RESUMEN
Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.
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Hormigas , Animales , Hormigas/fisiología , Barrera Hematoencefálica , Encéfalo/metabolismo , Drosophila , Conducta Social , Conducta AnimalRESUMEN
Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.
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Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Precondicionamiento Isquémico , Ácidos Cetoglutáricos/metabolismo , Animales , Isquemia/prevención & control , Ácido Quinurénico/metabolismo , Hígado/metabolismo , Ratones , Modelos Animales , Daño por Reperfusión Miocárdica/prevención & control , ParabiosisRESUMEN
Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.
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Adenosina Trifosfato , Neoplasias de la Mama , Ciclo del Ácido Cítrico , Desaceleración , Neoplasias Pulmonares , Metástasis de la Neoplasia , Neoplasias Pancreáticas , Animales , Ratones , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo del Ácido Cítrico/fisiología , Metabolismo Energético , Glucólisis , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Especificidad de Órganos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Biosíntesis de ProteínasRESUMEN
The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.
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Neoplasias de la Mama/metabolismo , Eliminación de Gen , Neoplasias Mamarias Experimentales/metabolismo , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Exones , Femenino , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación , Metástasis de la Neoplasia , Trasplante de Neoplasias , Empalme del ARNRESUMEN
APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.
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Apolipoproteína E4 , Encéfalo , Colesterol , Fibras Nerviosas Mielínicas , Oligodendroglía , Animales , Humanos , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Colesterol/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Autopsia , Células Madre Pluripotentes Inducidas , Neuronas/metabolismo , Neuronas/patología , Heterocigoto , Transporte Biológico , Homeostasis , Análisis de la Célula Individual , Memoria , Envejecimiento/genética , Perfilación de la Expresión Génica , Vaina de Mielina/metabolismo , Vaina de Mielina/patologíaRESUMEN
Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner. In the kidney, we visualize gluconeogenic flux and glycolytic flux in the cortex and medulla, respectively. Tricarboxylic acid cycle substrate usage differs across kidney regions; glutamine and citrate are used preferentially in the cortex and fatty acids are used in the medulla. In the brain, we observe spatial gradations in carbon inputs to the tricarboxylic acid cycle and glutamate under a ketogenic diet. In a carbohydrate-rich diet, glucose predominates throughout but in a ketogenic diet, 3-hydroxybutyrate contributes most strongly in the hippocampus and least in the midbrain. Brain nitrogen sources also vary spatially; branched-chain amino acids contribute most in the midbrain, whereas ammonia contributes in the thalamus. Thus, iso-imaging can reveal the spatial organization of metabolic activity.
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Encéfalo/metabolismo , Isótopos de Carbono/farmacocinética , Riñón/metabolismo , Isótopos de Nitrógeno/farmacocinética , Animales , Dieta , Enzimas , Gluconeogénesis , Ácido Glutámico/biosíntesis , Glucólisis , Masculino , Ratones Endogámicos C57BL , Imagen Molecular , Análisis de la Célula Individual , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Ácidos Tricarboxílicos/metabolismo , Flujo de TrabajoRESUMEN
Autophagy defects are a risk factor for inflammatory bowel diseases (IBDs) through unknown mechanisms. Whole-body conditional deletion of autophagy-related gene (Atg) Atg7 in adult mice (Atg7Δ/Δ) causes tissue damage and death within 3 mo due to neurodegeneration without substantial effect on intestine. In contrast, we report here that whole-body conditional deletion of other essential Atg genes Atg5 or Fip200/Atg17 in adult mice (Atg5Δ/Δ or Fip200Δ/Δ) caused death within 5 d due to rapid autophagy inhibition, elimination of ileum stem cells, and loss of barrier function. Atg5Δ/Δ mice lost PDGFRα+ mesenchymal cells (PMCs) and Wnt signaling essential for stem cell renewal, which were partially rescued by exogenous Wnt. Matrix-assisted laser desorption ionization coupled to mass spectrometry imaging (MALDI-MSI) of Atg5Δ/Δ ileum revealed depletion of aspartate and nucleotides, consistent with metabolic insufficiency underlying PMC loss. The difference in the autophagy gene knockout phenotypes is likely due to distinct kinetics of autophagy loss, as deletion of Atg5 more gradually extended lifespan phenocopying deletion of Atg7 or Atg12. Thus, autophagy is required for PMC metabolism and ileum stem cell and mammalian survival. Failure to maintain PMCs through autophagy may therefore contribute to IBD.
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Autofagia , Intestinos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Células Madre , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Proteína 7 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Supervivencia Celular , Ratones , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/metabolismoRESUMEN
The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival and improves the metabolic profile of Gpt2-null brain but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms that are required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.
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Personas con Discapacidad , Trastornos Motores , Alanina , Alanina Transaminasa , Animales , Humanos , Ratones , Ratones Noqueados , Neuronas , Ácido Pirúvico , Transaminasas/genéticaRESUMEN
Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase (PK), and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2-null mice (Pkm2(-/-)). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2(-/-) mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.
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Carcinoma Hepatocelular/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Metabolismo Energético/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/fisiopatología , Proliferación Celular/genética , Dieta Alta en Grasa , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Mutación de Línea Germinal , Crecimiento y Desarrollo/genética , Hepatocitos/citología , Homeostasis/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/fisiopatología , Masculino , Ratones , Isoformas de Proteínas , Proteínas de Unión a Hormona TiroideRESUMEN
Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.
