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The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.
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Granuloma/inmunología , Macrófagos/inmunología , Complejos Multiproteicos/metabolismo , Sarcoidosis/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular , Quinasa 4 Dependiente de la Ciclina/metabolismo , Progresión de la Enfermedad , Granuloma/tratamiento farmacológico , Humanos , Macrófagos/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Sarcoidosis/tratamiento farmacológico , Transducción de Señal , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Allergies are considered to represent mal-directed type 2 immune responses against mostly innocuous exogenous compounds. Immunoglobulin E (IgE) antibodies are a characteristic feature of allergies and mediate hypersensitivity against allergens through activation of effector cells, particularly mast cells (MCs). Although the physiological functions of this dangerous branch of immunity have remained enigmatic, recent evidence shows that allergic immune reactions can help to protect against the toxicity of venoms. Because bacteria are a potent alternative source of toxins, we assessed the possible role of allergy-like type 2 immunity in antibacterial host defense. We discovered that the adaptive immune response against Staphylococcus aureus (SA) skin infection substantially improved systemic host defense against secondary SA infections in mice. Moreover, this acquired protection depended on IgE effector mechanisms and MCs. Importantly, our results reveal a previously unknown physiological function of allergic immune responses, IgE antibodies, and MCs in host defense against a pathogenic bacterium.
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Inmunidad Adaptativa/inmunología , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Alérgenos/inmunología , Animales , Femenino , Hipersensibilidad/inmunología , Hipersensibilidad/microbiología , Mastocitos/microbiología , Ratones , Ratones Endogámicos C57BL , Piel/inmunología , Piel/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/microbiologíaRESUMEN
Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.
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Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Obesidad/genética , Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Adipogénesis , Animales , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Humanos , Ratones , Ratones Noqueados , Células Musculares/metabolismo , Proteínas Represoras/genéticaRESUMEN
OBJECTIVES: Bone remodelling is a highly dynamic process dependent on the precise coordination of osteoblasts and haematopoietic-cell derived osteoclasts. Changes in core metabolic pathways during osteoclastogenesis, however, are largely unexplored and it is unknown whether and how these processes are involved in bone homeostasis. METHODS: We metabolically and transcriptionally profiled cells during osteoclast and osteoblast generation. Individual gene expression was characterised by quantitative PCR and western blot. Osteoblast function was assessed by Alizarin red staining. immunoresponsive gene 1 (Irg1)-deficient mice were used in various inflammatory or non-inflammatory models of bone loss. Tissue gene expression was analysed by RNA in situ hybridisation. RESULTS: We show that during differentiation preosteoclasts rearrange their tricarboxylic acid cycle, a process crucially depending on both glucose and glutamine. This rearrangement is characterised by the induction of Irg1 and production of itaconate, which accumulates intracellularly and extracellularly. While the IRG1-itaconate axis is dispensable for osteoclast generation in vitro and in vivo, we demonstrate that itaconate stimulates osteoblasts by accelerating osteogenic differentiation in both human and murine cells. This enhanced osteogenic differentiation is accompanied by reduced proliferation and altered metabolism. Additionally, supplementation of itaconate increases bone formation by boosting osteoblast activity in mice. Conversely, Irg1-deficient mice exhibit decreased bone mass and have reduced osteoproliferative lesions in experimental arthritis. CONCLUSION: In summary, we identify itaconate, generated as a result of the metabolic rewiring during osteoclast differentiation, as a previously unrecognised regulator of osteoblasts.
