochondria after the various chemical treatments was either less than or equal to, and never greater than, proton conductance in the LFE mitochondria. The results suggest that there are subtle differences in membrane characteristics (e.g., lipids, integral membrane proteins) that affect proton conductance in broiler muscle mitochondria that may in turn play a role in the phenotypic expression of feed efficiency. http://www.ncbi.nlm.nih.gov/pubmed/19590084 Fri, 31 Jul 2009 00:00:00 -0700 Oxidative damage to cellular macromolecules is believed to underlie the development of many pathological states and aging. The agents responsible for this damage are generally thought to be reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radical. The main source of reactive species production within most cells is the mitochondria. Within the mitochondria the primary reactive oxygen species produced is superoxide, most of which is converted to hydrogen peroxide by the action of superoxide dismutase. The production of superoxide by mitochondria has been localized to several enzymes of the electron transport chain, including Complexes I and III and glycerol3phosphate dehydrogenase. In this chapter the current consensus view of sites, rates, mechanisms, and topology of superoxide production by mitochondria is described. A brief overview of the methods for measuring reactive oxygen species production in isolated mitochondria and cells is also presented. http://www.ncbi.nlm.nih.gov/pubmed/19513674 Sun, 31 May 2009 00:00:00 -0700 Pancreatic beta cells secrete insulin in response to raised blood glucose levels. This glucosestimulated insulin secretion (GSIS) depends on mitochondrial function and is regulated by the efficiency with which oxidative metabolism is coupled to ATP synthesis. Uncoupling protein2 (UCP2) affects this coupling efficiency and is therefore a plausible pathological and physiological regulator of GSIS. In this respect, it is important to be able to measure coupling efficiencies accurately. Here, we describe experimental protocols to determine the coupling efficiency of trypsinized INS1E cells, a popular beta cell model, and we present practical details of our RNA interference studies to probe the effect of UCP2 knockdown on this efficiency. We also introduce a method to determine coupling efficiencies noninvasively in attached cells and discuss theoretical and practical aspects of a modularkinetic approach to describe and understand cellular bioenergetics. http://www.ncbi.nlm.nih.gov/pubmed/19426881 Thu, 30 Apr 2009 00:00:00 -0700 Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, panneuronal or neurosecretory cellspecific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high panneuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespanextension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The lifeshortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulinlike peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILPlevels in fly heads and lead to a concomitant decrease in lifespan. http://www.ncbi.nlm.nih.gov/pubmed/19385039 Tue, 31 Mar 2009 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/18978830 Fri, 31 Oct 2008 00:00:00 -0700 Uncoupling protein 2 (UCP2) regulates glucosestimulated insulin secretion in pancreatic betacells. UCP2 content, measured by calibrated immunoblot in INS1E insulinoma cells (a pancreatic betacell model) grown in RPMI medium, and INS1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.04.4 ng/million cells (2.714.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein halflife of about 1 h. http://www.ncbi.nlm.nih.gov/pubmed/18692019 Tue, 30 Sep 2008 00:00:00 -0700 We assess the existence, mechanism, and functions of lessthanmaximal coupling efficiency of mitochondrial oxidative phosphorylation and its potential as a target for future antiobesity interventions. Coupling efficiency is the proportion of oxygen consumption used to make adenosine triphosphate (ATP) and do useful work. High coupling efficiency may lead to fat deposition low coupling efficiency to a decrease in fat stores. We review obligatory and facultative energy expenditure and the role of a futile cycle of proton pumping and proton leak across the mitochondrial inner membrane in dissipating energy. Basal proton conductance is catalyzed primarily by the adenine nucleotide translocase but can be mimicked by chemical uncouplers. Inducible proton conductance is catalyzed by specific uncoupling proteins. We discuss the opportunities and pitfalls of targeting these processes as a treatment for obesity by decreasing coupling efficiency and increasing energy expenditure, either directly or through central mechanisms of energy homeostasis. http://www.ncbi.nlm.nih.gov/pubmed/18407744 Sun, 31 Aug 2008 00:00:00 -0700 Pancreatic beta cells secrete insulin when blood glucose levels are high. Dysfunction of this glucosestimulated insulin secretion (GSIS) is partly responsible for the manifestation of type 2 diabetes, a metabolic disorder that is rapidly becoming a global pandemic. Mitochondria play a central role in GSIS by coupling glucose oxidation to production of ATP, a signal that triggers a series of events that ultimately leads to insulin release. Beta cells express a mitochondrial uncoupling protein, UCP2, which is rather surprising as activity of such a protein is anticipated to lower the efficiency of oxidative phosphorylation, and hence to impair GSIS. The mounting evidence demonstrating that insulin secretion is indeed blunted by UCP2 agrees with this prediction, and has provoked the idea that UCP2 activity contributes to beta cell pathogenesis and development of type 2 diabetes. Although this notion may be correct, the evolved function of UCP2 remains unclear. With this paper we aim to provide a brief account of the present state of affairs in this field, suggest a physiological role for UCP2, and highlight some of our own recent results. http://www.ncbi.nlm.nih.gov/pubmed/18433713 Sat, 31 May 2008 00:00:00 -0700 Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4hydroxynonenal, 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 4hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4Hydroxynonenalinduced 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 4hydroxynonenal 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 4hydroxynonenal and/or carboxyatractylate. http://www.ncbi.nlm.nih.gov/pubmed/18426390 Sat, 31 May 2008 00:00:00 -0700 Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 1020 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11), ANT (64) and other pathways (25). