The Role Of NR4A1 In Pancreatic β-cell Pyroptosis Induced By Lipotoxicity In Type 2 Diabetes Mellitus

BackgroundType 2 diabetes mellitus(T2DM)is one of the most common metabolic diseases,which is mainly caused by chronic insulin resistance and progressive decline of β-cell function,and lipotoxicity is an important factor.Pancreatic β-cells,its function and mass are the key to maintain glucose homeostasis,and chronic free fatty acids(FFA)can lead to pancreatic β-cell dysfunction.Long-term exposure to FFA can activate FFA receptors and cellular stress responses,including ceramide formation,lipiddroplet formation,endoplasmic reticulum stress,mitochondrial dysfunction and autophagy,which can lead to β-cell injury and impaired insulin secretion.Therefore,how to effectively protect pancreatic β cells from lipotoxic environment has been the focus of research.With the deepening of the research,it has been found that pyroptosis plays a important role in a variety of diseases,including type 2 diabetes.Proptosis is a Caspase-related programmed cell death mechanism,which triggers a series of inflammatory responses by releasing several inflammatory factors(such as IL-1 β and IL-18).This process is characterized by cell membrane rupture and the release of cell contents mediated by the GSDM protein family.Some studies have shown that there is overactivation of NLRP3 imflammasome in T2DM mouse model,and insulin resistance and metabolic disorder are improved after knocking down NLRP3.Among them,the activation of NLRP3 imflammasome is one of the important parts in the pyroptosis.Besides,inflammatory factors such as IL-1 β can affect insulin sensitivity through tumor necrosis factor(TNF)-dependent and independent pathways,and one of the characteristics of pyroptosis is the release of a large number of inflammatory factors.Therefore,pyroptosis can be used as a potential therapeutic target to treat T2DM,and the therapy targeting pyroptosis can help to slow down the progression of T2DM.NR4A1 belongs to the NR4A(nuclear receptors 4A)subfamily and is an orphan nuclear receptor whose natural ligand is unknown.Members of the NR4A subfamily play a role mainly by regulating gene expression under exogenous stimuli.Many stimuli,including nerve growth factor(NGF)and tumor necrosis factor(TNF),have been shown to induce transcription of NR4A family members in many different types of cells,including FFA.And NR4A1 is one of the important transcriptional regulators in pancreatic β-cell dysfunction induced by FFA.FFA can induce oxidative stress/endoplasmic reticulum stress in pancreatic β cells by increasing mitochondrial reactive oxygen species(ROS),which can be reversed by overexpression of NR4A1.At present,the related research on pancreatic pyroptosis under the condition of lipotoxicity in T2DM is still mainly focused on apoptosis,but there is no clear study on whether it can regulate pyroptosis and how to regulate it.Recently,it has been shown that NR4A1 is involved in the regulation of HIPPO-YAP pathway.NR4A1 promotes increased YAP degradation by preventing YAP from entering the nucleus,and it has been reported that YAP can bind to NLRP3,which leads to the inhibition of NLRP3 degradation.This process may be related to pyroptosis.Besides,Hippo pathway also plays an important role in T2DM.Long-term activation of Hippo pathway can lead to prosoplasia of pancreatic β-cells and pancreatic β-cell dysfunction,finally produce T2DM.At present,It is not clear whether NR4A1 can affect the occurrence and development of pyroptosis by regulating YAP,and whether this process is involved in the process of pyroptosis induced by lipotoxicity in T2DM.PurposesTo explore the effect of type 2 diabetes lipotoxicity on pyroptosis of pancreatic β-cell and whether NR4A1 can intervene in this process through the Hippo pathway.Methods1)Establishment of T2DM mouse model.The T2DM mouse model was induced by high-fat feeding and STZ,and then the high-fat feeding was continued.The weight of the mice was