Liraglutide Increases The Protective Effect Of MANF On Pancreatic ? Cells And Possible Mechanisms

Part1: MANF REDUCES THE DAMAGE OF ENDOPLASMIC RETICULUM STRESS TO PANCREATIC ? CELLS BY PROMOTING AUTOPHGYObjective: To investigate the effect of high glucose and fat on human serum MANF and whether MANF can promote autophagy and reduce ERS to protect pancreatic ? cells under the ERS induced by GLU,PA and TG.Methods: Serum samples from 60 healthy control(NC),40 subjects with simple hyperlipidemia(HLD)and 71 patients with type 2 diabetes mellitus(T2DM)were collected from the physical examination center and endocrine outpatient unit of the Second Affiliated Hospital at Chongqing Medical University.Serum MANF levels were detected by human-derived MANF ELISA kit.Immunohistochemical experiments were carried out to verify the expression of MANF in pancreatic islets by using sections of pancreatic from the research group's previous reserves.Meanwhile,in vitro experiments were conducted on mouse pancreatic ? cell line MIN6.After the MIN6 cells were treated with different concentrations of GLU,PA and TG for 24 h.MANF,ERS and autophagy related proteins,and apoptosis protein were detected by Western blotting.Cell proliferation and apoptosis phenomenon were detected using CCK8 and Hoechst combined with immunofluorescence,respectively.After the MIN6 cells were treated with PA for 24 h,the changes of autophagosomes and subcellular structures were examined using transmission electron microscopy(TEM).After PA or TG combined with autophagy inhibitor CQ,the proteins expression of MANF,ERS and autophagy pathway were detected via Western blotting.To further explore the relationship between MANF and autophagy and ERS,after the cells were treated with human-derived recombinant MANF protein combined with PA for 24 h,the expression changes of GRP78,mTOR and p-mTOR proteins were detected by Western blotting,and the autophagosomes and subcellular structures were detected using TEM.Results: Compared with the NC group,the expression of MANF in the HLD and T2 DM groups increased successively,and the difference was statistically significant(P <0.001).In the subgroup analysis of T2 DM,the median fasting glucose of non-normal distribution was defined as 8.23,and the MANF level greater than 8.23 was significantly higher than that of fasting glucose less than or equal to 8.23(P<0.05).In the pancreatic tissue sections of mice,the expression of MANF in the pancreas,especially in the islet tissues,was verified.In vitro,we found that with the increase of GLU,PA and TG concentrations,cell proliferation activity significantly decreased(P?0.01),and the expression of MANF protein increased in a concentration-dependent manner(P < 0.05),In addition,when high GLU and high PA were combined,the expression of MANF protein further increased,and the expression of C-cas-3 protein(P?0.05),ERS pathway proteins including IRE1,GRP78,ATF6,ATF4,CHOP(P ? 0.05),autophagy related proteins p62,LC3 B,and p-mTOR(P?0.05)all showed increase in a concentration-dependent manner.Double immunofluorescence staining showed that the fluorescence intensity of MANF and insulin increased in a concentration-dependent manner as the GLU and PA concentrations increased,while the combination of GLU and PA increased further.The fluorescence intensity of C-cas-3 was also increased in GLU and PA concentration dependence.Compared with the BSA group,the number of mitochondria in the PA group increased and damaged,and the endoplasmic reticulum expanded significantly.After CQ combined with PA and TG,the expression of MANF,ERS and autophagy-related proteins p62,LC3 B,and p-mTOR increased(P?0.05).After 24 h of treatment with recombinant human MANF protein combined with PA,we found that the expression of MANF protein was significantly increased(P?0.001),while the expression of p-mTOR(P?0.05)and GRP78 protein was decreased(P? 0.001).Compared with BSA group,the number of autophagosomes increased,the mitochondrial damaged and endoplasmic reticulum expanded in PA group.Compared with PA group,the number of autophagosomes increased in PA+MANF group,and no mitochondrial damage was observed.Conclusion: The serum MANF protein was increased in HLD and T2 DM patients.Meanwhile,GLU,PA and TG can induce the increased expression of MANF protein in MIN6 cells,inhibit autophagy,induce ERS,inhibit cell proliferation and promote its apoptosis.Which suggests MANF is involved in regulating autophagy and ERS.MANF may protect the cells by increasing compensatory expression during this process.After PA+MANF,the expression of p-mTOR and GRP78 proteins decreased,and the number of autophagosomes increased.It suggests that MANF can promote autophagy,reduce PA-induced ERS,and thus protect pancreatic ? cells.Part2: LIRAGLUTIDE BY REGULATING MANF TO PROMOTE AUTOPHGY AND REDUCE ENDOPLASMIC RETICULUM STRESS,PROTECTS PANCREATIC ? CELLObjective: To investigate the relationship between MANF and liraglutide(LRG),and whether LRG can promote autophagy and reduce ERS by regulating MANF,to protect pancreatic ? cells.Methods: After 24 h treatment of MIN6 cells with GLU,PA and TG combined with LRG,ERS,autophagy related proteins and apoptotic proteins were detected by Western blotting,cell proliferation was detected using CCK8,and apoptosis was detected via Hoechst combined with immunofluorescence.After 24 h treatment with PA+LRG,the changes of autophagosomes and subcellular structures were examined using TEM.After intervention with PA+CQ+LRG for 24 h,ERS and autophagy pathway proteins expression were tested by Western blotting,cell proliferation was determined using CCK8.Results: GLU,TG or PA combined with LRG group,compared with GLU,PA or TG group alone,cell proliferation activity increased significantly(P?0.05),while MANF protein expression increased(P?0.05),C-cas-3 protein expression decrease(P?0.05),ERS-related proteinIRE1,GRP78,XBP-1s,ATF4,CHOP expression decreased(P?0.01),p62 and p-m TOR expression decreased(P?0.01),LC3?/? protein expression increased(P?0.001).The immunofluorescence intensity of C-cas-3 was decreased.Compared with the PA group,the number of autophagosomes in the PA+LRG group increased.Compared with PA+CQ group,PA+CQ+LRG group,cell activity increase(P ? 0.05);MANF protein expression increased(P?0.001),ERS related proteins expression decreased(P?0.001),p62 and p-m TOR proteins expression decreased(P?0.001),LC3 ?/? protein expression increased(P?0.001).Conclusion: Liraglutide protects pancreatic ? cells by regulating MANF to promote autophagy,reduce ERS and apoptosis,and promote cell proliferation.