Figure 4.

Adcy2 expression and FFAR1-induced cAMP accumulation and GLP-1 secretion in enteroendocrine cells and cell lines. Panel A – Co-expression of Adcy2 with Ffar1 in enteroendocrine cells in murine ileum as determined by RNAscope in situ hybridization; A1 – zoom-in corresponding to box 1; A2 – zoom-in corresponding to box 2. Panel B – Colocalization of Adcy2 and peptide hormones in murine ileal enteroendocrine cells; left panel - multiplex RNAscope in situ hybridization of Adcy2, Ffar1, and Gcg – proglucagon/‘GLP-1’; middle panel – co-localization of Adcy2 (RNAscope) and PYY (Immunohistochemistry); right panel – colocalization of Adcy2 (RNAscope) and CCK (immunohistochemistry). Similar histological data including co-staining for 5HT for duodenum, Ileum and colon are shown in Supplementary Figure S5. Panel C – cAMP accumulation in STC-1 cells in response to AM5262 (black), AM5262 + Gq-inhibitor, YM254890 (red), PLC-inhibitor edelfosine (green) or PKC-inhibitor chelerythrine chloride, CTC (blue). Data represents mean ± SEM for 3 independent experiments performed in triplicate for AM5262 and 2 independent experiments performed in triplicates for the inhibitors. Panel D – GLP-1 secretion from STC-1 cells stimulated with 1 μM AM5262; mean ± SEM from 3 independent experiments. Panel E - GLP-1 secretion from GLUTag cells stimulated with AM5262 1 μM in the absence or presence of the specific Gq-inhibitor YM254890; mean ± SEM from 3 independent experiments. Statistical significance was calculated using two-tailed Mann–Whitney t test: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Panel F – Simplified schematic overview in enteroendocrine cell (EEC) of the proposed dual signal-transduction pathway activation by FFAR1 in response to second generation agonists (here AM5262) where Gq activates both the PLC/IP₃/Ca²⁺ pathway and the ADCY2/cAMP/PKA pathways acting in synergy to stimulate enteroendocrine hormone secretion (GLP-1) and that both signaling pathways are blocked by the Gq inhibitor YM254890.