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Glucólisis , Metanol , Ratones , Animales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Glucosa , Lípidos , Solventes , Isótopos , Fosfatos , Rayos LáserRESUMEN
BACKGROUND: Metabolic remodeling precedes most alterations during cardiac hypertrophic growth under hemodynamic stress. The elevation of glucose utilization has been recognized as a hallmark of metabolic remodeling. However, its role in cardiac hypertrophic growth and heart failure in response to pressure overload remains to be fully illustrated. Here, we aimed to dissect the role of cardiac PKM1 (pyruvate kinase muscle isozyme 1) in glucose metabolic regulation and cardiac response under pressure overload. METHODS: Cardiac-specific deletion of PKM1 was achieved by crossing the floxed PKM1 mouse model with the cardiomyocyte-specific Cre transgenic mouse. PKM1 transgenic mice were generated under the control of tetracycline response elements, and cardiac-specific overexpression of PKM1 was induced by doxycycline administration in adult mice. Pressure overload was triggered by transverse aortic constriction. Primary neonatal rat ventricular myocytes were used to dissect molecular mechanisms. Moreover, metabolomics and nuclear magnetic resonance spectroscopy analyses were conducted to determine cardiac metabolic flux in response to pressure overload. RESULTS: We found that PKM1 expression is reduced in failing human and mouse hearts. It is important to note that cardiomyocyte-specific deletion of PKM1 exacerbates cardiac dysfunction and fibrosis in response to pressure overload. Inducible overexpression of PKM1 in cardiomyocytes protects the heart against transverse aortic constriction-induced cardiomyopathy and heart failure. At the mechanistic level, PKM1 is required for the augmentation of glycolytic flux, mitochondrial respiration, and ATP production under pressure overload. Furthermore, deficiency of PKM1 causes a defect in cardiomyocyte growth and a decrease in pyruvate dehydrogenase complex activity at both in vitro and in vivo levels. CONCLUSIONS: These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response in the heart under hemodynamic stress.
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Proteínas Portadoras/genética , Susceptibilidad a Enfermedades , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Proteínas de la Membrana/genética , Miocitos Cardíacos/metabolismo , Hormonas Tiroideas/genética , Remodelación Ventricular/genética , Animales , Biomarcadores , Proteínas Portadoras/metabolismo , Respiración de la Célula , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática , Expresión Génica , Glucosa/metabolismo , Glucólisis , Insuficiencia Cardíaca/fisiopatología , Pruebas de Función Cardíaca , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.
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Metaboloma , Metabolómica , Metabolómica/métodos , Proteómica , Flujo de TrabajoRESUMEN
Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.
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Citometría de Flujo , Técnicas Analíticas Microfluídicas , Microfluídica , Neoplasias/diagnóstico , Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Ratones , Microfluídica/métodos , Neoplasias/genética , Células Neoplásicas Circulantes/patología , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
Lysosomes (or lytic bodies) were so named because they contain high levels of hydrolytic enzymes. Lysosome function and dysfunction have been found to play important roles in human disease, including cancer; however, the ways in which lysosomes contribute to tumorigenesis and cancer progression are still being uncovered. Beyond serving as a cellular recycling center, recent evidence suggests that the lysosome is involved in energy homeostasis, generating building blocks for cell growth, mitogenic signaling, priming tissues for angiogenesis and metastasis formation, and activating transcriptional programs. This review examines emerging knowledge of how lysosomal processes contribute to the hallmarks of cancer and highlights vulnerabilities that might be exploited for cancer therapy.
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Lisosomas/metabolismo , Neoplasias/metabolismo , Transducción de Señal/fisiología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Lisosomas/genética , Invasividad Neoplásica/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown. Here we show that Ras-transformed cells use macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.
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Aminoácidos/metabolismo , Transformación Celular Neoplásica , Proteína Oncogénica p21(ras)/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pinocitosis , Animales , Transporte Biológico , Carbono/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Femenino , Glutamina/metabolismo , Ratones , Ratones Desnudos , Células 3T3 NIH , Proteína Oncogénica p21(ras)/genética , Neoplasias Pancreáticas/genética , ProteolisisRESUMEN
Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and â¼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.
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Encéfalo/crecimiento & desarrollo , Mitocondrias/enzimología , Enfermedades del Sistema Nervioso/genética , Transaminasas/genética , Secuencia de Aminoácidos/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ciclo del Ácido Cítrico/genética , Homocigoto , Humanos , Ácidos Cetoglutáricos/metabolismo , Ratones , Mitocondrias/patología , Mutación Missense , Enfermedades del Sistema Nervioso/patología , Fenotipo , Ácido Pirúvico/metabolismo , Transaminasas/metabolismoRESUMEN
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
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Carbono/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/biosíntesis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carbono/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Femenino , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Purinas/biosíntesis , Serina/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Timidina/biosíntesis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
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Biopolímeros/metabolismo , Transformación Celular Neoplásica , Activadores de Enzimas/farmacología , Piruvato Quinasa/metabolismo , Animales , Biopolímeros/química , Western Blotting , Proliferación Celular , Humanos , Ratones , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Piruvato Quinasa/químicaRESUMEN
The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD.