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

描述（由申请人提供）：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1 α (HNF1A)的单倍不足引起。导致糖尿病表型的分子机制尚不清楚，因为HNF1A- MODY单倍体不足的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HNF1A-MODY等疾病提供了一套强大的工具。HNF1A以其在肝细胞和胰岛中的表达而闻名。我们假设HNF1A-MODY的病理生理是由肝细胞和胰腺细胞的基因表达变化引起的。已经从具有R272H突变和T564I突变的HNF1A-MODY患者中产生了两个iPSC系。这些克隆表达多能性因子，能够直接分化为肝系。缺乏直接的兄弟姐妹或亲本控制，由这些突变iPSC产生的细胞将与缺乏HNF1A突变的两个对照iPSC系进行比较。Aim 1将首先利用微阵列来鉴定突变型HNF1A-MODY肝细胞与对照肝细胞中基因转录的变化。靶向生物信息学评估将确定肝细胞中与葡萄糖稳态和胰岛素信号有关的关键靶标的改变。这些靶点将通过qRT-PCR和Western blots进行确认。将在功能水平上验证关键靶点的缺陷，例如测量葡萄糖摄取或氧化磷酸化能力的测定。Aim 2将首先通过体外和体内联合分化方案在小鼠中产生功能性胰腺β细胞。将对移植的HNF1A-MODY β细胞与对照β细胞比较：空腹血糖水平、基础胰岛素分泌和葡萄糖刺激胰岛素分泌。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胰岛素分泌减少是各自的预期结果。移植物将被移除，细胞将被纯化用于下游分析。首先，来自HNF1A-MODY和控制iPSCs的功能性胰腺内分泌细胞将通过各种测定来定量胰岛素的绝对表达、胰岛素裂解、胰岛素胞外分泌和钙信号。其次，从突变和对照β细胞中分离的mRNA将用于微阵列来鉴定基因表达的变化。靶向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关键靶标的改变。该建议将确定在hnf1a单倍体不足的肝细胞和胰腺β细胞中，哪些与葡萄糖稳态和胰岛素信号或产生相关的特定分子机制被改变。

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.