Liver proteomic response to hypertriglyceridemia in human-apolipoprotein C-III transgenic mice at cellular and mitochondrial compartment levels.

BACKGROUND: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the mitochondrial, sub-mitochondrial and cellular proteomic impact of hypertriglyceridemia in the hepatocytes of hypertriglyceridemic transgenic mice (overexpressing the human apolipoproteinC-III). METHODS: Quantitative proteomics (2D-DIGE) analysis was carried out on both "low-expressor" (LE) and "high-expressor" (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. RESULTS: The mitoproteome analysis has revealed a large-scale phenomenon in transgenic mice, i.e. a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. These data also demonstrate that the magnitude of proteomic changes strongly depends on the TG plasma level. Our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for TG de novo biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes has also been observed, suggesting that the cytoplasm of HTG mice is subjected to oxidative stress. Moreover, our results suggest that iron over-accumulation takes place in the cytosol of HE mice hepatocytes and may contribute to enhance oxidative stress and to promote cellular proliferation. CONCLUSIONS: These results indicate that the metabolic response to HTG in human apolipoprotein C-III overexpressing mice may support a high TG production rate and that the cytosol of hepatocytes is subjected to an important oxidative stress, probably as a result of FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes.

Uncoupling proteins: molecular, functional, regulatory, physiological and pathological aspects.

Uncoupling proteins are a subfamily of the mitochondrial anion carrier family. They are widespread in the whole eukaryotic world with a few exceptions and present tissue specific isoforms in higher organisms. They mediate purine nucleotide-sensitive free fatty acid-activated proton inward flux through the inner mitochondrial membrane. This proton flux occurs at the expense of the proton motive force build up by the respiration and weakens the coupling between respiration and ATP synthesis. In this chapter we describe current and reliable knowledge of uncoupling proteins. A new methodology allowing study of their activity and regulation during phosphorylating respiration is described. It has entitled us to assert that all uncoupling proteins share common mechanisms of activation and regulation. This is of the utmost importance in order to understand the physiological roles of UCPs as well as their participation in pathological processes since every role of the UCPs in every cell is an integral part of their function and regulation. The central role of reduction level of ubiquinone in the control of their regulation is well-argued. Their potential and reliable roles in thermogenesis, reactive oxygen species prevention and energy flow are discussed as well as their role in some pathological disorders.

Plasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: the logic of Aox1 gene localization.

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c1 together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.

Mitochondrial uncoupling proteins in unicellular eukaryotes.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.

Dynamics of the Dictyostelium discoideum mitochondrial proteome during vegetative growth, starvation and early stages of development.

In this study, a quantitative comparative proteomics approach has been used to analyze the Dictyostelium discoideum mitochondrial proteome variations during vegetative growth, starvation and the early stages of development. Application of 2-D DIGE technology allowed the detection of around 2000 protein spots on each 2-D gel with 180 proteins exhibiting significant changes in their expression level. In total, 96 proteins (51 unique and 45 redundant) were unambiguously identified. We show that the D. discoideum mitochondrial proteome adaptations mainly affect energy metabolism enzymes (the Krebs cycle, anaplerotic pathways, the oxidative phosphorylation system and energy dissipation), proteins involved in developmental and signaling processes as well as in protein biosynthesis and fate. The most striking observations were the opposite regulation of expression of citrate synthase and aconitase and the very large variation in the expression of the alternative oxidase that highlighted the importance of citrate and alternative oxidase in the physiology of the development of D. discoideum. Mitochondrial energy states measured in vivo with MitoTracker Orange CM Ros showed an increase in mitochondrial membrane polarization during D. discoideum starvation and starvation-induced development.

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

Regulation of Acanthamoeba castellanii alternative oxidase activity by mutual exclusion of purine nucleotides; ATP's inhibitory effect.

The effects of different adenine and guanine nucleotides on the cyanide-resistant respiration (i.e. alternative oxidase (AcAOX) activity) of mitochondria from the amoeba A. castellanii mitochondria were studied. We found that guanine nucleotides activate AcAOX to a greater degree than adenine nucleotides, and that nucleoside monophosphates were more efficient activators than nucleoside di- or triphosphates. The extent of the nucleotides' influence on AcAOX was dependent on the medium's pH and was more pronounced at pH 6.8, which is optimal for AcAOX activity. In contrast to other purine nucleosides, we demonstrate, for the first time, that ATP has an inhibitory effect on AcAOX activity. Since we also observed the inhibition by ATP in the mitochondria of another protozoon, such as Dictyostelium discoideum, and the yeast, Candida maltosa, it may be a regulatory feature common to all purine nucleotide-modulated non-plant AOXs. The physiological importance of this discovery is discussed. Kinetic data show that the binding of GMP (a positive allosteric effector) and the binding of ATP (a negative allosteric effector) to AcAOX are mutually exclusive. ATP's inhibition of the enzyme can be overcome by sufficiently high concentrations of GMP, and conversely, GMP's stimulation can be overcome by sufficiently high concentrations of ATP. However, an approximately three times lower concentration of GMP compared to ATP gives a half maximal effect on AcAOX activity. This is indicative of a higher binding affinity for the positive effector at the same or, at least overlapping, nucleotide-binding sites on AcAOX. These results suggest that AcAOX activity in A. castellanii mitochondria might be controlled by the relative intracellular concentrations of purine nucleotides.

