Effects And Mechanisms Study Of STAT3 Dictating β Cell Anontosis By Modulating PTEN On Hyperglycemia

ObjectivePancreatic β cell destruction is the primary cause of type 1 diabetes(T1D)and a pivotal factor underlying β cell mass reduction in type 2 diabetes(T2D).Both T1D and T2D are characterized by impaired insulin secretion and resultant hyperglycemia.Though diabetes can be controlled by certain drugs,none of these antidiabetic agents halts disease progression by targeting the endogenous β cells,which are exceptionally vulnerable to apoptotic damages induced by multiple stressors.Thus,preventing pancreatic β cell apoptosis is an attractive therapeutic approach to conquer serious hyperglycemia.However,the molecular mechanisms of β cell apoptosis are complex and not fully understood including the design of targeted therapies against these processes.Signal transduction and activator of transcription β(STAT3)is closely related to cell survival,proliferation,differentiation,and apoptosis,which affects the life activities of the body.Its research mainly focused on roles in immune regulation and malignant progression of cancer,and the relationship with metabolic diseases is also drawing scads of attention.STAT3 in hepatocytes,hypothalamic neurons,adipose tissue T lymphocytes,and skeletal muscle cells are key regulators affecting glucose metabolism and insulin signaling pathways.Although diverse functions of STAT3 have been explored,little study has detected the relationship between STAT3 and the regulation of β cell apoptosis has not been explored.This study is to investigate the effects and mechanism studies of STAT3 dictating β cell apoptosis by modulating PTEN and effects of targeting PTEN on inactivating STAT3-mutant hyperglycemiaSection 1 The function of STAT3 in the progression of β cell injuryMethodsIin the study,rat cell line INS-1 cells,mouse primary islet cells,STZ induced hyperglycemia mouse model and high-fat diet induced obesity model were used to evaluate the role of STAT3 on β cell damage.(1)Western blotting and Immunohistochemistry were used to detect the activation of STAT3 in β cell damage model in vitro and in vivo.(2)β cell-specific STAT3 knockout mice((3-STAT3KO)were generated.(3)OGTT and ITT were employed to determine β cell function after STZ/HFD treatment.(4)Immunofluorescence and Immunohistochemistry were employed to measure islet architecture and insulin secretion.(5)Small-interfering RNA was applied to knock down the specific STAT3 and Western blotting and Immunofluorescence were performed to analyze the INS-1 cell apoptosis and proliferation.(6)Western blotting and Immunofluorescence were employed to detect the cell apoptosis and proliferation of WT and β-STAT3KO mice.(7)qRT-PCR assay was performed to analyze the mRNA levels of β cell-related transcription factors.(8)Flow cytometer and H&E were used to analyze inflammatory infiltrate inside the pancreatic islets.Results(1)The effect of β cell injury on STAT3 activationGlucose,STZ,and H2O2 were applied to damage(3 cells,and the activation of STAT3 is inhibited in β cells,which showed a negative correlation between the degree of injury and pSTAT3 expression.After STZ induced hyperglycemia in C57BL/6J mice,results showed that hyperglycemia progression was negatively correlated with pSTAT3 expression in β cells.STAT3 and pSTAT3 expression in a high-fat diet(HFD)-induced obesity mouse model were explored,no obvious difference was observed in HFD-fed mice comparing to the control mice treated with normal chow.(2)The effect of STAT3 deletion on HFD-induced obesityLoss of STAT3 expression in β cells of P-STAT3KO mice indicated the effective disruption of STAT3 and significantly increased body weight compared to WT miceβ-STAT3KO mice and their littermates were fed a normal diet or an HFD for 16 weeks,both β-STAT3KO and WT mice exhibited impaired glucose tolerance and slightly increased blood glucose after HFD treatment,with compensated islet mass,normal βcell function,and integrated islets.