| 1. |
- Amisten, Stefan, et al.
(författare)
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ADP mediates inhibition of insulin secretion by activation of P2Y13 receptors in mice.
- 2010
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; Jul 1, s. 1927-1934
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Tidskriftsartikel (refereegranskat)abstract
- 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 co-incubation 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.
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| 2. |
- Högberg, Carl, et al.
(författare)
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Succinate independently stimulates full platelet activation via cAMP and PI3β kinase signaling.
- 2011
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 9:2, s. 361-372
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Tidskriftsartikel (refereegranskat)abstract
- Background: The citric cycle intermediate succinate has recently been identified as ligand for the G-protein coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most 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 signalling pathways of this receptor in platelets. Methods and Results: Using RT-PCR, we could demonstrate that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y(1) receptor. Light transmission aggregation experiments showed a dose-dependent aggregation induced by succinate reaching a maximum response at 0.5mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry showing increased surface expression of activated GPIIb/IIIa, and P-selectin. Intracellular SUCNR1 signalling was found to result in decreased cAMP levels, Akt phosphorylation mediated by PI3Kβ activation and receptor desensitisation. Further, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A(2) and ATP release. The 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.
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| 3. |
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| 4. |
- Tan, Chanyuan, et al.
(författare)
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ADP receptor P2Y(13) induce apoptosis in pancreatic beta-cells.
- 2010
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 67, s. 445-453
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Tan, Chanyuan
(författare)
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ADP regulation of insulin secretion and beta-cell apoptosis
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aims of this dissertation were to investigate the effects of extracellular purines on insulin secretion, and apoptosis in mouse pancreatic islets and β-cells; to examine if high glucose and free fatty acids induces β-cell apoptosis via autocrine effects of ADP acting on the P2Y13 receptor; and to investigate the modulation of extracellular purines in vascular smooth muscle cells (VSMCs) through ectonucleotidase or ATP release under the effect of high glucose. The expression of the ADP receptors P2Y1 and P2Y13 were shown in both mouse pancreatic islets and isolated β-cells using real-time PCR quantification. Insulin and glucagon secretion were measured both in mouse islets and in vivo. Results showed that ADP acting on P2Y1 receptors stimulated insulin secretion, while acting on P2Y13 receptors inhibited insulin secretion. In MIN6c4 cells, real-time PCR also revealed high expression of the ADP receptors P2Y1 and P2Y13. 2MeSADP induced calcium influx and inhibited cAMP production by activation of P2Y1 and P2Y13 respectively. ELISA and cell proliferation studies showed that 2MeSADP increased Caspase-3 activity and reduced cell proliferation. P2Y13 receptor antagonist MRS2211 could fully reverse both of these effects. Western blotting showed that activation of the cAMP/PKA/CREB pathway resulted in amplification of phosphorylation of Akt/PKB, leading to resistance to apoptosis, increase of β-cell viability and proliferation rate. ATP release in MIN6c4 cells was measured by bioluminescence. High glucose and palmitate potently elevated the extracellular ATP levels. The calcium-channel blocker nifedipine, VRAC-channel inhibitor NPPB, pannexin-1 blocker carbenoxolone, or silencing of MDR1 all resulted in a substantial decrease in high glucose/palmitate induced ATP release. Furthermore, high glucose and palmitate inhibited cAMP production, reduced cell proliferation in MIN6c4 and increased activated Caspase-3 in both mouse islets and MIN6c4 cells and all these effects were dependent on the P2Y13 receptor. Western blotting further showed that blocking the P2Y13 receptor resulted in enhanced CREB, Bad and IRS-1 phosphorylation. Real-time PCR study of ectonucleotidase expression in primary rat aortic VSMCs revealed the expression of CD39, CD39L1 and CD73. 48 hours treatment of high glucose significantly decreased CD39 gene expression and increased CD39L1 expression. Bioluminescence assays also revealed that high glucose caused VSMCs released of ATP and the ectonucleotidase inhibitor ARC67156 led to sustained levels of ATP. In conclusion, we have shown that activation of the P2Y13 receptor in mouse MIN6c4 cells exhibits a pronounced proapoptotic effect which is mediated by modulating the cAMP/ERKs/CREB/Akt pathway. Autocrine/paracrine effects of ADP acting on the P2Y13 receptor are involved in the proapoptotic effects of high glucose and free fatty acids. P2Y13 antagonist MRS2211, which increased the secretion of insulin and was able to protect the cells from ADP induced apoptosis in β-cells, could be a potential treatment of diabetes. High glucose also increases ATP release from VSMCs and accumulates more ADP by up-regulating the expression of CD39L1, thereby enhance vascular inflammation and VSMC proliferation via P2Y receptor activation.
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| 6. |
- Tan, Chanyuan, et al.
(författare)
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High glucose and free fatty acids induce beta cell apoptosis via autocrine effects of ADP acting on the P2Y(13) receptor.
- 2013
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Ingår i: Purinergic Signalling. - : Springer Science and Business Media LLC. - 1573-9546 .- 1573-9538. ; 9:1, s. 67-79
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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