RESUMO
We addressed the question of mitochondrial lactate metabolism using genetically-encoded sensors. The organelle was found to contain a dynamic lactate pool that leads to dose- and time-dependent protein lactylation. In neurons, mitochondrial lactate reported blood lactate levels with high fidelity. The exchange of lactate across the inner mitochondrial membrane was found to be mediated by a high affinity H+-coupled transport system involving the mitochondrial pyruvate carrier MPC. Assessment of electron transport chain activity and determination of lactate flux showed that mitochondria are tonic lactate producers, a phenomenon driven by energization and stimulated by hypoxia. We conclude that an overflow mechanism caps the redox level of mitochondria, while saving energy in the form of lactate.
RESUMO
Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Técnicas Biossensoriais , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Larva/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos/genéticaRESUMO
Glycolysis is the core of intermediate metabolism, an ancient pathway discovered in the heydays of classic biochemistry. A hundred years later, it remains a matter of active research, clinical interest and is not devoid of controversy. This review examines topical aspects of glycolysis in the brain, a tissue characterized by an extreme dependence on glucose. The limits of glycolysis are reviewed in terms of flux control by glucose transporters, intercellular lactate shuttling and activity-dependent glycolysis in astrocytes and neurons. What is the site of glycogen mobilization and aerobic glycolysis in brain tissue? We scrutinize the pervasive notions that glycolysis is fast and that catalysis is channeled through supramolecular assemblies. In brain tissue, most glycolytic enzymes are catalytically silent. What then is their function?
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Glicogênio/metabolismo , Glicólise/fisiologia , Ácido Láctico/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/química , Química Encefálica/fisiologia , Metabolismo Energético/fisiologia , Glucose/análise , Glucose/metabolismo , Glicogênio/análise , Humanos , Ácido Láctico/análise , Neurônios/química , Fatores de TempoRESUMO
Mitochondrial toxicity is a primary source of pre-clinical drug attrition, black box warning and post-market drug withdrawal. Methods that detect mitochondrial toxicity as early as possible during the drug development process are required. Here we introduce a new method for detecting mitochondrial toxicity based on MDA-MB-231 cells stably expressing the genetically encoded FRET lactate indicator, Laconic. The method takes advantage of the high cytosolic lactate accumulation observed during mitochondrial stress, regardless of the specific toxicity mechanism, explained by compensatory glycolytic activation. Using a standard multi-well plate reader, dose-response curve experiments allowed the sensitivity of the methodology to detect metabolic toxicity induced by classical mitochondrial toxicants. Suitability for high-throughput screening applications was evaluated resulting in a Z'-factor > 0.5 and CV% < 20 inter-assay variability. A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes. We envisage that the availability of this technology, based on a fluorescent genetically encoded indicator, will allow direct assessment of mitochondrial metabolism, and will make the early detection of mitochondrial toxicity in the drug development process possible, saving time and resources.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Mitocôndrias/efeitos dos fármacos , Testes de Toxicidade/métodos , Bioensaio , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Ácido Láctico/metabolismo , Sensibilidade e EspecificidadeRESUMO
Monocarboxylate transporter 4 (MCT4) is an H+-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mm, the reported Km of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective Km values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a Km for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous Km estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in high-lactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate.
Assuntos
Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Transporte Biológico/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Diclofenaco/farmacologia , Fluoresceínas/química , Edição de Genes , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Cinética , Macrófagos/citologia , Macrófagos/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Recent articles have drawn renewed attention to the housekeeping glucose transporter GLUT1 and its possible involvement in neurodegenerative diseases. Here we provide an updated analysis of brain glucose transport and the cellular mechanisms involved in its acute modulation during synaptic activity. We discuss how the architecture of the blood-brain barrier and the low concentration of glucose within neurons combine to make endothelial/glial GLUT1 the master controller of neuronal glucose utilization, while the regulatory role of the neuronal glucose transporter GLUT3 emerges as secondary. The near-critical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activity-dependent control by neurons may contribute to neurodegeneration. © 2017 Wiley Periodicals, Inc.