Biology subjects

Galaz, A.

Publications and source records attributed to Galaz, A..

4 recordsLinked to original sources

bioRxiv2020

Highly responsive single-fluorophoreindicator to explore lactate dynamics in high calcium environments

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 {Delta}F/F0 3.0 and a single binding site with a KD of 293 M. 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.

cell biology↗

bioRxiv2020

Temperature, but not excess of glycogen, regulate post-mortem AMPK activity in muscle of steer carcasses.

Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 {degrees}C down near 0 {degrees}C, and this pH decline is correlated with the enzymatic degradation of glycogen to lactate. This transformation occurs in an anaerobic context that includes the metabolic splice between glycogenolysis and glycolysis; and both processes are strongly upregulated by AMPK enzyme. In this study we reported changes (0.5 h and 24 h post-mortem) in muscle glycogen concentration, lactate and AMPK activity from 12 samples of Longissimus dorsi from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24 h post-mortem. Moreover, we evaluated changes in AMPK activity in samples from both categories incubated at 37, 25, 17 and 5 {degrees}C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our analyses show that enzymatic AMPK activity was significantly higher at 17 {degrees}C than at 37 {degrees}C or 25 {degrees}C (p<0.0001 and p<0.05 in samples from normal and high pH categories, respectively), and was near zero at 5 {degrees}C. On the other hand, AMPK activity did not change in relation with excess glycogen and we did not detect structural differences in the polymers present in samples from both categories. We concluded that post-mortem AMPK activity level is highly sensitive to temperature and not at in vitro changes in glycogen concentration. Their results suggest that that normal levels of pre-mortem muscle glycogen and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, trough of post-mortem AMPK signalling pathway.

biochemistry↗

bioRxiv2019

A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC

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.

cell biology↗

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↗