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

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

Uncoupling protein 1 inhibition by purine nucleotides is under the control of the endogenous ubiquinone redox state.

We studied non-esterified fatty acid-induced uncoupling of heterologously expressed rat UCP1 (uncoupling protein 1) in yeast mitochondria, as well as UCP1 in rat BAT (brown adipose tissue) mitochondria. The proton-conductance curves and the relationship between the ubiquinone reduction level and membrane potential were determined in non-phosphorylating BAT and yeast mitochondria. The ADP/O method was applied to determine the ADP phosphorylation rate and the relationship between the ubiquinone reduction level and respiration rate in yeast mitochondria. Our studies of the membranous ubiquinone reduction level in mitochondria demonstrate that activation of UCP1 leads to a purine nucleotide-sensitive decrease in the ubiquinone redox state. Results obtained for non-phosphorylating and phosphorylating mitochondria, as the endogenous ubiquinone redox state was gradually varied by a lowering rate of the ubiquinone-reducing or ubiquinol-oxidizing pathways, indicate that the endogenous ubiquinone redox state has no effect on non-esterified fatty acid-induced UCP1 activity in the absence of GTP, and can only regulate this activity through sensitivity to inhibition by the purine nucleotide. At a given oleic acid concentration, inhibition by GTP diminishes when ubiquinone is reduced sufficiently. The ubiquinone redox state-dependent alleviation of UCP1 inhibition by the purine nucleotide was observed at a high ubiquinone reduction level, when it exceeded 85-88%.

Mitochondrial comparative proteomics: strengths and pitfalls.

In this review, we describe the various techniques available to carry out valid comparative proteomics, their advantages and their disadvantages according to the goal of the research. Two-dimensional electrophoresis and 2D-DIGE are compared to shotgun proteomics and SILE. We give our opinion on the best fields of application in the domain of comparative proteomics. We emphasize the usefulness of these new tools, providing mass data to study physiology and mitochondrial plasticity when faced with a specific mitochondrial insufficiency or exogenic stress. We illustrate the subject with results obtained in our laboratory specifying the importance of an approach of comparative proteomics combined from mitochondria and from the cell, which makes it possible to obtain important information on the status of the mitochondrial function at the cellular level. Finally, we draw attention to the dangers of the extrapolation of proteomic data to metabolic flows which requires the greatest care.

Atypical myopathy in grazing horses: a first exploratory data analysis.

Over the last decade, atypical myopathy (AM) in grazing horses has emerged in several European countries. An exploratory analysis was conducted to determine horse- and pasture-level indicators or factors associated with AM in Belgium. Belgian cases of AM confirmed by histology (n=57) were compared to their healthy co-grazing horses (n=77) and to pastured horses not involved with AM as controls (n=386). The pastures where confirmed cases were grazing (42 pastures; 38 sites; 44 incidences of AM) were compared with those of the controls (216 pastures; 96 sites; no incidence of AM). Statistically significant (P< or =0.05) exploratory variables, identified by means of adjusted odds ratios, suggested that indicators or factors associated with individual horses (young age, inactivity, body condition poor to normal), management practices (permanent pasturing, spreading of manure) and pasture characteristics (humid, sloping pastures, accumulated dead leaves, presence of waterway) may increase the risk of AM. Specific interventions based on these factors might help to reduce the incidence of AM.

Mitochondrial UCPs: new insights into regulation and impact.

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.

History and clinical features of atypical myopathy in horses in Belgium (2000-2005).

BACKGROUND: The emergent nature of atypical myopathy or atypical myoglobinuria (AM) necessitates precise description of its clinical and epidemiologic features. PURPOSE: To define key features of AM to help practitioners recognize the disease and to advise owners to take preventive measures. ANIMALS: Belgian cases of AM confirmed by histology (CC horses; n = 57) from autumn 2000 to spring 2005 were included in the study. Co-grazing horses (Co-G horses; n = 77) that remained free of any abnormal clinical signs constituted a control group. METHODS: History, environmental characteristics, clinical signs, and laboratory results associated with AM were determined by a retrospective case series study. RESULTS: Young horses in poor or normal body condition were found to be at risk for AM. Pastures were characterized by poor natural drainage and vegetation of low nutritional value. Features of AM were seasonal occurrence, apparent link with weather conditions (ie, lack of solar radiation with no heavy frost and an excess of precipitation or relative humidity), sudden onset of clinical signs, and rapid death. Evaluation of serum creatine kinase activity indicated severe muscle destruction in CC horses and subclinical disease in a few Co-G horses. CONCLUSIONS: The association of AM with specific environmental conditions and individual animals suggests that young horses should not be pastured on bare premises subject to humidity when the weather has been very wet and cold for several days. Management of AM outbreaks should include control of Co-G horses who are apparently healthy.

