Regulation of Glucose-6-Phosphatase Gene Expression

描述（由申请人提供）：2型糖尿病的特征是胰岛素分泌、外周葡萄糖利用（PGU）和肝糖生成（HGP）的缺陷。在2型糖尿病患者中，胰岛素刺激PGU和抑制HGP的能力由于胰岛素抵抗而降低。此外，在1型糖尿病患者中，如果循环胰岛素水平较低，HGP可能会增加，当血糖控制不良导致胰岛素抵抗时，这是一个特别的问题。在1型和2型糖尿病中，HGP的增加被认为是糖异生率增加的结果，而不是糖原溶解，但最近的数据表明后者可能也很重要。在这两种情况下，两种代谢途径的最终反应，葡萄糖-6-磷酸水解为葡萄糖，都是由葡萄糖-6-磷酸酶催化的。在1型和2型糖尿病动物模型中，葡萄糖-6-磷酸酶催化亚基（G6Pase）基因表达升高。此外，肝脏过表达G6Pase足以诱导HGP率升高。这些观察结果表明，抑制G6Pase基因表达可能是降低糖尿病患者HGP的潜在策略。抑制G6Pase基因转录的药物制剂的基本原理开发将需要对基因转录调节的顺式作用元件和反式作用因子的详细了解。我们已经证明胰岛素对基础G6Pase基因转录的抑制作用需要两个启动子区域A和b。A区结合肝细胞核因子-1 (HNF-1)，但不直接介导胰岛素的作用。相反，HNF-1增强了胰岛素通过B区介导的作用。B区包含三个胰岛素反应序列（IRSs），命名为IRS 1-3。IRS 1和IRS 2结合胰岛素应答转录因子FKHR (FOXO1a)，而IRS 3结合一个未知的胰岛素应答因子。本资助申请的目的1旨在解决几个问题，即(i) HNF-1如何增强胰岛素对G6Pase基因转录的抑制作用？（ii）胰岛素如何抑制糖皮质激素刺激的G6Pase基因转录？（iii）胰岛素如何抑制camp刺激的G6Pase基因转录？（iv）什么是未识别的胰岛素反应因子结合IRS 3？在Aims 2和3中，我们提出分别表征介导佛波酯抑制作用和PGC-1刺激G6Pase基因转录作用的顺式作用元件和反式作用因子。我们已经证明，G6Pase启动子区域在-484和+66之间足以使激素调节的G6Pase融合基因在原位转录最大化。在第4项研究中，我们建议培养转基因小鼠，以确定相同的启动子区域是否可以赋予G6Pase融合基因在体内的发育，组织特异性和激素调节的表达模式，模仿内源性基因的表达模式。

DESCRIPTION (provided by applicant): Type 2 diabetes is characterized by defects in insulin secretion, peripheral glucose utilization (PGU) and hepatic glucose production (HGP). The ability of insulin to stimulate PGU and repress HGP in patients with type 2 diabetes is reduced as a consequence of insulin resistance. In addition, in Type 1 diabetics, HGP can increase if circulating insulin levels are low, a particular problem when poor glycemic control has led to the development of insulin resistance. In both type 1 and type 2 diabetes this increased HGP was thought to be a consequence of an increased rate of gluconeogenesis, rather than glycogenolysis, but more recent data suggest that the latter may also be important. In either case, the final reaction in both metabolic pathways, the hydrolysis of glucose-6-phosphate to glucose, is catalyzed by glucose-6-phosphatase. In animal models of both type 1 and type 2 diabetes glucose-6-phosphatase catalytic subunit (G6Pase) gene expression is elevated. Moreover, hepatic overexpression of G6Pase is sufficient to induce an increased rate of HGP. These observations suggest that the suppression of G6Pase gene expression may represent a potential strategy for reducing HGP in diabetic patients. The rationale development of a pharmaceutical agent that suppresses G6Pase gene transcription will require a detailed knowledge of the cis-acting elements and trans-acting factors through which transcription of the gene is regulated. We have shown that the inhibitory action of insulin on basal G6Pase gene transcription requires two promoter regions designated A and B. Region A binds hepatocyte nuclear factor-1 (HNF-1) but does not directly mediate the action of insulin. Instead, HNF-1 enhances the action of insulin mediated through Region B. Region B contains three insulin response sequences (IRSs) designated IRS 1-3. IRS 1 and 2 bind the insulin-responsive transcription factor FKHR (FOXO1a) whereas IRS 3 binds an unidentified insulin-responsive factor. Aim 1 of this grant application seeks to address several questions, namely (i) how does HNF-1 enhance the inhibitory action of insulin on G6Pase gene transcription? (ii) how does insulin inhibit glucocorticoid stimulated G6Pase gene transcription? (iii) how does insulin inhibit cAMP-stimulated G6Pase gene transcription? and (iv) what is the unidentified insulin-responsive factor binding IRS 3? In Aims 2 and 3 we propose characterizing the cis-acting elements and trans-acting factors that mediate the inhibitory effect of phorbol esters and the stimulatory effect of PGC-1 on G6Pase gene transcription, respectively. We have shown that the G6Pase promoter region between -484 and +66 is sufficient to confer maximal hormonally regulated G6Pase fusion gene transcription in situ. In Aim 4 we propose generating transgenic mice to determine whether this same promoter region can confer a developmental, tissue-specific and hormonally regulated pattern of G6Pase fusion gene expression in vivo that mimics that of the endogenous gene.