RESUMEN
BACKGROUND AND AIMS: Dysregulated cholesterol metabolism is a hallmark of atherosclerotic cardiovascular diseases, yet our understanding of how endogenous cholesterol synthesis affects atherosclerosis is not clear. The energy sensor AMP-activated protein kinase (AMPK) phosphorylates and inhibits the rate-limiting enzyme in the mevalonate pathway HMG-CoA reductase (HMGCR). Recent work demonstrated that when AMPK-HMGCR signaling was compromised in an Apoe-/- model of hypercholesterolemia, atherosclerosis was exacerbated due to elevated hematopoietic stem and progenitor cell mobilization and myelopoiesis. We sought to validate the significance of the AMPK-HMGCR signaling axis in atherosclerosis using a non-germline hypercholesterolemia model with functional ApoE. METHODS: Male and female HMGCR S871A knock-in (KI) mice and wild-type (WT) littermate controls were made atherosclerotic by intravenous injection of a gain-of-function Pcsk9D374Y-adeno-associated virus followed by high-fat and high-cholesterol atherogenic western diet feeding for 16 weeks. RESULTS: AMPK activation suppressed endogenous cholesterol synthesis in primary bone marrow-derived macrophages from WT but not HMGCR KI mice, without changing other parameters of cholesterol regulation. Atherosclerotic plaque area was unchanged between WT and HMGCR KI mice, independent of sex. Correspondingly, there were no phenotypic differences observed in hematopoietic progenitors or differentiated immune cells in the bone marrow, blood, or spleen, and no significant changes in systemic markers of inflammation. When lethally irradiated female mice were transplanted with KI bone marrow, there was similar plaque content relative to WT. CONCLUSIONS: Given previous work, our study demonstrates the importance of preclinical atherosclerosis model comparison and brings into question the importance of AMPK-mediated control of cholesterol synthesis in atherosclerosis.
RESUMEN
In solid tumors, drug concentrations decrease with distance from blood vessels. However, cellular adaptations accompanying the gradated exposure of cancer cells to drugs are largely unknown. Here, we modeled the spatiotemporal changes promoting chemotherapy resistance in breast cancer. Using pairwise cell competition assays at each step during the acquisition of chemoresistance, we reveal an important priming phase that renders cancer cells previously exposed to sublethal drug concentrations refractory to dose escalation. Therapy-resistant cells throughout the concentration gradient display higher expression of the solute carriers SLC38A7 and SLC46A1 and elevated intracellular concentrations of their associated metabolites. Reduced levels of SLC38A7 and SLC46A1 diminish the proliferative potential of cancer cells, and elevated expression of these SLCs in breast tumors from patients correlates with reduced survival. Our work provides mechanistic evidence to support dose-intensive treatment modalities for patients with solid tumors and reveals two members of the SLC family as potential actionable targets.
Asunto(s)
Neoplasias de la Mama , Neoplasias Mamarias Animales , Animales , Humanos , Femenino , Resistencia a Antineoplásicos/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mama/metabolismo , Transportador de Folato Acoplado a ProtónRESUMEN
Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.
Asunto(s)
Interleucina-4 , Activación de Macrófagos , Animales , Ratones , Colina/metabolismo , Citocinas/metabolismo , Interleucina-4/metabolismo , Macrófagos , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
The CD11c+ MHCII+ compartment within GM-CSF cultures consists of a MHCIIlow CD11bhigh population (GM-Macs) and a MHCIIhigh CD11bint population (GM-DCs), with different metabolic profiles. GM-Macs upregulate iNOS and produce nitric oxide (NO) upon TLR activation inhibiting mitochondrial respiration (OXPHOS) while promoting glycolytic metabolism in GM-DCs, which naturally do not express iNOS.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , Óxido Nítrico , Ratones , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Células Dendríticas/metabolismo , Diferenciación Celular , Ratones Endogámicos C57BLRESUMEN
Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Epirrubicina/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Dendritic cells (DCs) are key orchestrators of immunity and tolerance. It has become evident that DC function can be influenced by cellular metabolic programs. However, conclusions from early metabolic studies using in vitro GM-CSF DC cultures fail to correlate with bona fide DC populations. Here, we discuss the existing paradigms in the DC metabolism field, focusing on the limitations of the models utilized. Furthermore, we introduce alternative models to generate DCs in vitro that better emulate DCs found in vivo. Finally, we highlight new techniques to evaluate DC metabolism at the single-cell level. The combination of these two strategies could help advance the DC metabolism field towards a more physiological understanding, which is crucial for the development of effective DC-based therapies.
Asunto(s)
Células Dendríticas , Diferenciación CelularRESUMEN
Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 µM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.
Asunto(s)
Acilcoenzima A/fisiología , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Homeostasis/efectos de los fármacos , Macrófagos , Mitocondrias , Células 3T3 , Células A549 , Animales , Carnitina O-Palmitoiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Células HCT116 , Células Hep G2 , Humanos , Interleucina-4/metabolismo , Hígado/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection.
