Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13 C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization. Production of inflammatory mediators by M1-polarized macrophages is thought to rely on suppression of mitochondrial metabolism in favor of glycolysis. Refining this concept, here the authors define metabolic targets of nitric oxide as responsible for the mitochondrial rewiring resulting from polarization.

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title: Immunometabolism governs dendritic cell and macrophage function
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title: Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production☆☆☆
abstract: Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, “alternatively activated” macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes.

title: Succinate is an inflammatory signal that induces IL-1β through HIF-1α
abstract: Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the ‘GABA (γ-aminobutyric acid) shunt’ pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.

title: Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1
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title: The Monocarboxylate Transporter 4 Is Required for Glycolytic Reprogramming and Inflammatory Response in Macrophages*
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title: Microbial stimulation of different Toll-like receptor signalling pathways induces diverse metabolic programmes in human monocytes
abstract: Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

title: Trained Immunity: An Ancient Way of Remembering.
abstract: The innate arm of the immune system has generally been regarded as primitive and non-specific and, in contrast to adaptive immunity, not to possess memory. Here we review the growing body of evidence that innate immunity has an important capacity to adapt, a de facto innate immune memory (also termed trained immunity), and this provides broad protection against infections.

title: Tricarboxylic acid cycle metabolites in the control of macrophage activation and effector phenotypes
abstract: The tricarboxylic acid (TCA) cycle is a mitochondrial metabolic hub that coordinates the metabolism of carbohydrates, proteins, and fats into carbon dioxide and ATP. At specific points in the cycle, the diversion, import, or export of TCA metabolites allows for the dynamic regulation of a variety of tissue and/or cell‐specific phenotypic processes. Recent studies have identified that a number of TCA metabolites are important in controlling monocyte/macrophage phenotypes and effector functions while specific macrophage activation or polarization states functionally determine the relative utilization of each. This review focuses on the metabolic reprogramming of the TCA cycle in macrophages and how individual metabolites play a variety of context‐specific roles in determining physiologic and pathologic macrophage activation and homeostatic functions. We discuss the implications of these findings and address unanswered questions regarding the role of the TCA cycle in guiding macrophage‐dependent immune responses.

title: A broken krebs cycle in macrophages.
abstract: Macrophages undergo metabolic rewiring during polarization but details of this process are unclear. In this issue of Immunity, Jha et al. (2015) report a systems approach for unbiased analysis of cellular metabolism that reveals key metabolites and metabolic pathways required for distinct macrophage polarization states.

title: A key role of the mitochondrial citrate carrier (SLC25A1) in TNFα- and IFNγ-triggered inflammation.
abstract: The chronic induction of inflammation underlies multiple pathological conditions, including metabolic, autoimmune disorders and cancer. The mitochondrial citrate carrier (CIC), encoded by the SLC25A1 gene, promotes the export of citrate from the mitochondria to the cytoplasm, a process that profoundly influences energy balance in the cells. We have previously shown that SLC25A1 is a target gene for lipopolysaccharide signaling and promotes the production of inflammatory mediators. We now demonstrate that SLC25A1 is induced at the transcriptional level by two key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), and such induction involves the activity of the nuclear factor kappa B and STAT1 transcription factors. By studying the down-stream events following SLC25A1 activation during signals that mimic inflammation, we demonstrate that CIC is required for regulating the levels of nitric oxide and of prostaglandins by TNFα or IFNγ. Importantly, we show that the citrate exported from mitochondria via CIC and its downstream metabolic intermediate, acetyl-coenzyme A, are necessary for TNFα or IFNγ to induce nitric oxide and prostaglandin production. These findings provide the first line of evidence that the citrate export pathway, via CIC, is central for cytokine-induced inflammatory signals and shed new light on the relationship between energy metabolism and inflammation.

title: Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression*
abstract: Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases.

title: Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels*
abstract: Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1−/− mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.

