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

Author(s)

Rocha, Debora S. ; Manucci, Antonio C. ; Bruni-Cardoso, Alexandre ; Kowaltowski, Alicia J. ; Vilas-Boas, Eloisa A.

• Document type: Journal article
• Source: MOLECULAR METABOLISM; v. 83, p. 13-pg., 2024-04-03.

Abstract

Objective: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to dif ficulties in measuring primary islet respiration, which requires speci fic equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. Methods: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre -coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. Results: We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium in flux and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. Conclusions: Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic (3 -cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. (c) 2024 The Published Elsevier GmbH. This is an access article under the CC BY -NC license (AU)

• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 20/06970-5 - Mitochondrial ion transporters as sensors and regulators in energy metabolism
• Grantee: Alicia Juliana Kowaltowski
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 21/02481-2 - Energy metabolism regulation by mitochondrial Ca2+ transport in the liver
• Grantee: Eloisa Aparecida Vilas Boas
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 21/04781-3 - Energy metabolism regulation by beta-type estradiol receptor in hepatocytes and adipocytes submitted to energy overload
• Grantee: Débora Santos Rocha
• Support Opportunities: Scholarships in Brazil - Post-Doctoral