Asunto(s)
Células Dendríticas/inmunología , Ácidos Grasos/inmunología , Inmunidad Innata , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Tuberculosis/inmunología , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/inmunología , Animales , Células Dendríticas/microbiología , Células Dendríticas/patología , Ácidos Grasos/genética , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Noqueados , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/genética , Células TH1/microbiología , Células TH1/patología , Tuberculosis/genética , Tuberculosis/patologíaRESUMEN
Conjugated linoleic acid (CLA) is a prime substrate for intra-gastric nitration giving rise to the formation of nitro-conjugated linoleic acid (NO2-CLA). Herein, NO2-CLA generation is demonstrated within the context of acute inflammatory responses both in vitro and in vivo. Macrophage activation resulted in dose- and time-dependent CLA nitration and also in the production of secondary electrophilic and non-electrophilic derivatives. Both exogenous NO2-CLA as well as that generated in situ, attenuated NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and up-regulated Nrf2-regulated proteins. Importantly, both CLA nitration and the corresponding downstream anti-inflammatory actions of NO2-CLA were recapitulated in a mouse peritonitis model where NO2-CLA administration decreased pro-inflammatory cytokines and inhibited leukocyte recruitment. Taken together, our results demonstrate that the formation of NO2-CLA has the potential to function as an adaptive response capable of not only modulating inflammation amplitude but also protecting neighboring tissues via the expression of Nrf2-dependent genes.
Asunto(s)
Inmunoconjugados/metabolismo , Inflamación/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Óxido Nítrico/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Inmunoconjugados/inmunología , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Ácidos Linoleicos Conjugados/inmunología , Ácidos Linoleicos Conjugados/farmacología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/química , Óxido Nítrico/inmunología , Transducción de SeñalRESUMEN
Dendritic cells (DCs) determine the outcome of the immune response based on signals they receive from the environment. Presentation of antigen under various contexts can lead to activation and differentiation of T cells for immunity or dampening of immune responses by establishing tolerance, primarily through the priming of regulatory T cells. Infections, inflammation and normal cellular interactions shape DC responses through direct contact or via cytokine signaling. Although it is widely accepted that DCs sense microbial components through pattern recognition receptors (PRRs), increasing evidence advocates for the existence of a set of signals that can profoundly shape DC function via PRR-independent pathways. This diverse group of host- or commensal-derived metabolites represents a newly appreciated code from which DCs can interpret environmental cues. In this review, we discuss the existing information on the effect of some of the most studied metabolites on DC function, together with the implications this may have in immune-mediated diseases.
Asunto(s)
Microambiente Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Metaboloma , Animales , Biomarcadores , Células Dendríticas/clasificación , Células Dendríticas/citología , Metabolismo Energético , Humanos , Especificidad de Órganos/inmunología , Fenotipo , Transducción de SeñalRESUMEN
The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2(-)) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope-labeled NO2(-) and LC-MS/MS analysis of products reveals that NO2(-) also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide ((â¢)NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of (â¢)NO and NO2(-) shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2(-) propagation of (â¢)NO signaling and the regulation of both biomolecule function and signaling network activity via NO2(-)-dependent nitrosation and nitration reactions.
Asunto(s)
Macrófagos/química , Nitratos/química , Óxido Nítrico/química , Nitritos/química , Óxidos de Nitrógeno/química , Ácido Nitroso/química , Animales , Línea Celular , Glutatión/química , Glutatión/metabolismo , Concentración de Iones de Hidrógeno , Inflamación/inducido químicamente , Inflamación/metabolismo , Cinética , Ácidos Linoleicos Conjugados/química , Ácidos Linoleicos Conjugados/metabolismo , Ácidos Linoleicos Conjugados/farmacología , Lipopolisacáridos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Nitritos/farmacología , Isótopos de Nitrógeno , Óxidos de Nitrógeno/metabolismo , Nitrosación , Ácido Nitroso/metabolismo , Isótopos de OxígenoRESUMEN
Lipid nitration has been observed during oxidative/nitrative stress conditions generating a variety of biomolecules capable of modulating cellular responses. This concept has grown as a result of studies with nitro-derivatives of long-chain unsaturated fatty acids containing a nitroalkene group (nitro-fatty acids). This review focuses on the interactions of nitro-fatty acids with members of the peroxisome proliferator-activated receptors (PPARs) family. These nuclear receptors belong to a superfamily of ligand-activated transcription factors, which serve as sensors of lipophilic molecules and regulate the expression of a set of genes involved in glucose and lipid metabolism. Here we discuss how nitro-fatty acids bind and activate PPARs, including the current knowledge of the molecular interactions and cell signaling events involved as well as their therapeutic potential associated with chronic inflammation and metabolic disorders.