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production. http://www.ncbi.nlm.nih.gov/pubmed/18384278 Wed, 30 Apr 2008 00:00:00 -0700 We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloridehydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NADlinked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport. http://www.ncbi.nlm.nih.gov/pubmed/18395512 Wed, 30 Apr 2008 00:00:00 -0700 The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10 of the superoxide originates from the flavin site during reverse electron transport from succinate. http://www.ncbi.nlm.nih.gov/pubmed/18442479 Wed, 30 Apr 2008 00:00:00 -0700 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 wildtype and Ucp3knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wildtype and Ucp3knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3knockout 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. http://www.ncbi.nlm.nih.gov/pubmed/18251717 Mon, 31 Mar 2008 00:00:00 -0700 Studies of both survival after sepsis and sperm motility in human populations have shown significant associations with common European mitochondrial DNA haplogroups, and have led to proposals that mitochondria bearing haplogroup H have different bioenergetic capacities than those bearing haplogroup T. However, the validity of such associations assumes that there are no nonrandom influences of nuclear genes or other factors. Here, we removed the effect of any differences in nuclear genes by constructing transmitochondrial cybrids harbouring mitochondria with either haplogroup H or haplogroup T in cultured A549 human lung carcinoma cells with identical nuclear backgrounds. We compared the bioenergetic capacities and coupling efficiencies of mitochondria isolated from these cells, and of mitochondria retained within the cells, as a critical experimental test of the hypothesis that these haplogroups affect mitochondrial bioenergetics. We found that there were no functionallyimportant bioenergetic differences between mitochondria bearing these haplogroups, using either isolated mitochondria or mitochondria within cells. http://www.ncbi.nlm.nih.gov/pubmed/18280061 Fri, 29 Feb 2008 00:00:00 -0800 Mitochondria are incompletely coupled because of proton leaks that shortcircuit oxidative phosphorylation. Basal proton leak is unregulated and is associated with the presence (but not catalytic activity) of the adenine nucleotide translocase. Inducible proton leak is regulated and is catalysed by the adenine nucleotide translocase and specific uncoupling proteins (UCPs). UCP1 catalyses proton conductance in mammalian brown adipose tissue. It is activated by fatty acids, which overcome nucleotide inhibition. UCP2, UCP3 and UCPs from birds, fish and plants also catalyse proton conductance that is inhibited by nucleotides. However, they require activation by superoxide or other reactive oxygen species (ROS). The mechanism of proton transport by the UCPs is unresolved. UCPs may also transport fatty acids or fatty acyl peroxides. Several physiological functions of UCPs are postulated. (1) UCP1 is specialised for thermogenesis UCP3 and avian UCPs possibly share this function. (2) UCPs may attenuate ROS production and protect against oxidative damage, degenerative diseases and ageing. (3) UCP3 may catalyse fatty acid transport. (4) UCP2 has a signalling role in pancreatic beta cells, where it attenuates insulin secretion. Other roles remain to be discovered. http://www.ncbi.nlm.nih.gov/pubmed/18074632 Fri, 30 Nov 2007 00:00:00 -0800 Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic betacells (INS1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS1E mitochondria Affourtit and Brand (2006) Biochem. J. 393, 151159. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazolsensitive, oligomycinresistant respiration) was nearly four times higher in INS1E cells than in myoblasts. This relatively high leak activity was decreased more than 30 upon knockdown of UCP2 (uncoupling protein2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20 of INS1E respiration. UCP2 knockdown enhanced GSIS (glucosestimulated insulin secretion), consistent with a role for UCP2 in betacell physiology. We propose that the high mitochondrial proton leak in betacells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion. http://www.ncbi.nlm.nih.gov/pubmed/17824844 Fri, 30 Nov 2007 00:00:00 -0800 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 degrees C of liver mitochondria isolated from carp either maintained at 20 degrees C (warmacclimated, WA) or exposed to 8 degrees C (coldacclimated, CA) water temperature for 710 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 4hydroxytrans2nonenal, 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. http://www.ncbi.nlm.nih.gov/pubmed/17576568 Fri, 31 Aug 2007 00:00:00 -0700 An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H(2)O(2)) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, mediumsized mammals, birds). During succinate oxidation, H(2)O(2) production rates were generally lower in the longerlived species the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, whitefooted mouse, naked molerat, Damara molerat, guinea pig, baboon, little brown bat, Brazilian freetailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H(2)O(2) production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H(2)O(2) production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H(2)O(2) production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms. http://www.ncbi.nlm.nih.gov/pubmed/17596208 Fri, 31 Aug 2007 00:00:00 -0700 We have discovered that some weak uncouplers (typified by butylated hydroxytoluene) have a dynamic range of more than 10(6) in vitro: the concentration giving measurable uncoupling is less than one millionth of the concentration causing full uncoupling. They achieve this through a highaffinity interaction with the mitochondrial adenine nucleotide translocase that causes significant but limited uncoupling at extremely low uncoupler concentrations, together with more conventional uncoupling at much higher concentrations. Uncoupling at the translocase is not by a conventional weak acid/anion cycling mechanism since it is also caused by substituted triphenylphosphonium molecules, which are not anionic and cannot protonate. Covalent attachment of the uncoupler to a mitochondrially targeted hydrophobic cation sensitizes it to membrane potential, giving a small additional effect. The wide dynamic range of these uncouplers in isolated mitochondria and intact cells reveals a novel allosteric activation of proton transport through the adenine nucleotide translocase and provides a promising starting point for designing safer uncouplers for obesity