The Experimental Study On The Influence Of Peptide FLPNF Enhanced Autophagy On HIAPP Aggregation In INS-1 Cell Line And Its Molecular Mechanism

Objective: Diabetes mellitus is a common chronic disease that seriously threaten human health and lives globally.According to the most recent reports,the prevalence of adult diabetes in China is 10.9%,making China the country with the largest number of diabetes patients in the world.Type 2 diabetes(T2D)is the most common type and accounts for approximately 90-95% of all diabetes cases.Although much researches have been done on type 2 diabetes over the last two decades,its specific etiology and pathophysiology remains poorly understood.Current studies indicated that factors for T2 D development and progression can be divided into three main categories:(1)genetic susceptibility factors;(2)cytotoxicity caused by islet amyloid polypeptide;(3)risks factors related to life and diet style.Reportedly,the main pathophysiological features of the islets in patients with type 2 diabetes are islet β cell losses and dysfunction,insulin resistance,and formation of amyloid deposition within islet.Human islet amyloid polypeptide(hIAPP),the main component of islet amyloid deposition,which has been shown to induce islet β cell dysfunction and apoptosis,plays important roles in the development of diabetes.Therefore,preventing or reducing hIAPP deposition can reduce islet β cell apoptosis and improve insulin secretion function of islet β cell.The autophagy-lysosomal system,the way by which cells degrade misfolded proteins and damaged organelles,has been considered as the most important pathway for islet β cells to clear the hIAPP deposition.However,hIAPP could over-activate the mTOR complex 1(mTORC1),which inhibits autophagy flux by phosphorylating the ULK1(ATG1),resulting in further deposition of hIAPP.In addition,hIAPP could disrupt the lysosomal membrane which leads to the release of lysosomal enzymes and disturbance of autophagy-lysosomal system,resulting in β cell dysfunction and apoptosis and further deposition of hIAPP,thus aggravating the diabetes.While,a well-established mTOR inhibitor rapamycin has been shown to activate the autophagy to promote the degradation of hIAPP deposition,improve islet β cell function,and reduce cellular apoptosis through binding to the FRB domain of mTOR.Therefore,enhancing cellular autophagy activity is expected to reduce the cytotoxicity induced by hIAPP and to protectislet β cell function,and the autophagy agonists are considered as potential therapeutic agents for the treatment of T2 D.Another strategy to reduce hIAPP deposition is to prevent hIAPP misfolding by hIAPP aggregation inhibitor,which mainly include peptides inhibitors,natural small molecule compounds,metal ions,nanomaterials,malecular chaperone and so on.Compared to other types of inhibitors,peptide inhibitors have received more attention due to advantages such as less cytotoxicity,easier synthesis,and the ability to target the key region of hIAPP aggregation.It has been reported that the peptides SNNFGA,GAILSS,NYGAILSS,and NFGAILFF could effectively inhibit the aggregation of hIAPP in an in vitro buffer solution,while the intraperitoneal injection of peptide D-ANFLVH could significantly reduce the hIAPP deposition in the islet of hIAPP transgenic mice.However,the specific mechanism by which peptides inhibit hIAPP aggregation is still not clear.There are especially lack of studies on the effects of peptides on cellular biological functions such as autophagy.The peptide FLPNF(Phenylalanine-Leucine-Proline-Asparagine-Phenylalanine,Phe-Leu-Pro-Asn-Phe)is a five-amino-acids hIAPP aggregation inhibitor,which could effectively reduce the hIAPP aggregation in an in vitro buffer solution.Further label-free quantitative proteomics analysis shown that after exposure to peptide FLPNF,IGF2 was down-regulated and NPTX1 was up-regulated,both of which are downstream proteins of mTOR.And the DVL2,which could be degraded by autophagy,was also down-regulated.The changes of the level of above proteins were similar to