routinely monitored,and the glucose metabolism of the mice was detected by IPGTT and IPITT experiments.2)To detect the pyroptosis of pancreatic cells in T2DM mice.The mice pancreas were isolated,and the pyroptosis of pancreatic cells was observed by SEM and STM.3)To test whether PA can induce pyroptosis in MIN6 cells.MIN6 cells were treated with different concentrations of PA to screen for the best concentration,then MIN6 cells were treated with PA for different duration to screen for the best duration,both of which were based on the expression levels of GSDMD and GSDME.Subsequently,MIN6 cells were treated with the best concentration and time,and GSDMD-mediated pyroptosis pathway indicators(NLRP3,GSDMD,GSDMD-N,Caspase1,cleaved-Caspase1)and GSDME-mediated pyroptosis pathway indicators(GSMDE,GSDME-N,Caspase3,cleaved-Caspase3).The release of IL-1β was detected by ELISA.Cell viability was detected using CCK-8.LDH release assay and PI staining were used to detect cell membrane damage.The morphological changes of pyroptosis were observed by electron microscope.4)Screening of differentially expressed genes between T2DM patients and normals.The data of GSE29221 microarray(Skeletal muscle tissue of patients with T2DM and normal subjects)were analyzed by limma R package,and the differentially expressed genes were analyzed by GO and KEGG pathway.5)To test whether NR4A1 is involved in the process of PA-induced pyroptosis in MIN6 cells.The pancreatic tissue of NR4A1 knockout mice was stained with immunohistochemistry,and the changes of GSDMD and GSDME were observed.After silencing NR4A1 by siRNA and overexpressing NR4A1 by plasmid,Western blot was used to observe its effect on the pyroptosis pathway of MIN6 cells stimulated by PA,including Caspase1,cleaved-Caspase1,GSDMD,GSDMD-N,NLRP3 of GSDMD scorch death pathway and Caspase3,cleaved-Caspase3,GSDME,GSDME-N of GSDME pyroptosis pathway,and verified by TEM and IL-1β release.The cell viability was detected by CCK8 method.6)To explore whether NR4A1 can affect the process of cell pyrogenesis through Hippo pathway.NR4A1 was enriched by GO and KEGG pathway by clusterProfiler R package.Western blot was used to detect the changes of NR4A1,YAP,p-YAP and pyroptosis protein GSDMD and GSDME with the extension of PA stimulation duration.Then NR4A1 was overexpressed to observe the changes of related protein of NLRP3/Caspase1/GSDMD pyroptosis pathway and MST1,LATS1,p-LATS1,YAP and p-YAP of Hippo pathway.Cleaved-Caspase1,GSDMD,GSDMD-N,NLRP3 of GSDMD pyroptosis pathway and cleaved-Caspase3,GSDME,GSDME-N of GSDME pyroptosis pathwayResults1)Successful establishment of T2DM mouse model.Compared with wt mice,the somatotype and body weight of T2DM mice were larger than those of wt mice.IPGTT test confirmed the impairment of pancreatic β cells function and metabolic disorder in mice,and IPITT test confirmed insulin resistance in mice.2)Pyroptosis may exist in pancreatic β cells of T2DM mice.SEM showed protuberance on the surface of some pancreatic cells in T2DM mice,and STM showed vacuoles and continuous interruption of cell membrane in islet β cells.3)PA can induce pyroptosis in MIN6 cells.GSDMD and GSDME increased with the concentration and duration of PA respectively,and 0.4mM and 36 h were selected as the best concentration and duration.After MIN6 cells were treated with 0.4mMPA for 36 h,the expression of NLRP3,cleaved-Caspase1,GSDMD and GSDMD-N in GSDMD-mediated pyroptosis pathway was up-regulated,and the expression of GSDME and cleaved-Caspase3 in GSDME-mediated pyroptosis pathway was up-regulated,but the expression of GSDME-N was only slightly increased compared with the NC group.Compared with the NC group,the cell viability decreased significantly,the expression of IL-1 β increased,the release of LDH increased and PI staining was positive in PA treated group.Intracellular vacuoles,pore formation of cell membrane and cell rupture could be observed by TEM.4)NR4A1 expression increases significantly in patients with T2DM.Differential