Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

Inhibition of Succinate Dehydrogenase by Pesticides (SDHIs) and Energy Metabolism.

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.

FOXO1 transcription factor plays a key role in T cell-HIV-1 interaction.

HIV-1 is dependent on the host cell for providing the metabolic resources for completion of its viral replication cycle. Thus, HIV-1 replicates efficiently only in activated CD4+ T cells. Barriers preventing HIV-1 replication in resting CD4+ T cells include a block that limits reverse transcription and also the lack of activity of several inducible transcription factors, such as NF-κB and NFAT. Because FOXO1 is a master regulator of T cell functions, we studied the effect of its inhibition on T cell/HIV-1 interactions. By using AS1842856, a FOXO1 pharmacologic inhibitor, we observe that FOXO1 inhibition induces a metabolic activation of T cells with a G0/G1 transition in the absence of any stimulatory signal. One parallel outcome of this change is the inhibition of the activity of the HIV restriction factor SAMHD1 and the activation of the NFAT pathway. FOXO1 inhibition by AS1842856 makes resting T cells permissive to HIV-1 infection. In addition, we found that FOXO1 inhibition by either AS1842856 treatment or upon FOXO1 knockdown induces the reactivation of HIV-1 latent proviruses in T cells. We conclude that FOXO1 has a central role in the HIV-1/T cell interaction and that inhibiting FOXO1 with drugs such as AS1842856 may be a new therapeutic shock-and-kill strategy to eliminate the HIV-1 reservoir in human T cells.

Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect.

Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism.

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor's sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

Use of H2O2 to Cause Oxidative Stress, the Catalase Issue.

Addition of hydrogen peroxide (H2O2) is a method commonly used to trigger cellular oxidative stress. However, the doses used (often hundreds of micromolar) are disproportionally high with regard to physiological oxygen concentration (low micromolar). In this study using polarographic measurement of oxygen concentration in cellular suspensions we show that H2O2 addition results in O2 release as expected from catalase reaction. This reaction is fast enough to, within seconds, decrease drastically H2O2 concentration and to annihilate it within a few minutes. Firstly, this is likely to explain why recording of oxidative damage requires the high concentrations found in the literature. Secondly, it illustrates the potency of intracellular antioxidant (H2O2) defense. Thirdly, it complicates the interpretation of experiments as subsequent observations might result from high/transient H2O2 exposure and/or from the diverse possible consequences of the O2 release.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

UCPs, at the interface between bioenergetics and metabolism.

The first member of the uncoupling protein (UCP) family, brown adipose tissue uncoupling protein 1 (UCP1), was identified in 1976. Twenty years later, two closely related proteins, UCP2 and UCP3, were described in mammals. Homologs of these proteins exist in other organisms, including plants. Uncoupling refers to a deterioration of energy conservation between substrate oxidation and ADP phosphorylation. Complete energy conservation loss would be fatal but fine-tuning can be beneficial for processes such as thermogenesis, redox control, and prevention of mitochondrial ROS release. The coupled/uncoupled state of mitochondria is related to the permeability of the inner membrane and the proton transport mediated by activated UCPs underlies the uncoupling activity of these proteins. Proton transport by UCP1 is activated by fatty acids and this ensures thermogenesis. In vivo in absence of this activation UCP1 remains inhibited with no transport activity. A similar situation now seems unlikely for UCP2 and UCP3 and while activation of their proton transport has been described its physiological relevance remains uncertain and their influence can be envisaged as a result of another transport pathway that takes place in the absence of activation. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Positive feedback during sulfide oxidation fine-tunes cellular affinity for oxygen.

UNLABELLED: Sulfide (H2S in the gas form) is the third gaseous transmitter found in mammals. However, in contrast to nitric oxide (NO) or carbon monoxide (CO), sulfide is oxidized by a sulfide quinone reductase and generates electrons that enter the mitochondrial respiratory chain arriving ultimately at cytochrome oxidase, where they combine with oxygen to generate water. In addition, sulfide is also a strong inhibitor of cytochrome oxidase, similar to NO, CO and cyanide. The balance between the electron donor and the inhibitory role of sulfide is likely controlled by sulfide and oxygen availability. The present study aimed to evaluate if and how sulfide release and oxidation impacts on the cellular affinity for oxygen. RESULTS: i) when sulfide delivery approaches the maximal sulfide oxidation rate cells become exquisitely dependent on oxygen; ii) a positive feedback makes the balance between sulfide-releasing and -oxidizing rates the relevant parameter rather than the absolute values of these rates, and; iii) this altered dependence on oxygen is detected with sulfide concentrations that remain in the low micromolar range. CONCLUSIONS: i) within the context of continuous release of sulfide stemming from cellular metabolism, alterations in the activity of the sulfide oxidation pathway fine-tunes the cell's affinity for oxygen, and; ii) a decrease in the expression of the sulfide oxidation pathway greatly enhances the cell's dependence on oxygen concentration.

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase-1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only a few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre-LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex vivo muscle mFAO capacity and whole body fatty acid utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt-protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content, and upregulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

Increasing mitochondrial muscle fatty acid oxidation induces skeletal muscle remodeling toward an oxidative phenotype.