therapy. http://www.ncbi.nlm.nih.gov/pubmed/17608618 Fri, 31 Aug 2007 00:00:00 -0700 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(n6), and 20:4(n6) and decreases in 16:0, 16:1, 18:1, and 22:6(n3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein1 (UCP1) or the thermogenesis it mediatesimplying that they are secondary effectsor whether they are an integrated, independent part of the recruitment process. This question was addressed here using wildtype and UCP1ablated C57BL/6 mice acclimated to 24 degrees C or 4 degrees C. In wildtype 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 UCP1ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wildtype mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wildtype mice had little effect on phospholipid acyl composition in muscle mitochondria, but coldacclimation in UCP1ablated 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. http://www.ncbi.nlm.nih.gov/pubmed/17609311 Tue, 31 Jul 2007 00:00:00 -0700 This review focuses on some of the 'hot topics' that fall under the general heading 'mitochondria and aging'. For each selected topic, we highlight recent publications that have either addressed specific problems within the field or presented novel findings of interest regarding the links between mitochondria and aging. These include studies on the structure of complex I and the mechanisms of superoxide production by this complex work showing a novel site of hydrogen peroxide production within mitochondria that is modulated by caloric restriction explorations of the relationship between the rate of evolution of mitochondrial DNA and lifespan a demonstration that mitochondrial DNA mutations do not limit lifespan in mice and investigations of the effects of mitochondrial fission on aging. We also list other relevant articles of interest and suggest some key challenges for the field in the near future. http://www.ncbi.nlm.nih.gov/pubmed/17635416 Tue, 31 Jul 2007 00:00:00 -0700 We introduce a general test of the bioenergetic importance of mtDNA (mitochondrial DNA) variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mtDNA. We have applied this test to the hypothesis RuizPesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, 223226 that particular mtDNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups. http://www.ncbi.nlm.nih.gov/pubmed/17355224 Mon, 30 Apr 2007 00:00:00 -0700 One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H()) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H() leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H() leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenalinduced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed. http://www.ncbi.nlm.nih.gov/pubmed/17263904 Sun, 31 Dec 2006 00:00:00 -0800 The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential selflimiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3(3,5dinitro4hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited. http://www.ncbi.nlm.nih.gov/pubmed/16850251 Mon, 31 Jul 2006 00:00:00 -0700 The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4HNE (4hydroxy2nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4HNE analogue transcinnamate showed essentially the same characteristics as activation by 4HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDPsensitive proton conductance by palmitate and 4HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation. http://www.ncbi.nlm.nih.gov/pubmed/16451125 Fri, 31 Mar 2006 00:00:00 -0800 In this study, the effect of flight activity on mortality rates and lipoxidative damage in Drosophila was determined to identify mechanisms through which oxidative damage affects life span. The results showed that flies allowed flying throughout life had higher mortality rates and decreased median and maximum life spans compared to controls. The mortality rate of the flight activity group could be lowered, but not completely reversed by switching to control conditions and the accrued oxidative damage could not be eliminated. The levels of reactive oxygen species produced by mitochondria isolated from high activity and control flies did not differ significantly. However, the high activity flies had altered membrane fatty acid compositions, which made them prone to increased lipid peroxidation. The effect of flight activity on insect life span differs considerably from the beneficial effects of exercise in mammals these differences may be caused by physiological differences between the two taxa. http://www.ncbi.nlm.nih.gov/pubmed/16510857 Tue, 28 Feb 2006 00:00:00 -0800 Dietary restriction (DR) extends life span in diverse organisms and may do so by attenuating production of mitochondrial reactive oxygen species (ROS). However, measurements of ROS production from isolated mitochondria of organisms subjected to DR have produced inconsistent results. In the fruit fly Drosophila, DR does not reduce production of ROS from isolated mitochondria. In this study, we used Drosophila to test whether DR lowered mitochondrial density. We assessed mitochondrial densities of flies on DR and Control diets using (a) the activities of mitochondrial enzymes and (b) electron microscopy. Both methods showed no overall effect of DR on mitochondrial density however, mitochondrial enzyme activities and morphology differed significantly between DR and Control flies. We concluded that lifespan extension by DR in Drosophila is not mediated through a reduction in mitochondrial density. If DR in Drosophila extends life span by reducing ROS production, then it does so through mechanisms that operate only in vivo. http://www.ncbi.nlm.nih.gov/pubmed/16456193 Tue, 31 Jan 2006 00:00:00 -0800 In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling. Superoxide is able to give rise to other reactive oxygen species, which elicit deleterious effects primarily by oxidizing intracellular components, including lipids, DNA and proteins. Superoxideinduced lipid peroxidation leads to the production of reactive aldehydes, including 4hydroxynonenal. These aldehydic lipid peroxidation products are in turn able to modify proteins such as mitochondrial uncoupling proteins and the adenine nucleotide translocase, converting them into active proton transporters. This activation induces mild uncoupling and so diminishes mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of energy production. http://www.ncbi.nlm.nih.gov/pubmed/16403971 Sat, 31 Dec 2005 00:00:00 -0800 Pancreatic beta cells respond to rising blood glucose concentrations by increasing their oxidative metabolism, which leads to an increased ATP/ADP ratio, closure of K(ATP) channels, depolarization of the plasma membrane potential, influx of calcium and the eventual secretion of insulin. Such a signalling mechanism implies that the ATP/ADP ratio is flexible in beta cells (betacells), which is in contrast with other cell types (e.g. muscle and liver) that maintain a stable ATP/ADP poise while respiring at widely varying rates. To determine whether this difference in flexibility is accounted for by mitochondrial peculiarities, we performed a topdown metabolic control analysis to quantitatively assess how ATP/ADP is controlled in mitochondria isolated from rat skeletal muscle and cultured beta cells. We show that the ATP/ADP ratio is more strongly controlled (approx. 