Sulfide (H
2S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20
µM. Sulfide oxidation has ...been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (<
1
µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.
Succinate dehydrogenase (SDH) is one of the enzymes of the tricarboxylic acid cycle (Krebs cycle) and complex II of the mitochondrial respiratory chain. A class of fungicides (SDHIs) targets the ...complex II reaction in the SDH. A large number of those in use have been shown to inhibit SDH in other phyla, including humans. This raises questions about possible effects on human health and non-target organisms in the environment. The present document will address metabolic consequences in mammals; it is neither a review on SDH nor is it about the toxicology of SDHIs. Most clinically relevant observations are linked to a severe decrease in SDH activity. Here we shall examine the mechanisms for compensating a loss of SDH activity and their possible weaknesses or adverse consequences. It can be expected that a mild inhibition of SDH will be compensated by the kinetic properties of this enzyme, but this implies a proportionate increase in succinate concentration. This would be relevant for succinate signaling and epigenetics (not reviewed here). With regard to metabolism, exposure of the liver to SDHIs would increase the risk for non-alcoholic fatty liver disease (NAFLD). Higher levels of inhibition may be compensated by modification of metabolic fluxes with net production of succinate. SDHIs are much more soluble in lipids than in water; consequently, a different diet composition between laboratory animals and humans is expected to influence their absorption.
The Biology of Mitochondrial Uncoupling Proteins ROUSSET, Sophie; ALVES-GUERRA, Marie-Clotilde; MOZO, Julien ...
Diabetes (New York, N.Y.),
02/2004, Letnik:
53, Številka:
suppl 1
Journal Article, Conference Proceeding
Recenzirano
Odprti dostop
The Biology of Mitochondrial Uncoupling Proteins
Sophie Rousset ,
Marie-Clotilde Alves-Guerra ,
Julien Mozo ,
Bruno Miroux ,
Anne-Marie Cassard-Doulcier ,
Frédéric Bouillaud and
Daniel Ricquier
From ...the Centre National de la Recherche Scientifique, Unité Propre de Recherche 9078, Faculté de Médecine and Institut de
Recherches Necker-Enfants Malades (IRNEM), Paris, France
Address correspondence and reprint requests to Dr. D. Ricquier, Faculté de Médecine, Necker-Enfants Malades, CNRS UPR 9078,
7ème étage, 75730 Paris, Cedex 15, France. E-mail: ricquier{at}necker.fr
Abstract
Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They are found in
all mammals and in plants. They belong to the family of anion mitochondrial carriers including adenine nucleotide transporters.
The term “uncoupling protein” was originally used for UCP1, which is uniquely present in mitochondria of brown adipocytes,
the thermogenic cells that maintain body temperature in small rodents. In these cells, UCP1 acts as a proton carrier activated
by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP synthase. Activation of UCP1 enhances
respiration, and the uncoupling process results in a futile cycle and dissipation of oxidation energy as heat. UCP2 is ubiquitous
and highly expressed in the lymphoid system, macrophages, and pancreatic islets. UCP3 is mainly expressed in skeletal muscles.
In comparison to the established uncoupling and thermogenic activities of UCP1, UCP2 and UCP3 appear to be involved in the
limitation of free radical levels in cells rather than in physiological uncoupling and thermogenesis. Moreover, UCP2 is a
regulator of insulin secretion and UCP3 is involved in fatty acid metabolism.
ROS, reactive oxygen species
UCP, uncoupling protein
Footnotes
This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible
by an unrestricted educational grant from Les Laboratoires Servier.
Accepted April 2, 2003.
Received March 13, 2003.
DIABETES
The role of iron in the two major sites of adaptive thermogenesis, namely the beige inguinal (iWAT) and brown adipose tissues (BAT) has not been fully understood yet. Body iron levels and ...distribution is controlled by the iron regulatory peptide hepcidin. Here, we explored iron homeostasis and thermogenic activity in brown and beige fat in wild-type and iron loaded Hepcidin KO mice. Hepcidin-deficient mice displayed iron overload in both iWAT and BAT, and preferential accumulation of ferritin in stromal cells compared to mature adipocytes. In contrast to BAT, the iWAT of Hepcidin KO animals featured with defective thermogenesis evidenced by an altered beige signature, including reduced UCP1 levels and decreased mitochondrial respiration. This thermogenic modification appeared cell autonomous and persisted after a 48 h-cold challenge, a potent trigger of thermogenesis, suggesting compromised de novo adipogenesis. Given that WAT browning occurs in both mice and humans, our results provide physiological results to interrogate the thermogenic capacity of patients with iron overload disorders.