Adult skeletal muscle is a dynamic, remarkably plastic tissue, which allows myofibers to switch from fast/glycolytic to slow/oxidative types and to increase mitochondrial fatty acid oxidation (mFAO) capacity and vascularization in response to exercise training. mFAO is the main muscle energy source during endurance exercise, with carnitine palmitoyltransferase 1 (CPT1) being the key regulatory enzyme. Whether increasing muscle mFAO affects skeletal muscle physiology in adulthood actually remains unknown. To investigate this, we used in vivo electrotransfer technology to express in mouse tibialis anterior (TA), a fast/glycolytic muscle, a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA, its physiologic inhibitor. In young (2-mo-old) adult mice, muscle CPT1mt expression enhanced mFAO (+40%), but also increased the percentage of oxidative fibers (+28%), glycogen content, and capillary-to-fiber density (+45%). This CPT1mt-induced muscle remodeling, which mimicked exercise-induced oxidative phenotype, led to a greater resistance to muscle fatigue. In the context of aging, characterized by sarcopenia and reduced oxidative capacity, CPT1mt expression in TAs from aged (20-mo-old) mice partially reversed aging-associated sarcopenia and fiber-type transition, and increased muscle capillarity. These findings provide evidence that mFAO regulates muscle phenotype and may be a potential target to combat age-related decline in muscle function.

HIF-1α induction, proliferation and glycolysis of Theileria-infected leukocytes.

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 H2 O2 output, whereas virulent-infected macrophages produce high amounts of H2 O2 . Further increase in H2 O2 output leads to dampening of infected macrophage virulence, a characteristic of disease-resistant macrophages. At the same time exposure to H2 O2 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 H2 O2 drive virulent Theileria-infected macrophages towards attenuation it highlights how infection-induced pathology and redox balance are intimately linked.

Proteasomes associated with the Blm10 activator protein antagonize mitochondrial fission through degradation of the fission protein Dnm1.

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.

WITHDRAWN: Blm10-proteasomes antagonize mitochondrial fission through degradation of Dnm1.

This manuscript was withdrawn by the author.

The level of H2O2 type oxidative stress regulates virulence of Theileria-transformed leukocytes.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Oxidation of hydrogen sulfide by human liver mitochondria.

Hydrogen sulfide (H2S) is the third gasotransmitter discovered. Sulfide shares with the two others (NO and CO) the same inhibiting properties towards mitochondrial respiration. However, in contrast with NO or CO, sulfide at concentrations lower than the toxic (µM) level is an hydrogen donor and a substrate for mitochondrial respiration. This is due to the activity of a sulfide quinone reductase found in a large majority of mitochondria. An ongoing study of the metabolic state of liver in obese patients allowed us to evaluate the sulfide oxidation capacity with twelve preparations of human liver mitochondria. The results indicate relatively high rates of sulfide oxidation with a large variability between individuals. These observations made with isolated mitochondria appear in agreement with the main characteristics of sulfide oxidation as established before with the help of cellular models.

Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice.

The relationship between metabolism and reactive oxygen species (ROS) production by the mitochondria has often been (wrongly) viewed as straightforward, with increased metabolism leading to higher generation of pro-oxidants. Insights into mitochondrial functioning show that oxygen consumption is principally coupled with either energy conversion as ATP or as heat, depending on whether the ATP-synthase or the mitochondrial uncoupling protein 1 (UCP1) is driving respiration. However, these two processes might greatly differ in terms of oxidative costs. We used a cold challenge to investigate the oxidative stress consequences of an increased metabolism achieved either by the activation of an uncoupled mechanism (i.e. UCP1 activity) in the brown adipose tissue (BAT) of wild-type mice or by ATP-dependent muscular shivering thermogenesis in mice deficient for UCP1. Although both mouse strains increased their metabolism by more than twofold when acclimatised for 4 weeks to moderate cold (12°C), only mice deficient for UCP1 suffered from elevated levels of oxidative stress. When exposed to cold, mice deficient for UCP1 showed an increase of 20.2% in plasmatic reactive oxygen metabolites, 81.8% in muscular oxidized glutathione and 47.1% in muscular protein carbonyls. In contrast, there was no evidence of elevated levels of oxidative stress in the plasma, muscles or BAT of wild-type mice exposed to cold despite a drastic increase in BAT activity. Our study demonstrates differing oxidative costs linked to the functioning of two highly metabolically active organs during thermogenesis, and advises careful consideration of mitochondrial functioning when investigating the links between metabolism and oxidative stress.

Listeria monocytogenes transiently alters mitochondrial dynamics during infection.

Mitochondria are essential and highly dynamic organelles, constantly undergoing fusion and fission. We analyzed mitochondrial dynamics during infection with the human bacterial pathogen Listeria monocytogenes and show that this infection profoundly alters mitochondrial dynamics by causing transient mitochondrial network fragmentation. Mitochondrial fragmentation is specific to pathogenic Listeria monocytogenes, and it is not observed with the nonpathogenic Listeria innocua species or several other intracellular pathogens. Strikingly, the efficiency of Listeria infection is affected in cells where either mitochondrial fusion or fission has been altered by siRNA treatment, highlighting the relevance of mitochondrial dynamics for Listeria infection. We identified the secreted pore-forming toxin listeriolysin O as the bacterial factor mainly responsible for mitochondrial network disruption and mitochondrial function modulation. Together, our results suggest that the transient shutdown of mitochondrial function and dynamics represents a strategy used by Listeria at the onset of infection to interfere with cellular physiology.

Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations.

Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m-chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.