| BackgroundDiabetes mellitus (DM) is a common endocrine metabolic disease,which is increasingly concerned in the medical field, and the number of patients is rising rapidly, follwing the progress of society,improvement of the living condition, alteration of the life style. Diabetes has become another chronic non-communicable disease that severely threating people’s health following cardiocasscular disease and cancer. According to estimation of WHO, the number of DM patients in the world will rise up to 333 millions in 2025,and DM patients in China will be 50 millinons.However, the exact pathogenesis of diabetes remains unclear, restricting the development of clinical prevention and treatment.More and more researchs indicate islets injury inducing deficiency in insulin is important in the development of Diabetes,which is at the "core of the problem’.It has been reported many factors such as high glucose, free fatty acids and ribose can inhibit insulin secretion.Recently research indicated advanced glycosylation end products(AGEs) induceβ-cell apoptosis and insulin secretion decrease via oxidative stress.The body of DM, chronic kidney disease(CKD), obesity and metabolism syndrome are commonly at a state of oxidative stress. Reactive oxygen species(ROS) oxidizes their protein resulting in changing of construction and function,the oxidized protein include AGEs and advance oxidant protein products(AOPPs), which were found to be elevated in the plasma of DM, CKD, obesity and metabolism syndrome patients. AOPPs and AGEs is not only a marker of oxidant-mediated protein damage, but also it is a promoting factor for oxidative stress and inflammation. It has been identified that AOPPs or AGEs, which has similar construction and bioactivity, can contribute to some similar cytotoxicity and pathological progress,and we have confirmed AOPPs can induce some pathological change on vascular endothelial cell, by bind to RAGE(receptor of AGEs). However,the effect of AOPPs on islet remains unknown so far.The mechanism of islet injury and insulin secretion decrease is so complicated.Recently more and more research have indicated that in endocrine pancreas, the renin-angiotensin system (RAS) play a novel role in regulating islet glucose-stimulated insulin secretion and thus glucose homeostasis. A complete elements of RAS can also be detected in the endocrine pancreas(including canidae, rodent animals and human),using different methods. Angiotensin Receptor 1(AT1R) mainly express onβ-cell, Angiotensingen(AGT) in a-cell, and Angiotensin Converting Enzyme(ACE) in endothelial cell from islet microcirculation. In endocrine pancreas,it mainly behaves derectly regulating insulin secretion or through microcirculation inderectly. It is intriguing that the pancreatic RAS components are sensitive to various physiological and pathophysiological stimuli, including hypoxia, hyperglycaemia, and type 2 diabetes mellitus (T2DM). In diabetic animals,RAS inhibitors including angiotensin converting enzyme inhibitor (ACEI) and Angiotensin Receptors blocker(ARB), can improve islet morphosis and function, decrease the development of T2DM.And clinic researchs indicated RAS inhibitor can reduce the incidence of new-onset T2DM cases in hypertension.The relationships between AGEs and RAS have been studied,which is mainly about diabetic complication,such as diabetic retinopathy, diabetic nephropathy, diabetic Angiopathies.AGEs can activate local RAS system in many tissues. It has been reported AOPPs and AGEs can induce oxidative stress in many cells,such as vascular endothelial cell, renal tubular epithelial cell and mesangial cell.And recently we found that AOPPs can activate RAS in renal tubular epithelial cell.Summary,We suppose that AOPPs decrease islet insulin release through activating local RAS system mediated by oxidative stress.In order to study the role of AOPPs on islet insulin secretion and RAS activation,we cultured rat pancreatic islet cells as a model to observe the effect of AOPPs.MethodsRat Pancreatic islet were chosen as cell model to study effect of AOPPs on the RAS of islet and insulin secretion..1. Preparation of endotoxin free AOPPs-RSAAOPPs-RSA was prepared according to the method described by GUO ZJ et al. Briefly, endotoxin free RSA was incubated with HOCl at molar ratio 1:140 at room temperature for 30 min and then dialyzed against PBS to remove any free HOCl in the solution. AOPPs-RSA were determined by measuring absorbance at 