title: Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production
abstract: Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

title: Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation.
abstract: Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1(-/-) mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

title: Abolition of mitochondrial substrate‐level phosphorylation by itaconic acid produced by LPS‐induced Irg1 expression in cells of murine macrophage lineage
abstract: Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis‐aconitate. In mitochondria, itaconate can be converted by succinate‐coenzyme A (CoA) ligase to itaconyl‐CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS‐induced stimulation of Irg1 in murine bone marrow‐derived macrophages (BMDM) and RAW‐264.7 cells. In rotenone‐treated macrophage cells, stimulation by LPS led to impairment in substrate‐level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW‐264.7 cells, the LPS‐induced impairment in SLP was reversed by short‐interfering RNA(siRNA)—but not scrambled siRNA—treatment directed against Irg1. LPS dose‐dependently inhibited oxygen consumption rates (61‐91%) and elevated glycolysis rates (>21%) in BMDM but not RAW‐264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose‐dependently (0.5‐2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP‐induced depolarizations (3‐19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a “CoA trap” in the form of itaconyl‐CoA that negatively affects the upstream supply of succinyl‐CoA from the α‐ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate‐CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate‐CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1‐expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.—Németh, B., Doczi, J., Csete, D., Kacso, G., Ravasz, D., Adams, D., Kiss, G., Nagy, A. M., Horvath, G., Tretter, L., Mócsai, A., Csépányi‐Kömi, R., Iordanov, I., Adam‐Vizi, V., Chinopoulos, C. Abolition of mitochondrial substrate‐level phosphorylation by itaconic acid produced by LPS‐induced Irg1 expression in cells of murine macrophage lineage. FASEB J. 30, 286‐300 (2016). www.fasebj.org

title: Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection
abstract: Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions, principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through its inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1−/− mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1−/−, but not WT, mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, Mrp8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA sequencing analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, an Irg1 regulatory axis modulates inflammation to curtail Mtb-induced lung disease.

title: Itaconic acid mediates crosstalk between macrophage metabolism and peritoneal tumors
abstract: Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset–specific metabolites in situ, especially in the tumor setting. Here, we determined how a macrophage-specific (Mϕ-specific) metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show that peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation–mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue–resident macrophages (pResMϕ). Unbiased metabolomics identified itaconic acid, the product of immune-responsive gene 1–mediated (Irg1-mediated) catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMϕ of tumor-bearing mice. Administration of lentivirally encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of ROS in pResMϕ and ROS-mediated MAPK activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMϕ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patients’ ascites fluid expressed significantly elevated levels of IRG1. Therefore, IRG1 in pResMϕ represents a potential therapeutic target for peritoneal tumors.

title: Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells.
abstract: Previous studies show that cytotoxic activated macrophages cause a reproducible pattern of metabolic inhibition in viable tumor target cells. This includes inhibition of DNA synthesis, two oxidoreductases of the mitochondrial electron transport chain (NADH: ubiquinone oxidoreductase and succinate: ubiquinone oxidoreductase), and the citric acid cycle enzyme aconitase. This pattern of metabolic inhibition is induced by a cytotoxic activated macrophage associated biochemical pathway with L-arginine deimination activity that synthesizes L-citrulline from L-arginine and oxygenated nitrogen derivatives from the imino nitrogen removed from the guanido group of L-arginine. Here we report that macrophages activated in vivo by infection with bacillus Calmette-Guérin or in vitro by murine rIFN-gamma or murine IFN-alpha/beta (in the presence of the second signal LPS in all cases) develop inhibition of aconitase and the same two oxidoreductases of the mitochondrial electron transport chain as was documented earlier in target cells of cytotoxic activated macrophages. In addition, this pattern of metabolic inhibition which develops in cytotoxic activated macrophages is caused by the L-arginine-dependent effector mechanism. Inhibition of mitochondrial respiration by effectors of the L-arginine-dependent cytotoxicity system results in a compensatory increase in activity of the glycolytic pathway. We speculate that the pattern of metabolic inhibition induced in cytotoxic activated macrophages by the L-arginine-dependent effector system causes changes in the macrophage intracellular environment that increases resistance to certain facultative and obligate intracellular pathogens.