7.5fold) by proton leak in beta cells than in muscle. The comparatively high importance of proton leak in beta cell mitochondria (relative to phosphorylation) is evidenced furthermore by its relatively high level of control over membrane potential and overall respiratory activity. Modularkinetic analysis of oxidative phosphorylation reveals that these control differences can be fully explained by a higher relative leak activity in beta cell mitochondria, which results in a comparatively high contribution of proton leak to the overall respiratory activity in this system. http://www.ncbi.nlm.nih.gov/pubmed/16137248 Wed, 30 Nov 2005 00:00:00 -0800 The basal proton conductance of mitochondria causes mild uncoupling and may be an important contributor to metabolic rate. The molecular nature of the protonconductance pathway is unknown. We show that the proton conductance of muscle mitochondria from mice in which isoform 1 of the adenine nucleotide translocase has been ablated is half that of wildtype controls. Overexpression of the adenine nucleotide translocase encoded by the stresssensitive B gene in Drosophila mitochondria increases proton conductance, and underexpression decreases it, even when the carrier is fully inhibited using carboxyatractylate. We conclude that half to twothirds of the basal proton conductance of mitochondria is catalysed by the adenine nucleotide carrier, independently of its ATP/ADP exchange or fattyaciddependent protonleak functions. http://www.ncbi.nlm.nih.gov/pubmed/16076285 Mon, 31 Oct 2005 00:00:00 -0800 The topology of superoxide generation by snglycerol 3phosphate dehydrogenase and complex III in intact Drosophila mitochondria was studied using aconitase inactivation to measure superoxide production in the matrix, and hydrogen peroxide formation in the presence of superoxide dismutase to measure superoxide production from both sides of the membrane. Aconitase inactivation was calibrated using the known rate of matrix superoxide production from complex I. Glycerol phosphate dehydrogenase generated superoxide about equally to each side of the membrane, whereas centre o of complex III in the presence of antimycin A generated superoxide about 30 on the cytosolic side and 70 on the matrix side. http://www.ncbi.nlm.nih.gov/pubmed/16140258 Wed, 31 Aug 2005 00:00:00 -0700 The mitochondrial uncoupling proteins UCP2 and UCP3 may be important in attenuating mitochondrial production of reactive oxygen species, in insulin signalling (UCP2), and perhaps in thermogenesis and other processes. To understand their physiological roles, it is necessary to know what reactions they are able to catalyse. We critically examine the evidence for proton transport and anion transport by UCP2 and UCP3. There is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. However, they do not catalyse proton leak in the absence of such acute activation. They can also catalyse export of fatty acid and other anions, although the relationship of anion transport to proton transport remains controversial. http://www.ncbi.nlm.nih.gov/pubmed/16005426 Sun, 31 Jul 2005 00:00:00 -0700 Mitochondrial uncoupling proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25 after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC(50) for GDP is about 100 microM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force. http://www.ncbi.nlm.nih.gov/pubmed/16084485 Sun, 31 Jul 2005 00:00:00 -0700 Evidence for the physiological functions of UCP2 and UCP3 is critically reviewed. They do not mediate adaptive thermogenesis, but they may be significantly thermogenic under specific pharmacological conditions. There is strong evidence that the mild regulated uncoupling they cause attenuates mitochondrial ROS production, protects against cellular damage, and diminishes insulin secretion. Evidence that they export fatty acids physiologically is weak. UCP2 and UCP3 are important potential targets for treatment of aging, degenerative diseases, diabetes, and perhaps obesity. http://www.ncbi.nlm.nih.gov/pubmed/16098826 Sun, 31 Jul 2005 00:00:00 -0700 Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used antibreast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a shortterm treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMRbased metabolite analysis, we found that TMXtreated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly downregulates FAS expression and activity as indicated by the accumulation of malonylCoA, a known inhibitor of mitochondrial betaoxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonylCoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis. http://www.ncbi.nlm.nih.gov/pubmed/15985534 Tue, 31 May 2005 00:00:00 -0700 Mitochondria are a major source of superoxide, formed by the oneelectron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4hydroxytrans2nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxynonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a selflimiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate. http://www.ncbi.nlm.nih.gov/pubmed/15777023 Mon, 28 Feb 2005 00:00:00 -0800 Neither the route of electron transport nor the sites or mechanism of superoxide production in mitochondrial complex I has been established. We examined the rates of superoxide generation (measured as hydrogen peroxide production) by rat skeletal muscle mitochondria under a variety of conditions. The rate of superoxide production by complex I during NADHlinked forward electron transport was less than 10 of that during succinatelinked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin. This asymmetry was not explained by differences in protonmotive force or its components. However, when inhibitors of the quinonebinding site of complex I were added in the presence of ATP to generate a pH gradient, there was a rapid rate of superoxide production by forward electron transport that was as great as the rate seen with reverse electron transport at the same pH gradient. These observations suggest that quinonebinding site inhibitors can make complex I adopt