The Experimental Study On The Mechnism Of Diabetes Related To Insulin Gene Mutation

ObjectiveTo explore the moleculer mechanism of the (pre)proinsulin mutants related to human diabetes on (3-cell dysfunction. Construct5different biological characteristics human proinsulin mutants plasmids h-C(A6)Y, h-L(B11)P, h-C(B19)G, h-R(S6)C and h-F(B25)L by site-directed mutagenesis technology. Compare their effects on the coexistence of wild-type human proinsulin synthesis and secretion. Seculate the role of intramolecular disulfide bonds in insulin on dominant negative effect using m-DelCys mutation. Comparise endoplasmic reticulum stress (ESR)-related proteins of different mutants,and explore two interrelated mechanism of the mutants leading to β-cell failure providing theoretical and experimental basis for the future ultimately clarify the pathogenesis of diabetes and cure diabetes.Methods1. Construct and identify5different human proinsulin mutants plasmids by site-directed mutagenesis technology, and transfect them into INS-1cells. Detect the concentration of insulin in the cell culture fluid.(1)Design and synthesize primers, as pCMS-EGFP/h-WT a template, generate pCMS-EGFP/h-C (B19) G. h-L(B11)P、h-R(S6)C、h-F(B25)L and h-C(A6)Y5single-point mutation plasmid by site-directed mutagenesis technology. Verify the success of site-directed mutagenesis for each plasmid by agarose gel electrophoresis and sequencing.(2)Transfect each mutant and h-WT into INS-1cell by lipofactamine2000,and detect the concentration of insulin in the cell culture medium.2. Study on dominant negative effect and its mechanism of human mutant proinsulin to coexisted wild-type proinsulin, explore the effects of excessive proinsulin on endoplasmic reticulum stress. (1)Transfect recombinant plasmids containing five mutants cDNA and h-WT cDNA into INS-1cells, detect human proinsulin and insulin concentrations of cell lysates and culture medium by double antibody sandwich enzyme-linked immunoassay after transfection for48hours.(2)Co-transfect recombinant plasmids containing each mutant cDNA and h-WT cDNA into INS-1cells, detect the concentration of human insulin in culture medium after cotransfection for48hours.(3)Co-transfect recombinant plasmids containing h-WT/DelCys mutations cDNA into INS-1cells, detect the concentration of insulin and proinsulin outside and inside cell after cotransfection for48hours.(4)transfect recombinant plasmids containing five mutants、DelCys mutant and h-WT cDNA into INS-1cells respectively, and detect protein BiP、eIF2α and p-eIF2α in cell lysates by Western Blot after transfection for48hours.Results1. Identify mutations by agarose gel electrophoresis and sequencing, detecting the concentration of insulin in the INS-1cell culture medium after transfection for48hours.(1)Five site mutations are confirmed in the correct position by agarose gel electrophoresis and sequencing.(2)Successfully culture rat insulinoma INS-1cells, optimize transfection conditions for the plasmid: lipofactamine2000are1μg:2μL (24-well plates). the concentration of insulin in cells culture of h-C(B19)Q h-L(B11)P, h-C(A6)Y group are lower than that of h-R(S6)C、h-F(B25)L and h-WT group(P<0.05), and the concentration of insulin in h-R(S6)C, h-F(B25)L group have no significant difference with that of h-WT group (P>0.05).2. Study on dominant negative effect and its mechanism of human mutant proinsulin to coexisted wild-type proinsulin, explore the effects of excessive proinsulin on endoplasmic reticulum stress related protein.(1)The concentration of proinsulin in cells lysates of5mutants have no significant difference with that of h-WT group(P>0.05). The concentration of insulin in cell culture medium of h-C(B19)G、h-L(B11)P,、h-C(A6)Y are less than that of h-R(S6)C、h-F(B25)L and h-WT groups(P<0.05).(2)Compared with the h-WT/vector group, the concentration of insulin in cell culture medium of h-WT/H-C(B19)Q h-WT/h-L(B11)P, h-WT/h-C(A6)Y cotransfection groups are decreased significantly, respectively(P<0.05).(3)The concentration of human proinsulin and insulin in cells lysates and culture medium of h-WT/m-Delcys cotransfection group have no significant difference with that of h-WT/m-WT group(P>0.05).(4)Compared with the h-WT group, the expression of BiP protein and p-eIF2α/eIF2a in cells of transfected with h-C(B19)G, h-L(B11)P, h-C(A6)Y groups are increased(P<0.05), which in h-R(S6)C、h-F(B25)L group are no changed(P>0.05).Conclusions1. Synthesize mutant proinsulin plasmids of different biological characteristics by by site-directed mutagenesis technology.2. Proinsulin mutants plasmids can transiently transfect in INS-1cells, the transfection efficiency are stable.3. The secretion of insulin of h-WT are decreased by co-expression with h-C(B19)G, h-L(B11)P, h-C(A6)Y mutants associated with neonatal diabetes. 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. 4. Lots of abnormal proinsulin accumulate in the endoplasmic reticulum, which cause ERS and activate PERK-eIF2a-ATF4signaling pathway. These three mutations associated with neonatal diabetes may cause pancreatic β cell failure through a dominant negative effect and ERS..5. Neither mutations associated with adult diabetes have significant negative effects with coexpressed h-WT, nor activate ERS,which suggest that this kind of mutants may weaken the binding ability to receptor.