title: Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.
abstract: Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

title: Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression1
abstract: Comprehensive analysis of the gene expression profiles associated with human monocyte-to-macrophage differentiation and polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage differentiation is associated with activation of cell cycle genes, substantiating the underestimated proliferation potential of monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature of macrophage differentiation and polarization. 4) Lipid metabolism is a main category of modulated transcripts, with expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2-associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology.

title: Novel Markers to Delineate Murine M1 and M2 Macrophages
abstract: Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.

title: The chemokine system in diverse forms of macrophage activation and polarization.
abstract: Plasticity and functional polarization are hallmarks of the mononuclear phagocyte system. Here we review emerging key properties of different forms of macrophage activation and polarization (M1, M2a, M2b, M2c), which represent extremes of a continuum. In particular, recent evidence suggests that differential modulation of the chemokine system integrates polarized macrophages in pathways of resistance to, or promotion of, microbial pathogens and tumors, or immunoregulation, tissue repair and remodeling.

title: HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia.
abstract: Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.

title: HIF-1-mediated suppression of acyl-CoA dehydrogenases and fatty acid oxidation is critical for cancer progression.
abstract: Hypoxia-inducible factor 1 (HIF-1) mediates a metabolic switch that blocks the conversion of pyruvate to acetyl-CoA in cancer cells. Here, we report that HIF-1α also inhibits fatty acid β-oxidation (FAO), another major source of acetyl-CoA. We identified a PGC-1β-mediated pathway by which HIF-1 inhibits the medium- and long-chain acyl-CoA dehydrogenases (MCAD and LCAD), resulting in decreased reactive oxygen species levels and enhanced proliferation. Surprisingly, we further uncovered that blocking LCAD, but not MCAD, blunts PTEN expression and dramatically affects tumor growth in vivo. Analysis of 158 liver cancer samples showed that decreased LCAD expression predicts patient mortality. Altogether, we have identified a previously unappreciated mechanism by which HIF-1 suppresses FAO to facilitate cancer progression.

title: HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption.
abstract: The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

title: Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1.
abstract: Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.

title: Nitric Oxide‐Mediated Inhibition of the Mitochondrial Respiratory Chain in Cultured Astrocytes
abstract: The Ca2+‐independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon‐γ (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH‐ubiquinone‐1 reductase (complex I) and succinate‐cytochrome c reductase (complex II–III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate‐cytochrome c reductase activity, but NADH‐ubiquinone‐1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG‐monomethyl‐l‐arginine. The lipopolysaccharide/interferon‐γ treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide‐mediated neurotoxicity.

title: Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice
abstract: The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.

title: Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells.
abstract: TLR agonists initiate a rapid activation program in dendritic cells (DCs) that requires support from metabolic and bioenergetic resources. We found previously that TLR signaling promotes aerobic glycolysis and a decline in oxidative phosphorylation (OXHPOS) and that glucose restriction prevents activation and leads to premature cell death. However, it remained unclear why the decrease in OXPHOS occurs under these circumstances. Using real-time metabolic flux analysis, in the present study, we show that mitochondrial activity is lost progressively after activation by TLR agonists in inflammatory blood monocyte-derived DCs that express inducible NO synthase. We found that this is because of inhibition of OXPHOS by NO and that the switch to glycolysis is a survival response that serves to maintain ATP levels when OXPHOS is inhibited. Our data identify NO as a profound metabolic regulator in inflammatory monocyte-derived DCs.

title: The role of nitric oxide in metabolic regulation of Dendritic cell immune function.
abstract: Dendritic cells (DCs) are canonical antigen presenting cells of the immune system and serve as a bridge between innate and adaptive immune responses. When DCs are activated by a stimulus through toll-like receptors (TLRs), DCs undergo a process of maturation defined by cytokine & chemokine secretion, co-stimulatory molecule expression, antigen processing and presentation, and the ability to activate T cells. DC maturation is coupled with an increase in biosynthetic demand, which is fulfilled by a TLR-driven upregulation in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and cellular energy required for DC activation, and inhibition of glycolysis during initial activation impairs both the survival and effector function of activated DCs. Evidence shows that DC glycolytic upregulation is controlled by two distinct pathways, an early burst of glycolysis that is nitric oxide (NO) -independent, and a sustained commitment to glycolysis in NO-producing DC subsets. This review will address the complex role of NO in regulating DC metabolism and effector function.

