Effects Of The (Pre) Proinsulin Mutants On β-cell Dysfunction

ObjectiveTo explore the effects of the (pre)proinsulin mutants related to human diabetes onβ-cell dysfunction, (pre)proinsulin mutants, C(A7)Y, G(B8)S, G(C28)R, R(SP6)H and V(A3)L were observed. Detected the dominant-negative inhibition of wild-type proinsulin trafficking by mutants and investigated the action of aberrant disulfide on dominant negative effect. ERS and URP induced by mutants were examined. The study on the molecular pathogenesis of mutant (pre)proinsulin inducedβ-cell failure would provide valuable theoretical and experimental basis for clinical therapy.Method1. Observe the dominant-negative effect of co-existing wild-type proinsulin trafficking by mutants and investigated the action of aberrant disulfide on dominant negative effect.(1) 293T cells were co-transfected with wild type human preproinsulin and mutant preproinsulins [bearing substitutions C(A7)Y, G(B8)S, G(C28)R, R(SP6)H or V(A3)L]. After 48h, the media and cell were collected. Secreted human proinsulin was measured using human-specific proinsulin radioimmunoassay.(2) Co-transfected with wild type human preproinsulin and mutant preproinsulins [bearing substitutions Delcys]. Further incubated for 48h, collected the media and cells. Secreted human proinsulin was measured using human-specific proinsulin radioimmunoassay.2. Investigate the effects of mutants on ESR and UPR293T cells were transfected with vector expressing preproinsulin wild type (’WT’) or preproinsulin missense mutants C(A7)Y, G(B8)S, G(C28)R, R(SP6)H and V(A3). At 72h post-transfection, cells were collected and divided into 2 groups. In one group, the expression of spliced XBP-lmRNA and unspliced XBP-lmRNA in cell were detected with RT-PCR. In another group, Western Blot chest protein BiPn eIF2a and p-eIF2a.Results1. Observe the dominant-negative effect of co-existing wild-type proinsulin trafficking by mutants and investigated the action of aberrant disulfide on dominant negative effect.(1) Compared with WT which co-transfected with WT-human preproinsulin and WT-mouse preproinsulin) (135.84±1.89pM), mutants C(A7)Y(29.28±6.85pM) and G(B8)S(33.62±10.52pM) co-transfected with WT-hpreproinsulin blocked the secretion of co-expressed human proinsulin and there were significant differences(P<0.05). Mutants V(A3)L(132.80±5.39pM), R(SP6)H(135.84±2.11pM), G(C28)S(133.31±2.59pM) were unable to inhibit the secretion of WT-human proinsulin and there were no significant differences among them(P>0.05).(2) Compared with WT, mutant Delcys couldn’t effectively inhibit trafficking of co-expressed proinsulin-WT(P> 0.05).2. Investigate the effects of mutants on ESR and UPR(1) Compared with WT-mouse proinsulin, C(A7), G(B8)S and Delcys mutants increased XBP-1s/XBP-1u in 293T cells,and there were significant differences (P<0.05). While mutants V(A3)L, R(SP6)H, G(C28)S didn’t up-regulate the level of XBP-1s/XBP-lu (P>0.05).4. Compared with wild type, mutant C(A7)Y and G(B8)S up-regulate the expression of BiP and increased p-eIF2α/eIF2α(P<0.05). Mutant proinsulin G(C28)R, R(SP6)H and V(A3)L didn’t induced overexpression of BiP(P>0.05).Conclusion1. Expression of MIDY mutants C(A7)Y and G(B8)S blocked the secretion of co-expressed human proinsulin and induced ERS and UPR. Dominant-negative effect and ERS may be the key roles in molecular pathogenesis of mutant (pre)proinsulin inducedβ-cell failure.2. Mutant proinsulin G(C28)R, R(SP6)H, V(A3)L didn’t induced dominant-negative effect and ERS. Diverse mutations in the insulin gene should induced diabetes by different molecular pathogenesis.3. Although misfolded mutant proinsulin-DelCys activate ERS and UPR, it couldn’t effectively inhibit secretion of co-expressed proinsulin-WT. Thus, the evidence suggests that disulfide bond is very important and may linked to the mechanism of dominant-negative effect.