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OBJECTIVES: The activator protein-1 (AP-1) transcription factor component c-Fos regulates chondrocyte proliferation and differentiation, but its involvement in osteoarthritis (OA) has not been functionally assessed. METHODS: c-Fos expression was evaluated by immunohistochemistry on articular cartilage sections from patients with OA and mice subjected to the destabilisation of the medial meniscus (DMM) model of OA. Cartilage-specific c-Fos knockout (c-FosΔCh) mice were generated by crossing c-fosfl/fl to Col2a1-CreERT mice. Articular cartilage was evaluated by histology, immunohistochemistry, RNA sequencing (RNA-seq), quantitative reverse transcription PCR (qRT-PCR) and in situ metabolic enzyme assays. The effect of dichloroacetic acid (DCA), an inhibitor of pyruvate dehydrogenase kinase (Pdk), was assessed in c-FosΔCh mice subjected to DMM. RESULTS: FOS-positive chondrocytes were increased in human and murine OA cartilage during disease progression. Compared with c-FosWT mice, c-FosΔCh mice exhibited exacerbated DMM-induced cartilage destruction. Chondrocytes lacking c-Fos proliferate less, have shorter collagen fibres and reduced cartilage matrix. Comparative RNA-seq revealed a prominent anaerobic glycolysis gene expression signature. Consistently decreased pyruvate dehydrogenase (Pdh) and elevated lactate dehydrogenase (Ldh) enzymatic activities were measured in situ, which are likely due to higher expression of hypoxia-inducible factor-1α, Ldha, and Pdk1 in chondrocytes. In vivo treatment of c-FosΔCh mice with DCA restored Pdh/Ldh activity, chondrocyte proliferation, collagen biosynthesis and decreased cartilage damage after DMM, thereby reverting the deleterious effects of c-Fos inactivation. CONCLUSIONS: c-Fos modulates cellular bioenergetics in chondrocytes by balancing pyruvate flux between anaerobic glycolysis and the tricarboxylic acid cycle in response to OA signals. We identify a novel metabolic adaptation of chondrocytes controlled by c-Fos-containing AP-1 dimers that could be therapeutically relevant.
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Cartílago Articular , Osteoartritis , Proteínas Proto-Oncogénicas c-fos , Animales , Humanos , Ratones , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Colágeno/metabolismo , Modelos Animales de Enfermedad , Osteoartritis/patología , Factor de Transcripción AP-1/metabolismo , Proteínas Proto-Oncogénicas c-fos/genéticaRESUMEN
Fluorinated carbohydrates are important tools for understanding the deregulation of metabolic fluxes and pathways. Fluorinating specific positions within the sugar scaffold can lead to enhanced metabolic stability and subsequent metabolic trapping in cells. This principle has, however, never been applied to study the metabolism of the rare sugars of the pentose phosphate pathway (PPP). In this study, two fluorinated derivatives of d-sedoheptulose were designed and synthesized: 4-deoxy-4-fluoro-d-sedoheptulose (4DFS) and 3-deoxy-3-fluoro-d-sedoheptulose (3DFS). Both sugars are taken up by human fibroblasts but only 4DFS is phosphorylated. Fluorination of d-sedoheptulose at C-4 effectively halts the enzymatic degradation by transaldolase and transketolase. 4DFS thus has a high potential as a new PPP imaging probe based on the principle of metabolic trapping. Therefore, the synthesis of potential radiolabeling precursors for 4DFS for future radiofluorinations with fluorine-18 is presented.
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Heptosas , Azúcares , Humanos , Vía de Pentosa Fosfato , HalogenaciónRESUMEN
Fluorinated carbohydrates are valuable tools for enzymological studies due to their increased metabolic stability compared to their non-fluorinated analogues. Replacing different hydroxyl groups within the same monosaccharide by fluorine allows to influence a wide range of sugar-receptor interactions and enzymatic transformations. In the past, this principle was frequently used to study the metabolism of highly abundant carbohydrates, while the metabolic fate of rare sugars is still poorly studied. Rare sugars, however, are key intermediates of many metabolic routes, such as the pentose phosphate pathway (PPP). Here we present the design and purely chemical synthesis of a set of three deoxyfluorinated analogues of the rare sugars d-xylulose and d-ribulose: 1-deoxy-1-fluoro-d-ribulose (1DFRu), 3-deoxy-3-fluoro-d-ribulose (3DFRu) and 3-deoxy-3-fluoro-d-xylulose (3DFXu). Together with a designed set of potential late-stage radio-fluorination precursors, they have the potential to become useful tools for studies on the complex equilibria of the non-oxidative PPP.
