| ObjectiveRecently more and more attention has been payed to the relationship between reninangiotensin system(RAS) and type 2 diabetes. The role and its intervention value of RAS inthe development ofβcell dysfunction have become one of the research hot spots. Thisstudy aimed to investigate the effects of RAS blockade on isletβcell function in differentstages of type 2 diabetes, that is insulin resistance stage and overt diabetes stage, and itsimpact on the incidence of STZ-induced diabetes. This study also aimed to investigate themechanisms ofβcell dysfunction induced by RAS activation viaβcell line INS-1 cellscultivation and treatment in vitro.Methods(1) Effects and mechanisms of RAS blockade on isletβcell function in insulinresistance stage: Ninety 8-week male Wistar rats were randomly divided into normalcontrol group(n=15) and high fat diet group(n=75),which were given high fat diet for 16weeks and then divided into high fat contol group(n=30), perindopril intervention group(n=15) and telmisartan intervention group(n=30). Eight weeks later, to investigate theimpact of RSA blockade on the incidence of STZ-induced diabetes, fifteen rats wereselected randomly from high fat contol group and telmisartan intervention grouprespectively as high fat plus streptozotocin(STZ) intraperitoneal injection group(n=15) andtelmisartan plus STZ intraperitoneal injection group(n=15), which were given STZintraperitoneal injection at the dosage of 20mg/kg and whose random blood glucose was≥16.7mmol/l twice not in one day after one week were considered as diabetes. (2) Effectsand mechanisms of RAS blockade on isletβcell function in overt diabetic stage: Fifty8-week male Wistar rats were randomly divided into normal control group(n=10) anddiabetes model group(n=40), which were given high fat diet for 8 weeks and after that, STZ were intraperitoneal injected at the dosage of 30mg/kg. The diabetic criteria was asabove. Then the diabetic rats were divided into diabetes group(n=8), perindoprilgroup(n=10) and valsartan group(n=10), and treated for 8 weeks.(3)mechanisms ofβcelldysfunction induced by RAS activation:βcell line INS-1 cells were cultivated in vitro andtreated with glucose of different concentration for different time to investigate theexpression of AngiotensinⅡ(AngⅡ) receptor 1 (AT1R), and with AngⅡof differentconcentration for 24h to investigate its impact on the apoptosis, insulin signal moleculs andfunction of INS-1 cells. In animal experiments, the morphology of islet, insulin contentintraβcell, islet cell apoptosis, inflammation, oxidative stress, islet microvessel densityand RAS expression intra islet were detected by immunohistochemistry or RT-PCR. Theislet function was evaluated by intravenous glucose tolerance test(IVGTT). In vitro study,the expression of AT1R and insulin was detected by RT-PCR, the cytoactive, apoptosis andinsulin signal molecules were detected by MTT, immunofluorescence and flow cytometry,western-blot respectively.Results1. Effects and mechanisms of RAS blockade on isletβcell function ininsulin resistance stage1.1 insulin sensitivity and islet function of rats with long-term high fat dietIn compared with normal control group(NC, n=15), the glucose infusion rate(GIR) ofhigh fat control group(FC, n=15) was decreased significantly[(7.80±0.51) mg*kg-1*min-1vs (5.32±0.90) mg*kg-1*min-1, P＜0.01], indicating that FC group appeared obviousinsulin resistance. The islets of FC group were enlarged with decreased insulin relativeconcentration(IRC) intraβcell (P＜0.01).The insulin positive nucleus density(ICD) has adecreased tendency, but the variance was not statistically significant. Area under the curveof insulin(AUCI) from 0 to 10 min of FC group was lower in compared with that in NCgroup(P＜0.01), while the AUCI from 10 to 60 min was increased by 68.8%(P＜0.01),indicating the deficiency of insulin secretion in the first-stage and an abnormal highsecretion during the second-stage in FC group. 1.2 expression of RAS, inflammatin and apoptosis intra islets of insulin resistance ratsIn compared with NC group, the relative expression of AT1R in FC group wasincreased by 1.16 times(P＜0.01), the relative expression of IL-1βwas increased by 1.95folds(P＜0.01), the relative content of NF-KB was increased by 20.5%(P＜0.01), therelative concentration of Caspase-3 was increased by 19.1%(P＜0.01), and the number ofapoptotic cells in unit area of islet was increased by 2.43 times(P＜0.01).1.3 effects of RAS blockade on islet function in insulin resistance ratsAider intervention, the IRC of peridopril group(FP, n=15) and telmisartan group (FT,n=15) were all increased obviously(P＜0.05 or P＜0.01), the ICD was also increased, butthe variance was not statistically significant, AUCI(0-60) was decreased by 15.6% and17.3% respectively(all P＜0.01), AUCI(0-10) and AUCI(0-60) were also decreased, but thedifferences were not statistically significant, too. There were no significant differencesbetween FP and FT group.1.4 effects of RAS blockade on inflammation and apoptosis intra islet in insulinresistance ratsIn compared with FC group, the relative expression of AT1R in FP and FT group wasreduced significantly(all P＜0.01), the relative expression of IL-1βwas decreased by22.4%(P＜0.05) and 28.5%(P＜0.01) respectively, the relative content of NF-KB wasreduced by 20.1%, 22.8%(all P＜0.01), the relative concentration of Caspase-3 wasdecreased by 15.5%, 17.7%(all P＜0.01), and the number of apoptotic cells in unit area ofislet was reduced by 60.0%, 67.4%(all P＜0.01). There were no significant differencesbetween FP and FT group.1.5 effects of RAS blockade in insulin resistance stage on the incidence ofSTZ-induced diabetesIn compared with high fat plus STZ group(FS, n=15), the incidence of STZ-induceddiabetes of telmisartan plus STZ group(TS, n=15) was reduced signifcantly (P＜0.05),which were 80%(10/15) and 33%(5/15) respetctively. The difference of fasting bloodglucose between FS and TS group was also significant [(17.5±5.1)mmol/l vs(13.2±4.7)mmol/l, P＜0.05]. 