TY - JOUR

T1 - Free fatty acid receptor 1 stimulates cAMP production and gut hormone secretion through Gq-mediated activation of adenylate cyclase 2

AU - Petersen, Jacob Emil

AU - Pedersen, Maria Hauge

AU - Dmytriyeva, Oksana

AU - Nellemose, Emilie

AU - Arora, Tulika

AU - Engelstoft, Maja Storm

AU - Asher, Wesley B.

AU - Javitch, Jonathan A.

AU - Schwartz, Thue W.

AU - Trauelsen, Mette

N1 - Publisher Copyright: © 2023 The Author(s)

PY - 2023

Y1 - 2023

N2 - 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/Ca2+ 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 Gα 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 pro-glucagon 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/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR 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/Ca2+ 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.

AB - 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/Ca2+ 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 Gα 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 pro-glucagon 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/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR 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/Ca2+ 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.

KW - ADCY2

KW - Adenylate cyclase 2

KW - FFAR1

KW - GLP-1

KW - GPR40

KW - Gq

U2 - 10.1016/j.molmet.2023.101757

DO - 10.1016/j.molmet.2023.101757

M3 - Journal article

C2 - 37348738

AN - SCOPUS:85163164170

VL - 74

JO - Molecular Metabolism

JF - Molecular Metabolism

SN - 2212-8778

M1 - 101757

ER -