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Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets.

ABSTRACT: Background: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. Results:Activation of Gq/11-coupled receptor expressed in primary ? 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. Conclusions:Detection of the second messengers IP1, cAMP, and calcium, can be used to reliably analyze GPCR activation in intact islets.

SUBMITTER: Rothe J

PROVIDER: S-EPMC7023723 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets.

Röthe Juliane J Kraft Robert R Schöneberg Torsten T Thor Doreen D

Biological procedures online 20200215

<h4>Background</h4>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.<h4>Results</h4>Activa ...[more]

PMID: 32082084

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Project description:Pancreatic cancer is a highly lethal disease and has the worst prognosis of any major malignancy. G protein-coupled receptor GPR87 is reported to be overexpressed in multiple cancers. The clinical significance and biological role of GPR87 in pancreatic cancer, however, remain to be established.GPR87 expression in pancreatic cancer cell lines, paired patient tissues were determined using western blotting and Real-time PCR. Ninety-six human pancreatic cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the association between GPR87 expression and the clinicopathological characteristics of pancreatic cancer. Functional assays, such as anchorage-independent growth, chicken chorioallantoic membrane (CAM) assay, transwell matrix penetration assay, and Annexin V-FITC and PI staining and a xenograft tumor model were used to determine the oncogenic role of GPR87 in human pancreatic cancer progression. The effect of GPR87 on NF-?B signaling pathway was further investigated using the luciferase reporter assays, and by detection of the NF-?B signaling downstream genes.Herein, we reported that GPR87 was markedly overexpressed in pancreatic cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of GPR87 significantly correlated with patients' clinicopathologic features, including clinical stage and tumor-nodule-metastasis (TNM) classification. Pancreatic cancer patients with higher levels of GPR87 expression had shorter overall survival compared to patients with lower GPR87 levels. We gained valuable insights into the mechanism of GPR87 expression in pancreatic cancer cells by demonstrating that overexpressing GPR87 significantly enhanced, whereas silencing endogenous GPR87 inhibited, the proliferation, angiogenesis and increased resistance to gemcitabine-induced apoptosis of pancreatic cancer in vitro and tumorigenicity of pancreatic cancer cells in vivo. Finally, we demonstrated that GPR87 enhanced pancreatic cancer aggressiveness by activating NF-?B signaling pathway.Taken together, these findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.Our findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.

Dataset Information

Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets.

Publications

Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets.

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