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The activation of neurotoxic reactive astrocytes contributes to the pathogenesis of many neurodegenerative diseases. Itaconate, a product of cellular metabolism, is released from activated macrophage/microglia and has been shown to regulate inflammatory responses in several mammalian cells. This study was designed to investigate the impact of cell-permeable dimethyl itaconate (DI) on reactive astrocyte-dependent neurotoxicity. Primary murine astrocyte cells were isolated and stimulated with lipopolysaccharide (LPS) to generate reactive astrocytes. Treating these activated cells with DI was able to diminish the neurotoxic phenotype of reactive astrocytes, as we found reduced LPS-induced Nod-like receptor protein 3 (NLRP3) inflammasome activation and interleukin-1ß (IL-1ß) secretion. DI reduced the level of inflammasome components, attenuated inflammasome assembly and subsequently reduced caspase-1 cleavage and IL-1ß levels. Additionally, DI attenuated nuclear factor-kappa B (NF-κB) phosphorylation in LPS-activated astrocytes and also protected astrocytes from LPS-induced cytotoxicity, including a lowering of Bax and caspase3. DI-treated reactive astrocytes showed an elevated GSH/GSSG ratio and improved antioxidant defense factors including catalase and superoxide dismutase, while lipid peroxidation was reduced. We found that DI activated the nuclear factor 2 (NRF2) and heme oxygenase-1 (HO-1) pathway in astrocytes and thereby potentially control redox-regulation and the inflammatory state of astrocytes. Collectively, these results indicate the neuroprotective role of DI by reprogramming astrocytes from neurotoxic A1 to neuroprotective A2 states and thereby reveal a novel potential strategy for the treatment of neurodegenerative diseases.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Astrocitos/metabolismo , Inflamasomas/metabolismo , Lipopolisacáridos/toxicidad , Mamíferos , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFI , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas NLR , SuccinatosRESUMEN
BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.
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Hígado Graso/enzimología , Fumarato Hidratasa/metabolismo , Resistencia a la Insulina , Hígado/enzimología , Macrófagos/enzimología , MicroARNs/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Obesidad/enzimología , Animales , Carboxiliasas/genética , Carboxiliasas/metabolismo , Ciclo del Ácido Cítrico , Modelos Animales de Enfermedad , Hígado Graso/genética , Fumarato Hidratasa/genética , Fumaratos/metabolismo , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Factor 2 Relacionado con NF-E2/genética , Obesidad/genética , Estrés Oxidativo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Succinatos/metabolismoRESUMEN
We present the first two reported unrelated patients with an isolated sedoheptulokinase (SHPK) deficiency. The first patient presented with neonatal cholestasis, hypoglycemia, and anemia, while the second patient presented with congenital arthrogryposis multiplex, multiple contractures, and dysmorphisms. Both patients had elevated excretion of erythritol and sedoheptulose, and each had a homozygous nonsense mutation in SHPK. SHPK is an enzyme that phosphorylates sedoheptulose to sedoheptulose-7-phosphate, which is an important intermediate of the pentose phosphate pathway. It is questionable whether SHPK deficiency is a causal factor for the clinical phenotypes of our patients. This study illustrates the necessity of extensive functional and clinical workup for interpreting a novel variant, including nonsense variants.
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Vía de Pentosa Fosfato/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Anemia/complicaciones , Anemia/genética , Artrogriposis/genética , Preescolar , Colestasis/complicaciones , Colestasis/genética , Codón sin Sentido , Consanguinidad , Femenino , Heptosas/metabolismo , Humanos , Hipoglucemia/complicaciones , Hipoglucemia/genética , Masculino , Fenotipo , Fosfatos de Azúcar/metabolismoRESUMEN
Dynamic carbon re-routing between catabolic and anabolic metabolism is an essential element of cellular transformation associated with tumour formation and immune cell activation. Such bioenergetic adaptations are important for cellular function and therefore require tight control. Carbohydrate phosphorylation has been proposed as a rate-limiting step of several metabolic networks. The recent identification of a sedoheptulose kinase indicated that free sedoheptulose is a relevant and accessible carbon source in humans. Furthermore, the bioavailability of its phosphorylated form, sedoheptulose 7-phosphate, appears to function as a rheostat for carbon-flux at the interface of glycolysis and the pentose phosphate pathway. In the present paper, we review reports of sedoheptulose metabolism, compare it with glucose metabolism, and discuss the regulation of sedoheptulose kinase as mechanism to achieve bioenergetic reprogramming in cells.
