Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G-proteincoupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real-time-PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y 1 receptor. Light transmission aggregation experiments showed dose-dependent aggregation induced by succinate, reaching a maximum response at 0.5 mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb-IIIa and P-selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3-kinase-b activation, and receptor desensitization. Furthermore, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A 2 , and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y 12 receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A 2 generation, ATP release, and P2Y 12 activation.

Aims/hypotheses To investigate the effects of extracellular purines on insulin secretion from mouse pancreatic islets. Methods Mouse islets and beta cells were isolated and examined with mRNA real-time quantification, cAMP quantification and insulin and glucagon secretion. ATP release was measured in MIN6c4 cells. Insulin and glucagon secretion were measured in vivo after glucose injection. Results Enzymatic removal of extracellular ATP at low glucose levels increased the secretion of both insulin and glucagon, while at high glucose levels insulin secretion was reduced and glucagon secretion was stimulated, indicating an autocrine effect of purines. In MIN6c4 cells it was shown that glucose does induce release of ATP into the extracellular space. Quantitative real-time PCR demonstrated the expression of the ADP receptors P2Y 1 and P2Y 13 in both intact mouse pancreatic islets and isolated beta cells. The stable ADP analogue 2-MeSADP had no effect on insulin secretion. However, co-incubation with the P2Y 1 antagonist MRS2179 inhibited insulin secretion, while coincubation with the P2Y 13 antagonist MRS2211 stimulated insulin secretion, indicating that ADP acting via P2Y 1 stimulates insulin secretion, while signalling via P2Y 13 inhibits the secretion of insulin. P2Y 13 antagonism through MRS2211 per se increased the secretion of both insulin and glucagon at intermediate (8.3 mmol/l) and high (20 mmol/l) glucose levels, confirming an autocrine role for ADP. Administration of MRS2211 during glucose injection in vivo resulted in both increased secretion of insulin and reduced glucose levels. Conclusions/interpretation In conclusion, ADP acting on the P2Y 13 receptors inhibits insulin release. An antagonist to P2Y 13 increases insulin release and could be evaluated for the treatment of diabetes.

Pancreatic beta-cell loss represents a key factor in the pathogenesis of diabetes. Since the influence of purinergic signaling in beta-cell apoptosis has not been much investigated, we examined the role of the ADP receptor P2Y(13) using the pancreatic insulinoma-cell line MIN6c4 as a model system. Real time-PCR revealed high expression of the ADP receptors P2Y(1) and P2Y(13). Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced calcium influx/mobilization and inhibition of cAMP production by activation of P2Y(1) and P2Y(13), respectively. 2MeSADP reduced cell proliferation and increased Caspase-3 activity; both these effects could be fully reversed by the P2Y(13) receptor antagonist MRS2211. We further discovered that blocking the P2Y(13) receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in beta-cell survival. These results indicate that P2Y(13) is a proapoptotic receptor in beta-cells as the P2Y(13) receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.

While high levels of glucose and saturated fatty acids are known to have detrimental effects on beta cell function and survival, the signalling pathways mediating these effects are not entirely known. In a previous study, we found that ADP regulates beta cell insulin secretion and beta cell apoptosis. Using MIN6c4 cells as a model system, we investigated if autocrine/paracrine mechanisms of ADP and purinergic receptors are involved in this process. High glucose (16.7 mmol/l) and palmitate (100 μmol/l) rapidly and potently elevated the extracellular ATP levels, while mannitol was without effect. Both tolbutamide and diazoxide were without effect, while the calcium channel blocker nifedipine, the volume-regulated anion channels (VRAC) inhibitor NPPB, and the pannexin inhibitor carbenoxolone could inhibit both effects. Similarly, silencing the MDR1 gene also blocked nutrient-generated ATP release. These results indicate that calcium channels and VRAC might be involved in the ATP release mechanism. Furthermore, high glucose and palmitate inhibited cAMP production, reduced cell proliferation in MIN6c4 and increased activated Caspase-3 cells in mouse islets and in MIN6c4 cells. The P2Y 13 -specific antagonist MRS2211 antagonized all these effects. Further studies showed that blocking the P2Y 13 receptor resulted in enhanced CREB, Bad and IRS-1 phosphorylation, which are known to be involved in beta cell survival and insulin secretion. These findings provide further support for the concept that P2Y 13 plays an important role in beta cell apoptosis and suggest that autocrine/ paracrine mechanisms, related to ADP and P2Y 13 receptors, contribute to glucolipotoxicity.