the effect of inhibition of mTOR pathway by rapamycin,indicated that peptide FLPNF has effect on autophagy flux.However,the specific molecular mechanism by which the peptide FLPNF affects cellular autophagy activity remains unclear.Consequently,the hIAPP-INS-1 cells overexpressing hIAPP and rIAPP-INS-1 cells overexpressing rIAPP(rodent islet amyloid polypeptide)were constructed by virus transfection,and the aim of the present study was to confirm that the peptide FLPNF could increase autophagy flux and to clarify its underlying mechanisms.The protective effect of peptide FLPNF on hIAPP-INS-1 cells was also investigated,with the purpose of providing theoretical basis for the prevention and treatment of T2 D.Methods: 1.Synthesize the peptide FLPNF and peptide TAT-FLPNF(with cell-penetrating peptide TAT at N-terminal of FLPNF).The effects of the above peptides on LC3 expression in INS-1 were investigated by Western Blot.2.CCK-8 kits were used to detect the cellular viability of INS-1 cells after exposure to different concentration of peptide FLPNF(0-400μM)for different time(24-96h).3.INS-1 cells were transfected with hIAPP and rIAPP adenovirus to construct hIAPP-INS-1 and rIAPP-INS-1 cells,respectively.Western Blot was used to detect the expression level of LC3 protein in hIAPP-INS-1 cells after exposure to different concentrations peptides TAT-FLPNF,with the purpose of determining the optimal concentration of short peptides to enhance autophagy.4.By using autophagy inhibitor 3-MA,Western Blot and transmission electron microscopy were respectively used to detect the expression level of LC3 and P62 proteins and the number of intracellular autophagosomes in hIAPP-INS-1 cells treated with peptide TAT-FLPNF.5.By using autolysosome degradation inhibitor Bafilomycin A1,Western Blot was used to detect the expression level of LC3 and P62 proteins in hIAPP-INS-1 cells treated with peptide TAT-FLPNF;hIAPP-INS-1 cells expressing mRFP-GFP-LC3 were constructed by transfecting hIAPP-INS-1 cells with tandem mRFP-GFP-LC3 adenovirus,and the number of autophagosomes(mRFP+GFP+)and autolysosomes(m RFP+GFP-)in hIAPP-INS-1 cells after exposure to peptide TAT-FLPNF was detected using a confocal microscope.6.The expression level of the phosphorylated p70S6 K and phosphorylated S6 in hIAPP-INS-1 cells after exposure to peptide TAT-FLPNF were determined by Western blot to investigate the effect of peptide TAT-FLPNF on PI3K-Akt-mTOR-p70S6 K pathway.7.HEK-293-myr-Akt cells expressing a constitutively activated form of Akt were constructed by myr-Akt lentivirus transfection.After exposure to peptide TAT-FLPNF,Western Blot was used to detect the expression level of the phosphorylated GSK3β and phosphorylated S6 in both HEK-293 and HEK-293-myr-Akt cells,also the expression level of Akt and phosphorylated p70S6 K in hIAPP-INS-1 cell,to determine the effects of peptide TAT-FLPNF on mTORC1 and mTORC2 activity.8.HEK-293 cells overexpressing Flag-mTOR protein were constructed by transfection of Flag-labeled mTOR plasmid.Anti-Flag immunomagnetic beads were used to extract the mTOR protein.Magnetic beads containing mTOR protein were co-incubated with FITC-labeled peptide FLPNF.The intensity of FITC fluorescence signal on magnetic beads was detected by flow cytometry.9.Download the 3D structure of the FKBP12-rapamycin-FRB ternary complex from the RCSB Protein Data Bank(PDB ID: 1NSG),and use PyMOL 1.7.2.1 software to extract the 3D structure of the FRB domain;use ChemDraw 14.0.0.117 and PyMOL1.7.2.1 software to construct the 3D structure of peptide FLPNF;AutoDock Vina 1.1.2software was used to predict the binding of the peptide FLPNF to the FRB domain of mTOR.10.The FITC-labeled oligomer antibody FITC-A11 was used to detect the hIAPP oligomer to investigate the effect of peptide TAT-FLPNF on hIAPP oligomer deposition in hIAPP-INS-1 cells.11.The cellular viability was detected by using CCK-8 kits to determine the effect of peptide TAT-FLPNF on hIAPP-INS-1 cells viability.12.The expression level of Cleaved Caspase-3 in hIAPP-INS-1 cells after exposure to peptide TAT-FLPNF