(3)The effect of STAT3 deletion on STZ-induced hyperglycemia3MLD-STZ injections induced persistent blood glucose elevation in β-STAT3KO mice.while WT mice still exhibited normal plasma glucose levels.However,a single high dose of STZ treatment showed no difference in plasma glucose in mice betweenβ-STAT3KO and WT groups.3MLD-STZ treated β-STAT3KO mice significantly lost more body weight,showed delayed glucose clearance and impaired insulin secretion after glucose challenge.β-STAT3KO mice suffered from a remarkable decrease in β cell mass after 3MLD-STZ induction with distorted islet architecture.There was no significant change in the blood glucose of WT mouse after 3MLD-STZ treatment,while 5MLD-STZ treated WT mice showed rapidly increased blood glucose,impaired insulin secretion,architecturally distorted islets,inhibited compensatory proliferation and decreased β-cell ration.Using siSTAT3 to INS-1 cells,STZ exposure promotes INS-1 cell apoptosis and inhibits proliferation.The apoptosis rate of STZ-induce β-STAT3KO mice significantly increased,and the proliferation rate and the activity of transcription factors of β cells were inhibited,while immune cells did not participate in the processSection 2 Mechanism of STAT3 dictated β cell apoptosis by modulating PTEN-AKT axisMethodsIn the study,rat cell line INS-1,mouse primary islet cell,Human embryonic kidney cells 293FT cells and STZ induced hyperglycemia mouse model were used to investigate the mechanism of STAT3-PTEN signaling pathway regulating β-cell apoptosis.(1)qRT-PCR assay was performed to analyze the mRNA levels of glucose metabolism-related genes,insulin signaling pathway,and diabetes-regulated genes in WT and β-STAT3KO mice.(2)ELISA and qRT-PCR analysis were displayed to analyze MyD88 related inflammatory factors between STZ-induced WT and β-STAT3KO mice(3)Small-interfering RNA was used against STAT3 and Western blotting and qRT-PCR were performed to analyze the expression of PTEN.(4)Western blotting and Immunofluorescence were employed to explore PTEN protein levels in the islets ofβ-STAT3KO mice.(5)β-STAT3-PTENDKO(Ins2cre Stat3flox/flox Ptenflox/flox)mice were generated.(6)Random plasma glucose levels and glucose tolerance were analyzed in STZ-induced β-STAT3-PTENDKO mice,β-STAT3KO mice,and their littermates.(7)Immunofluorescence was performed to detect the islets structure and cell apoptosis of STZ-induced β-STAT3-PTENDKO mice,β-STAT3KO mice,and their littermates.(8)Luciferase reporter assay was used to examine the effects of PTEN on regulatory elements of islet-enriched PDX1 and MAFA.(9)qRT-PCR and Immunofluorescence were applied to analyze the transcription and translation levels of islet-enriched genes(10)Western blotting and Immunohistochemistry were performed to detect the downstream of STAT3-PTEN axis.(11)Random plasma glucose levels and glucose tolerance were analyzed in STZ-induced β-STAT3-PTENDKO mice,β-STAT3KO mice and their littermates treated with or without the AKT inhibitor.(12)Western blotting and qRT-PCR were used to investigate the expression of apoptosis-associated protein and islet-enriched genesResults(1)Searching of key factors regulating STAT3-driven β cell survival and function qRT-PCR results showed that the mRNA levels of Myd88 and Pten obviously increased in β-STAT3KO mice.ELISA and qRT-PCR analysis displayed no difference in MyD88 related inflammatory factors between STZ-induced WT and β-STAT3KO mice.MyD88 deficiency had no effect on β cell apoptosis induced by STAT3 knockdown and incubation with glucose.siRNA was used to against STAT3 and led to a remarkable accumulation of PTEN at both transcriptional and translational levels.STAT3 knockout led to elevated PTEN protein levels as was clearly seen in β cells of β-STAT3KO mice through western blotting and immunostaining,and PTEN protein further accumulated after STZ treatment in vivo.(2)The effect of STAT3-PTEN double knockout on STZ-induced hyperglycemiaLoss of STATS and PTEN expression in P cells of β-STAT3-PTENDKO mice indicated the effective disruption of STAT3 and PTEN.STAT3-PTEN double knockout in β cells reversed hyperglycemia progression in STZ-induced β-STAT3KO mice,improved oral glucose intolerance,protected islet structure,and β cell survival.