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

Abstract

Aims/hypothesis: Efficient 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. Methods: Pancreatic 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. Results: We 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/interpretation: We 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.