REGULATION OF GLUCOSE-6-PHOSPHATASE GENE EXPRESSION

II型非胰岛素依赖型糖尿病（NIDDM）以胰岛素分泌、外周葡萄糖利用（PGU）和肝糖生成（HGP）缺陷为特征。胰岛素在NIDDM患者中刺激PGU和抑制HGP的能力由于胰岛素抵抗而降低。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较低或血糖控制不良导致胰岛素抵抗，HGP会升高。持续的高血糖是许多糖尿病并发症的原因。在IDDM和NIDDM中，HGP的增加是糖异生速率增加的结果。糖异生途径的最后一步由葡萄糖-6-磷酸酶（G6Pase）催化。最近的数据表明，G6Pase催化亚基的过表达导致HGP的发生率增加。因此，抑制G6Pase催化亚基基因表达可能是降低糖尿病患者HGP的潜在策略。合理开发抑制G6Pase催化亚基基因表达的药物制剂，需要详细了解基因表达调控的顺式作用元件和反式作用因子。本资助申请的具体目标1和2提出分别表征介导cAMP刺激作用和phorbol酯对G6Pase催化亚基基因转录抑制作用的顺式作用元件和反式作用因子。这将通过融合基因策略与组织培养细胞系的转染以及转基因小鼠的产生来实现。从初步的研究来看，很明显，两种药物的充分作用需要多种顺式作用元素。此外，介导肝细胞cAMP刺激作用的顺式作用元件与介导肾细胞cAMP刺激作用的顺式作用元件不同。胰岛素对G6Pase催化亚基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通过一种未知的转录因子结合b区来增强胰岛素介导的作用。本应用的第三个特定目的是探索胰岛素作用于G6Pase基因转录的几个方面的机制。

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.