340 nm in acidic condition and were calibrated with chloramines-T in the presence of potassium iodide.2. Isolation and purification of rat islets and Cell cultureThe pancreatic islets were digested and isolated by perfusing collagenase P via pancreatic duct and purified by Ficoll-400 discontinuous density gradients. Islets were cultured in RPMI-1640 medium with 11mmol/1 D-glucose supplemented with 10% fetal calf serum (FCS),100U/ml penicillin,100mg/l streptomycin, 10mmol/1 HEPES,2mmol/l L-glutamine, 1mmol/l sodium pyruvate, and 50mol/lβ-mercaptoethanol,at 37℃,5% CO2.Islets become flatten after 2days culture, could be used in our experiment.3. Glucose-stimulated insulin release in islets(GSIS).The duplicate groups of 50 islets each,which diameters were between 150-200um,were cultured and treated.Wash cells one time,each groups were incubated during 2h at 3.3mmol/l glucose KRB solutios and during a 2nd hour at 16.7mmol/l glucose KRB solutions.The media of KRB were collected,and stored in-70℃for insulin measure. The insulin concentrations of the media were measured using a rat insulin enzyme-linked immunosorbent assay according to the manufacturer’sinstructions.The results were described as release index(content of insulin in 16.7mMglucose KRB/in 3.3mMglucose KRB.4.Analysis intracellular ROS level in islets.The levels of intracellular reactive oxygen species (ROS) was determined by measuring the fluorescence of 2’,7’-dichlorodrofluorescein diacetate. Fluorescence intensity were analysis on a flow cytometry(Excitation wave:488nm, Emission wave:525nm)Briefly, Islet cells were pre-incubated for 30min with O.lumol/L DCFH in PBS, then cells were treated with 200μg/ml AOPPs-RSA for 5,15,30,60min and untreated cells were considered as the control group. The time-dependent effect of AOPPs on ROS production were detceted.Likewise,after Islet cells were pre-incubated for 30 min with 0.1u mol/L DCFH in PBS. Islet cells were treated with 50,100,200μg/ml AOPPs-RSA or 200μg/ml unmodified RSA for 30min, The dose-dependent effect of AOPPs on ROS production were detceted. In inhibitor experiment, cells were pre-treated with apocynin (10μmol) or C-SOD(200 U/ml) for one hour at 37℃respectively before challenged by AOPPs-RSA. The ROS detctions were then repeated as described above. The insulin release experiments were then repeated as GSIS.5.Detection RAS expression of islet cells5.1. Detection the mRNA and protein expression of AT1R on islet cellsIslet cells were treated with 200μg/ml AOPPs-RSA for 6,12,24hours and untreated cells were considered as the control group. Total RNA and protein were extracted. AT1R mRNA and protein were detected by Quantitative real-time PCR and western bloting. The time-dependent effect of AOPPs on mRNA and protein expression of ATIR were detceted.Islt cells were also treated with 50,100,200μg/ml AOPPs-RSA or 200μg/ml unmodified RSA for 24 hours. Total RNA and protein were extracted. Quantitative real-time PCR and western bloting were selected for analyzing the dose-dependent effect of AOPPs on mRNA and protein expression of AT1R.5.2 Detection the mRNA expression of ACE on islet cellsIslet cells were treated with 200μg/ml AOPPs-RSA for 6,12,24 hours and untreated cells were considered as the control group. Total RNA were extracted. ACE mRNA were detected by Quantitative real-time PCR. The time-dependent effect of AOPPson mRNA expression of ACE were detceted.Islt cells were also treated with 50,100,200μg/ml AOPPs-RSA or 200μg/ml unmodified RSA for 24 hours. Total RNA were extracted. Quantitative real-time PCR were selected for analyzing the dose-dependent effect of AOPPs on mRNA expression of ACE.5.3 Detection the mRNA expression of AGT on islet cells Islet cells were treated with 200μg/ml AOPPs-RSA for 6,12,24hours and untreated cells were considered as the control group. Total RNA were extracted. AGT mRNA were detected by Quantitative real-time PCR. The time-dependent effect of AOPPs on mRNA expression of AGT were detceted.Islt cells were also treated with 50,100,200μg/ml AOPPs-RSA or 200μg/ml unmodified RSA for 24 hours. Total RNA were extracted. Quantitative real-time PCR were selected for analyzing the dose-dependent effect of AOPPs on mRNA expression of AGT.5.4 Measurement of AngⅡIslet cells were treated with 200μg/ml AOPPs-RSA for 6,12,24hours and untreated cells were considered as the control group. After incubation, cells were lysed and AngⅡlevels in the cell lysates were measured by