The STING-IRF3 Pathway Is Involved In Lipotoxic Injury Of Pancreatic β Cells In Type 2 Diabetes

BackgroundType 2 diabetes mellitus(T2DM)and obesity are often associated with lipotoxic conditions in multiple tissues.Insulin-producing β cells are susceptible to elevated lipid levels and ensuing lipotoxicity.Therefore,an in-depth study and identification of the molecular mechanisms related to the regulation of free fatty acids(FFAs)on β-cell mass and function can provide guidance for the development of novel therapeutic targets for T2DM.Initiation of the mitochondrial pathway of apoptosis and endoplasmic reticulum(ER)stress are important mechanisms involved in the lipotoxic injury of pancreatic β cells.However,the signaling molecules that mediate islet β-cell lipotoxic injury are still unclearRecently,it was reported that the stimulator of interferon genes(STING)-interferon regulatory factor 3(IRF3)signaling pathway can be activated by ER stress and mitochondrial damage.STING,encoded by transmembrane protein 173(TMEM173),is a cytosolic DNA sensor that plays an essential role in innate immune responses.When a pathogen invades a cell,a cyclic GMP-AMP synthase(cGAS)activates the downstream adaptor STING by generating a second messenger cGAMP(cyclic GMP-AMP).Activated STING in turn recruits and phosphorylates IRF3(P-IRF3).P-IRF3 enters the nucleus and binds to the promoter of target genes(such as IFN-α,IFN-β),thereby promoting the expression of inflammatory genes.Although previous studies have mostly focused on the role of the STING signaling pathway in the body’s resistance to viral invasion,emerging evidence suggests that STING can also recognize its own DNA leaked by damaged nuclei or mitochondria from the host cell.Thus,STING also plays an important role in the induction of auto-inflammation and various autoimmune diseases,and even in tumor immunity.A few studies have found that STING and its downstream signaling molecule,IRF3,play an important role in glucolipid metabolic disorders.Our previous study found that the STING-IRF3 signaling pathway is activated in the livers of mice fed a high-fat diet,and gene silencing of STING or IRF3 reversed liver inflammation,apoptosis,and dysfunction of glucose and lipid metabolism.In addition,studies have shown that metabolic dysfunction and insulin resistance induced by high-fat diets are improved in STING or IRF3 knockout mice.However,it is unknown whether STING is activated in pancreatic β cells in T2DM,and whether STING and its downstream IRF3 are associated with T2DM pancreatic β-cell dysfunction.To elucidate the possible involvement of the STING-IRF3 signaling pathway in lipotoxicity-induced β-cell injury,we examined the expression of STING and IRF3 in islets of db/db mice,as well as the effects of high levels of palmitic acid(PA)on the STING-IRF3 signaling pathway,inflammation,apoptosis,insulin synthesis,and secretion in INS-1 cells.Then we separately silenced STING and IRF3 in INS-1 cells using small interfering RNA(siRNA)to further explore the role of the STING-IRF3 signaling pathway in lipotoxicity-induced β-cell injury To explore the possible activation mechanism of the STING-IRF3 signaling pathway in INS-1 cells,we used STAT1 and CGAS siRNA to silence the genes to observe the changes in the STING-IRF3 signaling pathway respectivelyPurposesTo investigate whether the STING-IRF3 signaling pathway is involved in the lipotoxic injury of pancreatic β cells in T2DMMethods(1)Evaluation of glucose metabolism,lipid metabolism and islet function in db/db mice.The body weight and body fat content of the mice were routinely measured.GTT and ITT were used to detect glucose metabolism in mice.Serum triglyceride(TG),cholesterol(TC),free fatty acid(FFA),and serum insulin levels were measured by ELISA.(2)Evaluation of STING-IRF3 signaling pathway,inflammation and apoptosis in islets of db/db mouse.Immunofluorescence was used to semi-quantitate STING protein expression in islets which was co-localized with insulin.Isolate mouse islets and extract tissue proteins,Western blot was used to detect STING,P-IRF3,IFN-β,inflammation indicators(P65,TNF-α,IL-1β)and apoptosis indicators(BAX,cleaved Caspase3,caspase3,cleaved PARP,PARP).TUNEL staining was used to observe the apoptosis of pancreatic β cells(3)Effect of PA stimulation on inflammatory response,apoptosis and insulin synthesis and secretion in INS-1 cells.Western blot was used to detect changes in inflammation indicators P65,TNF-α,IL-1β and apoptosis indicators BAX,cleaved Caspase3,caspase3,cleaved PARP,PARP,and insulin.TUNEL staining was used to detect the apoptosis of INS-1 cells.The content of insulin in INS-1 cells was detected by immunofluorescence.ELISA was used to detect glucose-stimulated insulin secretion(4)Evaluation of activation of STING-IRF3 signaling pathway induced by PA in INS-1 cells.INS-1 cells were treated with PA at a concentration of 0.5 mmol/L for 24 hours.qPCR was used to detect the changes of STING mRNA levels.The following indicators were detected by Western blot:changes in STING,phosphorylated IRF3,and interferon beta.Nuclear translocation of phosphorylated IRF3 was detected by immunofluorescence.The IP technology detects the combination of STING and IRF3(5)To verify whether STING is involved in PA-mediated islet β-cell damage.The STING of INS-1 cells was knocked down with siRNA and then stimulated with 0.5mmol/L PA for 24 hours.Western blot was used to detect changes in STING,phosphorylated IRF3,IFN-β,inflammation indicators P65,TNF-α,IL-1β,and apoptosis indicators BAX,cleaved Caspase3,caspase3,cleaved PARP,PARP,and insulin.TUNEL staining was used to detect the apoptosis of INS-1 cells.ELISA was used to detect glucose-stimulated insulin secretion.