Methylene blue induced O2 consumption is not dependent on mitochondrial oxidative phosphorylation: Implications for salvage pathways during acute mitochondrial poisoning.

While administration of the cyclic redox agent methylene blue (MB) during intoxication by mitochondrial poisons (cyanide, hydrogen sulfide, rotenone) increases survival, the mechanisms behind these antidotal properties remain poorly understood. The objective of the studies presented in this paper was to characterize the interactions between the redox properties of MB, the intermediate metabolism and the mitochondrial respiration. We first show that intra-venous administration of micromolar levels of methylene blue in sedated and mechanically ventilated rats, increases not only resting oxygen consumption but also CO2 production (by ~ 50%), with no change in their ratio. This hypermetabolic state could be reproduced in a cellular model, where we found that the rate of electron transfer to MB was of the same order of magnitude as that of normal cellular metabolism. Notably, the large increase in cellular oxygen consumption caused by MB was relatively indifferent to the status of the mitochondrial respiratory chain: oxygen consumption persisted even when the respiratory chain was inhibited or absent (using inhibitors and cells deficient in mitochondrial oxidative phosphorylation); yet MB did not impede mitochondrial ATP production in control conditions. We present evidence that after being reduced into leuco-methylene blue (LMB) in presence of reducing molecules that are physiologically found in cells (such as NADH), the re-oxidation of LMB by oxygen can account for the increased oxygen consumption observed in vivo. In conditions of acute mitochondrial dysfunction, these MB redox cycling properties allow the rescue of the glycolysis activity and Krebs cycle through an alternate route of oxidation of NADH (or other potential reduced molecules), which accumulation would have otherwise exerted negative feedback on these metabolic pathways. Our most intriguing finding is that re-oxidization of MB by oxygen ultimately results in an in vivo matching between the increase in the rate of O2 consumed, by MB re-oxidation, and the rate of CO2, produced by the intermediate metabolism, imitating the fundamental coupling between the glycolysis/Krebs cycle and the mitochondrial respiration.

High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Glutamine stimulates translation of uncoupling protein 2mRNA.

Uncoupling protein 2 (UCP2) belongs to a family of transporters/exchangers of the mitochondrial inner membrane. Using cell lines representing natural sites of UCP2 expression (macrophages, colonocytes, pancreatic beta cells), we show that UCP2 expression is stimulated by glutamine at physiological concentrations. This control is exerted at the translational level. We demonstrate that the upstream open reading frame (ORF1) in the 5' untranslated region (5'UTR) of the UCP2 mRNA is required for this stimulation to take place. Cloning of the 5' UTR of the UCP2 mRNA in front of a GFP cDNA resulted in a reporter gene with which GFP expression could be induced by glutamine. An effect of glutamine on translation of a given mRNA has not been identified before, and this is the first evidence for a link between UCP2 and glutamine, an amino acid oxidized by immune cells or intestinal epithelium and playing a role in the control of insulin secretion.

Translation control of UCP2 synthesis by the upstream open reading frame.

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane. In vivo low expression of UCP2 contrasts with a high UCP2 mRNA level, and induction of UCP2 expression occurs without change in mRNA level, demonstrating a translational control. The UCP2 mRNA is characterized by a long 5' untranslated region (5'UTR), in which an upstream open reading frame (uORF) codes for a 36-amino-acid sequence. The 5'UTR and uORF have an inhibitory role in the translation of UCP2. The present study demonstrates that the 3' region of the uORF is a major determinant for this inhibitory role. In this 3' region, a single-base substitution that kept the codon sense unchanged significantly modified UCP2 translation, whereas some important amino acid changes had no effect. We discuss our results within the framework of the existing models explaining initiation of translation downstream of a uORF.

A new polymorphic site located in the human UCP1 gene controls the in vitro binding of CREB-like factor.

Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipose tissue (BAT) and generates heat by uncoupling respiration from ATP synthesis. A defect in BAT thermogenesis has been described in different models of rodent obesity. In humans, the implication of BAT in energy expenditure is still under discussion. A BclI polymorphism associated with fat gain over time has been described in the upstream region of the human UCP1 (hUCP1) gene. In this study, a new polymorphic site linked to the BclI site is described which results in a C to A point mutation, 89 bp downstream of the BclI site, ie at position -3737 bp. This site is located in the recently analysed regulatory region of the hUCP1 gene. The mutation disrupts a consensus site for the binding of ATF/CREB transcription factor family and inhibits the factor binding in vitro. However, transient transfection of a rodent brown adipocyte cell line shows that the isoproterenol (ISO) stimulation of the hUCP1 gene transcription is not significantly affected by this mutation. However, we postulate that the C/A substitution, in human, may contribute to a minor defect in the regulation of hUCP1 transcription and that would explain fat gain over time.

Carboxypeptidase E and thrombospondin-1 are differently expressed in subcutaneous and visceral fat of obese subjects.

The aim of this study was to identify candidate genes for visceral obesity by screening for genes strongly differentially expressed between human subcutaneous and visceral adipose depots. A cDNA microarray with human adipose-derived cDNAs was used as an initial screening to identify genes that are potentially differentially expressed between human subcutaneous and visceral abdominal fat tissues. For the two best candidates, carboxypeptidase E (CPE) and thrombospondin-1 (THBS1) (EST N72406), real-time RT-PCR was performed to confirm their depot specific expression in extremely obese individuals. Both genes appeared to be strongly differentially expressed, having a higher expression in the visceral depot than in the subcutaneous one. For THBS1, the difference in expression between the depots was greater in women than in men. The involvement of CPE and THBS1 in obesity allows us to suggest that the physiological processes controlled by these genes contribute to depot and gender-related differences in the metabolic complications of obesity.

Genetic and physiological analysis of the role of uncoupling proteins in human energy homeostasis.

The metabolic utilization of substrates results in ATP synthesis and energy loss as heat. In tissues and cells the mitochondria reoxidize reduced coenzymes and phosphorylate ADP. A significant proportion of the energy is released through thermogenesis by mitochondria. This is due to a less than perfect coupling of cellular respiration to ATP synthesis. Previous studies of brown adipocytes, which are cells specialized in regulatory thermogenesis, have shown that heat production is due to the regulated activity and synthesis of a particular proton transporter in the inner membrane of brown adipocyte mitochondria--uncoupling protein (UCP) 1. UCP homologues have recently been identified. UCP2 is widely expressed in human tissues, whereas UCP3 is expressed predominantly in human skeletal muscles. These novel UCPs represent genes which are potentially important for regulation of metabolic pathways and energy expenditure in humans. Biochemical and genetic studies support a role for these novel UCPs in metabolic regulations in humans. However, several physiological studies question such a role. Importantly, UCP2 and UCP3 seem to be able to control the activity of mitochondria in response to oxidants.

An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds.

The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.

Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation.

Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria. We generated a highly sensitive polyclonal antibody against human UCP2. Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(-/-) mice, leading to non-ambiguous identification of UCP2. We detected UCP2 in spleen, lung, stomach, and white adipose tissue. No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue. The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria. Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations. Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels. This is explained by translational regulation of the UCP2 mRNA. We have shown that an upstream open reading frame located in exon two of the ucp2 gene strongly inhibits the expression of the protein. This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions.

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance.

The coupling of oxygen consumption to ADP phosphorylation is incomplete, as is particularly evident in brown adipocyte mitochondria which use a regulated uncoupling mechanism to dissipate heat produced by substrate oxidation. In brown adipose tissue, uncoupling is effected by a specific protein in the inner mitochondrial membrane referred to as uncoupling protein-1 (UCP1). UCP1 gene disruption in mice has confirmed UCP1's role in cold-induced thermogenesis. Genetic analysis of human cohorts has suggested that UCP1 plays a minor role in the control of fat content and body weight. The recent cloning of UCP2 and UCP3, two homologues of UCP1, has boosted research on the importance of respiration control in metabolic processes, metabolic diseases and energy balance. UCP2 is widely expressed in different organs whereas UCP3 is mainly present in skeletal muscle. The chromosomal localization of UCP2 as well as UCP2 mRNA induction by a lipid-rich diet in obesity-resistant mice suggested that UCP2 is involved in diet-induced thermogenesis. A strong linkage between markers in the vicinity of human UCP2 and UCP3 (which are adjacent genes) and resting metabolic rate was calculated. UCPs are known or supposed to participate in basal and regulatory thermogenesis, but their exact biochemical and physiological functions have yet to be elucidated. UCPs may constitute novel targets in the development of drugs designed to modulate substrate oxidation. However, very recent data suggest an important role for the UCPs in the control of production of free radicals by mitochondria, and in response to oxidants.