the highly radicalproducing state that occurs during reverse electron transport. Despite complete inhibition of NADH: ubiquinone oxidoreductase activity in each case, different classes of quinonebinding site inhibitor (rotenone, piericidin, and high concentrations of myxothiazol) gave different rates of superoxide production during forward electron transport (the rate with myxothiazol was twice that with rotenone) suggesting that the site of rapid superoxide generation by complex I is in the region of the ubisemiquinonebinding sites and not upstream at the flavin or low potential FeS centers. http://www.ncbi.nlm.nih.gov/pubmed/15262965 Tue, 31 Aug 2004 00:00:00 -0700 The relationship between protonmotive force and superoxide production by mitochondria is poorly understood. To address this issue, the rate of superoxide production from complex I of rat skeletal muscle mitochondria incubated under a variety of conditions was assessed. By far, the largest rate of superoxide production was from mitochondria respiring on succinate this rate was almost abolished by rotenone or piericidin, indicating that superoxide production from complex I is large under conditions of reverse electron transport. The high rate of superoxide production by complex I could also be abolished by uncoupler, confirming that superoxide production is sensitive to protonmotive force. It was inhibited by nigericin, suggesting that it is more dependent on the pH gradient across the mitochondrial inner membrane than on the membrane potential. These effects were examined in detail, leading to the conclusions that the effect of protonmotive force was mostly direct, and not indirect through changes in the redox state of the ubiquinone pool, and that the production of superoxide by complex I during reverse electron transport was at least 3fold more sensitive to the pH gradient than to the membrane potential. http://www.ncbi.nlm.nih.gov/pubmed/15175007 Sat, 31 Jul 2004 00:00:00 -0700 Mitochondria are potent producers of cellular superoxide, from complexes I and III of the electron transport chain, and mitochondrial superoxide production is a major cause of the cellular oxidative damage that may underlie degradative diseases and aging. This superoxide production is very sensitive to the proton motive force, so it can be strongly decreased by mild uncoupling. Superoxide and the lipid peroxidation products it engenders, including hydroxyalkenals such as hydroxynonenal, are potent activators of proton conductance by mitochondrial uncoupling proteins such as UCP2 and UCP3, although the mechanism of activation has yet to be established. These observations suggest a hypothesis for the main, ancestral function of uncoupling proteins: to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of a small loss of energy. We review the growing evidence for this hypothesis, in mitochondria, in cells, and in vivo. More recently evolved roles of uncoupling proteins are in adaptive thermogenesis (UCP1) and perhaps as part of a signaling pathway to regulate insulin secretion in pancreatic beta cells (UCP2). http://www.ncbi.nlm.nih.gov/pubmed/15304252 Sat, 31 Jul 2004 00:00:00 -0700 The free radical theory of aging proposes that mitochondrial production of reactive oxygen species (ROS) determines the rate of aging. Supporting this hypothesis, longerlived species produce fewer ROS than shorterlived ones, and calorically restricted rodents live longer and produce fewer ROS than controls. We studied such correlation in Drosophila melanogaster in caloric restriction and in mutant flies overexpressing the mitochondrial adenine nucleotide translocase (ANT). Caloric restriction extended life span, but there was no significant difference in mitochondrial ROS production compared with controls. ANT overexpressers had significantly lower ROS production (because they had lower membrane potential), but their life span was not extended compared to wild type. Our results show two examples in which mitochondrial ROS production and life span are not correlated. http://www.ncbi.nlm.nih.gov/pubmed/15247051 Wed, 30 Jun 2004 00:00:00 -0700 Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein1 (UCP1), which mediate adaptive nonshivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxidestimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxidestimulated, GDPinhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocasecatalysed proton conductance than those from neverimmersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate). http://www.ncbi.nlm.nih.gov/pubmed/15146050 Mon, 31 May 2004 00:00:00 -0700 MOTIVATION: Microarray experiments measure complex changes in the abundance of many mRNAs under different conditions. Current analysis methods cannot distinguish between direct and indirect effects on expression, or calculate the relative importance of mRNAs in effecting responses. RESULTS: Application of modular regulation analysis to microarray data reveals and quantifies which mRNA changes are important for cellular responses. The mRNAs are clustered, and then we calculate how perturbations alter each cluster and how strongly those clusters affect an output response. The product of these values quantifies how an input changes a response through each cluster. Two published datasets are analysed. Two mRNA clusters transmit most of the response of yeast doubling time to galactose one contains mainly galactose metabolic genes, and the other a regulatory gene. Analysis of the response of yeast relative fitness to 2deoxyDglucose reveals that control is distributed between several mRNA clusters, but experimental error limits statistical significance. http://www.ncbi.nlm.nih.gov/pubmed/14976028 Fri, 30 Apr 2004 00:00:00 -0700 Two theories of how energy metabolism should be associated with longevity, both mediated via freeradical production, make completely contrary predictions. The 'rate of livingfreeradical theory' (Pearl, 1928 Harman, 1956 Sohal, 2002) suggests a negative association, the 'uncoupling to survive' hypothesis (Brand, 2000) suggests the correlation should be positive. Existing empirical data on this issue is contradictory and extremely confused (Rubner, 1908 Yan Sohal, 2000 Ragland Sohal, 1975 Daan et al., 1996 Wolf SchmidHempel, 1989. We sought associations between longevity and individual variations in energy metabolism in a cohort of outbred mice. We found a positive association between metabolic intensity (kJ daily food assimilation expressed as g/body mass) and lifespan, but no relationships of lifespan to body mass, fat mass or lean body mass. Mice in the upper quartile of metabolic intensities had greater resting oxygen consumption by 17 and lived 36 longer than mice in the lowest intensity quartile. Mitochondria isolated from the skeletal muscle of mice in the upper quartile had higher proton conductance than mitochondria from mice from the lowest quartile. The higher conductance was caused by higher levels of endogenous activators of proton leak through the adenine nucleotide translocase and uncoupling protein3. Individuals with high metabolism were therefore more uncoupled, had greater resting and total daily energy expenditures and survived longest supporting the 'uncoupling to survive' hypothesis. http://www.ncbi.nlm.nih.gov/pubmed/15153176 Fri, 30 Apr 2004 00:00:00 -0700 Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609613 and for increasing the binding of the activator retinoic acid to UCP1 Toms, Ledesma and Rial (2002) FEBS Lett. 526, 6365. