Study On Lipotoxic Damage Of β Cells Mediated By TMEM24 Dysfunction

Objective:Transmembrane protein 24(TMEM24)plays an important role in the regulation of insulin secretion.The purpose of this study was to investigate the effect of palmitic acid on TMEM24 and the effect of TMEM24 gene expression on the function of lipotoxic βcells.Methods:1.Effect of palmitic acid intervention on TMEM24:(1)βTC6 cells cultured in vitro were treated with three palmitic acid concentrations of 0.25mmol/l,0.5mmol/l and 1.0mmol/l for 24 hours,and compared with the control group.Then real-time fluorescence quantitative PCR(RT-q PCR)were used to detect the level of m RNA,Western blotting(WB)were used to detect the expression of protein and Enzyme-linked immunosorbent assay(ELISA)was used to detect the phosphorylated protein level of TMEM24 and the amount of insulin secretion in each group.(2)Three different intervention times were set and treated with PA of 1.0mmo/l for24 hours,36 hours and 48 hours,and compared with the blank group,Then,the m RNA level of TMEM24 was quantified by RT-q PCR,the protein expression of TMEM24 was detected by WB method,and the phosphorylation level of TMEM24 was measured by ELISA.2.The effect of TMEM24 gene expression on lipotoxic βcell function:(1)TMEM24 overexpression and knock-down virus were constructed,and then βTC6cells were transfected with lentivirus.After successful transfection,the cells were screened with appropriate concentration of puromycin,and then cultured,subcultured and cryopreserved.(2)PA of 1.0mmol/l interferes with lentivirus transfected cells to establish lipotoxicβcell model,and divided into NC+PA group,Vector+PA group,si RNA+PA group and TMEM24+/+ group.After the intervention,the apoptosis rate was detected by flow cytometry,the level of insulin secretion was detected by ELISA,the m RNA expression of Mafa and AKT was detected by RT-q PCR,and the protein contents of Mafa,AKT,phosphorylated AKT(p-AKT),PKC,phosphorylated PKC(p-PKC)and Phosphatidylinositol 4,5-bisphosphate were detected by WB.Results:1.PA decreased the expression and phosphorylation of TMEM24.(1)In the concentration gradient part,after 24 hours of intervention with different set concentrations,the results showed that PA reduced TMEM24 m RNA expression,protein expression,phosphorylated protein content,and glucose-stimulated insulin secretion(GSIS)levels in a concentration-dependent manner(P< 0.05).(2)In the time gradient part,1.0mmol/l PA was used as the intervention concentration,and the interventions were performed for different times.The results showed that PA reduced the m RNA expression,protein expression,phosphorylated protein content and the GSIS level in a time-dependent manner(P<0.05).(3)The correlation analysis between the m RNA and protein expression of TMEM24 and GSIS level in each group of concentration gradient and time gradient showed that the m RNA and protein expression of TMEM24 were positively correlated with GSIS level and had a strong correlation(P<0.05).2.TMEM24 has a certain antagonistic effect on β-cell lipotoxic dysfunction.(1)TMEM24 gene overexpression can ameliorate the decreased expression levels of Mafa,AKT,p-AKT,PI(4,5)P2 caused by high free fatty acids(P<0.05),and at the same time antagonize the activation of PKC protein in lipotoxic β cells,so that p-PKC content decreased(P<0.05).However,the opposite result appeared after knocking down the TMEM24 gene in β cells.(2)Overexpression of TMEM24 can reduce the apoptosis of β-cells caused by lipotoxicity,and significantly decrease the apoptosis rate(P<0.05),while the apoptosis rate of the gene is significantly increased after knocking down the gene(P<0.05).(3)Overexpression of TMEM24 can increase the amount of Glucose-stimulated insulin secretion(GSIS)in lipotoxic β cells(P<0.05),and GSIS is significantly impaired after knocking down this gene(P<0.05),but TMEM24 expression has no effect on the secretion of basal insulin.Conclusion:1.The lipotoxic damage caused by PA leads to the decrease of TMEM24 expression and the dysfunction of TMEM24.2.TMEM24 overexpression has a certain antagonistic effect on the lipotoxic dysfunction of β cells.3.Knockdown of TMEM24 can aggravate β-cell lipotoxic dysfunction caused by PA.