The Mechanism Of GPR40(FFA1) Receptor Regulating Pancreatic β Cell Insulin Secretion And The Ligand-independent Receptor Internalization

Free fatty acids (FFAs), have been discovered not only to act as a nutrient for human, but also activate various GPCRs to regulating the metabolism. Previous study revealed long chain fatty acids were endogenous ligands of GPR40and stimulate insulin releasing. Interestingly, GPR40promote insulin secretion at high concentrations of glucose and exhibite a enhancement of glucose-dependent insulin release. Moreover, GPR40has been observed to assist insulin secretion promoted by DPP-IV inhibitors. In this study, to investigated the underlying mechanism of GPR40-mediated enhancement of CREB activation, a cAMP response element (CRE)-driven luciferase assay system exhibited a remarkerble activition upon co-stimulation of FFAs and Forskolin or Exentin-4, the similar result was found in treating with GW9508and forskolin. Both FFAs and GW9508activation counld be blocked by GW1100. In addtion, results of three PDE inhibitor,IBMX, MMPX, Rolipram treatment were indicated that PDE1, but not PDE4was activating by LA to degrade cAMP. Then the Gs minipeptide was designed to investigate Gs subunit function have no effect on LA-mediated CREB activation, but UBO-QIC significantly block the synergistic effect. Furher we employ PKC inhibitor, cAMP ELISA kit, CREB mutants to detemine whether or not PKC involved in CREB transcription.The result revealed that LA induced CREB phorsphorylation at Ser133and Ser121through a Gaq/PKC pathway. Moreover, treatment of8-CPT-cAMP, an efficient activator of cAMP protein kinase, resulted in a remarkable increase in CRE-driven luciferase expression following co-treatment of LA. We next to test the LA mediated CREB activation on isolated islets from SD rat and Gq-/-mice, the same synegistic effect has been found to crosstalk between cAMP/PKA and Gaq/PKC on CREB transcription. Then, we found liragutide have palmitic acid modification and could active GPR40signalling. The co-transfect GPR40and GLP-1R HEK293cell stimulated by Liragutide (PA or LA modification) increasing more20%luciferase activity than GLP-1R transfect cell. These result provide a new insight of GPR40target drugs in type2diabetes design.In stably transfected HEK-293cells and beta-TC-6cells GPR40underwent ligand dependent and constitutive internalization. Data revealed that FITC-antiflag marked GPR40underwent internalization in the absence of ligands, suggested that the receptor from plasma membrane trafficing into cell not generated from the inside of cell. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3and GRK2expression revealed that arrestin-3and GRK2play an essential role in the regulation of agonist-mediated GPR40internalization, but are not involved in the regulation of constitutive GPR40internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40receptors were rapidly recycled back to the plasma membrane via a Rab4/Rab5positive endosomes, whereas the constitutively internalized GPR40receptors were recycled back to the cell surface through a Rab5positive endosomes.Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family. And the G-protein coupled receptor that bound with specific proteins in lipid raft could be designed to screen a ligand for preventing ligand-independent internalization.