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

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.

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 nearcritical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activitydependent control by neurons may contribute to neurodegeneration. V C 2017 Wiley Periodicals, Inc.

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 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.

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 Förster 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.