Role of dominant-negative effects in mutant INS-gene induced diabetes of youth

Abstract

Objective Dominant-negative effect refers to an interference pattern that the mutated protein does not only perform physiological function, but also inhibits activity of co-existing normal protein. The aim of this study is to investigate the role of dominant-negative effects in mutant INS-gene induced diabetes of youth (MIDY) caused by different types of insulin gene mutations. Methods The inhibition of co-expressed wild-type proinsulin secretion by three MIDY mutants was observed and a proinsulin-DelCys was introduced which carried MIDY mutations but couldn't form disulfide bond. There were 7 groups: 293T cells were co-transfected with wild-type human preproinsulin and mutant preproinsulins [as C(A7)Y group, G(B8)S group, R(SP6)H group and DelCys group]. Wild-type mouse preproinsulin and wild-type human preproinsulin were co-transfected as normal control group. Wild-type human preproinsulin and pCMS-EGFP plasmid were co-transfected as positive control group and wild-type mouse preproinsulin transfected 293T cells as negative control group. After 48 h, the media and cell were collected. Secreted human proinsulin was measured with human-specific proinsulin radioimmunoassay. Data were analyzed with one-way ANOVA and t test. Results There was no statistically significant difference between the groups in the comparison of intracellular proinsulin levels (F=0.58, P>0.05). The differences of human proinsulin in cell culture medium among groups were statistically significant (F=297.57, P 0.05). Conclusion MIDY mutants C(A7)Y and G(B8)S could induce the dominant-negative effects on co-existing wild-type proinsulin while R(SP6)H could not. Dominant-negative effect may be involved in the formation of intermolecular disulfide bond. Key words: Diabetes mellitus; β-cell failure; Dominant-negative effect; Proinsulin misfolding