(6)To verify whether IRF3 is involved in PA-mediated islet β-cell damage.IRF3 was knocked down in INS-1 cells with siRNA and then stimulated with 0.5 mmol/L PA for 24 hours.Western blot was used to detect changes in STING,P-IRF3,IFN-P,inflammation indicators P65,TNF-α,IL-1β,and apoptosis indicators BAX,cleaved Caspase3,caspase3,cleaved PARP,PARP,and insulin TUNEL staining was used to detect the apoptosis of INS-1 cells.ELISA was used to detect glucose-stimulated insulin secretion.(7)Investigate the possible mechanism of the activation STING-IRF3 signal pathway in INS-1 cells mediated by PA.STAT1 in INS-1 cells was knocked down with siRNA,and then stimulated with 0.5 mmol/L PA for 24 hours Western blot was used to detect the changes of STING.The cGAS of INS-1 cells was knocked down with siRNA and then stimulated with 0.5 mmol/L PA for 24 hours.Western blot deteced changes in STING,P-IRF3,IFN-β,inflammation indicators and apoptosis indicators,insulin,PI3K,P-AKTResults1.The db/db Mouse Is a T2DM Model with Significant Dyslipidemia.The body weight and body fat content of db/db mice were significantly higher than those of wt mice.GTT showed dysfunction of glucose metabolism and islet β-cell function in db/db mice.ITT experiments confirmed insulin resistance in db/db mice.The TG,TC,and FFA of db/db mice were significantly higher than those of wt mice,while the insulin content was lower than that of wt mice.2.STING-IRF3 pathway is activated in islets of db/db mice,accompanied by inflammation and apoptosis of islets.Immunofluorescence revealed that the level of STING in pancreatic β cells of db/db mice was obviously upregulated The mRNA and protein levels of STING in islets of db/db mice were elevated compared to those in wt mice.Furthermore,the levels of P-IRF3 and IFN-βwere also higher than those of wt mice.Additionally,the expression of phosphorylation of p65(P-p65)and inflammatory markers,such as IL-1β and TNF-α,in islets of db/db mice was higher than that of wt mice.At the same time,apoptotic markers(BAX,c-Cas3/Cas3,c-PARP/PARP)were elevated in islets of db/db mice.TUNEL staining also showed significant cell apoptosis3.PA induces inflammatory responses,apoptosis,insulin synthesis,and impairment of secretion in INS-1 cells.The P-p65 and inflammatory factors,including TNF-α and IL-1β,in PA-treated INS-1 cells were significantly higher than that in the control cells.PA-treated INS-1 cells showed obvious apoptosis,as indicated by elevation of apoptotic markers(BAX,c-Cas3/Cas3,c-PARP/PARP)and TUNEL staining.Insulin synthesis was also impaired after PA treatment,and PA suppressed the PI3K-AKT signaling pathway and GSIS,the former of which is closely related to insulin secretion.4.PA activates the STING-IRF3 signaling pathway in INS-1 cells.The results showed a significant increase in the expression of STING,both at the mRNA and protein levels.Also,PA promoted the binding of STING to its target IRF3 and induced the phosphorylation of IRP3.More P-IRP3 translocated into the nucleus after PA treatment.The level of IFN-β,which is a downstream target protein of STING-IRF3,was also higher than that in controls5.STING is involved in PA-induced lipotoxic injury of INS-1 cells.After transfection,STING was inhibited with or without PA stimulation,P-IRF3 and IFN-β were also suppressed.With inhibition of the STING-IRF3 signaling pathway,inflammatory factors and related indicators of apoptosis were also lower.TUNEL staining also suggested a reduction in apoptosis after STING knockdown.Impaired insulin synthesis,PI3K-AKT signaling,and GSIS were also ameliorated.6.IRF3 is involved in PA-induced lipotoxic injury of INS-1 cells.We knocked down IRF3 using IRF3 siRNA,and after 24 h of PA treatment,we observed similar results to those when STING was knocked down.With IRF3 knockdown,IFN-β expression was decreased;the levels of inflammatory cytokines and apoptotic markers were lower;TUNEL-positive cells were reduced;and the impaired synthesis and secretion of insulin were alleviated7.cGAS mediates PA-induced activation of the STING-IRF3 signaling pathway in INS-1 cells.With the inhibition of SIAT1,the expression of STING did not change significantly.However,after cGAS was silenced by siRNA,the levels of inflammatory cytokines and apoptosis markers were reduced,and the PI3K-AKT pathway and insulin synthesis were alleviatedConclusionThe STING-IRF3 pathway is involved in lipotoxicity-induced pancreatic β-cell damage during the pathological process of T2DM.Activation of the STING-IRF3 signaling pathway can affect insulin synthesis and secretion by inducing the inflammatory response and apoptosis of pancreatic β cells.cGAS may be a key factor that mediates PA-induced STING-IRF3 activation in INS-1 cells,while the transcription factor STAT1 has no significant effect on STING in INS-1 cells.