TLR4 Mediates β-cell Lipotoxic Damage By Inhibiting TMEM24

Purpose:1.To explore the relationship between the function of pancreatic β-cells caused by TLR4-mediated lipotoxicity and the function of transmembrane protein24(TMEM24).2.To study the relationship between TLR4 regulating TMEM24 and PIP2/IP3 pathway.Method:1.Correlation of lipotoxicity impaired βTC6 cell function with TLR4 and TMEM24: Taking the mouse insulinoma cell line βTC6 cells as the research object,we used 1.0mmol/L palmitic acid(PA)to interfere with βTC6 cells for 24 hours to establish a lipotoxicity model of β cells,observe the effect of TLR4 inhibitor(CLI095,1ug/ml)on the expression of TMEM24 in lipotoxic β cells;set TLR4agonist(LPS,1ug/ml)to interfere with β cells as a positive control.Experimental groupings: NC group,PA group,LPS group,DMSO group,CLI095 group,CLI095+PA group.After 24 hours of intervention,the ELISA method was used to detect the basal insulin secretion(BIS)and the glucose-stimulated insulin secretion(GSIS)of β cells,the flow cytometer was used to detect β cell apoptosis,and Western Blot was used to detect the protein levels of TLR4 and TMEM24.expression.2.The effect of silencing TLR4 on lipotoxicity regulation TMEM24-PIP2-IP3:Transfect βTC6 cells with TLR4 RNAi lentiviral vector(TLR4-si RNA)to down-regulate TLR4 gene expression,establish lipotoxic β-cell model,and observe the effect of down-regulating TLR4 expression on TMEM24-PIP2-IP3 in lipotoxicβ cells.Experimental groupings: NC group,PA group,TLR4 knockdown group,TLR4 knockdown + PA group.After 24 hours of intervention,the BIS and GSIS of β cells,β cell apoptosis,TLR4,TMEM24 protein and their upstream Kinase PKC,downstream PIP2 protein expression were detected by the same method as above;ELISA to detect intracellular IP3 level.3.Rescue effect of overexpression of TMEM24 on TLR4-mediated β cell lipotoxic damage: Using TMEM24 overexpression lentiviral vector to transfectβTC6 cells to up-regulate the expression of TMEM24 gene,establish lipotoxicβ-cell model or LPS-damaged β-cells,and observe the effect of up-regulated expression of TMEM24 on β-cell lipotoxicity damage and LPS damage.Experimental groupings: NC group,TMEM24 overexpression group,PA group,PA+TMEM24 overexpression group,LPS group,LPS + TMEM24 overexpression group.The BIS and GSIS of β cells,β cell apoptosis,TLR4,TMEM24 protein and their upstream Kinase PKC,downstream PIP2 protein expression were detected by the same method as above;ELISA to detect intracellular IP3 level.4.To preliminarily explore the interaction between TLR4 and TMEM24 protein in βTC6 cells: observe the changes in the localization and interaction of TLR4 and TMEM24 proteins in lipotoxic β-cell models or LPS-damaged β-cells,and observe the effect of inhibiting TLR4 activity on the localization and interaction of TLR4 and TMEM24 proteins in lipotoxic β-cells.Experimental groups: NC group,PA group,LPS group,CLI095 group,CLI095+PA group after 24 hours of intervention,laser confocal,co-immunoprecipitation(CO-IP)was used to observe the localization of TLR4 and TMEM24 and whether there is a direct interaction Possible.Result:1.PA intervention in βTC6 cells can induce the up-regulation of TLR4 expression,inhibit the expression of TMEM24,reduce the secretion of β-cell BIS and GSIS,and increase β-cell apoptosis;specifically inhibit the activity of TLR4,which can weaken the lipotoxic effect of PA;and specific activation TLR4 can also down-regulate the expression of β-cell TMEM24,inhibit the decrease of insulin secretion,and promote β-cell apoptosis.2.Silencing β-cell TLR4 expression can antagonize the down-regulation of TMEM24 expression caused by lipotoxicity,while up-regulating PIP2 expression,increasing intracellular IP3 levels,inhibiting PKC phosphorylation,increasing insulin secretion,and reducing β-cell apoptosis.The specific activation of TLR4 can lead to down-regulation of TMEM24 and its downstream PIP2 expression,lower intracellular IP3 levels,and up-regulate the phosphorylation level of PKC.3.Inducing the overexpression of TMEM24 in β cells can attenuate the lipotoxic effect,which is manifested by up-regulating the expression of PIP2,increasing the level of intracellular IP3,and inhibiting PKC phosphorylation,increasing insulin secretion,and reducing β cell apoptosis.Similarly,overexpression of TMEM24 can also antagonize the damaging effect of LPS on β cells.4.Using laser confocal microscope,it was observed that TLR4 and TMEM24 were co-localized in the cytoplasm.Co-immunoprecipitation suggested that TLR4 and TMEM24 might interact.Conclusion:1.PA promotes β cell apoptosis,promotes insulin secretion disorder,and inhibits the expression of TMEM24;2.TLR4 directly inhibits the expression of TMEM24 to mediate β-cell lipotoxic damage;3.TLR4 inhibits the expression of TMEM24 to mediate β-cell lipotoxic damage,which may act by down-regulating its downstream signal PIP2-IP3.