The Mechanism Study On Overexpression Of Insig-1and Silibinin Protect βCell Against Glucolipotoxicity

Part one:Generation of Insig-1stable cell line and study of the overexpressed Insig-1on β cell protection from glucolipotoxicity Chapter one:Construction of pcDNA3.1(+)-Insig-1recombinant expression plasmid and establishment of Insig-1stably-expressed INS-1cell lineObjective:To construct the pcDNA3.1(+)-Insig-1recombinant expression plasmid and to establish an Insig-1stably-expressed INS-1cell line.Method:Insig-1gene of the mouse was amplified from3T3-L1pre-adipocytes by RT-PCR and then cloned into eukaryotic expression vectors pcDNA3.1(+). After confirmation by PCR,double restriction enzyme digestion analysis and DNA sequencing, pcDNA3.1(+)-Insig-1was transfected into INS-1cell by lipofectamine2000. The positive cell line was then selected by G418. The expressions of Insig-1mRNA and protein in INS-1and pcDNA3.1(+)-Insig-1cell were detected by RT-PCR and western blot, respectively.Results:1.After amplification from3T3-L1cell, mouse Insig-1gene was inserted into pUCm-T vector;2. Detection by double restriction enzyme digestion analysis and DNA sequencing was done and then mouse Insig-1 gene was inserted into pcDNA3.1(+);3.The results of double restriction enzyme digestion analysis and DNA sequencing showed the generation of the expression plasmid of pcDNA3.1(+)-insig-1;4.The results of RT-PCR and Western blot showed that the Insig-1mRNA and protein of pcDNA3.1(+)-Insig-1cell were up-regulated compared to the INS-1cell. Hence,we established Insig-1stably expressed cell line successfully.Conclusion:The recombinant expression plasmid of pcDNA3.1(+)-Insig-1was successfully constructed and the cell line which expressed Insig-1was obtained. Chapter two:Study on the mechanism by which overexpressed Insig-1protects β cell against glucolipotoxicityObjective:To explore the mechanism by which overexpression of Insig-1leads to the protection of INS-1cell against glucolipotoxicity.Method:The INS-1and pcDNA3.1(+)-Insig-1cell were treated with standard (11.2mM) or high (25.0mM) glucose1640medium for0h,24h and72h. Intracellular lipid accumulation and medium FFA were detected by oil red O staining and Elisa. Cell viability was determined by MTT. Cell apoptosis was assessed by PI/Annexin V double staining. The ER stress IRE1α pathway relation protein expression (IRE1α,JNK,CHOP,BCL-2) was analyzed by Western blot analysis. Detection of insulin secretion relevant genes(IRS-2,PDX-1,UCP-2,Insulin,GLU-2) and FAS was done by real-time PCR and that of glucose stimulated insulin secretion (GSIS) was done by radio immunoassay.Results:1.FFA in medium and intracellular lipid accumulation of INS-1-Insig-1was lower than that in the INS-1group (p<0.05);2. No difference could be observed in cell viability in the two groups when exposed to either standard glucose for Oh,24h and72h or high glucose for24h (p>0.05),but a statistically significant decrease could be noted in the INS-1group compared to INS-1-Insig-1group (p<0.05).3. After being exposed to standard glucose concentration for Oh,24h and72h, there was absolutely no difference in the rate of apoptosis in the two groups. However, a significant reduction in the rate of apoptosis was seen in the INS-1-Insig-1group compared to INS-1group when exposed to high glucose concentration for24h and72h (24h:p<0.05,72h:p<0.01);4. ER stress IRE1α pathway related protein expression of INS-1-Insig-1was lower than that in INS-1group (p<0.05).Insulin secretion and insulin secretion relation genes expression of INS-1-Insig-1group were higher than that of INS-1group (p<0.05),but no difference was noted when both these groups were exposed to standard glucose concentration (p>0.05)Conclusion:These results suggest that Insig-1may play a critical role in protecting β cells against glucolipotoxicity by regulating the expression of SREBP-1c. Part two:Study whether silibinin protects INS-1cell against glucolipotoxicity via Insig/SREBP-lc pathwayChapter one:Study whether silibinin protects INS-1cell against glucolipotoxicityObjective:To explore the function of Insig/SREBP-lc pathway in the protection of INS-1cell against glucolipotoxicity by silibinin.Method:The INS-1and silibinin group were treated with standard (11.2mM) or high (25.0mM) glucose1640medium for0h,24h and72h. Intracellular lipid accumulation and medium FFA were detected by oil red O staining and ELISA. Cell viability was determined by MTT. Cell apoptosis was assessed by PI/Annexin V double staining and Hoeche33258staining. Glucose stimulated insulin secretion (GSIS) was detected by radioimmunoassay.Results:1.Silibinin significantly inhibited intracellular lipid accumulation induced by high glucose;2. Silibinin did not affect cell viability when INS-1cell was exposed to standard glucose concentration or high glucose concentration for24h (p>0.05). However, silibinin improved cell viability when INS-1was exposed to high glucose concentration for72h (p<0.05);3. Silibinin did not affect cell apoptosis rate when INS-1cell was exposed to standard glucose concentration or high glucose concentration for24h (p>0.05),but silibinin decreased apoptosis rate when INS-1was exposed to high glucose concentration for72h (p<0.05);4.Silibinin did not improve basal insulin secretion in any group (p>0.05),but it improved the insulin secretion induced by high glucose concentration (p<0.05)Conclusion:Silibinin decreased β cell apoptosis, improved GSIS and inhibited intracellular lipid accumulation, hence, protects β cell from glucolipotoxicity Chapter two:Study of the mechanism by which silibinin protects INS-1cell against glucolipotoxicityObjective:To explore the mechanism by which silibinin protects INS-1cell against glucolipotoxicity via Insig/SREBP-1c pathwayMethod:1.Both the INS-1and silibinin group were treated with standard (11.2mM) or high (25.0mM) glucose1640medium for0h,24h and72h. The protein expression of Insig-1, SREBP-1c and BCL-2expression were analyzed by Western blot analysis. Insulin secretion related genes(IRS-2,PDX-1,UCP-2,Insulin) and FAS were detected by real-time PCR.2. After Insig-1RNA interference, cell apoptosis was assessed by PI/Annexin V double staining.Resuls:The results of Western blot and real-time PCR showed that silibinin did not affect the protein and mRNA expression of either Insig-1,2,SREBP-1c or BCL-2(p>0.05),but significantly up-regulated Insig-1,2,BCL-2and down-regulated SREBP-lc in high glucose concentration (p<0.05). Furthermore, the Insig-2expression was lower than that in the control group when INS-1cell was exposed to high glucose concentration for24h (p<0.05). Silibinin did not have any effect on mRNA expression of IRS-2,PDX-1,UCP-2or insulin mRNA when INS-1was exposed to standard glucose concentration (p>0.05),but led to a significant up-regulation of PDX-1,UCP-2and insulin mRNA but down-regulation of IRS-1mRNA in high glucose concentration(p<0.05); 5. After Insig-1RNA interference, silibinin up-regulated the protein expression of Insig-1,2, BCL-2and down-regulated SREBP-lc and decreased the apoptosis rate induced by high glucose concentration.(p<0.05)Conclusion:Silibinin improved the INS-1glucolipotoxicity induced by high glucose concentration, the potential mechanism being the up-regulation of Insig-1,2, down-regulation of SREBP-1c and decreased expression of insulin secretion related genes and lipid synthesis genes.