Effects Of ACE2-Ang(1-7)-Mas Mediated Pancreatic Endothelial Function On Beta Cell Function In Rats

PartⅠEffects of ACE2-Ang(1-7)-Mas pathway on pancreatic islet microcirculation and beta cell function in type 2 diabetic model ratsObjectives:To detect the changes of pancreatic islet microcirculation and beta cell function by upregulating the activity of ACE2-Ang(1-7)-Mas pathway in type 2 diabetic model rats in vivo.Methods:Long term high-fat diet fed plus intraperitoneal injection of a low dose of streptozotocin in Wistar rats, to create a type 2 diabetic animal model. Rats were administered by Ang(1-7) (300μg·kg-1·d-1) with or without its receptor Mas antagonist A-779 for 4 wks. Pancreatic microcirculation was detected with microspheres and microvessel morphology was evaluated by endothelial biomarker von Willebrand factor(vWF) immunofluorescence, as well as vWF mRNA expressions were detected by RT-PCR. A glucose tolerance test (GTT) was performed and plasma insulin levels were determined to detect beta-cell function., and the content of insulin were detected by immunofluorescent staining.Results:Compared with nomal control group, most islets were impaired with disarrayed architecture, and islet microvessel blood flow were decreased by 54.9%(P<0.01); endothelial cells were reduced in number and even broken in morphology, and vWF protein and mRNA expression was down-regulated(P<0.05); the basic and first-phase insulin releasing were decreased significantly; fasting blood glucose,IPGTT 15min glucose and AUCG(area under the curve of glucose)were significantly increased. After Ang(1-7) intervention, islet microvessel blood flow as well as morphology were significantly improved in the whole pancreas and intra-islet. Ang(1-7) treatment potently increased vWF gene and protein expression. Ang(1-7) administration increased the basic insulin releasing by 9.6%(P>0.05) and first-phase insulin releasing by 44.3%(P<0.05). IPGTT 15min glucose and AUCG were significantly decreased. The positive effects of Ang(1-7) above were prevented by addition of A-779.Conclusions:Chronic Ang(1-7) treatment might improve pancreatic endothelial cell morphology, enhance islet microvessel blood flow in a model of type 2 diabetic rats in vivo. It might increase first-phase insulin releasing of beta cell, and lower the blood glucose in part.PartⅡACE2-Ang(1-7)-Mas mediated pancreatic endothelial effection on beta cell functionObjectives:To investigated the effect of ACE2-Ang(1-7)-Mas on endothelial cell function as well as beta cell survival and physiological function in pancreatic islets isolated from obesity model rats and type 2 diabetic model rats in vitro. Meanwhile to compare the different reaction of pancreatic islets to Ang(1-7) administration in these two model rats.Methods:Pancreatic islets were isolated from overnight-fasted adult Wistar rats (N/FC/DM) with collagenaseⅤand hand-picked using a stereomicroscope. N, normal control; FC, long term high fat-fed rats, is an obesity rat model; DM, long term high-fat diet fed plus intraperitoneal injection of a low dose of streptozotocin, is a type 2 diabetic rat model. Islets were incubated statically in RPMI 1640 medium supplemented with 10% FBS and antibiotics, and the quantity and viability were evaluated. To investigate the dose-response and time-response of Ang(1-7). Pancreatic islets isolated from FC rats and DM rats were incubated with Ang(1-7) at the most effective concentration and for the best time length, to determine its influences on NO release, insulin secretion, and Bcl-2/Bax expressions as well as cell apoptosis rate.Results:We found that Ang(1-7) lOnM was the most effective concentration to stimulate insulin secretion and islets viability of normal rats, and islets incubated with Ang(1-7) lOnM for 6h performed best. Ang(1-7) administration resulted in a significant increase of NO content (P<0.05) compared with control group in both FC and DM rats. Upon this, after incubation with Ang(1-7) 10nM for 6h, the basic insulin secretion of islets isolated from DM rats increased by 18.1%(P>0.05), while first-phase insulin secretion increased by 36.5%(P<0.05). Whereas the basic insulin secretion of islets isolated from FC rats decreased and first-phase insulin secretion increased in contrast with control group(P<0.01). These effects of Ang(1-7) above were prevented by addition of A-779. In addition, after administration with Ang(1-7) lOnM for 6h, Bcl-2/Bax was up-regulated in islets isolated from both FC and DM rats(P<0.05).Conclusions:Ang(1-7) administration could increase NO release and suppress islet cell apoptosis in both FC and DM rat islets in vitro. Ang(1-7) intervention might improve beta cell dysfunction of FC rats, while the effect on DM rats is limited.Parts III Effects of ACE-AngⅡ-ATIR and ACE2-Ang(1-7)-Mas countbalance on pancreatic islet cells and its possible mechanismObjective:To study the functional changes of endothelial cells and the impact on pancreaticβ-cell survival and function through simulating imbalance between the ACE-AngⅡ-AT1R and ACE2-Ang (1-7)-Mas pathways and restore the balance on isolated rat pancreatic islet cells in vitro.Methods:Pancreatic islets were isolated from overnight-fasted adult normal Wistar rats and cultured statically in vitro. Islets were induced apoptosis in high glucose culture medium, and were administrated with AngII(group 1), AngⅡ+Ang(1-7)(group 2), AngⅡ+Ang(1-7)+A-779(group 3), respectively. Endothelial vasodilation factor nitric oxide (NO) content was detected, and insulin content in the supernatant was evaluated by radioimmunoassay. Islet cells were collected by centrifugation and hepatocyte growth factor(HGF), pancreatic duodenal homeobox-1(PDX-1) mRNA and early apoptotic genes Bcl-2/Bax expression were analyzed by RT-PCR, and levels of apoptosis were deteced by Hoechst staining as well.Results:High glucose decreased islet endothelial cells NO synthesis and release significantly (P<0.01), reduced glucose-stimulated insulin secretion (GSIS)(P<0.05), induced apoptosis of pancreatic islet cells and inhibited HGF and PDX-1 mRNA expression (P<0.05). Upon this state, AngⅡintervention reduced NO content of islet endothelial cells compared with control group (P< 0.05), inhibitedβ-cell GSIS(P<0.05), decreased Bcl-2/Bax ratio of islet cells(P<0.05) and down-regulated HGF and PDX-1 mRNA expressions(P<0.05). While increased Ang(1-7) content in the culture medium, NO content was increased significantly than group 1,β-cell GSIS was enhanced(P<0.05), Bcl-2/Bax ratio of islet cells was increased(P<0.05), and HGF and PDX-1 mRNA expressions were up-regulated(P<0.05)as well. If added Ang(1-7) receptor Mas antagonist A-779 in the culture medium, NO content was decreased significantly than group 2,β-cell GSIS was lowered, Bcl-2/Bax ratio was decreased, and HGF and PDX-1 mRNA expressions were suppressed, suggesting that early apoptosis of pancreatic islet cells was triggered.Conclusion:The balance of ACE-AngⅡ-AT1R and ACE2-Ang (1-7)-Mas pathways in islets could regulateβcell function and proliferation through endothelial cells effects. Increased activity of ACE-AngⅡ-AT1R pathway might impair endothelial function, inhibiteβcell function and proliferation; while up-regulating ACE2-Ang (1-7)-Mas activity to restore the balance could improve endothelial cell function and ameliorateβ-cell dysfunction. The effects of endothelial cells onβcells may be through the regulation of HGF and PDX-1 mRNA expressions to influenceβcell proliferation and apoptosis, and insulin secretion.