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wildtype yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty aciddependent, GDPsensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Qcontaining and deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/14680474 Wed, 31 Mar 2004 00:00:00 -0800 Superoxide generated using exogenous xanthine oxidase indirectly activates an uncoupling protein (UCP)mediated proton conductance of the mitochondrial inner membrane. We investigated whether endogenous mitochondrial superoxide production could also activate proton conductance. When respiring on succinate, rat skeletal muscle mitochondria produced large amounts of matrix superoxide. Addition of GDP to inhibit UCP3 markedly inhibited proton conductance and increased superoxide production. Both superoxide production and the GDPsensitive proton conductance were suppressed by rotenone plus an antioxidant. Thus, endogenous superoxide can activate the proton conductance of UCP3, which in turn limits mitochondrial superoxide production. These observations provide a departure point for studies under more physiological conditions. http://www.ncbi.nlm.nih.gov/pubmed/14706836 Wed, 31 Dec 2003 00:00:00 -0800 One way to study lowabundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). Nmethylanthraniloyltagged ATP (MantATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (Kd) of 0.30.5 and 2392 microM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a Kd of greater than 100 microM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound 8(14)C ATP and 8(14)C ADP with Kds of 45 and 1618 microM, respectively. Mg2 (2 mM) reduced the apparent ATP affinity to 53 microM, an effect entirely explained by chelation of ATP with Mg2, Kd using calculated free ATP was 3 microM. A combination of gel filtration, Cu2phenanthroline crosslinking, and ultracentrifugation indicated that 7580 of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Manttagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls (b) UCP2 binds Mg2free nucleotides: the Kd for ATP is about 35 microM and for MantATP it is about 10 times lower and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form. http://www.ncbi.nlm.nih.gov/pubmed/14740889 Wed, 31 Dec 2003 00:00:00 -0800 Hyperglycemia causes many of the pathological consequences of both type 1 and type 2 diabetes. Much of this damage is suggested to be a consequence of elevated production of reactive oxygen species by the mitochondrial respiratory chain during hyperglycemia. Mitochondrial radical production associated with hyperglycemia will also disrupt glucosestimulated insulin secretion by pancreatic betacells, because pancreatic betacells are particularly susceptible to oxidative damage. Therefore, mitochondrial radical production in response to hyperglycemia contributes to both the progression and pathological complications of diabetes. Consequently, strategies to decrease mitochondrial radical production and oxidative damage may have therapeutic potential. This could be achieved by the use of antioxidants or by decreasing the mitochondrial membrane potential. Here, we outline the background to these strategies and discuss how antioxidants targeted to mitochondria, or selective mitochondrial uncoupling, may be potential therapies for diabetes. http://www.ncbi.nlm.nih.gov/pubmed/14749275 Wed, 31 Dec 2003 00:00:00 -0800 The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammalspecific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)(0.19), thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms. http://www.ncbi.nlm.nih.gov/pubmed/12943530 Sun, 30 Nov 2003 00:00:00 -0800 Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4hydroxy2transnonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondriatargeted derivative of the spin trap alphaphenylNtertbutylnitrone, which reacts rapidly with carboncentered radicals but is unreactive with superoxide and lipid peroxidation products. 44(1,1Dimethylethyl)oxidoiminomethylphenoxybutyltriphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chainbreaking antioxidant. MitoPBN did react with carboncentered radicals and also prevented lipid peroxidation by the carboncentered radical generator 2,2'azobis(2methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from ironsulfur center proteins, which then generates carboncentered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs. http://www.ncbi.nlm.nih.gov/pubmed/12972420 Sun, 30 Nov 2003 00:00:00 -0800 We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 8892 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 superoxideactivated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDPsensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak. http://www.ncbi.nlm.nih.gov/pubmed/14651968 Sun, 30 Nov 2003 00:00:00 -0800 Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with snglycerol 3phosphate as substrate twothirds mostly from glycerol 3phosphate dehydrogenase on the cytosolic side and onethird on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADHlinked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3phosphate was particularly sensitive to membrane potential, decreasing 70 when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively. http://www.ncbi.nlm.nih.gov/pubmed/14556858 Tue, 30 Sep 2003 00:00:00 -0700 Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4hydroxy2nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as transretinoic acid, transretinal and other 2alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenalinduced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDPsensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylatesensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production. http://www.ncbi.nlm.nih.gov/pubmed/12912909 Thu, 31 Jul 2003 00:00:00 -0700 The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have