ELISA. The time-dependent effect of AOPP on AngⅡproduction were detceted.Islt cells were also treated with 50,100,200μg/ml AOPPs-RSA or 200μg/ml unmodified RSA for 24 hours. After incubation, cells were lysed and AngⅡlevels in the cell lysates were measured by ELISA, for analyzing the dose-dependent effect of AOPPs on mRNA expression of AngⅡ.6.The effect of ACEI and ARB on the insulin secretionTo verify ACEI and ARB contribution on the insulin secretion, cells were co-treated with ACEI lisinopril(6 mmol/1) or ARB telmisartan(6 mmol/1) when challenged by AOPPs-RSA. The insulin release experiments were then repeated as described above.7. The effect of antioxidants on the RAS expressionsIn inhibitor experiments, cells were pre-treated with apocynin (10μmol) or C-SOD(200 U/ml) for one hour at 37℃respectively before challenged by AOPPs-RSA. The experiments were then repeated as described as 5. 8.StatisticsAll values are mean±SD. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS 13.0. Significance was defined as P<0.05.Results1. Glucose-stimulated insulin release in islets.After the stimulating islet cells with 200μg/ml AOPPs-RSA for 6,12,24 hours, insulin release index:the ratio of insulin secretion (16.7mM/3.3mM glucose), decreased gradually with the prolonging of time(P<0.05),release index decreased 34.8% and 67.8% at 12h and 24h respectively. Moreover, insulin release index decreased with the increasing of AOPPs doses,release index decreased 33.3% and 67.5% at 100ug/ml AOPPs-RSA and 200ug/ml AOPPs-RSA (P<0.05) respectively.2.Analysis intracellular ROS level in islets.After the stimulation of islet cells with various concentrations of AOPPs-RSA for indicated time. The production of ROS increase gradually with the prolonging of time (P<0.05) and the increasing of AOPPs doses (P<0.05).AOPPs induced ROS production was significantly suppressed by C-SOD and the NAD(P)H oxidase inhibitors apocynin(P<0.05), inhibition ratio were 73.6% and 54.1% respectively.AOPPs decreased insulin release index,and the effect was significantly suppressed by C-SOD and the NAD(P)H oxidase inhibitors apocynin(P<0.05),and insulin release index inreased 2.33 and 1.64 fold respectively3. The effect of AOPPs on the RAS expressions on cells3.1 Influence of AOPPs on islet ATIR mRNA and protein expression After the stimulating islet cells with 200μg/ml AOPPs-RSA for 6,12,24 hours, the expression of ATR1 mRNA increased gradually with the prolonging of time(P<0.05). Moreover, AT1R mRNA expression increased with the increasing of AOPPs doses (P<0.05). The protein expression of AT1R is similar. Unmodified RSA had no effect on AT1R mRNA and protein expression(P>0.05).3.2 Influence of AOPPs on ACE mRNA expressionAfter the stimulating islet cells with 200μg/ml AOPPs-RSA for 6,12,24hours, the expression of ACE mRNA increased gradually with the prolonging of time(P<0.05). Moreover, ACE mRNA expression increased with the increasing of AOPPs doses (P<0.05). Unmodified RSA had no effect on ACE mRNA expression(P>0.05).3.3 Influence of AOPPs on AGT mRNA expressionAfter the stimulating islet cells with 200μg/ml AOPPs-RSA for 6,12,24hours, the expression of AGT mRNA increased gradually with the prolonging of time(P<0.05). Moreover, AGT mRNA expression increased with the increasing of AOPPs doses (P<0.05). Unmodified RSA had no effect on AGT mRNA expression(P>0.05).3.4 Effects of AOPPs on AngⅡproductionAOPPs increased AngⅡproduction in a time- and dose-dependent manner(P<0.05).4.The effect of ACEI and ARB on the insulin secretionAOPPs decreased insulin release index was significantly suppressed by ACEI lisinopril and the ARB telmisartan (P<0.05), and insulin release index inreased 1.01 and 1.37 fold respectively,suggesting that AOPPs decrease islet insulin release also through RAS activation.5. The effect of antioxidants on the RAS expressionsAOPPs induced RAS production was significantly suppressed by C-SOD and the NAD(P)H oxidase inhibitors apocynin (P<0.05).ConclusionAOPPs decrease rat pancreatic islet cells insulin release in time-and dose-dependent manner, which may be through activating local RAS system mediated by oxidative stress.These results suggest that the accumulation of AOPP can induce islet cells RAS expression and following insulin secretion decreation,which involved the pathogenesis of diabetes.These datas can provide a new idea for the pathogenesis of diabetes and new way for treatment. |
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