NNF Center for Basic Metabolic Research, Program for Nutrient and Metabolite Sensing and Signaling, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
NNF Center for Basic Metabolic Research, Program for Nutrient and Metabolite Sensing and Signaling, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark; Departments of Psychiatry & Molecular Pharmacology and Therapeutics, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.
Mol Metab. 2023 Aug;74:101757. doi: 10.1016/j.molmet.2023.101757. Epub 2023 Jun 20.
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 IP/Ca and cAMP pathways. Whereas IP 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.
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.
Our results show that Gq signaling is responsible not only for the IP/Ca 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.
游离脂肪酸受体 1（FFAR1）在小肠内分泌细胞和胰腺β细胞中高度表达，FFAR1 激动剂分别作为 GLP-1 和胰岛素分泌激动剂发挥作用。最有效的是所谓的第二代合成激动剂，如 AM5262，与内源性长链脂肪酸不同，它能够通过 IP/Ca 和 cAMP 途径信号传递。虽然预期 IP 信号是主要与 Gq 偶联的 FFAR1，但 FFAR1 诱导的 cAMP 积累的机制仍不清楚，尽管最初被认为是 Gs 介导的。
当用 AM5262 刺激时，我们观察到 FFAR1 可以激活大多数 Gα 蛋白，除了令人惊讶的是 Gs 家族成员。AM5262 诱导的 FFAR1 介导的通过 cAMP 反应元件（CREB）的转录激活被特异性 Gq 抑制剂 YM253890 阻断。此外，在 Gq 缺陷细胞中，除非通过转染重新引入 Gq 或 G11，否则不会观察到 CREB 信号。通过 qPCR 我们确定，在表达前胰高血糖素的肠内分泌细胞中，腺苷酸环化酶 2（Adcy2）高度表达并富集，相对其他 9 种 Adcys。在 WT HEK293 细胞中，共转染 ADCY2 使 FFAR1 诱导的 cAMP 反应增加 4-5 倍，该效应完全被 YM253890 抑制。此外，在没有重新引入 Gq 或 G11 的 Gq 缺陷细胞中，共转染 ADCY2 没有影响。重要的是，尽管 AM5262/FFAR1 和异丙肾上腺素/β2 肾上腺素能受体（β2AR）诱导的 cAMP 产生在 Gs 缺陷细胞中丢失，但只有β2AR 反应可以通过 Gs 转染挽救，而共转染 ADCY2 是挽救 FFAR1 cAMP 反应所必需的。原位杂交显示，在整个肠道的肠内分泌细胞中，ADCY2 和 FFAR1 的高度表达具有高度的共表达。最后，在肠内分泌 STC-1 和 GLUTag 细胞系中，AM5262 诱导的 cAMP 积累和 GLP-1 分泌均被 YM253890 阻断。
我们的结果表明，Gq 信号不仅负责 IP/Ca，还负责 cAMP 反应，这两者共同构成了第二代 FFAR1 激动剂诱导的高效激素分泌所必需的，而 ADCY2 可能介导 Gq 驱动的 cAMP 反应。
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