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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
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Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets

Juliane Röthe1,2, Robert Kraft3, Torsten Schöneberg1

  • 11Rudolf Schönheimer Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

Biological Procedures Online
|February 22, 2020
PubMed
Abstract

Insights

Researchers developed a new method to study G protein-coupled receptor (GPCR) activation in intact pancreatic islets. This protocol reliably measures second messenger signals like IP1, cAMP, and calcium, crucial for understanding insulin secretion.

Area of Science:

  • Endocrinology
  • Cell Biology
  • Pharmacology

Background:

  • G protein-coupled receptors (GPCRs) are targets for modulating insulin secretion.
  • Discrepancies exist between cell line and primary islet responses due to islet complexity.
  • A robust method is needed to study GPCR signaling in intact pancreatic islets.

Purpose of the Study:

  • To establish a protocol for analyzing second messenger activation in mouse pancreatic islets.
  • To validate the protocol for detecting G protein-coupled receptor activity in primary islet cells.

Main Methods:

  • Utilized an IP1 accumulation assay to measure Gq/11-coupled receptor activation.
  • Employed fura-2-based fluorescence imaging to detect calcium signals.
  • Assessed intracellular cAMP levels using the developed protocol.

Main Results:

  • Gq/11-coupled receptor activation in beta cells increased IP1 levels, blocked by inhibitors.
  • Vasopressin and ghrelin receptors in alpha/delta cells did not significantly increase IP1 but induced calcium signals in intact islets.
  • The protocol successfully measured cAMP changes from Gs and Gi/o-coupled receptors.

Conclusions:

  • Second messengers IP1, cAMP, and calcium are reliable indicators of GPCR activation in intact islets.
  • The developed protocol enables accurate analysis of GPCR signaling pathways in pancreatic islets.
Keywords:
GPCRPrimary isletsSecond messengerβ cells

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