(3)STAT3-PTEN regulates β cell survival and function through AKT activationLuciferase reporter assays showed that knockdown of STAT3 resulted in attenuated promoter activity of PDX1 and MAFA,whereas the repressive activity was significantly diminished by pharmacological inhibition of PTEN with bpv(phen).STAT3-PTEN double knockout protected mice from STZ treatment and displayed an obvious increased of mRNA levels of β cell-related transcription factors compared toβ-STAT3KO mice,and the proportion of PDX1+β cells in islets was reestablished.Notably,STAT3-PTEN deficiency reversed AKT activity and protected p cells from apoptosis.We used the AKT specific inhibitor AZD5363 to against STAT3-PTEN axis.All the animals treated with AZD5363 had elevated glucose levels,glucose intolerance.β cell apoptosis,and inhibition of β cell-specific genes,and among them β-STAT3KO mice were most influenced.Section 3 Effect of PTEN inhibitor bpv(phen)on STAT3-inactivated hyperglycemia MethodsIn the study,a single high-dose of STZ induced hyperglycemia mouse model,three or five low-dose of STZ induced hyperglycemia mouse model were used to evaluate the therapeutic effect of the PTEN inhibitor bpv(phen)on hyperglycemia,and AKT inhibitors were used to explore the mechanism of bpv(phen)involved in the regulation of β cell survival and function.(1)bpv(phen)was used to evaluate the therapeutic effect on a single high-dose of STZ induced hyperglycemia and five low-dose of STZ induced hyperglycemia.(2)Random plasma glucose levels and body weight were analyzed in STZ-induced β-STAT3KO mice and their littermates after bpv(phen)treatment.(3)OGTT and ITT were employed to determine β cell function of STZ-inducedβ-STAT3KO mice after bpv(phen)treatment.(4)Immunofluorescence and Immunohistochemistry were performed to measure β cell mass and islet architecture after bpv(phen)treatment.(5)Western blotting and Immunofluorescence were employed to detect the cell apoptosis and proliferation of STZ-induced WT andβ-STAT3KO mice after bpv(phen)treatment.(6)Random plasma glucose levels and glucose tolerance were employed to determine β cell function of STZ-inducedβ-STAT3KO mice after bpv(phen)treatment with or without AZD5363.(7)Western blotting was employed to compare the difference between STZ-DKO-AZD5363 mice and in STZ-KO+bpv(phen)-AZD5363 mice.(8)Immunofluorescence was used to explore the downstream after bpv(phen)treatment with or without AZD5363Results(1)Therapeutic effect of PTEN inhibitor on common hyperglycemic miceWe explored the effect of bpv(phen)on classical β cell damaged model both induced by a high-dose and 5MLD of STZ in WT mice.However,the severely elevated blood glucose levels were not mitigated by bpv(phen)injection.(2)Therapeutic effect of PTEN inhibitor on STAT3-inactivated hyperglycemic micebpv(phen)treatment was able to restore levels of both blood glucose and plasma insulin to normality in 3MLD-STZ-injected β-STAT3KO mice.prevented β cell mass reduction,islet architecture disruption,abnormal β cell fraction,decreased insulin secretion,reduced P cell apoptosis and β cell-related genes inhibition.(3)Disruption of AKT abolishes alleviated hyperglycemia by inhibited PTEN in KO mice.We attempted to use the AKT inhibitor AZD5363 to arrest bpv(phen)’s function.AZD5363 treatment blocked the rescue of blood glucose and glucose intolerance by bpv(phen)treatment in 3MLD-STZ-induced β-STAT3KO mice,induced disruptions in islet structure,consequently increased 2 cell apoptosis and abolished PDX1 levels.ConclusionIn this study,we show that pancreatic STAT3 activation is severely inhibited in β cell damaged model and conditional ablation of STAT3 in β cells remarkably increases STZ-induced β cell apoptosis and hyperglycemia incidence.Using STAT3/PTEN double conditional knockout mice and along with pharmacological inhibitors of PTEN and AKT,our data further suggest that STAT3-PTEN signaling is critical for the progression of hyperglycemia by regulating AKT activity.Moreover,we also show that inactivation of PTEN by using small molecule inhibitor bpv(phen)protects β cells against STZ-induced destruction in STAT3 deficient mice,which offers an opportunity for precision intervention in inactiving STAT3 mutation hyperglycemia.