ADP regulation of insulin secretion and beta-cell apoptosis

Sammanfattning: 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.