Expression and function of the atypical purinergic receptor GPR17 in the endocrine pancreas

Background and aims: Islets of Langerhans control glucose homeostasis and metabolism by sensing blood glucose and nutrients. Factors secreted within the islet create a parallel tuning, defined autocrine-paracrine signaling, that modulates hormone release and cell viability. Studying the molecular actors of this signaling and their physiological significance may help in understanding diabetes pathogenesis thus providing novel therapeutic targets. G-protein coupled receptors (GPCRs) are expressed in islets of Langerhans, regulate hormone secretion and cell survival, and are emerging as new targets for type-2 diabetes therapies. In this work, we focused on the expression and role of the atypical orphan receptor GPR17, which is activated by uracil nucleotides and can be partially antagonized by ATP, a well known autocrine/paracrine signal in islets. We first verified the GPR17 expression in endocrine cell lines and in human islets, and then we investigated its possible role in islet physiology Materials and methods: The expression of GPR17 was analysed by RT-PCR analysis, western blot and immunofluorescence techniques in cell lines (αTC3, βTC3, the δ cell line RIN14B), in human isolated islets of Langerhans, and human pancreatic sections. The possible involvement of GPR17 in the control of hormone release and cell proliferation/viability was studied in cell lines and in human isolated by means of ELISA assays. Results: By means of RT-PCR, we detected GPR17 mRNA in human islets, in α and δ cell lines, but not in β cell lines (n=5). By means of western blot experiments, we confirmed GPR17 protein expression in α and δ cell lines and in human islets, where its abundance was upregulated by hyperglycemia (2.13+0.15 fold increase over normoglycemic conditions; p<0.05, n=4). To investigate the specific endocrine cells where GPR17 was expressed in vivo, we carried out double immunofluorescence and confocal analysis on human pancreas sections, using hormones as markers of the different cell populations. We confirmed the expression of GPR17 in a sub-population of δ cells but no expression was detected in β-cells, in physiological conditions. To understand the possible role of GPR17 in islet physiology we activated GPR17 with its agonist UDP-glucose, and we measured its effect on cell viability and hormone release in cell lines and human isolated islets. 3-day incubation of αTC3 with 100 µM UDP-G caused a significant increase in cell viability, measured by MTT test (108±2.56% UDP-G vs ctr, p<0.005 T-Test; n= 10). A similar behavior was observed in human islets, where we found a reduction of apoptosis in presence of 100 µM UDP-G (38±7% UDP-G vs ctr). Preliminary data on hormone release in static conditions indicated that incubation of human islets with 100 µM UDP-G, caused a decrease in the glucagon and somatostatin release in normoglycemia (37±10% and 25±5%, respectively)(Elisa assay, 2 islets preparations, duplicate) Conclusion: We demonstrated for the first time that GPR17 is expressed and functional in human islets of Langerhans. Studies are in progress to better define its role in islet cell physiology and pathology.