title: Nitric oxide prevents a pathogen permissive granulocytic inflammation during tuberculosis
abstract: Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

title: Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription
abstract: Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress response and also represses inflammation. However, the mechanisms how Nrf2 alleviates inflammation are still unclear. Here, we demonstrate that Nrf2 interferes with lipopolysaccharide-induced transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1β. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR analyses revealed that Nrf2 binds to the proximity of these genes in macrophages and inhibits RNA Pol II recruitment. Further, we found that Nrf2-mediated inhibition is independent of the Nrf2-binding motif and reactive oxygen species level. Murine inflammatory models further demonstrated that Nrf2 interferes with IL6 induction and inflammatory phenotypes in vivo. Thus, contrary to the widely accepted view that Nrf2 suppresses inflammation through redox control, we demonstrate here that Nrf2 opposes transcriptional upregulation of proinflammatory cytokine genes. This study identifies Nrf2 as the upstream regulator of cytokine production and establishes a molecular basis for an Nrf2-mediated anti-inflammation approach.

title: Cell-Permeating α-Ketoglutarate Derivatives Alleviate Pseudohypoxia in Succinate Dehydrogenase-Deficient Cells
abstract: Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH- or FH-deficient cells, HIF activation is due to HIF1α stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIFα prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, α-ketoglutarate. We designed and synthesized cell-permeating α-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate- or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular α-ketoglutarate. Introduction of α-ketoglutarate derivatives restores normal PHD activity and HIF1α levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction.

title: Substrate Fate in Activated Macrophages: A Comparison between Innate, Classic, and Alternative Activation
abstract: Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-13C2]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-γ or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1α activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1α, the switch of PFK2 isoenzymes still occurs in LPS/IFN-γ–activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1α-independent mechanisms.

title: Reversible inactivation of dihydrolipoamide dehydrogenase by Angeli's salt.
abstract: Dihydrolipoamide dehydrogenase (DLDH) is a key component of 3 mitochondrial α-keto acid dehydrogenase complexes including pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and branched chain amino acid dehydrogenase complex. It is a pyridine-dependent disulfide oxidoreductase that is very sensitive to oxidative modifications by reactive nitrogen species (RNS) and reactive oxygen species (ROS). The objective of this study was to investigate the mechanisms of DLDH modification by RNS derived from Angeli's salt. Studies were conducted using isolated rat brain mitochondria that were incubated with varying concentrations of Angeli's salt followed by spectrophotometric enzyme assays, blue native gel analysis, and 2-dimensional gel-based proteomic approaches. Results show that DLDH could be inactivated by Angeli's salt in a concentration dependent manner and the inactivation was a targeting rather than a random process as peroxynitrite did not show any detectable inhibitory effect on the enzyme's activity under the same experimental conditions. Since Angeli's salt can readily decompose at physiological pH to yield nitroxyl anion (HNO) and nitric oxide, further studies were conducted to determine the actual RNS that was responsible for DLDH inactivation. Results indicate that it was HNO that exerted the effect of Angeli's salt on DLDH. Finally, two-dimensional Western blot analysis indicates that DLDH inactivation by Angeli's salt was accompanied by formation of protein s-nitrosothiols, suggesting that s-nitrosylation is likely the cause of loss in enzyme's activity. Taken together, the present study provides insights into mechanisms of DLDH inactivation induced by HNO derived from Angeli's salt.

title: Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione.
abstract: Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

title: Effect of Peroxynitrite on the Mitochondrial Respiratory Chain: Differential Susceptibility of Neurones and Astrocytes in Primary Culture
abstract: The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO−), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO− (initial concentrations of 0.01–2.0 mM) caused, after a subsequent 24‐h incubation, a dose‐dependent decrease in succinate‐cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH‐ubiquinone‐1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO−. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO− in a dose‐dependent manner (40% at 0.5 mM ONOO−) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO−, but astrocytic glutathione was not affected up to 2 mM ONOO−. In isolated brain mitochondria, only succinate‐cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO−). We conclude that the acute exposure of ONOO− selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO−‐mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.