2. Effects and mechanisms of RAS blockade on isletβcell function inovert diabetic stage2.1 islet function of diabetic rats induced by high fat diet plus STZ intraperitonealinjectionIn compared with normal control group(NC, n=10), the islets of diabetes group (DM,n=8) were enlarged with disarrayed architecture, and IRC was decreased obviously(P＜0.01). First-phase insulin secretion, which was expressed as AUCI (0-10), was reducedby 67.0% (P＜0.01) and the early insulin secretion index(EISI) was decreased by 81.8%(P＜0.01).2.2 expression of RAS, oxidative stress, microvessel density and apoptosis intra islet ofdiabetic ratsThe relative expression of Angiotensinogen(AGT) mRNA of DM group was increasedsignificantly (P＜0.01) in compared with that in NC group. The relative content of inducedNO synthase(iNOS), which reflected the oxidative stress in islet, was increased by 10.3%(P＜0.01). The microvessel density(MVD) was decreased by 71.4% (P＜0.01) with theincreased expression of hypoxia inducing factor1α(HIF-1α) by 1.19 folds (P＜0.01). Thenumber of apoptotic cells in unit area of islet was increased by 2.14 times(P＜0.01).2.3 effects of RAS blockade on islet function in diabetic ratsAfter intervention, the IRC of ACEI group(AE, n=10) and ARB group(AR, n=10) wasall inreased obviously(P＜0.01) with the islet morphology improved. AUCI(0-10) wasincreased by 41.2% and 33.1% respectively(all P＜0.01), EISI was increased by 1.84 foldsand 1.74 folds(all P＜0.01), indicating that the first-phase insulin secretion was partlyrestored. There were no significant differences between AE and AR group.2.4 effects of RAS blockade on oxidative stress, MVD and apoptosis intra islet indiabetic ratsThe relative concentration of iNOS in AE and AR group was decreased by 16.5 % and18.2% (all P＜0.01) respectively in compared with that in DM group, the MVD wasincreased by 62.5% (P＜0.05) and 75.0%(P＜0.01) with the expression of HIF-1αdecreased by 27.2% and 29.0%(all P＜0.01) respectively. The number of apoptotic cells in unit areaof islet was reduced by 29.0% and 36.2% (all P＜0.01) respectively. There were nosignificant differences between AE and AR group.3. mechanisms ofβcell dysfunction induced by RAS activation3.1 expression of AT1R in INS-1 cells under different concentration glucose fordifferent timeIn compared with the expression of AT1R mRNA in INS-1 cells cultivated with 5.6mmol/l glucose for 24h(5.6mG(24h)), there was no significant difference in 5.6mG(48h)group. AT1R expression in 16.7mG(24h) group was increased, but the variance was notstatistically significant, while its expression in 16.7mG(48h) group was increase by 0.72times(P＜0.05); in 33.3mG (24h) and 33.3mG (48h) group, its expression was inceased by1.33 times and 2.6 times (all P＜0.01) respectively, indicating that with the increament ofglucose concentration and time, the expression of AT1R in INS-1 cells was also increasedaccordingly.3.2 insulin secretion function of INS-1 cells treated with AngⅡat differentconcentration for 24hIn compared with 5.6mG(24h) group, the basic insulin secretion of differentconcentration AngⅡ(0.1/1/10/100nmol/l) treatment group was not changed significantly,but the glucose stimulating insulin secretion(GSIS) was decreased by 7.9%(P＜0.05),21.1%(P＜0.01), 26.3%(P＜0.01) and 34.2%(P＜0.01) respectively.3.3 apoptosis of INS-1 cells treated with AngⅡat different concentration for 24hThe apoptosis rate was 5.7% in 5.6mG(24h) group, and 10.3% in the group with 0.1nmol/l AngⅡtreated for 24h, but the variance was not statistically significant. Theapoptosis rate in other groups(1/10/100nmol/l) was all increased significantly with theincreased concentration of AngⅡ.3.4 expression of insulin signal molecules of INS-1 cells treated with AngⅡat differentconcentration for 24hThe expression of insulin receptor substrate serine270(IRS-ser270) phosphorylationwas not detected in the 5.6mG(24h) group, while its expression could be detected after treated with different concentration AngⅡ(0.1/100nmol/l)for 24h, that was 0.31 and0.72(all P＜0.01) respectively. In compared with 5.6mG(24h) group, the expression ofprotein kinase B serine 473(PKB-ser473) phosphorylation was decreased by 20.1% and30.2%(all P＜0.01) after treated with 0.1 nmol/l and 100 nmol/l AngⅡfor 24h respectively.ConclusionsRAS had tight relation with pancreatic isletβcell dysfunction in the development ofdiabetes, glucose could up-regulate the activation of RAS intra islet depending on theconcentration and its duration. The activation of local RAS would provocate inflammation,oxidative stress, hemodynamics abnormality, apoptosis and insulin signal transductionimpairment intra islet, which ultimately induced damage toβcell survival and function. Toblock RAS in insulin resistance stage had protective effects on isletβcell function anddecreased the incidence of STZ-induced diabetes. Even in the stage of overt diabetes, RASblockade also had protective effects on isletβcell function. Its mechanisms may be thedecreased inflammtion and oxidative stress, the improvement of hemodynamics and insulinsignal transduction, and the reduced cell apoptosis intra islet.
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