Reversal Of Ang ?-Induced ?-cell Dedifferentiation Via Inhibition Of NF-?b Signaling

BackgroundDiabetes mellitus is causing serious threat to human health in 21 st century and the prevalence of T2DM is around 11.6%in China.According the IDF Diabetes Altas in 2017,the number of people with diabetes was 425 million worldwide,while diabetic patients reached 119.8 million in China.Pancreatic ?-cell failure underlies progressive development of type 2 diabetes,it commonly arises from apoptosis and dysfunction in? cells.Recent studies believes that the impaired-?-cell identity is a significant contributory factor of ?-cell failure.The loss of mature status of ? cells is detrimental to its identity,finally leading to ?-cell dysfunction.Jia et al found that impaired pancreatic beta cell compensatory function is the main cause of type 2 diabetes among Chinese with high genetic risk,according to a 9 year prospective cohort study in the Chinese population in 2016.Therefore,a better understanding of the mechanism of improving ?-cell function is mandatory in China.Increasing evidence shows that the pathological stimulation of RAS is associated with type 2 diabetes and the RAS(renin-Angiotensin system)inhibitor delay new onset-type 2 diabetes in high-risk populations.Angiotensin?(Ang?),a powerful RAS component,not only diminishes islet blood but also involves in increased oxidative stress,proinflammatory cytokines and islet fibrosis.Furthermore,Studies show that Ang? contributes to impaired ?-cell function in Ang?-infused mice,while the detrimental effect is independent of vasoconstriction,implying a complex mechanism underlying Ang?-induced ?-cell dysfunction.In all,previous studies shed some light on the relationship of RAS and development of type 2 diabetes,but the underlying role of RAS remains incompletely understood.Emerging evidence shows that the pancreatic ?-cell failure can develop through different mechanisms,including oxidative stress,endoplasmic reticulum stress,hypoxic stress,as well as induction of proinflammatory cytokines.In response to the stressors,? cells become dedifferentiated,reverting to a progenitor-like stage or converting into other types of pancreatic cell.Consistently,multiple studies have found?-cell dedifferentiation and increased progenitor cell markers expression in patients with type 2 diabetes.Hence,it has been recognized recently that loss of the fully differentiated(mature)state is a potential mechanism underlying compromised ?-cell function in type 2 diabetes.Recently,clinical trial and experimental evidence have shown that NF-Kb-mediated inflammation is associated with the cardioprotective effects of RAS inhibitors,and Ang? has been reported to stimulate NF-Kb activity in different types of cells.NF-Kb was reported to be a major transcription factor that positively regulate Angiotensin II type 1 receptor(AT1R)gene expression.The inactived NF-Kb complex is localized in the cytoplasm and includes the DNA binding p65 subunits and an inhibitory subunit I?B? which is bound to p65.In response to stimulation,phosphorylation of the I?B kinase(IKK)complex releases IkBa from the complex and promotes the nuclear translocation of p65,which regulates genes transcription.Moreover,inhibition of I?B-kinase,prevents Ang?-induced upregulation of proinflammatory cytokines in human islets.Taken together,these evidence suggests an interaction between RAS and NF-Kb in the development of diabetes.In summary,these findings allow us to formulate a hypothesis that the RAS activation plays an important role in ? cell dedifferentiation,while interference with RAS or NF-Kb signaling is an efficient way to reverse ?-cell dedifferentiation.In vitro,we conducted the studies on pancreatic ?-cell line in vitro,and parallel studies were performed with db/db mice,a model of type ? diabetes in vivo.Materials and Methods1.Cell cultureThe pancreatic ?