The Protective Effect Of GPR40 Agonist On Insulin Secretion Of ?cells With Lipotoxicity And Its Relationship Of TLR4/JNK/NF-?B Signaling

Objective: To investigate the protective effect of GPR40 agonist on insulin secretion of? cells with lipotoxicity and its relationship of TLR4/JNK/NF-?B signaling.Methods:The glucose-sensitive mouse beta pancreatic cell line ?TC6 cells were cultured in vitro;?TC6 cells were exposed to 0.5mmol/L of free fatty acid(FFAs)for 24 hours to mimick the conditions of lipotoxic ?-cell model.1.Observe the protective effect of GPR40 agonist on the fat toxicity of pancreatic beta cells: the cells were assigned to normal control group,FFAs group,TAK-875 group and FFAs+TAK-875 group,respectively intervened for 24 hours.Then,the morphology of beta cells was observed under the microscope,and insulin release was assessed with an insulin ELISA.2.Observe the protective effect of GPR40 agonist of TLR4/JNK/NF-?B signaling on the fat toxicity of pancreatic beta cells:the cells were assigned to normal control group,FFAs group,TAK-875 group and FFAs+TAK-875 group,respectively intervened for 24 hours.Then,the gene expression of TLR4?My D88?p-JNK?NF-?B?cleaved caspase3 were detected by RT-PCR;the protein erpression of TLR4?My D88?p-JNK?NF-?B?cleaved caspase3 were measured by Western Blot.3.Observe the protective effect of GPR40 agonist on the lipotoxicity of beta cells and its relationship of TLR4/JNK/NF-?B signaling: the cells were assigned to observe control group,FFAs group,TAK-875 group,FFAs+TAK-875 group,CLI-095 group,CLI-095+TAK-875 group,CLI-095+FFAs+TAK-875 group and CLI-095+FFAs group;CLI-095 should be pretreated for 4 hours,then respectively intervened for 24 hours.Then,? cells morphology was observed under the microscope;insulin release was assessed with an insulin ELISA;the gene expression of TLR4?My D88?p-JNK?NF-?B?cleaved caspase3 were detected by RT-PCR;the protein erpression of TLR4?My D88?p-JNK?NF-?B?cleaved caspase3 were measured by Western Blot.4.Statistically analyze the data by means of SPSS 20 software.Data to mean ±standard deviation(x±s),by single factor analysis of variance were completely designed under radomization(one-way,ANOVA)between the two groups using LSD test,P<0.05 believes that the difference was statistically significant.Results:1.The effect of GPR40 agonists on the morphology of pancreatic beta cells induced by lipid toxicity(1)After being treated with 0.50mmol/L FFAs for 24 hours,the morphological changes of ?TC6 cells were obvious,the cell protrusions disappeared,got rounded and shrunk,the nuclear concentrated,the cell lipid droplets the accumulation of intercellular fusion links significantly reduced,cell mass became disintegrated,showing a large number of translucent floating cells;(2)after being treated with TAK-875 for 24 hours,under the microscope,cell morphology improved,cells still kept being round,however,the number of adherent cells increased with intracellular particles multiplying,lipid droplet accumulation noticeably reducing,only the cell membrane was accompanied by a small amount of lipid droplets,and cell morphology improved significantly.2.GPR40 agonist can improve the insulin secretion function of beta cells,not only can increase the basal insulin secretion,but also increased the glucose stimulated insulin secretion(GSIS),and the differences were statistically significant(P < 0.05).3.After being treated with GPR40 agonists for 24 hours,the expression of TLR4,My D88,p-JNK,NF-?B m RNA and TLR4,My D88,p-JNK,NF-?B protein expression level decreased significantly,the differences were statistically significant(P < 0.05),at the same time,the expression of cleaved Caspase3 m RNA and cleaved Caspase3 protein expression level decreased,the difference was statistically significant(P < 0.05).4.After being treated with CLI-095 to inhibitate the activeness of TLR4,the protective effect of GPR40 agonist on the lipotoxicity of beta cells had been further enhanced, insulin secretion function improvement significantly,the expression of TLR4,My D88,p-JNK,NF-?B,cleaved Caspase3 m RNA and TLR4,My D88,p-JNK,NF-?B,cleaved Caspase3 protein expression level were decreased significantly,the differences were statistically significant(P < 0.05).Conclusions:1.After being treated with GPR40 agonists,the morphological changes of beta cells by the toxic injury can reduce.2.The effect of GPR40 agonist on the insulin secretion function of beta cells not only increased the basal insulin secretion,but also increased the glucose stimulated Insulin secretion(GSIS).3.The protective effect of GPR40 agonist on the lipotoxicity of beta cells maybe partly related of TLR4/JNK/NF-?B signaling.