SFEBES2018 Poster Presentations Diabetes & cardiovascular (27 abstracts)
Bright and specific far-red labels for visualizing endogenous glucagon-like peptide-1 receptors
Julia Ast 1, , Tom Podewin 4 , Anastasia Arvaniti 1, , Nick HF Fine 1, , Zania Stamataki 5 , Johannes Broichhagen 4 , Kai Johnsson 4 & David J Hodson 1,
1Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; 2Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK; 3COMPARE University of Birmingham and University of Nottingham Midlands, Birmingham, UK; 4Max Planck Institute for Medical Research, Department of Chemical Biology, Heidelberg, Germany; 5Institute of Immunology and Immunotherapy, Centre of Liver Research, University of Birmingham, Birmingham, UK.
The glucagon-like peptide-1 receptor (GLP-1R) is a G protein-coupled receptor (GPCR) expressed in various tissues such as brain and pancreas where it contributes to the regulation of energy expenditure and metabolism. Due to its involvement in glucose-dependent release of insulin from pancreatic beta cells, the GLP-1R has become a blockbuster target for the treatment of type 2 diabetes. Despite this, debate still exists about the exact distribution of the GLP-1R throughout the body, particularly at the protein level. Present approaches are limited by lack of antibodies against various GLP-1R epitopes, use of fluorescent agonists that induce internalization/degradation, poor signal or binding, and the requirement for fixed tissue. Here, we installed a Cy5 moiety onto the C-terminus of Exendin4(9-39) to produce a far-red fluorescent GLP-1R antagonist label, termed LUXendin. As expected, LUXendin was unable to generate cAMP in CHO-SNAP_GLP-1R cells unless the positive allosteric modulator BETP was co-applied. LUXendin strongly bound YFP-AD293-SNAP_GLP-1R but not YFP-AD293 cells with a Bmax=50 nM. At the same concentration, LUXendin produced intense membrane labelling in MIN6 beta cells and primary islets, with penetration in the latter approaching >100 μm imaged using conventional confocal microscopy. Again, no internalization of the GLP-1R was detected unless BETP was co-applied to allosterically activate the receptor. Co-staining for insulin, glucagon and somatostatin in LUXendin-treated islets revealed widespread GLP-1R expression. FACS analysis of islets from Ins1Cre;mTmGflox’d reporter mice demonstrated LUXendin staining in ~90% of non-beta cells, in contrast to transcriptomic and antibody studies where Glp1-r/GLP-1R was found to be almost absent in alpha cells (but abundant in delta cells). Thus, bright and highly specific antagonist labels allow sensitive detection and visualization of low levels of endogenous GLP-1R, with broad applicability to other GPCRs.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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