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）和肝葡萄糖生成（HGP）缺陷。2型糖尿病患者胰岛素刺激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，这种增加的HGP被认为是血管生成速率增加的结果，而不是糖原分解，但最近的数据表明后者也可能很重要。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-磷酸水解为葡萄糖，均由葡萄糖-6-磷酸酶催化。在1型和2型糖尿病的动物模型中，葡萄糖-6-磷酸酶催化亚单位（G6 β）基因表达升高。此外，肝脏G6 β过度表达足以诱导HGP的速率增加。这些观察结果表明，G6 β基因表达的抑制可能是一个潜在的策略，减少糖尿病患者的HGP。抑制G6 β基因转录的药物制剂的原理开发将需要详细了解基因转录调控的顺式作用元件和反式作用因子。 我们已经表明，胰岛素对基础G6 β基因转录的抑制作用需要两个启动子区域，分别命名为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通过区域B增强胰岛素介导的作用。区域B包含三个胰岛素应答序列（IRS），命名为IRS 1-3。IRS 1和2结合胰岛素应答转录因子FKHR（FOXO 1a），而IRS 3结合未鉴定的胰岛素应答因子。本授权申请的目的1寻求解决几个问题，即（i）HNF-1如何增强胰岛素对G6 β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G6 β基因转录？(iii)胰岛素如何抑制cAMP刺激的G6 β基因转录？以及（iv）结合IRS 3的未鉴定胰岛素反应因子是什么？在目标2和3中，我们提出分别表征介导佛波醇酯的抑制作用和PGC-1对G6 β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，G6 β启动子区在-484和+66之间足以使G6 β融合基因的原位转录达到最大的经神经调控。在目的4中，我们提出产生转基因小鼠，以确定是否可以赋予相同的启动子区域的发育，组织特异性和神经调控模式的G6 β融合基因表达在体内，模仿内源性基因。

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.