Impact Of Mutant (Pre)Proinsulin On The Function Of β Cells

Objective:This study aims to investigate the influence of mutant (pre)proinsulin which is related to the occurrence of human diabetes mellitus (DM) on the apoptosis of islet β cells. We evaluated the impact of6(pre)proinsulin mutants with different biological characteristics, namely C(A7)Y、G(B8)S、V(A3)L、R(SP6)H、G(C28)R and DelCys, on the apoptosis and proliferation of rat INS-1cells. We further explored the mechanism underlying the propoptotic effect of (pre)proinsulin mutants on βcells by comparing the action of different proinsulin mutants on the endoreticulum stress (ERS) and unfolded protein reaction (UPR) signaling pathway. This study provides theoretic base for further promotion of the therapeutic effect of DM.Methods:1. To evaluate the influence of (pre)proinsulin mutants on the apoptosis and proliferation of rat INS-1cells. Rat INS-1cells were respectively tranfected with m-WT, m-DelCys, m-C(A7)Y, m-G(B8)S, m-V(A3)L, m-R(SP6)H and m-G(C28)R plasmids, which accordingly contain cDNA of wild type proinsulin and6different (pre)proinsulin mutants.9groups were established that include m-WT group, m-DelCys group, m-C(A7)Y group, m-G(B8)S group, m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group, streptovirudin treated positive group and the negative group which only utilized the vector for transfection. The tranfection efficiency of INS-1cells was assessed by flow cytometry, the apoptotic rate of INS-1cells was determined by means of apoptosis detection kit (Annexin V-PE,7-AAD), and the proliferation status of INS-1cells was detected by cell counting kit (Cell Counting Kit-8) at48h after transfection.2. To investigate the influence of mutant (pre)proinsulin on ERS and UPR signaling pathway.The mRNA expression levels of ATF6and CHOP were determined by real time PCR (RT-PCR), and the amount of ATF6protein was detected by Western Blotting at72h after transfection.Results:1.Evaluation of the influence of (pre)proinsulin mutants on the apoptosis and proliferation of rat INS-1cells.(1)The transfection efficiency of each tansfected group was tested by flow cytometry at48h and72h after transfection. The results of each group are similar without statistical difference (F=0.966,P=0.464)(F=0.548,P=0.795).(2)The apoptosis of cells was detected at48h after transfection. The apoptotic rate of each group was lower than that of the streptovirudin treated group, and higher than that of the vector group. Besides, the apoptotic rate of m-DelCys group and m-C(A7)Y group are higher than that of m-WT group, m-G(B8)S group, m-V(A3)L group, m-R(SP6)H group and m-G(C28)R group with statistical difference (F=37.599, P=0.000).(3)The proliferation status of INS-1cells was analyzed. The proliferation rate of vector group, m-WT group, m-G(B8)S group, m-V(A3)L group, m-R(SP6)H group and m-G(C28)R group was higher than that of the streptovirudin treated group, and the result of each group was lower than that of the vector group with statistical difference (F=8.047,P=0.000).2.Investigation of the influence of mutant (pre)proinsulin on ERS and UPR signaling pathway.(1)The expression levels of mRNA and protein of ATF6were detected at72h after transfection. The mRNA level as well as protein amount of ATF6of INS-1cells from m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WTgroup were lower than those of the streptovirudin treated group. The according data of each group were higher than those of the vector group. The data of the m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WT group were lower than those of the m-DelCys group, m-C(A7)Y group and m-G(B8)S group. There was no statistical difference between the streptovirudin treated group, m-DelCys group, m-C(A7)Y group and m-G(B8)S group (F=7.197,P=0.000). Neither was there statistical difference between the m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WTv group (F=7.641,P=0.000).(2)The mRNA expression level of CHOP was determined at72h after transfection. The mRNA level of CHOP of m-G(B8)Sgroup, m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WTgroup was lower than that of the streptovirudin treated group. The mRNA level of CHOP of each group was higher than that of the vector group. The mRNA level of CHOP of m-G(B8)Sgroup, m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WTgroup was lower than that of the m-DelCys group and m-C(A7)Y group. There was no statistical difference between the streptovirudin treated group, m-DelCys group, m-C(A7)Y group and m-G(B8)S group. Neither was there statistical difference between the m-V(A3)L group, m-R(SP6)H group, m-G(C28)R group and m-WTgroup (F=7.741,P=0.000) Conclusion:1.The (pre)proinsulin mutants, DelCys, C(A7)Y, G(B8)S, V(A3)L, R(SP6) and G(C28)R, were transfected into INS-1cells by means of cation liposome tansfection method. The transfection efficiency of each group was similar and all were higher than40%.2.All the (pre)proinsulin mutants, DelCys, C(A7)Y, G(B8)S, V(A3)L, R(SP6) and G(C28)R induce the apoptosis and attenuate the proliferation of βcells. Among them, the Delcys and C(A7) Y mutants show stronger effect than the G(B8)S, V(A3)L, R(SP6)H and G(C28)R mutants, which is probably because of the misfolding of proinsulin caused by Delcys and C(A7)Y mutantion or the probability of other mechanisms elicited by G(B8)S, V(A3)L, R(SP6)H, G(C28)R to attenuate apoptosis and enhance proliferation.3.The (pre)proinsulin mutants, DelCys, C(A7)Y, G(B8)S, V(A3)L, R(SP6) and G(C28)R disturb the accurate folding, cause the misfolding of proinsulin and lead to ERS mainly by way of ATF6signaling pathway to activate UPR. ATF6signaling pathway plays an important role in the process of ERS induced by (pre)proinsulin mutants, DelCys, C(A7)Y and G(B8)S.4.CHOP mediates the apoptosis inducing effect of (pre)proinsulin mutants, DelCys, C(A7)Y, G(B8)S, V(A3)L, R(SP6) and G(C28)R on INS-1cells, and is a vital factor underlying the ERS and apoptosis induced by m-DelCys and m-C(A7)Y mutant.