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: 9134732
• 负责人: Megan R Determan
• 金额: $ 3.9万
• 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 2017-05-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134732
• 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; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Health; Hepatic; Hepatocyte; Hepatomegaly; Histocompatibility Testing; Human; Hyperglycemia; In Vitro; Individual; 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; Protocols documentation; Research; Research Training; Role; Siblings; Steatohepatitis; Structure of beta Cell of islet; System; Time; Tissues; Transcriptional Regulation; Western Blotting; autosomal dominant mutation; basal insulin; base; blood glucose regulation; diabetic; fasting glucose; functional disability; gene function; gene product; glucose uptake; hepatic nuclear factor 1; human tissue; in vivo; induced pluripotent stem cell; insulin secretion; insulin signaling; mouse model; mutant; overexpression; pluripotency; prevent; 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 ß 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（由申请人提供）：HNF 1A-MODY是人类单基因糖尿病的最常见形式，由转录因子肝细胞核因子1 α（HNF 1A）的单倍不足引起。导致糖尿病表型的分子机制仍不清楚，因为HNF 1A-MODY的单倍不足小鼠模型未能产生任何表型。人类诱导多能干细胞（iPSC）领域和人类肝脏和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疾病（如HNF 1A-MODY）提供了一套强大的工具。HNF 1A特别已知其在肝细胞和胰岛中的表达。我们推测HNF 1A-MODY的病理生理学是由肝细胞和胰腺癌细胞中基因表达的变化引起的。已经从具有R272 H突变和T564 I突变的HNF 1A-MODY患者产生了两个iPSC系。这些克隆表达多能性因子并且能够定向分化为肝谱系。缺乏直接的同胞或亲本对照，将从这些突变iPSC产生的细胞与缺乏HNF 1A突变的两个对照iPSC系进行比较。目的1将首先利用微阵列来鉴定突变型HNF 1A-MODY肝细胞与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葡萄糖稳态和胰岛素信号相关的改变的关键靶标。将通过qRT-PCR和蛋白质印迹法确认这些靶标。将在功能水平上验证关键靶点的缺陷，例如用于测量葡萄糖摄取或氧化磷酸化能力的测定。目的2将首先通过体外和体内组合的分化方案在小鼠中产生功能性胰腺β细胞。与对照β细胞相比，将测定移植的HNF 1A-MODY β细胞的空腹葡萄糖水平、基础胰岛素分泌和葡萄糖刺激的胰岛素分泌。高血压、基础胰岛素分泌减少和葡萄糖刺激的胰岛素分泌减少是基于HNF 1A-MODY临床特征的相应预期结果。取出移植物，纯化β细胞用于下游分析。首先，来自HNF 1A-MODY和对照iPSC的功能性胰腺内分泌细胞将通过各种测定进行处理，以定量绝对胰岛素表达、胰岛素裂解、胰岛素胞吐和钙信号传导。其次，从突变体和对照β细胞分离的mRNA将用于微阵列以鉴定基因表达的变化。有针对性的生物信息学方法将确定改变的关键目标有关的葡萄糖稳态和胰岛素加工。该建议将确定在HNF 1A-单倍不足的肝细胞和胰腺β细胞中与葡萄糖稳态和胰岛素信号传导或产生相关的特定分子机制被改变。