title: Characterisation of the active/de-active transition of mitochondrial complex I☆
abstract: Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39 kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD+/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

title: S-Nitrosation of Mitochondrial Complex I Depends on Its Structural Conformation*
abstract: Nitric oxide is known to cause persistent inhibition of mitochondrial respiration as a result of S-nitrosation of NADH: ubiquinone oxidoreductase (complex I) (Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) ). Little is known about whether such nitrosation occurs in physiological conditions and, if so, what are the possible cellular mechanisms. We have now found that the conformational state (active/deactive transition (Vinogradov, A. D. (1998)  Acta 1364, 169–185)) of mitochondrial complex I is an important factor for the interaction of the enzyme with nitrosothiols and peroxynitrite. Only the deactivated, idle form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. In contrast, the active form of the enzyme was insensitive to such treatment. Neither form of complex I was inhibited by nitric oxide itself. Our data suggest that the process of active/deactive transition plays an important role in the regulation of complex I activity and cellular respiration by nitric oxide. The implications of this finding for hypoxic or pathophysiological conditions in vivo are discussed.

title: Inducible nitric oxide synthase-derived extracellular nitric oxide flux regulates proinflammatory responses at the single cell level
abstract: The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2−/− mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2high cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2high cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2high cells, NO flux, and the inflammatory status of other cells, such as Cox2high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors.

title: Sorting cells alters their redox state and cellular metabolome
abstract: A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

title: An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates.
abstract: A spectrophotometric assay for the pyruvate dehydrogenase complex has been developed, which measures NADH production by coupling it to the reduction of a tetrazolium dye via an intermediate electron carrier. The procedure uses only commercial reagents. Dependence on pyruvate, coenzyme A, and NAD is complete, and activities are comparable to those obtained by other procedures with both purified (rat kidney mitochondria) and crude (mouse brain homogenate) preparations. These dye-coupled procedures are potentially applicable to assays of other NADH-producing dehydrogenases in crude preparations.

title: ‘Idealized’ State 4 and State 3 in Mitochondria vs. Rest and Work in Skeletal Muscle
abstract: A computer model of oxidative phosphorylation (OXPHOS) in skeletal muscle is used to compare state 3, intermediate state and state 4 in mitochondria with rest and work in skeletal muscle. ‘Idealized’ state 4 and 3 in relation to various ‘experimental’ states 4 and 3 are defined. Theoretical simulations show, in accordance with experimental data, that oxygen consumption (V’O2), ADP and Pi are higher, while ATP/ADP and Δp are lower in rest than in state 4, because of the presence of basal ATP consuming reactions in the former. It is postulated that moderate and intensive work in skeletal muscle is very different from state 3 in isolated mitochondria. V’O2, ATP/ADP, Δp and the control of ATP usage over V’O2 are much higher, while ADP and Pi are much lower in the former. The slope of the phenomenological V’O2-ADP relationship is much steeper during the rest-work transition than during the state 4-state 3 transition. The work state in intact muscle is much more similar to intermediate state than to state 3 in isolated mitochondria in terms of ADP, ATP/ADP, Δp and metabolic control pattern, but not in terms of V’O2. The huge differences between intact muscle and isolated mitochondria are proposed to be caused by the presence of the each-step activation (ESA) mechanism of the regulation of OXPHOS in intact skeletal muscle. Generally, the present study suggests that isolated mitochondria (at least in the absence of Ca2+) cannot serve as a good model of OXPHOS regulation in intact skeletal muscle.

title: Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis
abstract: Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I– or complex II–mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6–8 h to complete.

title: Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.
abstract: The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

title: A roadmap for interpreting (13)C metabolite labeling patterns from cells.
abstract: Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.