Endocrine regulation of uncoupling proteins and energy expenditure.

Regulatory thermogenesis occurs upon exposure to the cold or during food intake. Among a variety of mechanisms leading to heat production, uncoupling of respiration in brown adipocyte mitochondria appears to be a major contributor to resistance to the cold in rodents. This uncoupling mechanism is due to the activity of uncoupling protein-1 (UCP-1), a specific carrier present in the inner membrane of mitochondria. The recent identification of UCP-2 and UCP-3, two homologues of the brown fat UCP, suggested that respiration uncoupling could contribute to thermogenesis in most tissues. Activity and expression of the three UCP's are stimulated by several neuromediators and hormones such as noradrenaline, tri-iodothyronine and leptin.

Transcriptional activation of the human ucp1 gene in a rodent cell line. Synergism of retinoids, isoproterenol, and thiazolidinedione is mediated by a multipartite response element.

Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipocytes and generates heat production by uncoupling respiration from ATP synthesis. The activatory effects of norepinephrine and retinoic acid (RA) on rodent ucp1 gene transcription have been well characterized. These effects are mediated by a 211-base pair (bp) enhancer which is also sufficient to restrict expression to brown adipose tissue. The molecular mechanisms controlling the transcription of the human ucp1 gene are unknown. In order to study the transcriptional regulation of the human gene, we set up chloramphenicol acetyltransferase constructs containing the entire or deleted 5' regions upstream of the transcriptional start site of the gene. These constructs were transiently transfected in a mouse cell line. A 350-bp hormone response region showing a significant homology with the rat ucp1 enhancer and located between the BclI polymorphic site and an AatII site (bp -3820/-3470) was detected. This region was sufficient to mediate the stimulation by RA and by combined treatments (RA + isoproterenol (ISO), RA + thiazolidinedione (TZD), or RA + ISO + TZD). The highest stimulation, a 26-fold increase in basal activity, was obtained by RA + ISO + TZD treatment. In contrast to the rodent gene, under our conditions, the effect of ISO and/or TZD is dependent on RA stimulation. Analysis of 105 bp inside the 350-bp element by site-directed mutagenesis and gel retardation experiments demonstrated that a multipartite response element mediates the drug stimulation. This region binds RARs and RXRs nuclear factors, CREB/ATF factors, and also PPARgamma despite the absence of a consensus peroxisome-proliferator response element. The activation of the human ucp1 gene transcription by certain hormones or drugs, and the identification of the cis-elements involved, will help to identify new compounds activating fat oxidation and energy expenditure in humans.

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP.

Animal and plant uncoupling protein (UCP) homologues form a subfamily of mitochondrial carriers that are evolutionarily related and possibly derived from a proton/anion transporter ancestor. The brown adipose tissue (BAT) UCP1 has a marked and strongly regulated uncoupling activity, essential to the maintenance of body temperature in small mammals. UCP homologues identified in plants are induced in a cold environment and may be involved in resistance to chilling. The biochemical activities and biological functions of the recently identified mammalian UCP2 and UCP3 are not well known. However, recent data support a role for these UCPs in State 4 respiration, respiration uncoupling and proton leaks in mitochondria. Moreover, genetic studies suggest that UCP2 and UCP3 play a part in energy expenditure in humans. The UCPs may also be involved in adaptation of cellular metabolism to an excessive supply of substrates in order to regulate the ATP level, the NAD(+)/NADH ratio and various metabolic pathways, and to contain superoxide production. A major goal will be the analysis of mice that either lack the UCP2 or UCP3 gene or overexpress these genes. Other aims will be to investigate the possible roles of UCP2 and UCP3 in response to oxidative stress, lipid peroxidation, inflammatory processes, fever and regulation of temperature in certain specific parts of the body.

Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2.

