The Protective Effects Of Clec11a On Lipotoxicity Induced Islets Injury And Potential Mechnism

Part ? Effect of lipotoxicity on the expression of Clec11 a in mouse isletsBackground and objective: Obesity is the leading cause of chronic metabolic diseases such as hypertension,type 2 diabetes,cardiovascular and cerebrovascular diseases,which has become a major public health problem that threatens human health worldwide.In the obesity,excess fat deposits in skeletal muscle,liver and islets,causing insulin resistance and beta cell dysfunction,thereby mediating the occurrence of diabetes.Previous studies have suggested that pancreatic ?-cells will respond to endoplasmic reticulum stress,oxidative stress and other reactions after long-term exposure to fatty acid,which will trigger ?-cell dysfunction.However,the molecular mechanism of lipotoxicity-induced islet cell damage has not been fully elucidated.Therefore,this study explored and verified differentially expressed genes in islet cells under lipotoxicity to explore specific molecular therapeutic targets for islet damage.Methods: C57 BL / 6 mice(five weeks of age)were fed with high-fat diet for 22 weeks to build diet-induced obesity(DIO)mice model.Mice of the same age were fed normal diet as the control group.Body weight,abdominal fat,plasma free fatty acid,triglyceride,total cholesterol,and insulin levels were measured.Oral glucose tolerance test and insulin tolerance test were performed.Differently expressed genes were detected by RNA-seq.Quantitative real-time PCR and immunofluorescence staining were performed to confirm the expression.In vitro,mouse islets,islet ? cell line MIN6 cells,and mouse islet stellate cells(ISCs)were treated with different concentrations of palmitic acid(PA)for different periods of time.Western blot was used to detect expression of the differently expressed gene in mouse islets,MIN6 cells and ISC cultured with PA.Results: 1.Body weight,abdominal fat deposition,plasma free fatty acids,total cholesterol,fasting blood glucose,and plasma insulin levels of DIO mice were significantly higher than those of controls.OGTT and ITT experiments showed that DIO mice had apparently impaired glucose tolerance and insulin resistance.2.Transcriptome sequencing of the islets showed that the expression of Clec11 a gene in DIO mice was significantly lower than that of control mice,which was verified by q PCR experiment result.3.Immunofluorescence staining showed that Clec11 a protein was expressed in normal mouse islets,whereas the expression was weaker in DIO mouse islets.4.The expression of Clec11 a in islets reached a peak at 12 h and then decreased when islets of mice were treated with 0.5m M PA.The expression of Clec11 a was significantly lower than that of the control group after 48 hours.The results of PA treatment for 12 hours with different concentrations showed that the expression of Clec11 a in the 0.5m M PA treatment group was significantly higher than those in other groups.After 48 hours treatment of PA with different concentrations,the expression of Clec11 a gradually decreased with the increase of PA concentration.5.Treatment of MIN6 cells and ISC with different concentrations of PA for 48 hours showed that the higher concentration of PA,the lower the expression of Clec11 a protein.When both cells were treated with 0.5m M PA,it was found that Clec11 a expression reached a peak at 0.5 hours with subsequent decline.Conclusion: Clec11 a is expressed in mouse islet cells,whereas the expression is significantly decreased in the islets of DIO mice.In addition,the expression of Clec11 a can be inhibited by long-term palmitic acid culture in islets,MIN6 cells and ISC cells of normal mice.The above results suggest that lipiotoxicity can inhibit the expression of Clec11 a in islet cells.The effect of Clec11 a on islet function is worthy of further study.Part ? Effects and mechanism of Clec11 a protein on mice isletBackground and objective: The elevated level of free fatty acids in circulation can impair islet ?-cell function and induce type 2 diabetes.However,the molecular mechanism of pancreatic ?-cells dysfunction induced by lipotoxicity has not been fully elucidated.In the first part,the low expression of Clec11 a gene was found in the islets of obese mice,while it moderated in DIO mice based on transcriptome sequencing.Therefore,it is proposed that the low expression of Clec11 a gene is related to high-fat-induced islet dysfunction.Our aim was to explore the role and mechanism of Clec11 a in mouse islet.Methods: Mouse islets,MIN6 cells and ISC,were treated with different concentrations of recombinant Clec11 a protein.The proliferation of MIN6 cells and ISC was observed by CCK-8 assay and Ki67 immunofluorescence staining to obtain the optimal concentration of r Clec11 a,which can be used in the subsequent experiment.Insulin secretion function was measured by insulin release test under high glucose stimulation.The BODPIY staining was performed to detect the lipid drop in cells.The proliferation marker PCNA and lipid metabolism-related genes expression were detected by q PCR.The relevant signaling pathways were detected by western blot.Results: 1.CCK8 results showed that the proliferation capacities of MIN6 cells and ISC treated with r Clec11 a protein were significantly higher than those of the control group.Immunofluorescence staining showed that the number of Ki67 positive cells in the 10ng/ml r Clec11 a treated group was significantly higher than that in the control group.In both normal and high-fat environments,the PCNA gene m RNA levels of r Clec11a-treated group were significantly higher than those of the control group.2.Mice islet