expression analysis showed that 72 genes were up-regulated and 196 genes were down-regulated.In the aspect of GO analysis of differentially expressed genes,biological process(GO)analysis showed that differentially expressed genes were mainly enriched in cell structure,protein modification and regulation of growth and development.Cell localization(GO)analysis showed that it was mainly concentrated in extracellular matrix and membrane structure.The results of molecular function(GO)analysis showed that it was mainly concentrated in the composition and connection of extracellular matrix components and the regulation of receptor activity.KEGGpathway analysis showed that differentially expressed genes were significantly enriched in ECM-receptor interaction,cytokine-receptor interaction,chemokine signal pathway and PI3K-Akt pathway.The expression of NR4A1,a member of NR4A subfamily,was up-regulated and significantly different in patients with T2DM.5)NR4A1 is involved in the regulation of PA-inducedpyroptosis in MIN6 cells.Compared with the control group,the immunohistochemical results showed that the expression of GSDMD was slightly increased,and the change of GSDME was not obvious in NR4A1 knockout mice.When the NR4A1 of MIN6 cells was knocked out and stimulated by PA,compared with the NC group and PA treatment group,in the protein of GSDMD pyroptosis pathway,cleaved-Caspase1,GSDMD-N were significantly up-regulated.Only cleaved-Caspase3 was significantly increased and GSDME was slightly increased in GSDME pyroptosis pathway.And most of them could be reversed by overexpression of NR4A1.After NR4A1 silencing,the release of IL-1β of MIN6 cells treated with PA was higher than that of PA group,and the release of IL-1β after NR4A1 overexpression was lower than that of PA group.The results of CCK8 assay showed that the cell viability of NR4A1 knockout+PA treatment group was weaker than that of PA treatment group,while that of NR4A1 overexpression group was stronger than that of PA treatment group.TEM showed that the cells in the NR4A1 knockout+PA treatment group were completely ruptured and a large number of cell contents were exuded,while in the PA treatment group,there were only vacuoles and the formation of cell membrane pores.The pore formation was observed in the NR4A1 overexpression+PA treatment group,but the pore diameter was smaller than that in the PA treatment group.6)NR4A1 can affect the process of cell pyroptosis under the condition of lipotoxicity by Hippo-YAP.The results of NR4A1-GO enrichment analysis showed that extracellular matrix,cell adhesion and pancreatic β cell proliferation were mainly mediated on BP,and located in extracellular matrix and membrane structure on CC.The functions of connection and composition of extracellular matrix,binding of cell adhesion molecules and binding secretion of proteins were mainly performed on MF.KEGG results showed that the pathways with the highest enrichment degree of NR4A1 were mainly T2DM,PI3-Akt pathway,calcium signal pathway and so on.The results of Western blot experiment showed that with the increase of PA treatment duration,the expression of GSDMD pyroptosis pathway protein(GSDMD,GSDMD-N,NLRP3)gradually increased,the expression of p-YAP decreased gradually,the expression of NR4A1 increased at first and then decreased,and YAP decreased slightly and then increased.After PA treatment of MIN6 cells,except for the increase of pyroptosis protein,Hippo pathway was activated(MST1,p-LATS and p-YAP down-regulated,LATS and YAP up-regulated),while NR4A1 overexpression+PA stimulation group could reverse the above expression except MST1、LATS1个p-LATS1.ConlusionPyroptosis may exist in pancreatic β cells of T2DM mouse model.PA can induce Pyroptosis of MIN6 cells.There is a difference in the expression of NR4A1 between T2DM patients and normal controls,and the high expression of NR4A1 can promote the phosphorylation of YAP through negative feedback,and finally inhibit the occurrence and development of pyroptosis.