-cell lines Min6 were cultured in RPMI 1640 at 37°C in a humidified atmosphere of 95%air and 5%C02.We conducted experiments when the cells reach 70%confluence.2.Animal StudiesEight-week-old male db/db mice and wild-type littermate controls(db/m mice)were purchased from the Model Animal Research Center of Nanjing University.3.Intraperitoneal glucose tolerance tests(IPGTT)After overnight fasting with free access to water,mice were intraperitoneally injected with a 20%glucose solution(1 g/kg body weight).The glucose concentrations of 2 ul total blood from the tail vein at 0,15,30,60,and 90 min were collected and detected using a glucometer.The serum was collected from eye canthus blood at 0,15,30 min and insulin was tested by mouse insulin ELISA kit according to the manufacturer's instructions.4.Glucose-stimulated insulin secretion(GSIS)Glucose-starved preparation in Krebs-Ringer bicarbonate HEPES(KRBH)buffer for 45 minutes.The cells were incubated in the basal glucose conditioned KRBH buffer containing 3mM glucose and then incubated in glucose stimulation KRBH buffer containing 25mM glucose for 60min respectively.Before treating with different mediums,the cells were washed twice with PBS,and the mediums were collected for ultra-sensitive ELISA analysis.5.Enzyme linked immunosorbent assay(ELISA)The medium or serum was then assayed for insulin or IL6 using their specific ELISA kit.The absorbance was read at 450nm using an Enzyme-labeled Instrument.6.Real-time polymerase chain reaction(RT-PCR)Gene expression was determined by relative gene expression using the 2-??Ct methodology.7.Western blot analysis(WB)Western blotting and immunohistochemistry Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting were performed as previously described using primary antibodies against FoxO1,Pdx-1,Ngn3,phospho-I?B?(p-I?B?),I?B?,phospho-p65(p-p65),p65,?-actin and HRP labeled secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG.8.Immunofluorescence analysisImmunostaining was performed as followed on pancreatic ? cells and on fixed wax-embedded pancreatic sections(5mm).To assess the protein expression of insulin?glucogon?Pdx1?Fox01?Ngn3.The images of the double staining were captured with a Nikon Y-TV55 fluorescent microscope.Basically,we measured positive stain area divided by total islet area using Image-Pro analyzer software.9.Statistical analysisData are expressed as means ± standard error.Statistical analyses were performed using Prism7.0.For statistical significance of different experimental groups,we used one-way,or two-way analysis of variance(ANOVA)and P<0.05 to declare a statistically significant differenceResults1.The protective effect of RAS inhibitor on glucotoxicity-induced compromised identity of pancreatic ? cell linesGlucotocity induced ?-cell compromised function and identiy,including decreased messenger RNA(mRNA)expression of Pdxl,Insl and upregulated dedifferentiated markers Ngn3 and Oct4 and decreased insulin secretion.While the protective effect of RAS inhibitor on ? cell improved GSIS and markedly suppressed dedifferentiaition2.The dedifferentiated effect of Ang? on pancreatic ? cell lines and NF-?b signalingThe deleterious effect of Ang? was dependent on NF-?b signaling in ? cells.We observed that sc-514,an IkB-kinase-2 inhibitor,markedly decreased mRNA expression of Ngn3,Oct4,IL6 and reversed the identity of ?cell.Ang? induced the activation of NF-Kb,leading to dedifferentiation and dysfunction in ? cells.We also found that increased concentration of Ang? activated mRNA and protein of NF-Kb signaling in a dose-related manner.Moreover,it was consistent with the deleterious ?-cell identity.3.The effect of sc-514 and Irbesartan on pancreatic ? cell in Ang? infused-db/db miceAng? induced dedifferentiated state of ? cell in db/db mice.Irbesartan slightly restored the loss of insulin expression,protein expression of Pdxl and[?-cell function.NF-Kb inhibitor,sc-514 decreased the expression of Ngn3 and reversed dedifferentiated? cells,leading to improved ?-cell function.ConclusionOur study,for the first time,found that RAS might inducep-cell dedifferentiation via AT1R activation,which was promoted by NF-Kb signaling.Therefore,blocking RAS or NF-?b signaling efficiently reversed the dedifferentiated status of ? cells,suggesting a potential therapy for patients with type 2 diabetes.