Research On The Inhibitory Effect Of HMG-CoA Reductase Inhibitors On The Function Of Pancreatic Islet β Cells In Rat And Its Mechanisms

(Part 1) Isolating method of rat pancreatic islets and its influentialfactorsObjective To assess the factors that effect the activity of isolated islets, and to construct a steady and effective isolating method of rat pancreatic islets. Methods Pancreatic islets were isolated by the collagenase method. The activity of isolated rat pancreatic islets and its influcential factors under different exprimental conditions were evaluated by the glucose-stimulated insulin secretion test (performed by the 37°C batch incubation method), the insulin was measured by RIA. Results The level of insulin secretion stimulated by 25mmol/L glucose was 5-fold higher than that of 2.8mmol/L glucose. The glucose-stimulated insulin secretion was improved by BSA;There is a pronounced decrease(about 25%)of insulin secretion in the 7 days cultured islets group, however, no difference was observed between the freshly isolated islets group and the l~5days cultured islets groups. Compared to the groups of 5.5mmol/L and 25mmol/L glucose in the culture medium, the 11.1 mmol/L glucose group showed higher insulin release. Conclusion The isolated rat pancreatic islets have sufficient biological activity, BSA, the glucose concentration of RPMI1640 medium and the cultured hours are related to the activity of isolated islets.(Part2) Research on the effect of HMG-CoA reductase inhibitors on glucose-stimulated insulin secretion and its mechanisms frompancreatic islet 0 cells in ratObjective To evaluate the effect of HMG-CoA reductase inhibitors on glucose-stimulated insulin secretion(GSIS) and its mechanisms from pancreatic islet P cells in rat. Methods Freshly isolated or 24 hours cultured rat pancreatic islets were used in this experiment.By performing the glucose-stimulated insulin secretion test(37°C batch incubation method), we observed l)the effect of 100 u mol/L pravastatin,fluvastatin,simvastatin and atorvastatin on ATP content from pancreatic islet P cells in rat;2) the effect of 100 u mol/L pravastatin,fluvastatin,simvastatin and atorvastatin on GSIS from pancreatic islet P cells in rat;3) the relationship between the dose and the effect of pravastatin,fluvastatin,simvastatin and atorvastatin on GSIS;4)the effect of 24 hours' washing-out on the GSIS;The insulin was measured by RIA.;Results l)Not only 30 minutes'exposure ,but also 24 hours'culture,100 u mol/L simvastatin and atorvastatin significantly reduced the ATP content and GSIS in pancreatic islet P cells(P<0.05);2)Cultured with 100 u mol/L fluvastatin for 24 hours, the ATP content and the GSIS were also pronounced inhibited(P<0.05);3) Exposured to 100 u mol/L pravastatin for both 30 minutes and 24 hours , however, this inhibition on the ATP content and GSIS was notobserved;4)Both simvastatin(with a concentration of 30 u mol/L) and atorvastatin (with a concentration of 100 u mol/L) dose-dependently decreased the GSIS;5)The inhibiton on ATP content and GSIS was related to statins' lipophilicity;Conclusion1) the lipophilic HMG-CoA reductase inhibitors, simvastatin, atorvastain as well as fluvastatin, at a higher serum concentration, have an ability to inhibit glucose-induced insulin secretion via decreasing [ATP]j in pancreatic islet B cells;2) HMG-CoA reductase inhibitors reduce the GSIS in a dose-dependent manner;3) Cautions should be taken when statins are prescribed clinically for patients with advanced age, renal or heptic dysfunction, especially in type 2 diabetic patients.(Part3) Research on the effect of HMG-CoA reductase inhibitors oninsulin synthesis and its mechanism from pancreatic islet ft cells in ratObjective To evaluate the effect of statins on insulin synthesis and its mechanism from pancreatic islet P cells in rat. Methods l)Cultured with 100 u mol/L pravastatin,fluvastatin,simvastatin and atorvastatin for 24 hours respectively,the alteration of insulin content from rat's pancreatic islet 3 cell was measured by RIA;2)The effect of 100 n mol/L pravastatin,fluvastatin,simvastatin and atorvastatin on the expression of insulin mRNA from rat's pancreatic islet P cell was accessed by SYBER Green I fluorescent quantitative PCR;3)The MIN6 cells wascotransfected with the human insulin promoter-Luciferase vector and the interalcontrol phRL-TK Vector to analyze the effect of statins on the activity of insulin promoter by Dual-luciferase Assay;Results 1) Exposured to 100 n mol/L statins for 24 hours, the insulin content of pancreatic islet P cell was significantly decreased by fluvastatin, atorvastatin and simvastatin(P<0.05);2)Cultured for 24 hours, 100 u mol/L fluvastatin.simvastatin as well as atorvastatin inhibited the expression of insulin mRNA from rat's pancreatic islet ^ cell(P<0.05);3)The hydrophilic pravastatin, however, showed no effect on both insulin content and the expression of insulin mRNA from rat's pancreatic islet ￡ cell;4) 100 y mol/L atorvastatin showed remarkable inhibition on the activity of insulin promoter(P<0.05),however,this inhibitory effect was not observed in pravastatin, fluvastatin and simvastatin groups;Conclusion Lipophilic simvastatin, atorvastatin and fluvastatin, in a higher concentration, suppress the synthesis of insulin via inhibiting the expression of insulin mRNA of pancreatic islet ^ cell, this negative effect is relative to its inhibition on the activity of insulin promoter.