| 008P Nottingham, UK
7th Focused Meeting on Cell Signalling
A chemogenetic approach to study the short chain fatty acid receptor FFA2
Introduction: FFA2 and FFA3 are two G protein-coupled receptor subtypes activated by short chain fatty acids (SCFAs), endogenous ligands generated as a by-product of the breakdown of starches by the gut microbiota. Understanding the precise physiological roles of these receptors and their potential therapeutic utility has proven to be challenging. This reflects: 1) the co-expression of FFA2 and FFA3 in some tissues, 2) conflicting and contradictory results generated in transgenic mouse knock-out models and 3) that available FFA2 antagonists act only at the human and not rodent receptor orthologues. To address these challenges we have generated a transgenic knock-in mouse line expressing a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) version of human FFA2 receptor (hFFA2-DREADD) such that it no longer responds to SCFAs but instead to a range of non-endogenously generated ligands, including sorbic acid (SA) . The pharmacology of the modified receptor was assessed and colonic crypt cultures compared between the transgenic line and wild type mice (C57BL/6) for ligand-induced release of glucagon-like peptide-1 (GLP-1).
Methods: GLP-1 release assays were performed as previously described  using colonic crypts derived from C57BL/6 or hFFA2-DREADD expressing mice. Results were analysed as % of GLP-1 release, taking the response induced by 10μM 3-isobutyl-1-methylxanthine as 100%. Antagonists were incubated 15min prior to agonist treatment. Results represent the mean±SEM and derive from at least three independent experiments. * P<0.05; *** P<0.001 for significance versus vehicle data; $$$ P<0.001 for significance versus SA data, one-way ANOVA followed by Bonferroni post hoc test.
Results: 1mM SA was able to induce GLP-1 release in colonic crypts derived from hFFA2-DREADD mice (table) but not C57BL/6. This effect was similar to that obtained by treatment with a SCFA, propionate (10mM), in C57BL/6 mice (49.4±9.8**), suggesting that the mutated DREADD receptor retains the pharmacological properties of the wild type receptor in this tissue. In addition, the SA-mediated effect was inhibited by two selective human FFA2 antagonists, CATPB and GLPG0974 (table), confirming that this effect of SA is mediated exclusively by FFA2 receptor activation.
|hFFA2-DREADD||Vehicle||SA||SA + CATPB||SA + GLPG0974|
|GLP-1 release (%)||18.3±1.5||50.7±4.3***||29.0±3.5*;$$$||23.1±2.5$$$|
Conclusions: These animals provide means to further assess roles of FFA2 and a model to predict efficacy of human specific antagonists of FFA2 prior to studies in humans.
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