Röthe Juliane, Kraft Robert, Schöneberg Torsten, Thor Doreen
1Rudolf Schönheimer Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.
IFB AdiposityDiseases, University Medical Center, Leipzig, Germany.
Biol Proced Online. 2020 Feb 15;22:4. doi: 10.1186/s12575-019-0116-y. eCollection 2020.
Targeting G protein-coupled receptors (GPCRs) in pancreatic cells is feasible to modulate glucose-induced insulin secretion. Because pancreatic islets consist of several cell types and GPCRs can couple to more than one G-protein family, results obtained in pancreatic cell lines do not always match the response in primary cells or intact islets. Therefore, we set out to establish a protocol to analyze second messenger activation in mouse pancreatic islets.
Activation of Gq/11-coupled receptor expressed in primary β cells increased the second messenger IP1 in an accumulation assay. Applying a Gq/11 protein inhibitor completely abolished this signal. Activation of the V1 vasopressin and ghrelin receptors, predominantly expressed in the less abundant alpha and delta cells, was not sufficient to induce a significant IP1 increase in this assay. However, fura-2-based fluorescence imaging showed calcium signals upon application of arginine vasopressin or ghrelin within intact pancreatic islets. Using the here established protocol we were also able to determine changes in intracellular cAMP levels induced by receptors coupling to Gs and Gi/o proteins.
Detection of the second messengers IP1, cAMP, and calcium, can be used to reliably analyze GPCR activation in intact islets.
在胰腺细胞中靶向G蛋白偶联受体（GPCRs）来调节葡萄糖诱导的胰岛素分泌是可行的。由于胰岛由多种细胞类型组成，且GPCRs可与不止一个G蛋白家族偶联，因此在胰腺细胞系中获得的结果并不总是与原代细胞或完整胰岛中的反应相匹配。因此，我们着手建立一种分析小鼠胰岛中第二信使激活的方法。
在原代β细胞中表达的Gq/11偶联受体的激活在累积试验中增加了第二信使IP1。应用Gq/11蛋白抑制剂完全消除了该信号。主要在数量较少的α细胞和δ细胞中表达的V1血管加压素和胃泌素释放肽受体的激活在该试验中不足以诱导IP1显著增加。然而，基于fura-2的荧光成像显示在完整胰岛中应用精氨酸血管加压素或胃泌素释放肽时会出现钙信号。使用此处建立的方法，我们还能够确定与Gs和Gi/o蛋白偶联的受体诱导的细胞内cAMP水平的变化。
检测第二信使IP1、cAMP和钙可用于可靠地分析完整胰岛中的GPCR激活。
