Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant proinsulin, but also the co-expressed WT-proinsulin, forming misfolded disulfide-linked proinsulin complexes. This impaired the intracellular trafficking of WT-proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.

胰岛素基因突变是新生儿糖尿病（NDM）的第二大最常见原因。它也是与年轻人发病有关的基因之一（MODY）。我们旨在研究不同INS的分子行为可能与糖尿病表型的临床范围相关的基因变异。在这项研究中，我们集中研究了两个以前未鉴定的引起MODY的突变体，即胰岛素原p.Gly44Arg [G（B20）R]和p.Pro52Leu [P（B28）L]（在一个法国家族中鉴定出的新型突变体），以及NDM引起胰岛素原-p。（Cys96Tyr）[C（A7）Y]。我们发现这些胰岛素原突变体在内质网（ER）中显示受损的氧化折叠，阻止了ER出口，ER应激和细胞凋亡。重要的是，胰岛素原突变体形成异常的分子间二硫键，不仅与突变体胰岛素原有关，而且还与野生型胰岛素原共表达，形成错折叠的二硫键连接的胰岛素原复合物。这损害了WT-胰岛素原的细胞内运输并限制了具有生物活性的成熟胰岛素的产生。值得注意的是 尽管所有三个突变体在折叠，运输和显性负行为方面均表现出相似的缺陷，但这些缺陷的程度似乎不同。具体而言，与MODY突变体G（B20）R和P（B28）L部分影响共表达的WT-胰岛素原的折叠和运输相比，NDM突变体C（A7）Y几乎完全阻断了ER的出口。 WT-胰岛素原，降低胰岛素产生，诱导更严重的内质网应激和细胞凋亡。因此，我们证明了不同胰岛素原突变体之间细胞生物学行为的差异与由不同胰岛素表型引起的糖尿病表型谱有关。与部分影响共表达WT-胰岛素原的折叠和运输的MODY突变体G（B20）R和P（B28）L相比，NDM突变体C（A7）Y几乎完全阻断了WT-胰岛素的ER出口。胰岛素，减少胰岛素的产生，引起更严重的内质网应激和细胞凋亡。因此，我们证明了不同胰岛素原突变体之间细胞生物学行为的差异与由不同胰岛素表型引起的糖尿病表型谱有关。与部分影响共表达WT-胰岛素原的折叠和运输的MODY突变体G（B20）R和P（B28）L相比，NDM突变体C（A7）Y几乎完全阻断了WT-胰岛素的ER出口。胰岛素，减少胰岛素的产生，引起更严重的内质网应激和细胞凋亡。因此，我们证明了不同胰岛素原突变体之间细胞生物学行为的差异与由不同胰岛素引起的糖尿病表型的频谱相关INS基因突变。