Biology subjects

Aburto, C.

Publications and source records attributed to Aburto, C..

3 recordsLinked to original sources

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↗

bioRxiv2025

Acute stimulation of glucose metabolism by H2O2 sustains theNADPH steady-state under oxidative stress

Oxidative stress reprograms metabolic flux from glycolysis to the pentose phosphate pathway. Recently, it has been proposed that NADPH acts as a key molecule in pentose phosphate pathway regulation by exerting negative feedback through tonic inhibition of glucose-6-phosphate dehydrogenase. Interestingly, recent studies show that NADPH levels remain stable during acute exposure to hydrogen peroxide in the presence of glucose, ruling out NADPH-dependent feedback inhibition. We hypothesize that hydrogen peroxide triggers a feedforward activation mechanism, increasing NADPH production even before any detectable NADPH depletion. To probe this hypothesis, we used a panel of genetically encoded fluorescent indicators to monitor glucose, NADPH, F1,6BP, and pyruvate in single cells with high temporal resolution. Our results reveal that hydrogen peroxide rapidly activates glucose transport and consumption rates, enabling cells to preserve NADPH steady-state levels during early oxidative stress. Notably, this response precedes NADPH depletion, implying an anticipatory phenomenon that boosts NADPH production prior to its consumption. Furthermore, hydrogen peroxide induced an acute perturbation of F1,6BP steady-state and an increase of pyruvate accumulation. The pharmacological inhibition of the PPPs gateway enzymes, glucose-6-phosphate dehydrogenase and transketolase, abolished the hydrogen peroxide-dependent alterations in F1,6BP steady-state levels and pyruvate accumulation, respectively. These findings suggest that a substantial fraction of glucose-derived carbon flux is diverted to the pentose phosphate pathway under oxidative stress, underscoring the importance of feedforward control in maintaining redox balance.

biochemistry↗

bioRxiv2025

The activation of pentose phosphate pathway flux by hydrogen peroxide is not regulated by NADPH-mediated feedback inhibition

Background and PurposeOxidative stress induces a rerouting of metabolic flux from glycolysis to the pentose phosphate pathway. One proposed mechanism involves negative feedback via tonic inhibition of glucose-6-phosphate dehydrogenase by NADPH. However, recent evidence shows that NADPH levels do not decrease five seconds after hydrogen peroxide (H2O2) treatment. This finding is inconsistent with the canonical model wherein feedback inhibition loop is modulated by NADPH-depletion. This inconsistency prompts us to test the involvement of feedback inhibition at high temporal resolution. Experimental ApproachWe employed genetically encoded fluorescent indicators for H2O2 (HyPerRed) and NADPH (iNap1) expressed in epithelial HEK293 cells. These tools enable simultaneous real-time, single-cell monitoring of NADPH and H2O2. Key ResultsGlucose sustains NADPH levels under acute oxidative stress in the first seconds following H2O2 exposure. This result contradicts the reported feedback inhibition, which is considered one of the fundamental mechanisms to explain the acute rerouting of glycolysis to PPP. Furthermore, pharmacological inhibition of G6PDH suggests that the PPP is the primary source of cytosolic NADPH under oxidative stress. Monitoring NADPH levels following G6PDH inhibition allows for the assessment of the NADPH consumption flux. This parameter is low under baseline conditions, but rises dramatically under oxidative stress. Conclusion and ImplicationsOur results support an anticipatory phenomenon that maintains NADPH levels under acute H2O2 exposure, thereby discarding the proposed feedback inhibition loop. This work offers a new perspective on the regulatory nuances of a metabolic pathway implicated in aging, cancer and a plethora of pathological conditions associated with the deleterious consequences of oxidative stress.

cell biology↗