and MIN6 cells were treated with different concentrations of r Clec11 a for 48 hours.The insulin secretion of islets in the r Clec11a-treated group was significantly higher than that in the control group.There was no statistical difference on the insulin content in the islets among groups.The insulin secretion and content of MIN6 were not affected by r Clec11 a administration.3.Western blot results showed that the phosphorylation levels of Erk and Akt signaling pathway were significantly increased in islets,MIN6 cells and ISC of mice after r Clec11 a treatment compared with the control.When Erk signaling pathway inhibitor U0216 was used,islet cell proliferation and PCNA gene levels decreased.However,this phenomenon was not observed after using the Akt signaling pathway inhibitor.4.The BODPIY staining of lipids in MIN6 cells and ISC was significantly enhanced after PA treatment for 48 hours,while r Clec11 a treatment did not significantly moderate PA-induced lipid droplet deposition.The levels of Sterol regulatory element-binding protein 1(SREBP-1c),Fatty acid synthase(Fasn)and perilipin-2(Plin2)in PA treated group were significantly higher than those in control group.The levels of SREBP-1c,Fasn and Plin2 genes in Clec11 a combined PA treatment group were significantly lower than those in PA treatment group.5.The phosphorylation level of Fox O1 signaling pathway in r Clec11 a treatment group was significantly higher than that of the control group.The phosphorylation of Fox O1 signaling pathway was significantly lower than that of the control group after using the Akt signaling pathway inhibitor MK2206.Conclusion: The treatment of r Clec11 a can improve the insulin secretion capability and promote the proliferation of islet cells,as well as regulate the expression of lipid metabolism genes.Part III Expression and function of Clec11 a in human islets and ?-cellBackground and objective: In our previous study,it was observed that the expression of Clec11 a decreased in the islets of obese mice.Clec11 a can promote the proliferation of islet cells and involves in the regulation of intracellular lipid metabolism under lipotoxicity.Depth analysis of the single-cell sequencing data published in the GEO database suggests that Clec11 a expression may be present in a variety of cells in human islets.However,the location and function of Clec11 a in human islets have not been explored.Therefore,our aim was to explore the expression and effect of Clec11 a in human islets and Endo C-?H1 cells.Methods: Human pancreatic islets and Endo C-?H1 cell were treated with recombinant Clec11 a protein or si RNA transfection.Immunofluorescence staining was used to observe the expression and localization of Clec11 a in human islets and Endo C-?H1 cells.WST-1 assay,Ki67 and Ed U staining were performed to observe cell proliferation.Insulin release test under high glucose stimulation was done to detect insulin secretion capability.Quantitative PCR was performed to detect insulin-related gene expression.Palmitic acid treatment was administrated to mimic high-fat environment.Results: 1.Immunofluorescence staining showed that Clec11 a expressed in Endo C-?H1 cells,which was co-localized with insulin.2.WST-1 results showed that the proliferation of Endo C-?H1 cells in the r Clec11a-treated group was significantly higher than that in the control group.Immunofluorescence staining showed that the number of Ki67 positive cells in the r Clec11 a protein-treated group was significantly higher than that in the control group 3.Insulin secretion of Endo C-?H1 cells in the r Clec11 a protein-treated group was significantly higher than the control group under high glucose stimulation.The level of intracellular insulin increased with the increasing concentration of r Clec11 a.Compared with the si RNA negative control group,the insulin secretion capacity of Endo C-?H1 cells in the si RNA-Clec11 a transfection group was significantly reduced.However,there was no significant difference in intracellular insulin content.4.q PCR results show that the levels of PCNA,insulin,IAPP and Maf A genes in Endo C-?H1 cells of the r Clec11 a protein-treated group were significantly increased compared with those in control group.Moreover,the protein expression of Pdx1 was also significantly higher than that in control group.5.Insulin secretion and levels of insulin and Maf A genes in Endo C-?H1 cells significantly decreased in the 1.5m M PA-treated group.The insulin secretion capacity of Endo C-?H1 cells in r Clec11 a combined with PA treatment group had no significant change compared with that of the PA treatment group,but the level of insulin gene significantly increased.6.Immunofluorescence staining showed that Clec11 a was expressed in human pancreatic islets and co-stained with insulin.The insulin secretion of islets in the r Clec11a-treated group was significantly improved compared with that in the control group.There was no statistical difference among the insulin content in islets.Conclusion: 1.Clec11 a expressed in both human islets and ?-cells,which is co-localized with insulin expression.2.Clec11 a protein can promote the proliferation and increase the secretion capacity of ?-cells.After interfering with the expression of Clec11 a gene,insulin secretion of ?-cells was significantly inhibited.Therefore,Clec11 a is an important intermediate molecule that regulates insulin secretion in human islets ?-cells.3.Long-term high fat culture inhibits the insulin secretion of islet ?-cells.Although r Clec11 a treatment did not rescue the suppression of insulin secretion induced by lipotoxcity,it can rescue the downregulation of insulin gene expression partially.