Inhibitory effect of UDP-glucose on cAMP generation and insulin secretion.

Type-2 diabetes (T2D) is a global disease caused by the inability of pancreatic ß-cells to secrete adequate insulin. However, the molecular mechanisms underlying the failure of ß-cells to respond to glucose in T2D remains unknown. Here, we investigated the relative contribution of UDP-glucose (UDP-G), a P2Y14-specific agonist, in the regulation of insulin release using human isolated pancreatic islets and INS-1 cells. P2Y14 was expressed in both human and rodent pancreatic ß-cells. Dose-dependent activation of P2Y14 by UDP-G suppressed glucose-stimulated insulin secretion (GSIS) and knockdown of P2Y14 abolished the UDP-G effect. 12-h pretreatment of human islets with pertussis-toxin (PTX) improved GSIS and prevented the inhibitory effect of UDP-G on GSIS. UDP-G on GSIS suppression was associated with suppression of cAMP in INS-1 cells. UDP-G decreased the reductive capacity of nondiabetic human islets cultured at 5 mm glucose for 72 h and exacerbated the negative effect of 20 mm glucose on the cell viability during culture period. T2D donor islets displayed a lower reductive capacity when cultured at 5 mm glucose for 72 h that was further decreased in the presence of 20 mm glucose and UDP-G. Presence of a nonmetabolizable cAMP analog during culture period counteracted the effect of glucose and UDP-G. Islet cultures at 20 mm glucose increased apoptosis, which was further amplified when UDP-G was present. UDP-G modulated glucose-induced proliferation of INS-1 cells. The data provide intriguing evidence for P2Y14 and UDP-G's role in the regulation of pancreatic ß-cell function.

Novel vasocontractile role of the P2Y14 receptor: characterization of its signalling in porcine isolated pancreatic arteries.

BACKGROUND AND PURPOSE: The P2Y14 receptor is the newest member of the P2Y receptor family; it is G(i/o) protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y14 receptors were functionally expressed in porcine isolated pancreatic arteries. EXPERIMENTAL APPROACH: Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA2 mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit. KEY RESULTS: Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y14 receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y6 receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA2, PGF(2α) and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN. CONCLUSIONS AND IMPLICATIONS: P2Y14 receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²âº levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA2, PGF(2α) and endothelin-1.