Mechhanism Of TXNIP Promoting ?-cell Dysfunction By Mediating Autopagy Disorde

Part 1:TXNIP EFECTS ISLET DYSFUNCTION CAUSED IN HIGH-FAT-GLU DIET VIA AUTOPHAGYObjective:To investigate the relationship between txnip and islet cell secretory dysfunction and islet autophagy dysfunction.Methods:12 healthy 4-week-old male C57BL/6J mice?WT?and 12TXNIP knockout mice(TXNIP^-/-),each group randomly divided into two feeding methods:SD group?ordinary diet?and HFD group?high-fat and high-glucose diet?.Afte fed for 16 weeks,GTT and ITT tests were performed.Islet cells were isolated,Pancreatic tissue specimens were collected and tested for TXNIP,P62,and LC3B protein expression levels.Results:The expression of TXNIP in pancreatic tissue of C57BL/6J mice was increased after high fat and high glucose feeding?p<0.01?.Compared with the homologous general food group,fasting blood glucose and the area under blood glucose curve of ITT or GTT increased?p<0.01?.Compared with those of C57BL/6J mice fed the same manner,TXNIP^-/-mice had lower fasting blood glucose,and the area under blood glucose curve of ITT or GTT were decreased(?p<0.05?.The expression levels of autophagy-related proteins P62,LC3B and LC3?/? in the pancreas of mice increased in the HFD group compared with the normal food group?p<0.01?.Compared with the C57BL/6J HFD group,they were lower in the TXNIP^-/-HFD group?p<0.05?.Conclusion:High-fat-glu diet can increase the expression of TXNIP in pancreatic tissue,decrease the level of insulin secretion,increase insulin resistance,and lead to the accumulation of autophagic substrate protein in pancreatic islet cells.Txnip deletion can effectively resist islet autophagy dysfunction and islet cell dysfunction induced by high fat and high glucose.PART2:MECHANISM OF TXNIP MEDIATED AUTOPHAGY AND DYSFUNCTION OF ISLET CELLSObjective:To investigate the effect of TXNIP on islet cell autophagy and its related mechanismMethods:Routinely cultured MIN6 mouse islet cells were infected with lentivirus to construct three groups of cells: TXNIP overexpression?LV-TXNIP?,negative control virus?LV-GFP?and TXNIP expression inhibition?LV-TXNIP-siRNA?.And Western Blot method was used to test infection efficiency.Each group was treated with free fatty acids?FFA?,autophagy agonist rapamycin,and inhibitor bafilomycin A1.Autophagy bodies were detected by transmission electron microscopy.The expression of TXNIP,P62,LC3 B,Atg3,Atg5,Atg12,Atg16 and m TOR pathway were detected by Western blot.Results:Compared with LV-GFP group,TXNIP,P62,Atg12,Atg16 protein expression was significantly increased in LV-TXNIP group?P<0.01?,Atg3,Atg5,and LC3?/?,m TOR phosphorylation levels were increased?p<0.05?.The expression of TXNIP,P62,Atg3,and Atg5 in LV-TXNIP-si RNA group was significantly decreased?p<0.01?,the expression of Atg16 was decreased?p<0.05?,LC3?/? was increased?p<0.01?,and Atg12 and m TOR phosphorylation levels were absent obvious change.After rapamycin treatment of MIN6 cells,the expression of P62 decreased?P<0.01?and the ratio of LC3?/? increased?p<0.01?.Compared with LV-GFP group,P62 increased in LV-TXNIP group?p<0.01?.),LC3?/? ratio increased?p<0.05?.The ratios of P62,LC3 B and LC3?/? increased after FFA treatment in all groups?p<0.05?.Compared with LV-GFP group,LV-TXNIP group showed more autophagic bodies under electron microscopy and P62 and LC3 B expression Increased?p<0.05?,LC3?/? ratio did not alter significantly;Compared with LV-GFP group,the autophagosomes,P62 expression in LV-TXNIP-siRNA group were reduced,but LC3?/? ratio was increased?p<0.05?.After the effect of autophagy inhibitor bafilomycin A1,the expression of P62 and LC3?/? in LV-GFP-TXNIP group was up-regulated compared with LV-GFP group?p<0.05?.Conclusion:Overexpression of TXNIP promoted the up-regulation of autophagy-associated protein expression,autophagic substrate protein,and autophagosomal accumulation in MIN6 cells.After silence,the expression of autophagy bodies and autophagy-related proteins was reduced.This indicates that the upregulation of TXNIP can lead to the dysfunction of islet cell autophagy conversion and distribution.This may be related to its activation of m TOR signaling pathway,inhibition of autophagy lysosomes degradation.