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Metabolismo de los Hidratos de Carbono , Proteínas Quinasas/metabolismo , Fosfatos de Azúcar/metabolismo , Animales , Humanos , Activación de Macrófagos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Fosfatos de Azúcar/químicaRESUMEN
AIMS: Previous risk assessment scores for patients with coronary artery disease (CAD) have focused on primary prevention and patients with acute coronary syndrome. However, especially in stable CAD patients improved long-term risk prediction is crucial to efficiently apply measures of secondary prevention. We aimed to create a clinically applicable mortality prediction score for stable CAD patients based on routinely determined laboratory biomarkers and clinical determinants of secondary prevention. METHODS AND RESULTS: We prospectively included 547 patients with stable CAD and a median follow-up of 11.3 years. Independent risk factors were selected using bootstrapping based on Cox regression analysis. Age, left ventricular function, serum cholinesterase, creatinine, heart rate, and HbA1c were selected as significant mortality predictors for the final multivariable model. The Vienna and Ludwigshafen Coronary Artery Disease (VILCAD) risk score based on the aforementioned variables demonstrated an excellent discriminatory power for 10-year survival with a C-statistic of 0.77 (P < 0.001), which was significantly better than an established risk score based on conventional cardiovascular risk factors (C-statistic = 0.61, P < 0.001). Net reclassification confirmed a significant improvement in individual risk prediction by 34.8% (95% confidence interval: 21.7-48.0%) compared with the conventional risk score (P < 0.001). External validation of the risk score in 1275 participants of the Ludwigshafen Risk and Cardiovascular Health study (median follow-up of 9.8 years) achieved similar results (C-statistic = 0.73, P < 0.001). CONCLUSION: The VILCAD score based on a routinely available set of risk factors, measures of cardiac function, and comorbidities outperforms established risk prediction algorithms and might improve the identification of high-risk patients for a more intensive treatment.
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Biomarcadores/sangre , Enfermedad de la Arteria Coronaria/mortalidad , Anciano , Austria/epidemiología , Enfermedad de la Arteria Coronaria/sangre , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Medición de Riesgo , Factores de RiesgoRESUMEN
Studies indicate that the radiotracer 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) can be metabolized beyond 2-[18F]FDG-6-phosphate (2-[18F]FDG-6-P), but its metabolism is incompletely understood. Most importantly, it remains unclear whether downstream metabolism affects tracer accumulation in vivo. Here we present a fingerprint of 2-[18F]FDG radiometabolites over time in cancer cells, corresponding tumor xenografts and murine organs. Strikingly, radiometabolites representing glycogen metabolism or the oxPPP correlated inversely with tracer accumulation across all examined tissues. Recent studies suggest that not only hexokinase, but also hexose-6-phosphate dehydrogenase (H6PD), an enzyme of the oxidative pentose phosphate pathway (oxPPP), determines 2-[18F]FDG accumulation. However, little is known about the corresponding enzyme glucose-6-phosphate dehydrogenase (G6PD). Our mechanistic in vitro experiments on the role of the oxPPP propose that 2-[18F]FDG can be metabolized via both G6PD and H6PD, but data from separate enzyme knockdown suggest diverging roles in downstream tracer metabolism. Overall, we propose that tissue-specific metabolism beyond 2-[18F]FDG-6-P could matter for imaging.
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BACKGROUND: Low serum butyrylcholinesterase activity was associated with all-cause and cardiovascular mortality in a community-based study; however, there are no data from investigations of the long-term effects of butyrylcholinesterase on mortality in patients with diagnosed coronary artery disease (CAD). We therefore assessed the effect of butyrylcholinesterase activity on the outcomes of patients with CAD. METHODS AND RESULTS: We prospectively included 720 patients in our study: 293 patients with stable CAD and 427 patients with acute coronary syndrome. During a median follow-up of 11.3 years corresponding to 6469 overall person-years, 278 deaths (38.6%) were recorded. We detected a significant and independent protective effect of butyrylcholinesterase on all-cause mortality [adjusted hazard ratio (HR) for a 1-SD increase, 0.62; 95% CI, 0.54-0.71; P < 0.001] and cardiovascular mortality (adjusted HR, 0.64; 95% CI, 0.54-0.76; P < 0.001) in a Cox proportional hazards regression analysis. The 10-year survival rates were 42%, 74%, and 87% in the first, second, and third tertiles of butyrylcholinesterase activity. The presentation of CAD affected the effect of butyrylcholinesterase on mortality (P for interaction = 0.012), with a stronger association found in patients with stable CAD (adjusted HR, 0.56; 95% CI, 0.45-0.70; P < 0.001). CONCLUSIONS: Our study demonstrates a strong inverse association between butyrylcholinesterase activity and long-term outcome in patients with known CAD. Because butyrylcholinesterase added predictive information after adjustment for established cardiovascular risk factors, additional underlying pathophysiological mechanisms and the potential applicability of butyrylcholinesterase activity for secondary risk prediction needs to be addressed in future studies.
