ALDOSE REDUCTASE EXPRESSION IN DIABETES

本申请的总体目标是描述该机构的特征(S) 调节视网膜中醛糖还原酶(AR2，EC1.1.1.21)基因的表达 色素上皮(RPE)细胞在体外，据信参与了 糖尿病视网膜病变的体内研究进展。这样做的理由是 提出的基础是AR2在人类免疫缺陷病毒的起源中所起的假定作用 糖尿病的慢性并发症，以及越来越多的证据表明AR2 参与生理渗透调节，并受其调节。这个 多元醇假说认为糖尿病并发症(如 视网膜病变、神经病变和肾病)的部分原因是直接的 或由Ar2，a从葡萄糖中产生山梨醇的间接后果 依赖于NADPH的单体醛酮还原酶家族的成员。 尽管它在多元醇假说中起着关键作用，但人们对它知之甚少。 AR2基因表达的调控。从这方面的初步研究 实验室检测了培养的RPE细胞中AR2mRNA的变化 受到高渗透介质的影响。暴露在300毫米的24小时内 葡萄糖、3-O-甲基葡萄糖或甘露醇、AR2基因表达增加40倍 随后山梨醇含量的增加。四种RPE细胞系中的 经过广泛研究，有三个人证明了这种“正常”的AR2诱导 对渗透胁迫的反应，但第四个(RPE 91)表现出结构性 AR2mRNA的高水平表达及其加速和夸大 山梨醇的积累。AR2的异常表达 由这第四个RPE细胞株展示，如果在糖尿病患者中存在， 可能是糖尿病并发症的易感因素。 基于这些考虑，本申请提出了四个具体的 目标： 1)定量检测RPE中稳态AR2mRNA水平的变化 细胞对葡萄糖、3-O-甲基葡萄糖和甘露醇的反应 并将该基因的表达水平与AR2蛋白和 山梨醇。 2)确定AR2基因转录的改变，和/或 MRNA的稳定性导致了稳态AR2mRNA水平的变化。 3)鉴定AR2基因的启动子，并鉴定 调节渗透诱导的顺式作用调控序列 抄写。 4)探讨AR2在RPE 91中异常表达的机制。

The overall aim of this application is to characterize the mechanism(s) regulating aldose reductase (AR2, EC1.1.1.21) gene expression in retinal pigment epithelial (RPE) cells in vitro, believed involved in the development of diabetic retinopathy in vivo. The rationale for this proposal rests on the postulated role of AR2 in the genesis of the chronic complications of diabetes, and the growing evidence that AR2 participates in, and is modulated by, physiological osmoregulation. The polyol hypothesis asserts that diabetic complications (such as retinopathy, neuropathy and nephropathy) result, in part, from the direct or indirect consequences of sorbitol production from glucose by AR2, a member of the monomeric, NADPH-dependent aldoketoreductase family. Despite its pivotal role in the polyol hypothesis, little is known about the regulation of AR2 gene expression. Preliminary studies from this laboratory have examined the change in AR2 mRNA in cultured RPE cells subjected to hyperosmotic media. Within 24 hours of exposure to 300 mM glucose, 3-O-methyl glucose or mannitol, AR2 mRNA increased 40 fold with a subsequent increase in sorbitol content. Of the four RPE cell lines studied extensively, three demonstrated this "normal" AR2 induction in response to osmotic stress, but a fourth (RPE 91) exhibited constitutive high level expression of AR2 mRNA and accelerated and exaggerated accumulation of sorbitol. Aberrant expression of AR2 such as that exhibited by this fourth RPE cell line, if present in diabetic patients, could predispose to the development of diabetic complications. Based on these considerations, this application proposes four specific aims: 1) To quantitate the change in the steady-state AR2 mRNA level in RPE cells in response to exposure to glucose, 3- O-methylglucose and mannitol and to correlate the mRNA levels with changes in AR2 protein and sorbitol. 2) To determine whether alterations in AR2 gene transcription, and/or mRNA stability cause the changes in steady-state AR2 mRNA level. 3) To characterize the promoter of the AR2 gene, and identify the cis-acting regulatory sequences which regulate osmotic induction of transcription. 4) To determine the mechanism of aberrant AR2 expression in RPE 91.