The Preliminary Study Of FFAs Aggravating The Damage Of Pancreatic Beta Cell By Affecting HIAPP Conformation And Endoplasmic Reticulum UPR

Objective:The incidence of diabetes is increasing year by year,of which T2 DM is the main types.It is expected that there will be 439 million people suffering from diabetes in 2030.With the improvement of living standards,obese patients increased gradually,and obesity leads to increased free fatty acids(Free Fatty Acids,FFAs)content in plasma and hyperlipidemia.It is well known that hyperlipidemia can cause beta cell dysfunction.Long term exposure to high levels of FFAs can cause cell apoptosis,but the mechanism remains unclear.Recent studies have shown that beta cell apoptosis is associated with endoplasmic reticulum stress induced by glucose / lipid toxicity and islet amyloid deposition.Human islet amyloid polypeptide(hIAPP)is a hormone associated with glucose metabolism.hIAPP and insulin are co-secreted by pancreatic beta cells.It's not clear the physiological function of hIAPP.However the amyloid deposition of this polypeptide was found in islets of most patients with type 2 diabetes mellitus.Several studies have suggested that the presence of amyloid deposition followed the conformational changes of these protein,especially the increase in ?-sheet.The increase in ?-sheet may cause the unusual degradation of these proteins,which may initiate the endoplasmic reticulum UPR pathway to remove excess amyloid,eventually may lead to endoplasmic reticulum stress and cell apoptosis depended on the endoplasmic reticulum stress.These lead to the lost of islet beta cells.Therefore,it is important to explore the mechanisms by which the interaction between FFAs and hIAPP can lead to the damage of beta cells,which may be beneficial to control/ delay the development of T2 DM.Methods:Part I: Chemical ExperimentshIAPP alone or FFAs+hIAPP was incubated with ThT at 37?,the fluorescence intensity wasdetected to reflect the hIAPP fibrosis level by using the multifunctional microplate reader.hIAPP alone or PA+hIAPP was incubated at 37?for 7 h,then the formation of hIAPP fibrosis was observed under transmission electron microscopy.The conversion of hIAPP conformation from alpha helix or random coil to ?-sheet was assayed by using CD experiment.The prepared SUVs(the ratio of peptide/ lipid 1:10)made of POPC,POPS or POPC/POPS was incubated with hIAPP,PA+ hIAPP and ThT at 37?,the fluorescence intensity was detected by using the multifunctional microplate reader.Part II: Cellular ExperimentsThe transfection efficiency was observed under fluorescence microscope after the empty plasmid and the plasmid expressing hIAPP were transfected into the INS-1 cells for 48 h.The empty plasmid and the plasmid expressing hIAPP were transfected into the INS-1 cells for 24 h,then the cells were treated with the indicated concentration of PA for 24 h or 48 h.Cell viability was measured by MTT method.The relative expression of hIAPP,GRP78,Chop and ATF6 mRNA were detected by using real time RT-PCR.After the INS-1 cells transfected with the empty plasmid and the plasmid expressing hIAPP was treated with the different concentrations of PA for48 h,cells were collected,fixed with 70% ethanol and washed with PBS,then incubated with PI staining for 30 min.The apoptotic sub G1 peak was detected by flow cytometry.Results:1.It was found that 200 or 400 ?M FFAs could increase the formation of hIAPP fibrosis using Thioflavine T Fluorometric Assay.Among them,200 ?M PA not only promoted the formation of hIAPP fibrosis,but also increased the stability of hIAPP fibrosis.An increase in the formation of fibrosis hIAPP induced by PA was observed under transmission electron microscopy.PA promoted the conversion of hIAPP conformation from alpha helix or random coil to ?-sheet in the CD experiment.Under the condition of artificial plasma membrane,the formation of hIAPP fibrosis was increased in the presence of the negative lipid POPS,and POPS cooperatd with PA to promote the formation of hIAPP aggregation.2.The plasmid expressing hIAPP were transfected into INS-1 cells,the transfection efficiency was 65%.The relative expression level of hIAPP mRNA was increased obviously in INS-1 cells transfected with hIAPP plasmid.The cells transfected with hIAPP plasmid,compared to the control group(empty plasmid),cell survival rate was significantly decreased after treatment with PA.Additionally,the relative expression of GRP78 / Bip,ATF6 and CHOP mRNA related to endoplasmic reticulum stress in the cells expressing hIAPP were obviously increased and cell apoptosis rate was significantly increased compared to those of control cells.In the cellstransfected with hIAPP plasmid,compared to hIAPP alone,PA + hIAPP significantly decreased cell viability,and significantly increased endoplasmic reticulum stress and apoptosis rate.Conclusion:1.FFAs,especially PA,can change hIAPP conformation,promote conversion of hIAPP conformation from alpha helix or random coil to ?-sheet,and the formation of hIAPP fibrosis.2.Under the condition of artificial plasma membrane,the formation of hIAPP fibrosis is increased in the presence of the negative lipid POPS,and POPS cooperates with PA to promote the formation of hIAPP aggregation.3.The conversion of hIAPP conformation and the formation of hIAPP fibrosis induced by PA triggers endoplasmic reticulum stress,which induces cell apoptosis and aggravates the damage of pancreatic beta cells.