Objective: Free fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where
FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists
such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2þ and cAMP pathways. Whereas
IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear,
although originally proposed to be Gs mediated.
Methods and results: When stimulated with AM5262, we observe that FFAR1 can activate the majority of the Ga proteins, except - surprisingly
- members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by
the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by
transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in proglucagon
expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293
cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of
either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/b2 adrenergic receptor (b2AR) induced cAMP production was lost
in Gs-deficient cells, only the b2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the
FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells
throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion
were both blocked by YM253890.
Conclusions: Our results show that Gq signaling is responsible not only for the IP3/Ca2þ but also the cAMP response, which together are
required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the
Gq-driven cAMP response.