| Objective Under the background of lipid toxicity,to verify the activation of the G-protein-coup led receptor 119(GPR119),can activate the MST1-FoxO1 through the Ca2+-CaM,eventually induce the dysfunction of the pancteatic ? cell.Methods1.INS-1 cells were incubated by the palmitic acid(PA);INS-1 cells survival rate were detected by the MTT tests;WB of Caspase-3 and confirm PA to regulation function.The Caspase-3's expression under the PA were detected by WB.2.INS-1 cells were incubated by the GPR119 agonists MBX;INS-1 cells survival rate were detected by the MTT tests;Using flow cytometry testing INS-1 cells apoptosis rate,using cells immunohistochemical method to identify the expression of GPR119 parts and expression;WB of GPR119,phospho-MST1,clMST1,MST1,phospho-FoxO1,FoxO1,Caspase-3,clC3 and Pdx1;qPCR analyzed mRNA leves of BCL-2,Bax,Pdxl and Insulin;TG,FFA and Insulin contene in INS-1 cells was examined by enzyme colorimetry.3.INS-1 cells were incubated by the Ca2+ chelating agent EGTA and CaM inhibitors CPZ;INS-1 cells survival rate were detected by the MTT tests;Using flow cytometry testing INS-1 cell apoptosis rate;WB of phospho-MST1,clMST1,MST1,phospho-FoxO1 and FoxO1.4.Normal and PA-pretreated INS-1 cells 12 hrs were transfected witpcDNA3-MST1 expression plasmid and MST1 shRNA plasmid,then incubated for 72 hrs,WB of phospho-MST1,elMST1,MST1,phospho-FoxO1 FoxO1,Caspase-3,clC3 and Pdx1.5.80 C57BL/6J mice were divided into 10 groups,5 groups with high-fat diet,5 groups with low-fat diet,Feed them for 18 weeks continuely,recording food intake and weight.Insulin tolerance and glucose tolerance in the 12 week were detected.Verify the animal model successful or not.6.GPR119 over-expression vector intervention trial in the C57BL/6J.Transducted the reconstructed pCDH GFP,pCDH GPR119,shCtrl,shGPR119 plasmid into the 293T cells by liposome transfection,then packing,amplification,got the Lentiviral transduction particles,Tail vein injection technology was used with recombinant lentivirus LV-GFP,LV-GPR119,LV-shCtr1.LV-shGPR119 into eight groups of mice respectively(high fat/low fat feeded over 18 weeks).A week later To obtain The pancreas,by HE staining and transmission electron microscope to observe the morphology and ultramicroscopic structure of the pancreas islet and? cell;FFA content in pancreas tissue was examined by enzyme colorimetry;WB of GPR119,phospho-MST1,clMST1,MST1,phospho-FoxO1,FoxO1,Caspase-3,clC3 and Pdx1.Results1.PA increased Caspase-3 expression in a dose-dependent or time-dependent mannerINS-1 cells were incubated with 250.0 uM/L and 500.0 uM/L PA for 12 hrs,PA increased Caspase-3 expression in a dose-dependent manner;INS-1 cells were incubated with 250.0 uM/L PA at 0 hr,1 hr,3 hrs,6 hrs,12 hrs,24 hrs,48 hrs,respectively.PA increased Caspase-3 expression in a time-dependent manner.2.INS-1 cells were incubated with 250.0 uM/L PA for 12 hrs,then GPR119 agonist MBX 4.0uM/L incubation INS-1 cell 10 min.MBX inhibits phosphorylation of MST1 and FoxO1.qPCR results showed that MBX reduced Bax expression,increased Pdxl and Insulin expression;cellular TG and FFA content were decreased.3.WB results show that the change of the extracellular Ca2+ did not effect the phosphorylation of MST1 expression.INS-1 cells were incubated with 250.0 uM/L PA for 12 hrs,CaM inhibitors CPZ 100.0 uM/L continued to incubate for 60 min,CPZ increased phosphorylation of MST1 and FoxO1.4.INS-1 cells were incubated with PA for 12 hrs,pcDNA3-MST1 expression plasmid and MST1 shRNA plasmid transfection after incubation 72 hrs,WB results show that the MST1 overexpression of transfection increased phosphorylation of MST1 expression and phosphorylation of FoxO1 expression;MST1 shRNA significantly inhibited the phosphorylation of MST1 and FoxO1.5.High fat Induced C57BL/6J mice,tail vein injection of GPR119 overexpression lentivirus,GPR119 shRNA lentivirus,after one week,the severity of pancreatic ? cell's injury in the GPR119 over-expression mice groups were much more than the GPR119-shRNA mice group,through the observation of the HE staining and the transmission electron microscope.Besides,the serum triglyceride and free fatty acid concentration decreased,WB results showed that GPR119 over-express inhibited phosphorylation of MST1 and FoxO1 in the mice pancreatic.Conclusion1.Under the background of lipid toxicity,the activation of the GPR119 alleviated the dysfunction of the pancreatic beta cells?2.Under the background of lipid toxicity,GPR119 can regulate MST1-FoxO1 expression through the Ca2+-CaM in INS-1 cells probably.3.Under the background of lipid toxicity,GPR119 can alleviate pancreatic beta cell dysfunction through the MST1-FoxO1 pathways. |
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