Generation of Wolfram syndrome mice, aiming at development of new therapeutics for diabetes through preserving pancreatic beta cells

Wolfram综合征是一种常染色体隐性遗传病，与青少年发病的糖尿病、视神经萎缩、感音神经性耳聋和尿崩症有关。我们利用定位克隆技术鉴定出了一个导致这种疾病的基因，并将其命名为WFS1。我们还发现WFS1蛋白定位于内质网膜。此外，WFS1与酵母Hrd3p具有一定的序列相似性，参与er相关的折叠错误蛋白的降解。因此，尽管WFS1蛋白似乎在内质网应激反应中起作用，但其确切功能仍有待阐明。为了阐明这种新蛋白的功能和Wolfram综合征的病理生理，我们培育了缺乏WFS1基因的小鼠（WFS1- ko）。雄性WFS1-KO小鼠的血糖水平在12周左右开始升高，并在24周显著高于野生型小鼠（267±33 vs 146±34 mg/dl）。这与胰岛β细胞的损失有关；WFS1-KO小鼠全胰腺胰岛素含量比野生型小鼠低30倍（11±4 vs 287±34 ng/mg胰腺）。使用抗WFS1蛋白抗体对胰腺进行免疫组化分析，发现WFS1蛋白在外分泌组织中缺失，但在内分泌β细胞中强烈表达。WFS1蛋白在胰高血糖素阳性细胞中不表达，大多数生长抑素和胰腺多肽阳性细胞缺乏WFS1免疫反应性。在WFS1-KO小鼠中，β细胞数量随着年龄的增长而下降，而a细胞则有所增加，并分散在整个胰岛中。这些结果强烈提示WFS1蛋白在β细胞的存活和/或再生和胰岛β细胞的进行性损失中发挥关键作用，是糖尿病的主要原因。

Wolfram syndrome is an autosomal recessive disorder associated with juvenile onset diabetes mellitus, optic atrophy, sensorineural deafness and diabetes insipidus. We identified, employing positional cloning, a gene responsible for this disorder and designated it WFS1. We also showed WFS1 protein to be localized in the ER membrane. In addition, WFS1 shares some sequence similarity with yeast Hrd3p, which is involved in ER-associated degradation of malfolded proteins. Thus, though WFS1 protein appears to play a role in the ER-stress response, its precise function remains to be clarified. To elucidate the functions of this novel protein and the pathophysiology of Wolfram syndrome, we have generated mice lacking the WFS1 gene (WFS1-KO). WFS1-KO mice are normal in appearance, growth and fertility Blood glucose levels in male WFS1-KO mice start to rise at around 12 weeks and are significantly higher than those of wild-type mice at 24 weeks (267±33 vs 146±34 mg/dl). This is associated with loss of islet β cells; whole pancreatic insulin content in WFS1-KO mice was 30-times lower than that in wild-type mice (11±4 vs 287±34 ng/mg pancreas). Immunohistochemical analysis of the pancreas using an anti-WFS1 protein antibody revealed that WFS1 protein is absent from exocrine tissue, but is strongly expressed in endocrine β cells. WFS1 protein is not expressed in glucagon-positive cells and the majority of somatostatin-, and pancreatic polypeptide-positive cells are devoid of WFS1 immunoreactivity. In WFS1-KO mice, β cell number fell with age, while a cells were somewhat increased and were scattered throughout the islet. These results strongly suggest WFS1 protein to play a critical role in survival and/or regeneration of β cells and progressive loss of islet β cells to be the major cause of diabetes in this disorder.