In mammalian brown adipose tissue, thermogenesis is explained by uncoupling mitochondrial respiration from ATP synthesis. Uncoupling protein-1 (UCP1) is responsible for this uncoupled state, because it allows proton re-entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain. Proton transport by UCP1 is regulated negatively by nucleotides and positively by fatty acids. Adrenergic stimulation of brown adipocytes stimulates lipolysis and therefore enhances uncoupling and thermogenesis. Adrenergic stimulation also boosts ucp1 gene transcription. Since retinoic acid also promotes ucp1 gene transcription and its structure makes it a possible activator of UCP1, we hypothesized that retinoic acid, like noradrenaline, could have a dual action and trigger the activity of the protein UCP1 itself. Here we show that retinoic acid strongly increases proton transport by UCP1 in brown adipose tissue mitochondria and that it is much more potent than fatty acids. These data are corroborated with yeast mitochondria where UCP1 was introduced by genetic manipulation. The yeast expression system allows the comparison of the UCP1 with the newly described homologues UCP2 and UCP3. The search for regulators of UCP2 has demonstrated that it is positively regulated by retinoids in a pH-dependent manner.

Structural and functional study of a conserved region in the uncoupling protein UCP1: the three matrix loops are involved in the control of transport.

It has been reported that the region 261-269 of the uncoupling protein from brown adipose tissue mitochondria, UCP1, has an important role in the control of its proton translocating activity. Thus the deletion of residues Phe267-Lys268-Gly269 leads to the loss of the nucleotide regulation of the protein, while the complete deletion of the segment leads to the formation of a pore. The region displays sequence homology with the DNA-binding domain of the estrogen receptor. The present report analyzes the structure, by NMR and circular dichroism, of a 20 amino acid residue peptide containing the residues of interest. We demonstrate that residues 263-268 adopt an alpha-helical structure. The helix is at the N-terminal end of the sixth transmembrane domain. The functional significance of this helix has been examined by site-directed mutagenesis of the protein expressed recombinantly in yeasts. Alterations in the structure or orientation of the region leads to an impairment of the regulation, by nucleotides and fatty acids, of the transport activity. UCP1 is one member of the family formed by the carriers of the mitochondrial inner membrane. The family is characterized by a tripartite structure with three repeated segments of about 100 amino acid residues. Two of the mutations have also been performed in the first and second matrix loops and the effect on UCP1 function is very similar. We conclude that the three matrix loops contribute to the formation of the gating domain in UCP1 and propose that they form a hydrophobic pocket that accommodates the purine moiety of the bound nucleotide.

UCP1, UCP2 and UCP3: are they true uncouplers of respiration?

The thermogenesis in brown adipose tissue (BAT) is due to the activity of a mitochondrial uncoupling protein (UCP1). This protein allows the protons pumped by the respiratory chain to re-enter the matrix without ATP synthesis. Therefore respiration is dramatically increased and produces only heat. The discovery of genes showing strong similarities with the UCP1 gene and expressed in other tissues raised the possibility that these proteins participate in the proton leak observed in mitochondria, and therefore participate in the regulation of energy expenditure. The recombinant expression of UCP1, UCP2 and UCP3 in yeast allows the comparison of the coupling state of yeast mitochondria in the presence or absence of these proteins.

Contributions of studies on uncoupling proteins to research on metabolic diseases.

The coupling of O2 consumption to ADP phosphorylation in mitochondria is partial. This is particularly obvious in brown adipocyte mitochondria which use a regulated uncoupling mechanism generating heat production from substrate oxidation, and catalysing thermogenesis in rodents or infants in response to cold, and arousing hibernators. In the case of brown adipose tissue, the uncoupling mechanism is related to a specific protein in the inner mitochondrial membrane referred to as UCP1. Although the biological importance of UCP1 in human adults is not demonstrated, genetic analysis of various human cohorts suggested a participation of UCP1 to control of fat content and body weight. Very recently, the cloning of UCP2 and UCP3, two homologues of UCP1, has renewed the field of research on the importance of respiration control in metabolic processes and metabolic diseases. UCP2 is widely expressed in organs, whereas UCP3 is mainly present in muscles. These proteins may explain why the coupling of respiration to ADP phosphorylation is less than perfect. Their biological importance should be studied. They also represent new putative targets for drugs against metabolic diseases such as obesity.