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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?

Lactate is an energy substrate and intercellular signaling molecule with multiple bodily functions. Lactate has physiological roles in neurogenesis, axon integrity, memory consolidation, immune response, exercise, adipose tissue lipolysis, etc, and is involved in inflammation, cancer and neurodegeneration. The FRET lactate indicator Laconic has been instrumental in the discovery of mechanisms involved in neurometabolic coupling, and has advanced the understanding of lactate transport, glycolysis and mitochondrial physiology. However, the low fluorescent response and the complex saturation kinetics of Laconic limit its use for high-throughput screening and quantitation. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed the first single-fluorophore indicator for lactate. The sensor exhibited an intensiometric fluorescence increase of ΔF/F0 3.0 and a single binding site with a KD of 293 μM. The fluorescence is not affected by other monocarboxylates or pH. However, it is sensitive to Ca2+ in the nanomolar range. Targeting of the sensor to the endoplasmic reticulum revealed that this organelle presents a high permeability for lactate. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. This indicator, which we have termed CanlonicSF, is well suited to explore lactate dynamics in environments with micromolar Ca2+ or higher, such as the endoplasmic reticulum and the extracellular space.

The ratio between the cytosolic concentrations of lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a Forster resonance energy transfer (FRET)-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable for use as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in the fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules acetate and β-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK 293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed robust stability over time and was found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.

The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

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 controls the decision between respiration and anaplerosis/gluconeogenesis in an ultrasensitive fashion. The functionality of the sensor in living tissue is demonstrated in the brain ofDrosophila melanogasterlarvae. 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.

Postmortem muscle temperature affects the rate of pH decline in a linear manner from 37.5°C to 0–2°C. The pH decline is correlated with the enzymatic degradation of glycogen to lactate and this process includes the metabolic coupling between glycogenolysis and glycolysis, and that are strongly upregulated by the AMPK. In this study, we used 12 samples previously characterized by have different muscle glycogen concentration, lactate and AMPK activity, selected from 38 steers that produced high final pH (>5.9) and normal final pH ( 0.05) and we did not detect structural differences in the polymers present in samples from both categories (p > 0.05), suggesting that postmortem AMPK activity may be highly sensitive to temperature and not toin vitrochanges in glycogen concentration (p > 0.05). Our results allow concluding that normal concentrations of muscle glycogen immediately at the time of slaughter (0.5 h) and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, through the postmortem AMPK signalling pathway.

MCT4 is an H+-coupled transporter expressed in metastatic cancer cells, macrophages, and other highly glycolytic cells, where it extrudes excess lactate generated by the Warburg phenomenon or by hypoxia. Intriguingly, its reported Kmfor lactate, obtained with pH-sensitive probes, is more than an order of magnitude higher than physiological lactate. Here we examined MCT4-rich MDA-MB-231 cells using the FRET sensor Laconic and found a median Kmfor lactate uptake of only 1.7 mM, while parallel estimation in the same cells with a pH probe gave a Kmof 27 mM. The median Kmof MCT4 for lactate was 0.7 mM in MCT4-expressing HEK293 cells and 1.2 mM in human macrophages, suggesting that high substrate affinity is a robust property of the transporter. Probed with the FRET sensor Pyronic, MCT4 showed a Kmfor pyruvate of only 4.2 mM in MDA-MB-231 cells, as opposed to > 150 mM reported previously. We conclude that prior estimates of MCT4 affinity based on pH probes were severely biased by the confounding action of pH regulatory mechanisms. Numerical simulation showed that MCT4, but not MCT1 or MCT2, endows cells with the capability of lactate extrusion in high lactate environments. The revised kinetic properties and novel transport assays may help in developing small-molecule MCT4 blockers for research and therapy.

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 Forster 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.