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bioRxiv · 10.1101/2023.07.25.550538

A practical and robust method to evaluate metabolic fluxes in pancreatic islets

Rocha, D. S.·Manucci, A. C.·Bruni-Cardoso, A.·Kowaltowski, A. J.·Vilas-Boas, E. A.
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Abstract

Aims/hypothesisEfficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. MethodsPancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca2+) influx in response to different modulators by fluorescence microscopy. ResultsWe provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca2+ influx and insulin secretion in response to glucose. Conclusions/interpretationWe established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. Research in context What is already known about this subject?O_LIPancreatic beta cells efficiently couple oxidative phosphorylation and ATP production with insulin secretion; mitochondrial ATP production is crucial for proper insulin secretion. C_LIO_LIMost studies of beta cell respiration use cell lines instead of primary islets, which are a much more robust model to evaluate beta cell function. C_LIO_LIThe few works with primary islet respiration use specific equipment and consumables that are expensive, complicated, and poorly reproducible. C_LI What is the key question?O_LIIs it possible to develop a practical method to evaluate metabolic fluxes and ATP production in isolated islets, using the standard Seahorse Extracellular Flux Technology? C_LI What are the new findings?O_LIWe optimized rodent islet isolation and functional analysis protocols using standard extracellular flux analysis equipment and consumables. C_LIO_LIOur method allows for increased islet yield and robust islet respiration measurements. C_LI How might this impact on clinical practice in the foreseeable future?O_LIQuantitative measurements of metabolic fluxes and oxidative phosphorylation are the cornerstone of new discoveries in beta cells, and can contribute toward the establishment of new cellular protocols, such as for cell transplantation, as well as the development of new pharmacological agents targeted to these cells. C_LI

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Rocha, D. S., Manucci, A. C., Bruni-Cardoso, A., Kowaltowski, A. J., Vilas-Boas, E. A.. 2023-07-26. A practical and robust method to evaluate metabolic fluxes in pancreatic islets. https://doi.org/10.1101/2023.07.25.550538

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