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.

Edited by Mike Shipston 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 K m 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 K m 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 K m 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 K m 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 highlactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate. Cancer cells ferment glucose to lactate in the presence of oxygen, a phenomenon originally described by Otto Warburg and colleagues in the 1920s and later found to promote tumor growth and malignancy (1-4). In addition to fostering glycolysis by end product removal, cytosolic alkalinization, and NADH recycling, the co-extrusion of lactate and protons causes inter

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.

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 NH + 4 , a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH + 4 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 NH + 4 and in the somatosensory cortex of anesthetized mice in response to i.v. NH + 4 . Unexpectedly, NH + 4 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 NH + 4 diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH + 4 is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH + 4 behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.B rain tissue is almost exclusively energized by the oxidation of glucose. However, during neuronal activation, there is a larger increase in local glucose consumption relative to oxygen consumption (1). As this mismatch occurs in the presence of normal or augmented oxygen levels, it has been termed aerobic glycolysis, paralleling the signal detected by functional magnetic resonance imaging (2). Aerobic glycolysis and its associated lactate surge are causally linked to diverse functions of the brain in health and disease (3-10). Two signals are known to trigger aerobic glycolysis in brain tissue: glutamate and K + , which are released by active neurons and stimulate glycolysis in astrocytes (11,12).Neurons produce as much NH + 4 as they produce glutamate, both molecules being stoichiometrically linked in the glutamateglutamine cycle (13). Brain tissue NH + 4 increases within seconds of neural activation (14-16) and is quickly released to the interstitium (17, 18) to be captured by astrocytes through K + channels and transporters (19). It is well established that chronic exposure to pathological levels of NH + 4 such as those observed during liver failure has a major impact on brain metabolism, but it is not known whether this molecule may affect energy metabolism at physiological levels, particularly within the time scale of synaptic transmission. A previous study showed a reversible rise in brain tissue la...

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.