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

摘要 糖尿病(DM)是一种广泛存在的糖稳态代谢紊乱，其特征是 胰岛素分泌的绝对或相对不足。内质网功能障碍，尤其是 不受调控的内质网应激是β细胞病理的关键介质，导致β细胞质量和β- 细胞功能。因此，负责维持内质网的分子机制中的遗传缺陷 动态平衡导致单基因形式的糖尿病，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是由WFS1基因突变引起的，临床上首发于此 作为青少年起病的糖尿病，继发于内质网应激介导的β细胞功能障碍。我们的初步数据显示 WFS1缺失的β细胞表现为胰岛素分泌受损和胰岛素含量降低。此外，我们的数据 WFS1基因敲除与内质网钙离子浓度降低、胞浆钙离子浓度升高及细胞内钙离子浓度升高有关 β细胞死亡增加。最值得注意的是，我们的初步研究表明，在体外增加WFS1的表达 增加胰岛素的产生，从而表明WFS1在β细胞中对代谢具有保护作用 挑战，如高血糖。因此，这项提案试图从功能上确定工作组第一工作组在 评估WFS1调节胰岛素的假说对胰腺β细胞活性和β细胞功能的影响 通过调节关键的内质网稳态分子和内质网钙离子转运体的产生和分泌。 为了解决这一假设，这一建议试图确定WFS1在饮食诱导的β细胞中的作用 高脂饮食条件下β细胞特异性WFS1过度表达小鼠的代谢应激研究 (目标1)。WFS1介导的胰岛素调节机制(S)将在体外进行研究。这项提议还包括 通过评价WFS1对β-细胞内钙离子调节的稳定性，探讨WFS1对细胞内钙的调节机制(S) WFS1表达背景下的主要ER钙转运体(目标2)。识别性和功能性 利用高通量将进一步研究干扰ER钙离子的WFS1等位基因的特征 WFS1突变文库的功能分析。通过这种方式，这项建议旨在评估治疗 WFS1和WFS1介导的胰岛素调节通路在饮食诱导的糖尿病中的潜力， 同时也在内质网钙稳态的背景下表征WFS1突变。因此，这项研究 有可能利用内质网应激介导的疾病的原型来扩大我们对 内质网功能障碍在更常见的糖尿病中触发β细胞病理的机制，如 类型2 DM。

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.