reexamined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acidstimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/12672801 Sat, 31 May 2003 00:00:00 -0700 PGC1 alpha is a coactivator involved in adaptive thermogenesis, fattyacid oxidation and gluconeogenesis. We describe the identification of several isoforms of a new human PGC1 alpha homologue, cloned independently and named PGC1 beta. The human PGC1 beta gene is localized to chromosome 5, has 13 exons and spans more than 78 kb. Two different 5' and 3' ends due to differential splicing were identified by rapid amplification of cDNA ends PCR and screening of human cDNA libraries. We show that PGC1 beta variants in humans, mice and rats are expressed predominantly in heart, brown adipose tissue, brain and skeletal muscle. PGC1 beta expression, unlike PGC1 alpha, is not upregulated in brown adipose tissue in response to cold or obesity. Fasting experiments showed that PGC1 alpha, but not PGC1 beta, is induced in liver and this suggests that only PGC1 alpha is involved in the hepatic gluconeogenesis. No changes in PGC1 beta gene expression were observed associated with exercise. Human PGC1 beta1a and 2a isoforms localized to the cell nucleus and, specifically, the isoform PGC1 beta1a coactivated peroxisomeproliferatoractivated receptorgamma, alpha and the thyroid hormone receptor beta1. Finally, we show that ectopic expression PGC1 beta leads to increased mitochondrial number and basal oxygen consumption. These results suggest that PGC1 beta may play a role in constitutive adrenergicindependent mitochondrial biogenesis. http://www.ncbi.nlm.nih.gov/pubmed/12678921 Sat, 31 May 2003 00:00:00 -0700 OBJECTIVE: Uncoupling of isolated mitochondria by nonsteroidal antiinflammatory drugs (NSAIDs) has been considered relevant to the development of gastrointestinal (GI) side effects. We investigated the occurrence of NSAIDinduced uncoupling of mitochondria in intact cells (rat thymocytes) compared with the effects of a selective cyclooxygenase 2 (COX2) inhibitor. METHODS: Oxygen consumption and mitochondrial membrane potential were simultaneously measured amperometrically and by distribution of radioactive tracer molecules, respectively, in the presence and absence of pharmacologically relevant concentrations of the NSAIDs indomethacin and diclofenac and the selective COX2 inhibitor SC236. Analysis of data by a technique related to topdown elasticity analysis permitted assessment of the influence of these compounds on individual components of cellular energy metabolism. RESULTS: Indomethacin, diclofenac, and SC236 increased proton leak in isolated mitochondria. Both diclofenac and SC236 significantly stimulated proton leak in intact cells and simultaneously inhibited substrate oxidation and ATP turnover. Oxygen consumption rates of isolated cells remained unchanged over a wide concentration range of the drugs, despite significant effects on subsystems of cellular energy metabolism. CONCLUSION: NSAIDs and selective COX2 inhibitors have significant and equally directed effects on cellular energy metabolism. They both uncouple mitochondrial respiration and inhibit substrate oxidation and ATP turnover. However, the topical effect and selective COX2 inhibition may not be sufficient to cause NSAIDlike damage to the GI tract. http://www.ncbi.nlm.nih.gov/pubmed/12746918 Wed, 30 Apr 2003 00:00:00 -0700 A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissuespecific depression of substrate oxidation. http://www.ncbi.nlm.nih.gov/pubmed/12676751 Mon, 31 Mar 2003 00:00:00 -0800 Five markers of different kinds of oxidative damage to proteins glutamic semialdehyde, aminoadipic semialdehyde, N (epsilon)(carboxymethyl)lysine, N (epsilon)(carboxyethyl)lysine and N (epsilon)(malondialdehyde)lysine and phospholipid fatty acyl composition were identified and measured in skeletal muscle mitochondria isolated from mice genetically engineered to underexpress or overexpress uncoupling protein 3 (UCP3). Mitochondria from UCP3underexpressing mice had significantly higher levels of oxidative damage than wildtype controls, suggesting that UCP3 functions in vivo as part of the antioxidant defences of the cell, but mitochondria from UCP3overexpressing mice had unaltered oxidative damage, suggesting that mild uncoupling in vivo beyond the normal basal uncoupling provides little protection against oxidative stress. Mitochondria from UCP3underexpressing mice showed little change, but mitochondria from UCP3overexpressing mice showed marked changes in mitochondrial phospholipid fatty acyl composition. These changes were very similar to those previously found to correlate with basal proton conductance in mitochondria from a range of species and treatments, suggesting that high protein expression, or some secondary result of uncoupling, may cause the observed correlation between basal proton conductance and phospholipid fatty acyl composition. http://www.ncbi.nlm.nih.gov/pubmed/12193161 Thu, 31 Oct 2002 00:00:00 -0800 We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/12237311 Thu, 31 Oct 2002 00:00:00 -0800 Superoxide activates nucleotidesensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 14821486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCPcatalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxideinduced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10(6'ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 22(triphenylphosphonio)ethyl3,4dihydro2,5,7,8tetramethyl2H1benzopyran6ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxideinduced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6hydroxy2,5,7,8tetramethylchroman 2carboxylic acid (TROLOX) or of the mitochondrially targeted but redoxinactive analogs decyltriphenylphosphonium or 4chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotidesensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide did not need to be exported or to cycle across the inner membrane to cause uncoupling. We conclude that superoxide (or its products) exerts its uncoupling effect by activating the proton transport mechanism of uncoupling proteins at the matrix side of the mitochondrial inner membrane. http://www.ncbi.nlm.nih.gov/pubmed/12372827 Thu, 31 Oct 2002 00:00:00 -0800 DNA microarrays produce large amounts of data. Complex changes in gene expression are revealed sometimes thousands of mRNAs change between experiments. Here we apply modular regulation analysis to microarray data to reveal and quantify the mRNA changes that are important for cellular responses. The mRNAs are sorted into clusters. How strongly a perturbation alters each cluster is multiplied by