title: Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells.
abstract: Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

title: A Single Adaptable Cochaperone-Scaffold Complex Delivers Nascent Iron-Sulfur Clusters to Mammalian Respiratory Chain Complexes I-III.
abstract: The iron-sulfur (Fe-S) cluster of the Rieske protein, UQCRFS1, is essential for Complex III (CIII) activity, though the mechanism for Fe-S cluster transfer has not previously been elucidated. Recent studies have shown that the co-chaperone HSC20, essential for Fe-S cluster biogenesis of SDHB, directly binds LYRM7, formerly described as a chaperone that stabilizes UQCRFS1 prior to its insertion into CIII. Here we report that a transient subcomplex involved in CIII assembly, composed of LYRM7 bound to UQCRFS1, interacts with components of an Fe-S transfer complex, consisting of HSC20, its cognate chaperone HSPA9, and the holo-scaffold ISCU. Binding of HSC20 to the LYR motif of LYRM7 in a pre-assembled UQCRFS1-LYRM7 intermediate in the mitochondrial matrix facilitates Fe-S cluster transfer to UQCRFS1. The five Fe-S cluster subunits of Complex I also interact with HSC20 to acquire their clusters, highlighting the crucial role of HSC20 in the assembly of the mitochondrial respiratory chain.

title: Regulation of Cellular Caveolin-1 Protein Expression in Murine Macrophages by Microbial Products
abstract: Previously, we reported that expression of caveolin-1 in elicited peritoneal mouse macrophages was up-regulated by remarkably low (1.0-pg/ml) concentrations of Escherichia coli O111 lipopolysaccharide (LPS). Here we report that increases in caveolin-1 expression are manifested by different types of LPS, LPS-mimetic taxol, and heat-killed E. coli and to a much lesser extent by zymosan, polysaccharide-peptidoglycan, and heat-killed Staphylococcus aureus. Rhodobacter sphaeroides lipid A (RsDPLA) could not induce caveolin-1 expression in macrophages. Interestingly, polymyxin B (5 μg/ml) and RsDPLA show only a limited capacity to inhibit LPS-induced caveolin-1 expression. These findings suggest that expression of caveolin-1 in response to LPS may only partially be dependent upon lipid A. Recombinant tumor necrosis factor alpha marginally induces caveolin-1, suggesting that the ability of LPS to regulate caveolin-1 is not mediated primarily through an autocrine/paracrine mechanism involving this cytokine. Under conditions in which cellular levels of caveolin-1 are profoundly induced, no significant changes in TLR4 expression are observed. Of interest, caveolin-1 appears to localize to two cellular compartments, one associated with lipid rafts and a second associated with TLR4. Gamma interferon treatment inhibits the induction of caveolin-1 by LPS in macrophages. Inhibition of the p38 kinase-dependent pathway, but not the extracellular signal-regulated kinase pathway, effectively reduced the ability of LPS to mediate caveolin-1 up-regulation. Lactacystin, a potent inhibitor of the proteasome pathway, significantly modulates LPS-independent caveolin-1 expression, and lactacystin inhibits LPS-triggered caveolin-1 responses. These studies suggest that caveolin-1 up-regulation in response to LPS is likely to be proteasome dependent and triggered through the p38 kinase pathway.

title: Two functionally independent pathways for lipopolysaccharide-dependent activation of mouse peritoneal macrophages.
abstract: We have investigated the effects of human LPS-binding protein (LBP) and human bactericidal/permeability-increasing protein (BPI) on LPS-dependent activation of mouse thioglycolate-elicited peritoneal macrophages in vitro, in comparison with human PBMCs. Confirming earlier published studies, BPI inhibited, and LBP enhanced, the ability of LPS to stimulate PBMC production of the cytokines TNF-alpha and IL-6. In marked contrast to these results, under identical conditions of in vitro culture, both LBP and BPI suppressed, in a dose-dependent manner, the ability of LPS to stimulate cytokine production in mouse macrophages. Further, while human BPI also suppressed LPS-dependent NO secretion in mouse macrophages, human LBP had no inhibitory effect on NO secretion under conditions that inhibited TNF-alpha secretion. These data provide the first direct evidence that mouse macrophages may utilize two independent pathways in response to LPS, thus leading to different phenotypic responses.

title: Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor.
abstract: Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.