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP.

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.

Mitochondrial uncoupling proteins: new insights from functional and proteomic studies.

Mitochondria are the major sites of ATP synthesis through oxidative phosphorylation, a process that is weakened by proton leak. Uncoupling proteins are mitochondrial membrane proteins specialized in inducible proton conductance. They dissipate the proton electrochemical gradient established by the respiratory chain at the expense of reducing substrates. Several physiological roles have been suggested for uncoupling proteins, including roles in the control of the cellular energy balance and in preventive action against oxidative stress. This review focuses on new leads emerging from comparative proteomics about the involvement of uncoupling protein in the mitochondrial physiology. A brief overview on uncoupling proteins and on proteomics applied to mitochondria is also presented herein.

Uncoupling protein 1 affects the yeast mitoproteome and oxygen free radical production.

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.

Reduced cardiac output is associated with decreased mitochondrial efficiency in the non-ischemic ventricular wall of the acute myocardial-infarcted dog.

Cardiogenic shock is the leading cause of death among patients hospitalized with acute myocardial infarction (MI). Understanding the mechanisms for acute pump failure is therefore important. The aim of this study is to examine in an acute MI dog model whether mitochondrial bio-energetic function within non-ischemic wall regions are associated with pump failure. Anterior MI was produced in dogs via ligation of left anterior descending (LAD) coronary artery, that resulted in an infract size of about 30% of the left ventricular wall. Measurements of hemodynamic status, mitochondrial function, free radical production and mitochondrial uncoupling protein 3 (UCP3) expression were determined over 24 h period. Hemodynamic measurements revealed a > 50% reduction in cardiac output at 24 h post infarction when compared to baseline. Biopsy samples were obtained from the posterior non-ischemic wall during acute infarction. ADP/O ratios for isolated mitochondria from non-ischemic myocardium at 6 h and 24 h were decreased when compared to the ADP/O ratios within the same samples with and without palmitic acid (PA). GTP inhibition of (PA)-stimulated state 4 respiration in isolated mitochondria from the non-ischemic wall increased by 7% and 33% at 6 h and 24 h post-infarction respectively when compared to sham and pre-infarction samples. This would suggest that the mitochondria are uncoupled and this is supported by an associated increase in UCP3 expression observed on western blots from these same biopsy samples. Blood samples from the coronary sinus measured by electron paramagnetic resonance (EPR) methods showed an increase in reactive oxygen species (ROS) over baseline at 6 h and 24 h post-infarction. In conclusion, mitochondrial bio-energetic ADP/O ratios as a result of acute infarction are abnormal within the non-ischemic wall. Mitochondria appear to be energetically uncoupled and this is associated with declining pump function. Free radical production may be associated with the induction of uncoupling proteins in the mitochondria.

New Features on the Environmental Regulation of Metabolism Revealed by Modeling the Cellular Proteomic Adaptations Induced by Light, Carbon, and Inorganic Nitrogen in Chlamydomonas reinhardtii.

Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism is a crucial issue. Light, carbon and nitrogen availability have been reported to induce important metabolic adaptations. So far, the influence of these variables has essentially been studied while varying only one or two environmental factors at the same time. The goal of the present work was to model the cellular proteomic adaptations of the green microalga Chlamydomonas reinhardtii upon the simultaneous changes of light intensity, carbon concentrations (CO2 and acetate), and inorganic nitrogen concentrations (nitrate and ammonium) in the culture medium. Statistical design of experiments (DOE) enabled to define 32 culture conditions to be tested experimentally. Relative protein abundance was quantified by two dimensional differential in-gel electrophoresis (2D-DIGE). Additional assays for respiration, photosynthesis, and lipid and pigment concentrations were also carried out. A hierarchical clustering survey enabled to partition biological variables (proteins + assays) into eight co-regulated clusters. In most cases, the biological variables partitioned in the same cluster had already been reported to participate to common biological functions (acetate assimilation, bioenergetic processes, light harvesting, Calvin cycle, and protein metabolism). The environmental regulation within each cluster was further characterized by a series of multivariate methods including principal component analysis and multiple linear regressions. This metadata analysis enabled to highlight the existence of a clear regulatory pattern for every cluster and to mathematically simulate the effects of light, carbon, and nitrogen. The influence of these environmental variables on cellular metabolism is described in details and thoroughly discussed. This work provides an overview of the metabolic adaptations contributing to maintain cellular homeostasis upon extensive environmental changes. Some of the results presented here could be used as starting points for more specific fundamental or applied investigations.

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria.

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.

Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast.

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.