Whole-brain neuronal MCT2 lactate transporter expression links metabolism to human brain structure and function.

Brain activity is constrained by local availability of chemical energy, which is generated through compartmentalized metabolic processes. By analyzing data of whole human brain gene expression, we characterize the spatial distribution of seven glucose and monocarboxylate membrane transporters that mediate astrocyte-neuron lactate shuttle transfer of energy. We found that the gene coding for neuronal MCT2 is the only gene enriched in cerebral cortex where its abundance is inversely correlated with cortical thickness. Coexpression network analysis revealed that MCT2 was the only gene participating in an organized gene cluster enriched in K[Formula: see text] dynamics. Indeed, the expression of K[Formula: see text] subunits, which mediate lactate increases with spiking activity, is spatially coupled to MCT2 distribution. Notably, MCT2 expression correlated with fluorodeoxyglucose positron emission tomography task-dependent glucose utilization. Finally, the MCT2 messenger RNA gradient closely overlaps with functional MRI brain regions associated with attention, arousal, and stress. Our results highlight neuronal MCT2 lactate transporter as a key component of the cross-talk between astrocytes and neurons and a link between metabolism, cortical structure, and state-dependent brain function.

Dietary Fiber Modulates the Release of Gut Bacterial Products Preventing Cognitive Decline in an Alzheimer's Mouse Model.

Fiber intake is associated with a lower risk for Alzheimer´s disease (AD) in older adults. Intake of plant-based diets rich in soluble fiber promotes the production of short-chain fatty acids (SCFAs: butyrate, acetate, propionate) by gut bacteria. Butyrate administration has antiinflammatory actions, but propionate promotes neuroinflammation. In AD patients, gut microbiota dysbiosis is a common feature even in the prodromal stages of the disease. It is unclear whether the neuroprotective effects of fiber intake rely on gut microbiota modifications and specific actions of SCFAs in brain cells. Here, we show that restoration of the gut microbiota dysbiosis through the intake of soluble fiber resulted in lower propionate and higher butyrate production, reduced astrocyte activation and improved cognitive function in 6-month-old male APP/PS1 mice. The neuroprotective effects were lost in antibiotic-treated mice. Moreover, propionate promoted higher glycolysis and mitochondrial respiration in astrocytes, while butyrate induced a more quiescent metabolism. Therefore, fiber intake neuroprotective action depends on the modulation of butyrate/propionate production by gut bacteria. Our data further support and provide a mechanism to explain the beneficial effects of dietary interventions rich in soluble fiber to prevent dementia and AD. Fiber intake restored the concentration of propionate and butyrate by modulating the composition of gut microbiota in male transgenic (Tg) mice with Alzheimer´s disease. Gut dysbiosis was associated with intestinal damage and high propionate levels in control diet fed-Tg mice. Fiber-rich diet restored intestinal integrity and promoted the abundance of butyrate-producing bacteria. Butyrate concentration was associated with better cognitive performance in fiber-fed Tg mice. A fiber-rich diet may prevent the development of a dysbiotic microbiome and the related cognitive dysfunction in people at risk of developing Alzheimer´s disease.

Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur.

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.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect.

Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments.

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.

Fluid Brain Glycolysis: Limits, Speed, Location, Moonlighting, and the Fates of Glycogen and Lactate.

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?

Nanomolar nitric oxide concentrations quickly and reversibly modulate astrocytic energy metabolism.

Nitric oxide (NO) is an intercellular messenger involved in multiple bodily functions. Prolonged NO exposure irreversibly inhibits respiration by covalent modification of mitochondrial cytochrome oxidase, a phenomenon of pathological relevance. However, the speed and potency of NO's metabolic effects at physiological concentrations are incompletely characterized. To this end, we set out to investigate the metabolic effects of NO in cultured astrocytes from mice by taking advantage of the high spatiotemporal resolution afforded by genetically encoded Förster resonance energy transfer (FRET) nanosensors. NO exposure resulted in immediate and reversible intracellular glucose depletion and lactate accumulation. Consistent with cytochrome oxidase involvement, the glycolytic effect was enhanced at a low oxygen level and became irreversible at a high NO concentration or after prolonged exposure. Measurements of both glycolytic rate and mitochondrial pyruvate consumption revealed significant effects even at nanomolar NO concentrations. We conclude that NO can modulate astrocytic energy metabolism in the short term, reversibly, and at concentrations known to be released by endothelial cells under physiological conditions. These findings suggest that NO modulates the size of the astrocytic lactate reservoir involved in neuronal fueling and signaling.

Glia in brain energy metabolism: A perspective.

Early views of glia as relatively inert, housekeeping cells have evolved, and glia are now recognized as dynamic cells that not only respond to neuronal activity but also sense metabolic changes and regulate neuronal metabolism. This evolution has been aided in part by technical advances permitting progressively better spatial and temporal resolution. Recent advances in cell-type specific genetic manipulation and sub-cellular metabolic probes promise to further this evolution by enabling study of metabolic interactions between intertwined fine neuronal and glial processes in vivo. Views of glia in disease processes have also evolved. Long considered purely reactive, glia and particularly microglia are now seen to play active roles in both promoting and limiting brain injury. At the same time, established concepts of glial energetics are now being linked to areas such as learning and neural network function, topics previously considered far removed from glial biology.

