CCK’s Functions On Islets β Cells And The Underlying Mechanisms In β-arrestin-1 Knockout Mice

Arrestins play important roles in regulating downstream signaling pathways of G protein-coupled receptors (GPCRs). Upon binding with GPCRs, arrestins block G protein-mediated signaling pathway, and mediate endocytosis of G protein-coupled receptors. Arrestins also transfer the GPCR-mediated signaling through a non-G protein-dependent pathway. Besides GPCRs, arrestin can bind many other receptor proteins and a variety of signaling proteins.Mammalian express four arrestin subtypes. Among these, the expressions of β-arrestin-1 and P-arrestin-2 in mammals are very extensive. Their functions are very important. The use of β-arrestin-1 and β-arrestin-2 gene knockout mice are very extensive to study their physiological function. Currently, β-arrestin-1 knockout mice and β-arrestin-2 knockout mice have been used in many fields, involving many physiological problems:like the receptor downstream pathways on various systerms, the organ developmental problems, the diseases or therapeutic outcomes and and so on. Compared to the studying on β-arrestin-2 knockout mice, the β-arrestin-1 mice have scarcely been investigated.Our laboratory experiments on pancreatic β cell line MIN6 cells showed that, β-arrestin-1 plays an important role in the CCK’s protection effect on pancreatic β cell apoptosis. In order to get more physiological conclusions, we selected β-arrestin-1 knockout mouse and primary pancreatic β cells. The mouse model confirmed the β-arrestin-1’s role in CCK promoting insulin secretion and protecting islet β cell against apoptosis. The knowledge of the biased signal pathway after CCK1 receptor activation is of great importance in developing of new anti-diabetic drugs.Research purposesStudy the effects and the underlying mechanism of CCK-8s on insulin secretion and islet β cell apoptosis with β-arrestin-1 knockout mice.Research methods1. Preparation of knockout mice and wild-type mice(1) Homozygous knockout mice backcross with C57 mice.(2) The F1 generation of heterozygous’ breeding.(3) RT-PCR to distinguish between wild-type and non-wild-type of the F2 generation.(4) Real-time Quantitative PCR (RT-PCR) detecting system was used to identify the F2 β-arrestin-1 heterozygous and β-arrestin-1 KO mice.(5) Use the above-described method to expand populations, select littermate wild-type and homozygous β-arrestin-1 knockout mice for further animal experiments.2. Animal experiments(1) Using an ELISA kit to detect CCK-8-stimulated insulin secretion from β-arrestin-1 knockout mouse islets and wild type mouse islets.(2) Using an ELISA kit to detect CCK-8s-stimulated IP3 and cAMP signaling in β-arrestin-1 knockout mouse islet and wild type mouse islets.(3) With STZ-induced diabetic models in β-arrestin-1 knockout mice and wild type mice, giving CCK-8s treatment to detect glucose tolerance test (GTT).(4) Western blot to detect whether β-arrestin-1 knockout mice and wild type mice islets apoptosis is mediated through p90RSK-phosphor-Bad pathway.Result1. Compared with wild-type mouse islet, CCK-8s-induced insulin secretion from β-arrestin-1 knockout mouse islet is reduced.2. After stimulation by CCK-8s, the accumulation of cAMP is suppressed compared with wild-type mouse islet.3. Compared with wild-type mice, CCK-8s did not increase glucose tolerance in STZ-pretreated β-arrestin-1 knockout mice.4. Compared with WT mouse islets, CCK-8s-induced p90RSK-phosphor-Bad activation was significantly inhibited in β-arrestin-1 knockout mouse islets.Conclusionβ-arrestin-1 mediated CCK1 receptor signaling pathway plays an important role in both CCK-8s-induced insulin secretion and the CCK’s protective effect against pancreatic β cell apoptosis.