GENE EXPRESSION IN DIABETES BY SUBTRACTIVE CLONING

这是RO1 DK45935的竞争性续订。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。利用消减克隆和聚合酶链式反应-差异显示，我们已经从2型糖尿病患者的肌肉中发现了11个基因表达的变化，并在2型糖尿病的动物模型ob/ob小鼠中发现了17个变化。此外，我们还鉴定了其中几个基因在糖尿病和胰岛素抵抗中的调节作用和可能的作用，包括糖原磷酸化酶、线粒体基因组基因、延伸因子1α、香叶基香叶基焦磷酸合成酶和新的GTP酶Rad。我们还发现了两个可能影响基因表达的2型糖尿病候选基因的变异：一个是Frederick‘s共济失调基因的三联体扩张，另一个是PPAR Gamma的新的激活突变，PPAR Gamma是一种已知的通过作用于基因表达而调节胰岛素敏感性的基因。在接下来的一段时间里，我们建议使用正常和突变的PPARGamma作为工具来识别胰岛素抵抗/敏感性途径，并使用更新和更强大的DNA芯片阵列技术来扩大对基因表达变化的搜索，以关注基因表达在糖尿病病理生理学中的作用。其具体目的是：L)通过构建和鉴定在脂肪组织和肌肉中表达正常和突变PPARγ的转基因小鼠，将这些基因表征为潜在的肥胖/糖尿病基因；2)确定在肌肉和脂肪中过表达正常和突变PPARγ的小鼠各种组织中基因表达的变化；3)确定肌肉特异性敲除PPARγ对胰岛素敏感性和TZD诱导的基因表达变化的影响；4)利用DNA芯片分析确定人类2型糖尿病患者肌肉中基因表达的变化，并表征这些变化与代谢控制、糖尿病类型(仅限于2型糖尿病的患者可能反映原发缺陷或因胰岛素抵抗引起的特定变化)的关系；以及胰岛素增敏TZD对2型糖尿病患者基因表达变化的影响；以及5)分析其他胰岛素抵抗状态，尤其是肥胖，以及根据是否存在胰岛素抵抗而划分的糖尿病父母的正常血糖后代的基因表达变化。

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.