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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.
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Dysregulated central-energy metabolism is a hallmark of brain aging. Supplying enough energy for neurotransmission relies on the neuron-astrocyte metabolic network. To identify genes contributing to age-associated brain functional decline, we formulated an approach to analyze the metabolic network by integrating flux, network structure and transcriptomic databases of neurotransmission and aging. Our findings support that during brain aging: (1) The astrocyte undergoes a metabolic switch from aerobic glycolysis to oxidative phosphorylation, decreasing lactate supply to the neuron, while the neuron suffers intrinsic energetic deficit by downregulation of Krebs cycle genes, including mdh1 and mdh2 (Malate-Aspartate Shuttle); (2) Branched-chain amino acid degradation genes were downregulated, identifying dld as a central regulator; (3) Ketone body synthesis increases in the neuron, while the astrocyte increases their utilization, in line with neuronal energy deficit in favor of astrocytes. We identified candidates for preclinical studies targeting energy metabolism to prevent age-associated cognitive decline.
Assuntos
Astrócitos , Metabolismo Energético , Astrócitos/metabolismo , Metabolismo Energético/genética , Transmissão Sináptica , Perfilação da Expressão Gênica , Glucose/metabolismoRESUMO
Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and in vivo, acquired with genetically-encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K+ coincides with a Warburg effect mediated by NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4+. The compounded outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na+ pump. We conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.
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Encéfalo/fisiologia , Glicólise/fisiologia , Animais , Astrócitos/metabolismo , Respiração Celular/fisiologia , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismoRESUMO
Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.
Assuntos
Ácido Láctico/metabolismo , Macrófagos/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Células THP-1/metabolismoRESUMO
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?
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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
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
Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors in vitro and in vivo. A short theta burst triggered immediate Na+ entry, followed by a delayed stimulation of the Na+/K+ ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na+ pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na+ pump stimulation. Experiments revealed that the Na+ pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca2+, but by a mechanism commanded by the Na+ pump.
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Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Astrócitos/citologia , Metabolismo Energético , Glucose/metabolismo , Glicólise , Homeostase , Camundongos Endogâmicos C57BL , Neurônios/citologiaRESUMO
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.
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Encéfalo/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/patologia , Metabolismo Energético/fisiologia , Transportador de Glucose Tipo 1/deficiência , Humanos , Doenças Neurodegenerativas/patologia , Neurônios/patologiaRESUMO
Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.