GENE EXPRESSION IN DIABETES BY SUBTRACTIVE CLONING

通过消减克隆表达糖尿病的基因

• 批准号: 6050951
• 负责人: C RONALD KAHN
• 金额: $ 66.04万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6050951
• 关键词: clinical research; diabetes mellitus genetics; differential display technique; gene expression; genetic regulation; genetically modified animals; human subject; insulin dependent diabetes mellitus; insulin sensitivity /resistance; laboratory mouse; laboratory rat; microarray technology; molecular cloning; noninsulin dependent diabetes mellitus; obesity; striated muscles; subtraction hybridization; thiazoles; transfection

This is a competitive renewal of RO1 DK45935. The overall goal of this project is to identify changes in gene expression in skeletal muscle of humans and rodents with Type 2 diabetes mellitus and to define the role of such changes in the pathophysiology of diabetes. Using subtraction cloning and PCR-differential display, we have identified 11 alterations in gene expression in muscle from patients with Type 2 diabetes and 17 alterations in an animal model of Type 2 diabetes, the ob/ob mouse. In addition, we have characterized aspects of regulation and possible roles for several of these genes in diabetes and insulin resistance, including glycogen phosphorylase, genes of the mitochondrial genome, elongation factor 1alpha, geranylgeranyl pyrophosphate synthase, and the novel GTPase Rad. We have also identified variations in two candidate genes for Type 2 diabetes that can influence the expression of genes: one is a triplet expansion in the Frederick's ataxia gene and the other is a novel activating mutation in PPARgamma, a known regulator of insulin sensitivity via its action on gene expression. In the coming period we propose to focus on the role of gene expression in the pathophysiology of diabetes using normal and mutant PPARgamma as tools to identify insulin resistance/ sensitivity pathways, and to expand the search for alterations in gene expression using newer and more powerful DNA chip array technology. The specific aims are to: l) Characterize normal and mutant PPARgamma as potential obesity/diabetes genes by construction and characterization of transgenic mice expressing these genes in adipose tissue and muscle; 2) Identify changes in gene expression of various tissues of mice overexpressing normal and mutant PPARgamma in muscle vs. fat with and without thiazolidinedione (TZD) treatment; 3) Determine the effect of a muscle-specific knockout of PPARgamma on insulin sensitivity and TZD-induced changes in gene expression; 4) Identify alterations in gene expression in muscle from humans with Type 2 diabetes using DNA chip analysis and characterize the relationship of these changes to metabolic control, type of diabetes (those limited to Type 2 diabetes may reflect primary defects or specific alterations due to insulin resistance); and the effect of insulin-sensitizing TZDs on the alterations in gene expression in the Type 2 diabetic; and 5) Analyze alterations in gene expression in other insulin resistant states, particularly obesity, and in normoglycemic offspring of diabetic parents subgrouped by presence or absence of insulin resistance.

这是RO 1 DK 45935的竞争性更新。该项目的总体目标是确定2型糖尿病患者和啮齿动物骨骼肌中基因表达的变化，并确定这种变化在糖尿病病理生理学中的作用。使用消减克隆和PCR差异显示，我们已经确定了11个基因表达的改变，在肌肉中的2型糖尿病患者和17个改变，在2型糖尿病的动物模型，ob/ob小鼠。此外，我们的特点方面的监管和可能的作用，这些基因在糖尿病和胰岛素抵抗，包括糖原磷酸化酶，线粒体基因组的基因，延伸因子1 α，香叶基香叶基焦磷酸合酶，和新的GTADRad。我们还确定了2型糖尿病的两个候选基因的变异，这两个基因可以影响基因的表达：一个是弗雷德里克共济失调基因中的三联体扩增，另一个是PPARgamma中的新激活突变，PPARgamma是一种已知的胰岛素敏感性调节因子，通过其对基因表达的作用。在未来的一段时间内，我们建议集中在糖尿病的病理生理学中的基因表达的作用，使用正常和突变的PPARgamma作为工具，以确定胰岛素抵抗/敏感性途径，并扩大寻找基因表达的改变，使用更新和更强大的DNA芯片阵列技术。具体目标是：1）通过构建和表征在脂肪组织和肌肉中表达这些基因的转基因小鼠，将正常和突变的PPARgamma表征为潜在的肥胖/糖尿病基因; 2）鉴定在有和没有噻唑烷二酮（TZD）处理的情况下，在肌肉相对于脂肪中过表达正常和突变的PPARgamma的小鼠的各种组织的基因表达的变化; 3）确定肌肉特异性敲除PPARgamma对胰岛素敏感性和TZD诱导的基因表达变化的影响;四、使用DNA芯片分析确定2型糖尿病患者肌肉中基因表达的改变，并描述这些变化的关系代谢控制，糖尿病类型（局限于2型糖尿病的那些可能反映由于胰岛素抵抗引起的原发性缺陷或特定改变）;以及胰岛素增敏TZD对2型糖尿病中基因表达改变的影响;和5）分析其他胰岛素抵抗状态，特别是肥胖症，糖尿病父母的血糖正常后代根据是否存在胰岛素抵抗进行亚组。