Biology subjects

Contreras-Baeza, Y.

Publications and source records attributed to Contreras-Baeza, Y..

4 recordsLinked to original sources

bioRxiv2026

Opportunistic neuronal fuelling

Neurons can utilize either glucose or lactate, yet fuel selection during periods of activity remains unresolved. By combining optical monitoring of cytosolic pyruvate and NADH with mathematical modelling, we systematically evaluated mitochondrial fuelling in cultured neurons and acute brain slices. Under substrate concentrations typical of resting brain tissue, neurons rely almost exclusively on glucose, with a minor contribution from pyruvate. However, when extracellular lactate rises to levels mimicking tissue activity, glycolysis is inhibited and lactate becomes a major substrate, irrespective of ongoing neuronal activity. Ultimately, neuronal fuel selection is dictated not by internal energy demand, but by extracellular lactate availability, which fluctuates with local glial metabolism and systemic states such as exercise. These findings redefine our understanding of short-term metabolic flexibility in the brain and underscore the significant yet understudied role of extracellular pyruvate.

physiology↗

bioRxiv2025

Two-Point Calibration Protocol for the FRET Indicator Pyronic in Neurons

SignificancePyruvate is a nodal intermediate in cellular metabolism, positioned at the crossroads between glycolysis and fermentative metabolism. It is exchanged between the intracellular and extracellular compartments through the proton-coupled monocarboxylate transporters and between the cytosol and mitochondria through the mitochondrial pyruvate carrier, where it serves as a primary carbon source for respiration. AimOur goal is to present a detailed protocol for quantifying cytosolic pyruvate concentration in neurons at single-cell resolution using a minimally invasive, two-point calibration approach with the FRET-based genetically-encoded fluorescent indicator Pyronic. ApproachThis protocol is based on a non-invasive pharmacological two-point calibration approach, where Pyronics dynamic range ({Delta}RMAX) is established by using trans-acceleration exchange to deplete intracellular pyruvate (RMIN), and by inducing Pyronic saturation (RMAX) through the combination of inhibition of pyruvate export, stimulation of its production, and blockade of its mitochondrial consumption. The protocol also incorporates the previously published KD values for Pyronic obtained from in vitro experiments. This procedure does not require the use of detergents to permeabilize the cells. ResultsImplementing this protocol enables the measurement of absolute cytosolic pyruvate concentrations. This quantitative parameter facilitates comparisons of pyruvate metabolism across different cells, samples and experimental batches, thereby enabling the comparison between a plethora of experimental conditions. ConclusionsThe FRET-based fluorescent indicator Pyronic can be reliably calibrated using a minimally invasive, pharmacology-based two-point calibration protocol in neurons, thus providing a robust and quantitative method to study pyruvate metabolism under various physiological and pathological scenarios.

neuroscience↗

bioRxiv2024

Lactate-carried Mitochondrial Energy Overflow

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. One Sentence SummaryMitochondrial lactate production

biochemistry↗

bioRxiv2019

MCT4 is a high affinity transporter capable of exporting lactate in high-lactate environments

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 Km for 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 Km for lactate uptake of only 1.7 mM, while parallel estimation in the same cells with a pH probe gave a Km of 27 mM. The median Km of 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 Km for 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.

physiology↗