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Butirilcolinesterasa/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/enzimología , Síndrome Coronario Agudo/mortalidad , Anciano , Enfermedad de la Arteria Coronaria/enzimología , Enfermedad de la Arteria Coronaria/mortalidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
Macrophages undergo extensive metabolic rewiring upon activation which assist the cell in roles beyond energy production and synthesis of anabolic building blocks. So-called immunometabolites that accumulate upon immune activation can serve as co-factors for enzymes and can act as signaling molecules to modulate cellular processes. As such, the Krebs-cycle-associated metabolites succinate, itaconate and alpha-ketoglutarate (αKG) have emerged as key regulators of macrophage function. Here, we describe that 2-hydroxyglutarate (2HG), which is structurally similar to αKG and exists as two enantiomers, accumulates during later stages of LPS-induced inflammatory responses in mouse and human macrophages. D-2HG was the most abundant enantiomer in macrophages and its LPS-induced accumulation followed the induction of Hydroxyacid-Oxoacid Transhydrogenase (HOT). HOT interconverts αKG and gamma-hydroxybutyrate into D-2HG and succinic semialdehyde, and we here identified this enzyme as being immune-responsive and regulated during the course of macrophage activation. The buildup of D-2HG may be further explained by reduced expression of D-2HG Dehydrogenase (D2HGDH), which converts D-2HG back into αKG, and showed inverse kinetics with HOT and D-2HG levels. We tested the immunomodulatory effects of D-2HG during LPS-induced inflammatory responses by transcriptomic analyses and functional profiling of D-2HG-pre-treated macrophages in vitro and mice in vivo. Together, these data suggest a role for D-2HG in the negative feedback regulation of inflammatory signaling during late-stage LPS-responses in vitro and as a regulator of local and systemic inflammatory responses in vivo. Finally, we show that D-2HG likely exerts distinct anti-inflammatory effects, which are in part independent of αKG-dependent dioxygenase inhibition. Together, this study reveals an immunometabolic circuit resulting in the accumulation of the immunomodulatory metabolite D-2HG that can inhibit inflammatory macrophage responses.
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Antiinflamatorios , Glutaratos , Macrófagos , Receptor Toll-Like 4 , Animales , Antiinflamatorios/farmacología , Glutaratos/farmacología , Humanos , Ácidos Cetoglutáricos/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , RatonesRESUMEN
Metabolism plays a key role in controlling immune cell functions. In this review, we will discuss the diversity of plaque resident myeloid cells and will focus on their metabolic demands that could reflect on their particular intraplaque localization. Defining the metabolic configuration of plaque resident myeloid cells according to their topologic distribution could provide answers to key questions regarding their functions and contribution to disease development.
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Aterosclerosis , Placa Aterosclerótica , Humanos , MacrófagosRESUMEN
The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells' metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells' metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.
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The epidermis is a multi-layered epithelium that consists mainly of keratinocytes which proliferate in its basal layer and then differentiate to form the stratum corneum, the skin's ultimate barrier to the environment. During differentiation keratinocyte function, chemical composition, physical properties, metabolism and secretion are profoundly changed. Extrinsic or intrinsic stressors, like ultraviolet (UV) radiation thus may differently affect the epidermal keratinocytes, depending on differentiation stage. Exposure to UV elicits the DNA damage responses, activation of pathways which detoxify or repair damage or induction of programmed cell death when the damage was irreparable. Recently, rapid diversion of glucose flux into the pentose phosphate pathway (PPP) was discovered as additional mechanism by which cells rapidly generate reduction equivalents and precursors for nucleotides - both being in demand after UV damage. There is however little known about the correlation of such metabolic activity with differentiation state, cell damage and tissue localization of epidermal cells. We developed a method to correlate the activity of G6PD, the first and rate-limiting enzyme of this metabolic UV response, at cellular resolution to cell type, differentiation state, and cell damage in human skin and in organotypic reconstructed epidermis. We thereby could verify rapid activation of G6PD as an immediate UVB response not only in basal but also in differentiating epidermal keratinocytes and found increased activity in cells which initiated DNA damage responses. When keratinocytes had been UVB irradiated before organotypic culture, their distribution within the skin equivalent was abnormal and the G6PD activity was reduced compared to neighboring cells. Finally, we found that the anti-diabetic and potential anti-aging drug metformin strongly induced G6PD activity throughout reconstructed epidermis. Activation of the protective pentose phosphate pathway may be useful to enhance the skin's antioxidant defense systems and DNA damage repair capacity on demand.
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Estrés Oxidativo , Preparaciones Farmacéuticas , Piel , Rayos Ultravioleta , Adulto , Diferenciación Celular , Células Cultivadas , Humanos , Queratinocitos , Preparaciones Farmacéuticas/metabolismo , Piel/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.