Current technical approaches to brain energy metabolism.

Neuroscience is a technology-driven discipline and brain energy metabolism is no exception. Once satisfied with mapping metabolic pathways at organ level, we are now looking to learn what it is exactly that metabolic enzymes and transporters do and when, where do they reside, how are they regulated, and how do they relate to the specific functions of neurons, glial cells, and their subcellular domains and organelles, in different areas of the brain. Moreover, we aim to quantify the fluxes of metabolites within and between cells. Energy metabolism is not just a necessity for proper cell function and viability but plays specific roles in higher brain functions such as memory processing and behavior, whose mechanisms need to be understood at all hierarchical levels, from isolated proteins to whole subjects, in both health and disease. To this aim, the field takes advantage of diverse disciplines including anatomy, histology, physiology, biochemistry, bioenergetics, cellular biology, molecular biology, developmental biology, neurology, and mathematical modeling. This article presents a well-referenced synopsis of the technical side of brain energy metabolism research. Detail and jargon are avoided whenever possible and emphasis is given to comparative strengths, limitations, and weaknesses, information that is often not available in regular articles.

NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells.

Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and Epithelial Factors.

Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc.

Near-critical GLUT1 and Neurodegeneration.

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.

Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Sodium signaling and astrocyte energy metabolism.

The Na(+) gradient across the plasma membrane is constantly exploited by astrocytes as a secondary energy source to regulate the intracellular and extracellular milieu, and discard waste products. One of the most prominent roles of astrocytes in the brain is the Na(+) -dependent clearance of glutamate released by neurons during synaptic transmission. The intracellular Na(+) load collectively generated by these processes converges at the Na,K-ATPase pump, responsible for Na(+) extrusion from the cell, which is achieved at the expense of cellular ATP. These processes represent pivotal mechanisms enabling astrocytes to increase the local availability of metabolic substrates in response to neuronal activity. This review presents basic principles linking the intracellular handling of Na(+) following activity-related transmembrane fluxes in astrocytes and the energy metabolic pathways involved. We propose a role of Na(+) as an energy currency and as a mediator of metabolic signals in the context of neuron-glia interactions. We further discuss the possible impact of the astrocytic syncytium for the distribution and coordination of the metabolic response, and the compartmentation of these processes in cellular microdomains and subcellular organelles. Finally, we illustrate future avenues of investigation into signaling mechanisms aimed at bridging the gap between Na(+) and the metabolic machinery. GLIA 2016;64:1667-1676.

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons.

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders.

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Higher transport and metabolism of glucose in astrocytes compared with neurons: a multiphoton study of hippocampal and cerebellar tissue slices.

Glucose is the most important energy substrate for the brain, and its cellular distribution is a subject of great current interest. We have employed fluorescent glucose probes, the 2-deoxy-D-glucose derivates 6- and 2-([N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose) (2-NBDG), to measure transport and metabolism of glucose in acute slices of mouse hippocampus and cerebellum. In the hippocampus, 6-NBDG, which is not metabolized and hence indicates glucose transport, was taken up faster in astrocyte-rich layers (Stratum radiatum [S.r.], Stratum oriens [S.o.]) than in pyramidal cells. Metabolizable 2-NBDG showed larger signals in S.r. and S.o. than in Stratum pyramidale, suggesting faster glucose utilization rate in the astrocyte versus the neuronal compartment. Similarly, we found higher uptake and temperature-sensitive metabolism of 2-NBDG in Bergmann glia when compared with adjacent Purkinje neurons of cerebellar slices. A comparison between 6-NBDG transport and glucose transport in cultured cells using a fluorescence resonance energy transfer nanosensor showed that relative to glucose, 6-NBDG is transported better by neurons than by astrocytes. These results indicate that the preferential transport and metabolism of glucose by glial cells versus neurons proposed for the hippocampus and cerebellum by ourselves (in vitro) and for the barrel cortex by Chuquet et al. (in vivo) is more pronounced than anticipated.

Non-preferential fuelling of the Na(+)/K(+)-ATPase pump.

There is abundant evidence that glycolysis and the Na(+)/K(+)-ATPase pump are functionally coupled, and it is thought that the nature of the coupling is energetic, with glycolysis providing the ATP that fuels the pump. This notion has been instrumental to current models of brain energy metabolism. However, structural and biophysical considerations suggest that the pump should also have access to mitochondrial ATP, which is much more abundant. In the present study, we have investigated the source of ATP that fuels the Na(+) pump in astrocytes, taking advantage of the high temporal resolution of recently available FRET nanosensors for glucose, lactate and ATP. The activity of the Na(+) pump was assessed in parallel with the Na(+)-sensitive dye SBFI AM (Na(+)-binding benzofuran isophthalate acetoxymethyl ester). OXPHOS (oxidative phosphorylation) inhibition resulted in bulk ATP depletion and a 5-fold stimulation of glycolytic flux, in spite of which Na(+) pumping was inhibited by 90%. Mathematical modelling of ATP dynamics showed that the observed pump failure is inconsistent with preferential fuelling of the Na(+) pump by glycolytic ATP. We conclude that the nature of the functional coupling between the Na(+) pump and the glycolytic machinery is not energetic and that the pump is mainly fuelled by mitochondrial ATP.