Regulation of Glucose-6-Phosphatase Gene Expression

描述(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)和肝脏葡萄糖产生(HGP)缺陷。由于胰岛素抵抗，2型糖尿病患者胰岛素刺激PGU和抑制HGP的能力降低。此外，在1型糖尿病患者中，如果循环胰岛素水平低，HGP会增加，当血糖控制不佳导致胰岛素抵抗时，这是一个特别的问题。在1型和2型糖尿病中，这种升高的HGP被认为是糖异生速率增加的结果，而不是糖原分解速度增加的结果，但最近的数据表明，后者也可能是重要的。在任何一种情况下，两种代谢途径中的最终反应，即葡萄糖-6-磷酸水解物为葡萄糖，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-磷酸酶催化亚单位(G6Pase)基因表达均升高。此外，肝脏G6Pase的过度表达足以诱导HGP发生率增加。这些观察结果表明，抑制G6Pase基因的表达可能是降低糖尿病患者HGP的一种潜在策略。开发一种抑制G6Pase基因转录的药物的基本原理需要详细了解调节基因转录的顺式作用元件和反式作用因子。我们发现，胰岛素对基础G6Pase基因转录的抑制作用需要A和B两个启动子区域，A区域与肝细胞核因子-1(HNF-1)结合，但不直接介导胰岛素的作用。相反，HNF-1增强了通过B区介导的胰岛素的作用。B区包含三个胰岛素反应序列(IRS)，命名为IRS1-3。IRS1和IRS2结合胰岛素反应转录因子FKHR(FOXO1a)，而IRS3结合未知的胰岛素反应因子。这项拨款申请的目的1试图解决几个问题，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？以及(Iv)什么是未知的胰岛素反应因子结合IRS3？在目标2和目标3中，我们分别描述了介导佛波酯抑制作用和PGC-1对G6Pase基因转录的刺激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之间足以提供最大的激素调节的G6Pase融合基因的原位转录。在目标4中，我们建议建立转基因小鼠，以确定这个相同的启动子区域是否能够赋予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，模拟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.