β-arrestin-1Is An Important Component In Regulating CCK Promoted Antiapoptotic Effects And Insulin Secretion In Pancreatic β Cells

Cholecystokinin(CCK), a gastro-intestinal hormone, is secreted by intestinal I cells after meal and regulates important digestive and metabolitic functions. In pancreatic islets, CCK managed insulin secretion and beta cell mass majorly through CCKAR, a G protein coupled receptor, while the downstream signaling of CCKAR in pancreatic beta cells were not well defined. Here, we demonstrated that CCK-8s activated CCKAR and induced both IP3and cAMP accumulation, which coordinately regulated efficient insulin secretion. Apart from activation of G protein signaling, CCK also promotes complex formation of CCKAR and β-arrestinl. Exploiting RNA interference and β-arrestinl knockout (β-arrestinl-/-) mice, we revealed that beta-arrestin-1was key mediators of both CCK promoted insulin secretion and its protective effects against apoptosis in pancreatic β cells. The anti-apoptotic effects of β-arrestin-1was through arrestin mediated cytoplasmic long term ERK activation, which activate p90RSK-phosphor-BAD antiapoptotic pathway and inhibit the caspase-3cleavage. Further, we observed that knockdown of β-arrestin-1decreased both CCK-8s induced cAMP and IP3accumulation statistically, suggesting a crosstalk between β-arrestin-1and G protein mediated pathways. The importance of β-arrestin-1function downstream of CCKAR in pancreatic beta cells could be appreciated for development of newly anti-diabetic drugs.Objective To investigate β-arrestin-1as an important component in regulating CCK promoted anti-apoptotic effects and insulin secretion in pancreatic β cells.Methods1β-arrestin-1is an important component in regulating CCK promoted insulin secretion in pancreatic P cells(1) Western blotting analysis is used to check endogenous CCKAR and β-arrestinl expression and subcellular fractionation concentration in pancreatic islets.(2) ELISA kit was used to detect the insulin response after being treated with CCK-8s in pancreatic islet β cells.(3) ELISA kit was used to detect the IP3response after being treated with CCK-8s in pancreatic islet β cells.(4) ELISA kit was used to detect the cAMP response after being treated with CCK-8s in pancreatic islet β cells.2β-arrestin-1is an important component in regulating CCK promoted anti-apoptotic effects in pancreatic P cells(1) We used Hoechst33342staining and Annexin V-FITC/PI apoptosis detection kit to detect the apoptosis of pancreatic islet β cells and HEK293cells which was transfected with CCKAR DNA.(2) Western blotting analysis is used to check the activation of p90RSK-phospho-BAD antiapoptotic pathway.(3) SLIC was used to subclone CCKAR into pCDNA3.1with the N-terminal signal peptide followed by flag tag.(4) Immunoprecipitation was used to detect the interaction between β-arrestin-1and CCKAR in HEK293cells.(5) β-arrestin-1expression was silenced in MIN6cells through transfection with chemically synthesized, double-strand19-nucleotide siRNA with2-nucleotide3’dTdT overhangs in deprotected and desalted form.RESULTS1. β-arrestin-1is an important component in regulating CCK promoted insulin secretion in pancreatic β cells(6) We firstly confirmed the expression of CCKAR in mouse islet β cells. We treated isolated mouse islet with100pM CCK-8s for20minutes, a4fold increase of insulin secretion was observed, which was blocked by1μM lorglumide, a selective CCKAR antagonist, we isolated primary mice β cells from islets and treated the cells with CCK-8s. Again, application of100pM CCK-8s led to about5fold increase of insulin secretion in primary mice β cells, which can be blocked by lorglumide.(1)100pM CCK-8s induce4folds IP3accumulation compared to basal, a little less than IP3induced by carbonylcholine. Similarly,100pM CCK-8s induced6folds cAMP accumulation than basal, a little less than the cAMP accumulation stimulated by lOnM GLP-1. We applied the PLC inhibitor U73122and PKA inhibitor H89to selectively block each signaling pathways. The application of Gq downstream inhibitor U73122or Gs downstream PKA inhibitor H89partially reduced the insulin secretion to60%and50%respectively, while the combination usage of the inhibitors totally suppress the CCK-8s induced insulin secretion.2. p-arrestin-1is an important component in regulating CCK promoted antiapoptotic effects in pancreatic P cells(1) High glucose exposure lead to85%apoptotic cells after72hours and this effect can be reduced to65%with application of GLP-1, the agonist of GLP-1R.Apoptosis cells reduced from85%to63%with100pM CCK-8s treatment.(2) CCK-8s led to a concentration-dependent ERK activation which reached to a plateau after100pM. Pretreatment MIN6cells with1μM specific CCKAR antagonist lorglumide blocked the ERK activation. Application of CCK-8s stimulated two phases ERK1/2activation; one was rapid which peaked at2minutes; the other was a slow and sustained phase with a peak at20minutes. Rapid p-ERK1/2mainly located in nucleus, however, the majority of late phase ERK1/2phosphroylation remained in cytosolic fraction.(3) Upon CCK-8s stimulation, we found that the kinetic pattern of ERK activation in β-arrestinl knockdown MIN6cells varied significantly. The early phase (2min) of ERK activation was decreased by about30%, whereas the long-lasting (20min) ERK activation was90%diminished.(4) The association between CCKAR and β-arrestinl increased after CCK-8s treatment and the interaction peaked at15minutes, agreeing with the temporal course of sustained ERK activation. (5) β-arrestinl knockdown significantly suppressed the p90RSK (Thr573) and BAD (Serl12) phosphorylation induced by CCK-8s. High glucose induced cleavage of caspase-3was significantly blocked by CCK-8s.(6) CCK-8s treatment reduced apoptotic cells from86%to68%, while this protective effect was not detectable after beta-arrestin-1knockdown.ConclusionGq and Gs signaling pathway concerted contribute to the CCK-8promoted insulin secretion and β-arrestinl is an important component in regulating CCK promoted insulin secretion in pancreatic β cells.CCK-8s protect pancreatic β cells by activating p90RSK-phospho-BAD anti apoptotic pathway and β-arrestinl is an important component in regulating CCK promoted antiapoptotic effects in pancreatic β cells.