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.
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.
Iron, zinc and folate statuses of 45 women were determined during pregnancy around 12, 20, 28, 32 and 36 weeks, and again 2 months after delivery. Analyses of plasma ferritin, Hb, MCV, folate and zinc in plasma and whole blood were performed. Iron supplementation was recommended from mid-pregnancy but 13 of the participants did not use the iron supplements. This group had significantly decreased levels of plasma ferritin and MCV at the end of pregnancy, but none developed anemia. Two months post partum the plasma ferritin of the unsupplemented group had normalized and was in the same range as in the supplemented group. The concentrations of zinc in plasma and whole blood and the calculated levels of red cells were low even at the first examination around 12 weeks of gestation, compared with non-pregnant women. Throughout the course of pregnancy the plasma zinc levels continued to decrease, while the whole blood and red cell levels showed a significant rise. At term of gestation almost half the women had subnormal plasma folate levels (L. casei), which persisted during the post partum follow-up. The corresponding value for red cell folate was 10% below normal values at term and 30% subnormal 2 months after parturition. These findings stress the importance of extending the observation period to include also the lactating period, in order to judge the need for folate supplementation.
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.
Earlier results regarding the in vitro and in vivo effects of zinc on delta-aminolaevulinic acid dehydratase (ALAD) activity in red blood cells were confirmed in healthy human subjects after oral intake of zinc for 12 weeks. In one group of seven healthy adults, oral intake of zinc for 12 weeks. In one group of seven healthy adults, oral intake of zinc, as the sulphate salt (2.07 mmol zinc/day) for 6 weeks, resulted in a 44% increase in the activity of ALAD in erythrocytes. Plasma zinc levels also increased during the experimental period and reached a maximum of 29 mumol after 6 weeks and remained constant thereafter. In another group, the intake of zinc in a lower dose, (0.69 mmol zinc/day) for 12 weeks, showed a similar tendency, although the increase in the enzyme activity, as well as the plasma zinc levels, was relatively much less. The plasma copper levels decreased significantly in the second group after the zinc intake and reached a low value of 11 mumol at the end of the experimental period. The Cu:Zn ratio also decreased considerably towore valuable indicator of zinc status than plasma zinc levels alone.
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