how strongly each cluster affects an output, to obtain coefficients that describe how much of the change in the output is transmitted through each mRNA cluster. An example published dataset is analysed to reveal that the response ('relative fitness') of yeast to 2deoxyDglucose is not transmitted by a single mRNA cluster, but instead many clusters contribute to the overall response. The method is applicable to microarray, transcriptome, proteome and metabolome data. http://www.ncbi.nlm.nih.gov/pubmed/12241077 Sat, 31 Aug 2002 00:00:00 -0700 Experiments were performed to test the hypothesis that recombinant human uncoupling protein2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotideinhibited uncoupling that is observed in kidney mitochondria. For this, sarkosylsolubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90 pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, Nmethylanthraniloyltagged purine nucleoside di and triphosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by leastsquares nonlinear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 35 microM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with 814CATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 25 microM affinity. Affinities for ADP and GDP were approx. 10fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosylsolubilized bacterial inclusion bodies by a twostep treatment with C12E9 detergent and hydroxyapatite (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity (c) binds GDP with the same affinity as GDP inhibits superoxidestimulated uncoupling by kidney mitochondria and (d) exhibits a different nucleotide preference than kidney mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/12030845 Wed, 31 Jul 2002 00:00:00 -0700 In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twentyseven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown. http://www.ncbi.nlm.nih.gov/pubmed/12171066 Wed, 31 Jul 2002 00:00:00 -0700 A complete description of the regulation of metabolism in even a single cell will be very hard to achieve, enormous and indigestible. However, there are two powerful ways to simplify the complexity. Firstly, related processes and intermediates can be grouped into a small number of modules, and the regulation of the simplified system can be studied. Secondly, control analysis can be used. With these simplifications to illuminate the important regulatory features, even a full description could be made intellectually and experimentally accessible without distorting the essential regulatory features. Modular control analysis is powerful because it can quantify the relative importance of different flows of regulatory information through any metabolic, physiological, signalling or transcriptional network. It can answer global questions about the importance of different pathways mediating any change to a system. It has been used to analyse how cadmium, a poison with multiple effects, changes oxidative phosphorylation in isolated mitochondria, and to quantify the regulation of energy metabolism in hepatocytes. It has been used to measure how energy metabolism is regulated during mitogen stimulation of thymocytes, quantifying the relative importance of different signalling pathways and how each pathway contributes to the activation of energy metabolism. Recently, we have applied modular control analysis to modern DNA microarray expression profiling to measure the importance of different groups of mRNA transcripts in mediating physiological responses. http://www.ncbi.nlm.nih.gov/pubmed/12023818 Tue, 30 Apr 2002 00:00:00 -0700 The ability of native uncoupling protein3 (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 (UCP3tg) and from UCP3 knockout (UCP3KO) mice. The concentration of UCP3 in UCP3tg mitochondria was approximately 3 microg/mg protein, approximately 20fold higher than the wild type value. UCP3tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3tg 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 UCP3KO mice. Nevertheless, UCP3KO 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 UCP3tg 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. http://www.ncbi.nlm.nih.gov/pubmed/11707458 Mon, 31 Dec 2001 00:00:00 -0800 Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxideinduced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/11780125 Mon, 31 Dec 2001 00:00:00 -0800 Cells isolated from the hepatopancreas of estivating snails (Helix aspersa) have strongly depressed mitochondrial respiration compared with controls. Mitochondrial respiration was divided into substrate oxidation (which produces the mitochondrial membrane potential) and ATP turnover and proton leak (which consume it). The activity of substrate oxidation (and probably ATP turnover) decreased, whereas the activity of proton leak remained constant in estivation. These primary changes resulted in a lower mitochondrial membrane potential in hepatopancreas cells from estivating compared with active snails, leading to secondary decreases in respiration to drive ATP turnover and proton leak. The respiration to drive ATP turnover and proton leak decreased in proportion to the overall decrease in mitochondrial respiration, so that the amount of ATP turned over per O2 consumed remained relatively constant and aerobic efficiency was maintained in this hypometabolic state. At least 75 of the total response of mitochondrial respiration to estivation was caused by primary changes in the kinetics of substrate oxidation, with only 25 or less of the response occurring through primary effects on ATP turnover. http://www.ncbi.nlm.nih.gov/pubmed/11792646 Mon, 31 Dec 2001 00:00:00 -0800 Western blots detected uncoupling protein 3 (UCP3) in skeletalmuscle mitochondria from wildtype but not UCP3 knockout mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200700fold less than the concentration of UCP1 in brownadiposetissue mitochondria from warmadapted hamster (2484 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brownadiposetissue mitochondria from warmadapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7fold those found in rodent skeletalmuscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than emptyvector controls, particularly at concentrations that were 7fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of nonnative protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50 of the physiological level of UCP1. Low levels of UCP1 gave palmitatedependent and GDPsensitive proton conductance but higher levels of UCP1 caused an additional GDPinsensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein. http://www.ncbi.nlm.nih.gov/pubmed/11743882 Fri, 30 Nov 2001 00:00:00 -0800