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

WFS1 在胰腺 β 细胞活力和功能中的功能表征

• 批准号: 9328414
• 负责人: Damien Abreu
• 金额: $ 3.07万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328414
• 关键词: Address; Adolescent; Alleles; Anabolism; Animals; B Cell Proliferation; B-Lymphocytes; Beta Cell; Biochemical; Biological Assay; Cell Death; Cell Line; Cell Separation; Cell Survival; Cell physiology; Cells; Clinical; Data; Diabetes Mellitus; Diet; Disease; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Etiology; Exhibits; Fluorescence; Functional disorder; Genetic; Genotype; Glucose; Glucose Metabolism Disorders; Goals; High Fat Diet; Homeostasis; Impairment; In Vitro; Insulin; Islets of Langerhans; Knockout Mice; Knowledge; Lead; Libraries; Mediating; Mediator of activation protein; Membrane; Mendelian disorder; Metabolic; Metabolic stress; Molecular; Monitor; Morbidity - disease rate; Mus; Mutagenesis; Mutation; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Outcome; Pancreas; Pathology; Pathway interactions; Patients; Periodicity; Phenotype; Pilot Projects; Production; Proteins; Publishing; Regulation; Reporter; Reporting; Research; Rodent; Role; STEM research; Secondary to; Structure of beta Cell of islet; Techniques; Testing; Therapeutic; Time; Tungsten; Variant; WFS1 gene; Wolfram Syndrome; Work; base; biological adaptation to stress; blood glucose regulation; comparative; diabetic; endoplasmic reticulum stress; glucose tolerance; human disease; impaired glucose tolerance; insulin secretion; insulin tolerance; insulinoma; interest; islet; knock-down; mortality; new therapeutic target; novel; overexpression; prevent; prototype; stable cell line; stem; ubiquitin ligase

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.

抽象的 糖尿病（DM）是一种葡萄糖稳态的广泛代谢障碍，其特征是 胰岛素产生的绝对或相对缺乏。内质网（ER）功能障碍，特别是 不受管制的ER应激是β细胞病理学的关键介体，导致β细胞质量和β-的下降 细胞功能。彼此之间，分子机制中负责维持ER的遗传缺陷 稳态导致糖尿病的单基因形式，例如Wolfram综合征。这个常染色体隐性， 进行性神经退行性疾病源于WFS1基因座的突变，该突变在临床上表现出 作为少年发作的糖尿病，继发于ER应力介导的β细胞功能障碍。我们指示的初步数据 β细胞耗尽了WFS1暴露的胰岛素分泌受损并减少胰岛素含量。此外，我们的数据 证明WFS1的敲低与ER Ca2+降低有关，胞质Ca2+增加和 增加β细胞死亡。最值得注意的是，我们的试点研究表明，体外WFS1表达增加 增加胰岛素的产生，从而表明WFS1在β细胞中具有保护作用 诸如高葡萄糖之类的挑战。因此，该建议试图在功能上表征WFS1在 通过评估WFS1调节胰岛素的假设，胰腺β细胞活力和β细胞功能 通过调节关键ER稳态分子和ER Ca2+转运蛋白的生产和分泌。 为了解决这一假设，该提案旨在确定WFS1在饮食引起的β细胞中的作用 通过研究有条件的β细胞特异性WFS1过表达小鼠的代谢应激 （目标1）。 WFS1介导的胰岛素调节的机制将在体外研究。这项建议也是如此 试图通过评估β细胞中WFS1介导的Ca2+调节的机制来评估 在WFS1表达的背景下，主要的ER Ca2+转运蛋白（AIM 2）。识别和功能 将使用高吞吐量进一步研究破坏ER Ca2+的WFS1等位基因的表征 WFS1突变库的功能评估。通过这种方式，该建议旨在评估治疗 WFS1和WFS1介导的胰岛素调节途径的潜力在饮食诱导的糖尿病的背景下， 同时还表征了在ER Ca2+稳态的背景下进行WFS1突变。因此，这项研究 有可能利用ER压力介导的疾病的原型，以扩大我们对 ER功能障碍会触发更常见形式的糖尿病形式的β细胞病理的机制，例如 2型DM。