A Study of the Cellular Mechanisms Causing Progression to Diabetes in HNF1A-MODY

HNF1A-MODY 进展为糖尿病的细胞机制研究

• 批准号: 8648262
• 负责人: Megan R Determan
• 金额: $ 4.43万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-04 至 2019-09-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648262
• 关键词: American; Amputation; Beta Cell; Bioinformatics; Biological Assay; Calcium Signaling; Candidate Disease Gene; Cell Line; Cells; Cessation of life; Clinical; Communities; Defect; Development; Diabetes Mellitus; Diagnosis; Disease; Dominant-Negative Mutation; Endocrine; Exhibits; Exocytosis; Fibroblasts; Functional disorder; Gene Expression; Gene Expression Alteration; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Hepatic; Hepatocyte; Hepatomegaly; Histocompatibility Testing; Human; Hyperglycemia; In Vitro; Infection; Insulin; Islets of Langerhans; Kidney Diseases; Knockout Mice; Liver Glycogen; Measures; Messenger RNA; Mitochondria; Modeling; Molecular; Mus; Mutate; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Oxidative Phosphorylation; Pancreas; Patients; Phenotype; Population; Process; Production; Proteins; Protocols documentation; Research; Research Proposals; Research Training; Role; Siblings; Skin; Steatohepatitis; Structure; Structure of beta Cell of islet; System; Time; Tissues; Transcriptional Regulation; Western Blotting; basal insulin; base; blood glucose regulation; diabetic; fasting glucose; functional disability; gene function; glucose uptake; hepatic nuclear factor 1; human tissue; in vivo; induced pluripotent stem cell; insulin secretion; insulin signaling; mouse model; mutant; overexpression; pluripotency; prevent; public health relevance; stem cell differentiation; stem cell technology; theories; tool; transcription factor

DESCRIPTION (provided by applicant): HNF1A-MODY is the most prevalent form of monogenic diabetes in humans and results from a haploinsufficiency of the transcription factor hepatocyte nuclear factor 1 alpha (HNF1A). The molecular mechanisms resulting in a diabetic phenotype remain unclear as haploinsufficient mouse models of HNF1A- MODY have failed to produce any phenotypes. The recent advances in the human induced pluripotent stem cell (iPSC) field and human hepatic and pancreatic endocrine differentiation fields provide a powerful set of tools for assessing diseases, such as HNF1A-MODY, at the molecular and cellular level. HNF1A is particularly known for its expression in the hepatocyte and the pancreatic islet. We hypothesize that the pathophysiology of HNF1A-MODY results from gene expression changes in the hepatocyte and in the pancreatic ? cell. Two iPSC lines have been generated from HNF1A-MODY patients with a R272H mutation and a T564I mutation. These clones express pluripotency factors and are capable of directed differentiation to the hepatic lineage. Lacking direct sibling or parental controls, cells generated from these mutant iPSCs will be compared to two control iPSC lines lacking mutations in HNF1A. Aim 1 will first utilize microarrays to identify changes in gene transcription in the mutant HNF1A-MODY hepatocytes versus control hepatocytes. A targeted bioinformatics assessment will identify altered key targets relating to glucose homeostasis and insulin signaling in the hepatocyte. These targets will be confirmed by qRT-PCR and Western blots. Key targets will be validated for deficiencies at the functional level, such as assays for measuring glucose uptake or oxidative phosphorylation capacity. Aim 2 will first generate functional pancreatic beta cells by a combined in vitro and in vivo differentiation protocol in mice. Engrafted HNF1A-MODY beta cells will be assayed compared to control beta cells for: fasting glucose levels, basal insulin secretion, and glucose-stimulated insulin secretion. Hyperglycemia, decreased basal insulin secretion, and diminished glucose-stimulated insulin secretion are the respective anticipated results based on the clinical profile of HNF1A-MODY. Grafts will be removed and beta cells will be purified for downstream analyses. First, functional pancreatic endocrine cells from HNF1A-MODY and control iPSCs will be processed via various assays to quantitate absolute insulin expression, insulin cleavage, insulin exocytosis, and calcium signaling. Second, mRNA isolated from mutant and control beta cells will be utilized for microarrays to identify changes in gene expression. A targeted bioinformatics approach will identify altered key targets relating to glucose homeostasis and to insulin processing. This proposal will establish which specific molecular mechanisms relating to glucose homeostasis and insulin signaling or production are altered in the HNF1A-haploinsufficient hepatocyte and pancreatic beta cell.

描述(申请人提供)：HNF1A-MODY是人类中最常见的单基因糖尿病形式，由转录因子肝细胞核因子1α(HNF1A)单倍体不足引起。导致糖尿病表型的分子机制尚不清楚，因为单倍体不足的HNF1A-MODY小鼠模型未能产生任何表型。人类诱导多能干细胞(IPSC)领域和人肝胰腺内分泌分化领域的最新进展为在分子和细胞水平上评估HNF1A-MODY等疾病提供了一套强大的工具。HNF1A因其在肝细胞和胰岛的表达而广为人知。我们假设HNF1A-MODY的病理生理学是肝细胞和胰腺中基因表达变化的结果。手机。已从具有R272H突变和T564I突变的HNF1A-MODY患者中产生了两个IPSC株。这些克隆表达多能性因子，并能够定向分化为肝脏谱系。由于缺乏直接兄弟姐妹或父母控制，从这些突变的IPSC产生的细胞将与两个缺乏HNF1A突变的对照IPSC株进行比较。目的1将首先利用微阵列来识别突变的HNF1A-MODY肝细胞和对照肝细胞中基因转录的变化。有针对性的生物信息学评估将确定与肝细胞中的葡萄糖稳态和胰岛素信号有关的改变的关键靶点。这些靶标将通过qRT-PCR和Western blotts进行确认。将验证关键目标在功能水平上的不足，例如测量葡萄糖摄取或氧化磷酸化能力的分析。AIM 2将首先通过体外和体内相结合的分化方案在小鼠身上产生具有功能的胰岛β细胞。移植的HNF1A-MODYβ细胞将与对照β细胞进行比较：空腹血糖水平、基础胰岛素分泌和葡萄糖刺激的胰岛素分泌。根据HNF1A-MODY的临床特征，高血糖、基础胰岛素分泌减少和葡萄糖刺激的胰岛素分泌减少是各自的预期结果。移植物将被移除，β细胞将被提纯用于下游分析。首先，来自HNF1A-MODY和对照IPSCs的功能性胰腺内分泌细胞将通过各种方法进行处理，以量化绝对胰岛素表达、胰岛素裂解、胰岛素胞吐和钙信号。其次，从突变的和对照的β细胞中分离出来的信使核糖核酸将被用于微阵列，以识别基因表达的变化。一种有针对性的生物信息学方法将确定与葡萄糖稳态和胰岛素处理相关的改变的关键靶点。这项建议将确定在HNF1a单倍体不足的肝细胞和胰岛β细胞中，哪些与葡萄糖稳态和胰岛素信号或产生有关的特定分子机制发生了变化。