Microglia fulfill important immunological functions in the brain by responding to pathological stresses and modulating their activities according to pro- or anti-inflammatory stimuli. Recent evidence ...indicates that changes in metabolism accompany the switch in microglia activation state, favoring glycolysis over oxidative phosphorylation when cells exhibit a pro-inflammatory phenotype. Carbon monoxide (CO), a byproduct of heme breakdown by heme oxygenase, exerts anti-inflammatory action and affects mitochondrial function in cells and tissues. In the present study, we analyzed the metabolic profile of BV2 and primary mouse microglia exposed to the CO-releasing molecules CORM-401 and CORM-A1 and investigated whether CO affects the metabolic adaptation of cells to the inflammatory stimulus lipopolysaccharide (LPS). Microglia respiration and glycolysis were measured using an Extracellular Flux Analyzer to provide a real-time bioenergetic assessment, and biochemical parameters were evaluated to define the metabolic status of the cells under normal or inflammatory conditions. We show that CO prevents LPS-induced depression of microglia respiration and reduction in ATP levels while altering the early expression of inflammatory markers, suggesting the metabolic changes induced by CO are associated with control of inflammation. CO alone affects microglia respiration depending on the concentration, as low levels increase oxygen consumption while higher amounts inhibit respiration. Increased oxygen consumption was attributed to an uncoupling activity observed in cells, at the molecular level (respiratory complex activities) and during challenge with LPS. Thus, application of CO is a potential countermeasure to reverse the metabolic changes that occur during microglia inflammation and in turn modulate their inflammatory profile.
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•Microglia cells change their metabolic profile when exposed to LPS.•CO prevents the LPS-induced depression of microglia respiration and ATP levels.•CO action relies on mechanisms involving uncoupling and modulation of glycolysis.
Cellular bioenergetics requires an intense ATP turnover that is increased further by hypermetabolic states caused by cancer growth or inflammation. Both are associated with metabolic alterations and, ...notably, enhancement of the Warburg effect (also known as aerobic glycolysis) of poor efficiency with regard to glucose consumption when compared to mitochondrial respiration. Therefore, beside this efficiency issue, other properties of these two pathways should be considered to explain this paradox: (1) biosynthesis, for this only indirect effect should be considered, since lactate release competes with biosynthetic pathways in the use of glucose; (2) ATP production, although inefficient, glycolysis shows other advantages when compared to mitochondrial respiration and lactate release may therefore reflect that the glycolytic flux is higher than required to feed mitochondria with pyruvate and glycolytic NADH; (3) Oxygen supply becomes critical under hypermetabolic conditions, and the ATP/O2 ratio quantifies the efficiency of oxygen use to regenerate ATP, although aerobic metabolism remains intense the participation of anaerobic metabolisms (lactic fermentation or succinate generation) could greatly increase ATP/O2 ratio; (4) time and space constraints would explain that anaerobic metabolism is required while the general metabolism appears oxidative; and (5) active repression of respiration by glycolytic intermediates, which could ensure optimization of glucose and oxygen use.
Brown adipose tissue expresses uncoupling protein 1 (UCP1), which dissipates energy as heat, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit ...respiration uncoupling by UCP1. Our molecular simulations predict that GDP and GTP bind UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with conserved residues R92 and E191. We identify a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. In yeast spheroplast respiration assays, both I187A and W281A mutants increase the fatty acid-induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by fatty acids even at high concentrations of purine nucleotides. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.
The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation ...of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys25 and Cys259 as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.
Summary
Within 2 h of infection by Theileria annulata sporozoites, bovine macrophages display a two‐ to fourfold increase in transcription of hypoxia inducible factor (HIF‐1α). Twenty hours ...post‐invasion sporozoites develop into multi‐nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper‐invasive and disease‐causing leukaemia‐like cell. Once immortalized Theileria‐infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF‐1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF‐1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF‐1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high Reactive Oxygen Species (ROS) environment. We propose a model where uninfected macrophages have low levels of H2O2 output, whereas virulent‐infected macrophages produce high amounts of H2O2. Further increase in H2O2 output leads to dampening of infected macrophage virulence, a characteristic of disease‐resistant macrophages. At the same time exposure to H2O2 sustains HIF‐1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF‐1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukaemic‐like cell, we propose that manipulating ROS levels, HIF‐1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2O2 drive virulent Theileria‐infected macrophages towards attenuation it highlights how infection‐induced pathology and redox balance are intimately linked.
The mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in mammals. NMR experiments in DPC micelles identified two crucial residues K56 and K269 for fatty acid ...binding and UCP1 activation. Using mitochondrial respiration, we revisit those residues in a physiological context. We show that mutation of K56 and K269 does not alter the uncoupling activity of UCP1 supporting the damaging properties of DPC on UCP1 and related carriers.
Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long‐chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure–function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomyces cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.
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