PhD Scientific Days 2023

Budapest, 22-23 June 2023

Pathology - Posters C

The Effect of Glutaminolysis Inhibitors on Synaptosomal Membrane Potential

Gergely Pallag, Department of Biochemistry and Molecular Biology, Semmelweis University, Budapest
Christos, Chinopoulos, Department of Biochemistry and Molecular Biology, Semmelweis University, Budapest

Text of the abstract

Title
The effect of glutaminolysis inhibitors on synaptosomal membrane potential

Introduction
Altered metabolism is a hallmark of cancer cells. Their increased nutrient and energy needs are met mainly in the form of glucose and glutamine. Glutamine-driven mitochondrial substrate-level phosphorylation can partially compensate for reduced ATP synthesis when glycolysis and oxidative phosphorylation are impaired. The isolated nerve terminal (synaptosome) is the simplest preparation that allows mitochondrial bioenergetics to be studied in a physiological milieu. For that reason, synaptosomes from healthy brains can serve as a control prior to examining glutaminolysis in brain tumor cell lines.

Aims
- to find substrates that can generate membrane potential in in situ mitochondria in isolated nerve terminals
- to examine the effect of inhibitors of glutaminolysis on membrane potential

Methods
Animals: Mice were of mixed 129 Sv and C57BL/6 background.
Synaptosomes were prepared from adult male and female mice by Percoll gradient.
The mitochondrial membrane potential of in situ mitochondria of synaptosomes was qualitatively estimated fluorimetrically by loading synaptosomes with potentiometric fluorescent dye tetramethylrhodamine methyl ester.

Result
Glucose (Gluc), lactate (Lac), pyruvate (Pyr), or their combinations (Gluc + Lac, Gluc + Pyr, Gluc + Lac) were used to generate membrane potential.
Among the inhibitors, the most remarkable depolarization of polarised synaptosomes was induced by arsenite. However, this was only seen when a single substrate was used, in their combination the depolarization was considerably reduced.
Aminooxyacetate induced moderate depolarization only when Gluc, Lac, or their combination was present, not in the presence of Pyr. The glutaminase inhibitor CB-839 had a comparable effect on membrane potential to AOAA. Another glutaminase inhibitor BPTES also contributed to a mild depolarization, albeit to a lesser extent.
Glutamine transporter inhibitor GPNA, alanine aminotransferase inhibitor L-Cycloserine, and glutamine analog DON did not change the membrane potential.

Conclusion
Given that heterogenous metabolic pathways can contribute to cellular bioenergetics, the potential effects of glutaminolysis inhibitors on glutamine-independent pathways should be taken into consideration.

Funding
ÚNKP-22-3-II