Functional Characterization of WFS1 in Pancreatic beta-cell Viability and Function

摘要 糖尿病（DM）是一种广泛存在的葡萄糖稳态代谢紊乱，其特征在于： 胰岛素产生的绝对或相对缺乏。内质网（ER）功能障碍，特别是 不受调节的ER应激，是β细胞病理学的关键介质，其引起β细胞质量和β- 细胞功能因此，负责维持ER的分子机制中的遗传缺陷 体内平衡导致单基因形式糖尿病，例如Wolfram综合征。这种常染色体隐性遗传 进行性神经退行性疾病源于WFS 1基因座的突变，其首先在临床上表现为 作为青少年发病的糖尿病，继发于ER应激介导的β细胞功能障碍。我们的初步数据显示 消耗了WFS 1的β细胞表现出胰岛素分泌受损和胰岛素含量降低。此外，我们的数据 表明WFS 1的敲低与ER Ca 2+降低、胞浆Ca 2+增加和 增加β细胞死亡。最值得注意的是，我们的初步研究表明，在体外增加WFS 1的表达， 增加胰岛素的产生，从而表明WFS 1在β细胞中对代谢的保护作用。 比如高血糖。因此，本提案试图从职能上描述第一工作队在以下方面的作用： 通过评估WFS 1调节胰岛素的假设来评估胰腺β细胞活力和β细胞功能 通过调节关键的ER稳态分子和ER Ca 2+转运蛋白来产生和分泌。 为了解决这一假设，该建议试图确定WFS 1在饮食诱导的β细胞中的作用。 通过研究高脂饮食条件下β细胞特异性WFS 1过表达小鼠的代谢应激 (Aim 1）。将在体外研究WFS 1介导的胰岛素调节机制。该提案还 试图通过评估WFS 1的稳定性来确定β细胞中WFS 1介导的Ca 2+调节的机制。 WFS 1表达背景下的主要ER Ca 2+转运蛋白（目的2）。鉴定与功能 破坏ER Ca 2+的WFS 1等位基因的表征将使用高通量进一步研究 WFS 1突变文库的功能测定。通过这种方式，该建议旨在评估治疗效果。 WFS 1和WFS 1介导的胰岛素调节途径在饮食诱导的糖尿病背景下的潜力， 同时还表征了ER Ca 2+稳态背景下的WFS 1突变。因此，这项研究 有可能利用ER应激介导的疾病的原型来扩大我们对 ER功能障碍在更常见的糖尿病形式中触发β细胞病理学的机制，例如 2型糖尿病。

ABSTRACT Diabetes mellitus (DM) is a widespread metabolic disorder of glucose homeostasis characterized by an absolute or relative deficiency in insulin production. Endoplasmic reticulum (ER) dysfunction, particularly unregulated ER stress, is a key mediator of β-cell pathology that causes a decline in both β-cell mass and β- cell function. Accordingly, genetic defects in the molecular machinery responsible for maintaining ER homeostasis result in monogenic forms of diabetes, such as Wolfram syndrome. This autosomal recessive, progressive neurodegenerative disorder stems from mutation of the WFS1 locus, which manifests clinically first as juvenile-onset diabetes, secondary to ER stress-mediated β-cell dysfunction. Our preliminary data indicate that β-cells depleted of WFS1 exhibit impaired insulin secretion and reduced insulin content. Further, our data demonstrate that knockdown of WFS1 is associated with decreased ER Ca2+, increased cytosolic Ca2+ and increased β-cell death. Most notably, our pilot studies indicate that increasing WFS1 expression in vitro increases insulin production, thereby suggesting a protective role for WFS1 in β-cells against metabolic challenges such as high glucose. This proposal therefore seeks to functionally characterize the role of WFS1 in pancreatic β-cell viability and β-cell function by evaluating the hypothesis that WFS1 regulates insulin production and secretion through the regulation of key ER homeostasis molecules and ER Ca2+ transporters. To address this hypothesis, this proposal seeks to determine the role of WFS1 in β-cells under diet-induced metabolic stress through the study of conditional β-cell-specific WFS1 overexpression mice on a high fat diet (Aim 1). The mechanism(s) of WFS1-mediated insulin regulation will be investigated in vitro. This proposal also seeks to determine the mechanism(s) of WFS1-mediated Ca2+ regulation in β-cells by evaluating the stability of major ER Ca2+ transporters in the context of WFS1 expression (Aim 2). Identification and functional characterization of WFS1 alleles that disrupt ER Ca2+ will be further investigated using high throughput functional assays of WFS1 mutation libraries. In this manner, this proposal aims to evaluate the therapeutic potential of WFS1 and WFS1-mediated insulin regulation pathways in the context of diet-induced diabetes, while also characterizing WFS1 mutations in the context of ER Ca2+ homeostasis. This research therefore holds the potential to capitalize on a prototype of ER stress-mediated disease to expand our understanding of the mechanisms by which ER dysfunction triggers β-cell pathology in more common forms of diabetes, such as type 2 DM.