was determined by Western Blot.13.Glucose-stimulated insulin secretion of hIAPP-INS-1 cells after exposure to peptide TAT-FLPNF was determined by using insulin ELISA kits.Results: 1.The peptides TAT-FLPNF(P <0.001)and FLPNF(P <0.01)significantly increased the LC3-II / LC3-I ratio in INS-1 cells;while the cell-penetrating peptide TAT did not affect the LC3-II / LC3-I ratio.The peptide TAT-FLPNF can further increase the LC3-II / LC3-I ratio(P <0.05)compared with the peptide FLPNF.2.INS-1 cells were cultured at a concentration of 0-400μM of peptide TAT-FLPNF for 24-96 hours.However,there was no difference in cell viability between groups.3.After exposure to peptide TAT-FLPNF for 24 hours,the LC3-II / LC3-I ratio in hIAPP-INS-1 cells gradually evaluated with the increasing of concentration of peptide,and reaching a peak when the peptide concentration was 200μM(P <0.0001).4.The peptide TAT-FLPNF significantly increased the LC3-II / LC3-I ratio in hIAPP-INS-1 cells(P <0.001),while significantly reduced the intracellular P62 proteinlevel(P <0.05).This effect can be suppressed by autophagy inhibitor 3-MA.In addition,the peptide TAT-FLPNF significantly increased the autophagosomes number in hIAPP-INS-1 cells(P <0.01).5.The peptide TAT-FLPNF combined with Bafilomycin-A1 further increased the LC3-II / LC3-I ratio(P <0.001)and P62 protein level in hIAPP-INS-1 cells(P <0.05)compared to Bafilomycin-A1 alone.The peptide TAT-FLPNF significantly increased the number of autophagosomes(mRFP+GFP+)(P <0.05)and autolysosomes(mRFP+GFP-)(P <0.001)in hIAPP-INS-1 cells.6.The peptide TAT-FLPNF significantly reduced p-p70S6K(Thr389)(P <0.0001)and p-S6(Ser240 / 244)(P <0.001)protein level in hIAPP-INS-1 cells,and this effect is not affected by p-p70S6K(Thr389)dephosphorylation inhibitor OA.7.The peptide TAT-FLPNF significantly reduced the level of p-S6(Ser240 / 244)protein in HEK-293-myr-Akt cells(P <0.0001)and did not affect the level of p-GSK3β(Ser9)protein.Furthermore,peptide TAT-FLPNF significantly reduced p-p70S6K(Thr389)(P <0.01)and p-S6(Ser240 / 244)(P <0.0001)protein level in hIAPP-INS-1cells,without affecting p-Akt(Ser473)protein levels.8.FITC fluorescence signals of magnetic beads that incubated with FITC-FLPNF significantly increased.AutoDock analysis shown that the peptide FLPNF binds to the FRB domain through π-π stacking and hydrogen bonding,and the peptide FLPNF interacts with the FRB domain applies a dramatically similar position to that seen for rapamycin in the FKBP12-rapamycin-FRB ternary complex.9.The hIAPP oligomer deposition in INS-1 cells overexpressing hIAPP significantly increased(P <0.0001),while no oligomer deposition in INS-1 cells overexpressing rIAPP was detected.The peptide TAT-FLPNF significantly reduced hIAPP oligomer deposition in hIAPP-INS-1 cells(P <0.001).10.The peptide TAT-FLPNF significantly increased the cellular viability of hIAPP-INS-1 cells(P <0.05).11.The peptide TAT-FLPNF significantly reduced the Cleaved Caspase-3 protein level in hIAPP-INS-1 cells(P <0.05).12.The insulin secretion of INS-1 cells over-expressing hIAPP decreased significantly under high glucose(20mM)condition(P <0.0001).The peptideTAT-FLPNF induced a 1.26-fold increase(P <0.0001)in insulin secretion from hIAPP-INS-1 cells under high glucose(20mM)condition.While there was no difference in insulin secretion from hIAPP-INS-1 cells with or without peptide TAT-FLPNF treatment under low glucose(1.1mM)condition.Conclusion: Compared with peptide FLPNF,the TAT-FLPNF had stronger autophagy enhancing activity;peptide TAT-FLPNF did not affect cellular viability in short-term culture;the optimal concentration of peptide TAT-FLPNF to enhance autophagy of hIAPP-INS-1 cells is 200μM;the peptide FLPNF inhibited mTORC1 activity by binding to the FRB domain of mTOR without affecting mTORC2 activity,thereby enhancing cell autophagy;the peptide TAT-FLPNF significantly reduced hIAPP oligomers in hIAPP-INS-1 cells;the peptide TAT-FLPNF significantly reduced hIAPP-INS-1cell apoptosis,and improved glucose-stimulated insulin secresion function of hIAPP-INS-1cells.