REGULATION OF GLUCOSE-6-PHOSPHATASE GENE EXPRESSION

II型非胰岛素依赖型糖尿病（NIDDM）的特征在于胰岛素分泌、外周葡萄糖利用（PGU）和肝葡萄糖产生（HGP）的缺陷。 胰岛素抵抗导致NIDDM患者胰岛素刺激PGU和抑制HGP的能力降低。 此外，在I型胰岛素依赖型糖尿病（IDDM）中，如果循环胰岛素水平低或血糖控制不良导致胰岛素抵抗的发展，HGP可能会增加。 持续高血糖是许多与糖尿病相关的并发症的原因。在胰岛素依赖型糖尿病和非胰岛素依赖型糖尿病中，HGP的增加是肿瘤发生率增加的结果。 葡萄糖-6-磷酸酶（G6 β）是葡萄糖代谢途径的最后一步。 最近的数据表明，G6 β催化亚基的过度表达导致HGP的发生率增加。因此，G6 β催化亚基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催化亚基基因表达的药剂的合理开发将需要详细了解调控基因表达的顺式作用元件和反式作用因子。本授权申请的具体目标1和2提出分别表征介导cAMP的刺激作用和佛波酯对G6 β催化亚基基因转录的抑制作用的顺式作用元件和反式作用因子。 这将使用融合基因策略结合组织培养细胞系的转染以及转基因小鼠的产生来实现。 从初步研究中可以明显看出，两种药物的充分作用需要多个顺式作用元件。 此外，在肝细胞中介导cAMP刺激作用的顺式作用元件与在肾细胞中介导这种作用的顺式作用元件不同。胰岛素对G6 β催化亚基基因转录的抑制作用需要两个启动子区，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通过结合区域B的未鉴定的转录因子增强胰岛素介导的作用。 本申请的第三个具体目的是探索胰岛素对G6 β基因转录作用机制的几个方面。

Type II, non-insulin dependent diabetes mellitus (NIDDM) 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 NIDDM is reduced as a consequence of insulin resistance. In addition, in Type I, insulin-dependent diabetes mellitus (IDDM), HGP can increase if circulating insulin levels are low or because poor glycemic control has led to the development of insulin resistance. Persistent hyperglycemia is the cause of many of the complications associated with diabetes. In both IDDM and NIDDM this increased HGP is a consequence of an increased rate of gluconeo-genesis. The final step of the gluconeogenic pathway is catalyzed by glucose-6-phosphatase (G6Pase). Recent data has shown that overexpression of the G6Pase catalytic subunit results in an increased rate of HGP. Thus, the suppression of G6Pase catalytic subunit gene expression may represent a potential strategy for reducing HGP in diabetic patients. The rational development of a pharmaceutical agent that suppresses G6Pase catalytic subunit gene expression will require a detailed knowledge of the cis-acting elements and trans-acting factors through which expression of the gene is regulated. Specific Aims 1 and 2 of this grant application propose to characterize the cis-acting elements and trans-acting factors that mediate the stimulatory effect of cAMP and the inhibitory effect of phorbol esters on G6Pase catalytic subunit gene transcription, respectively. This will be achieved using a fusion gene strategy in conjunction with the transfection of tissue culture cell lines as well as the generation of transgenic mice. From preliminary studies it is apparent that multiple cis-acting elements are required for the full effect of both agents. In addition, the cis-acting elements that mediate the stimulatory effect of cAMP in liver cells are distinct from those that mediate this effect in kidney cells. The inhibitory action of insulin on G6Pase catalytic subunit 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 an unidentified transcription factor that binds Region B. The third Specific Aim of this application proposes to explore several aspects of the mechanism of insulin action on G6Pase gene transcription.