ALDOSE REDUCTASE EXPRESSION IN DIABETES

本申请的总体目的是表征机制 调节视网膜中的醛糖还原酶（AR 2，EC1.1.1.21）基因表达 色素上皮（RPE）细胞在体外，认为参与了 体内糖尿病视网膜病变的发展。 这样做的理由 这一提议基于AR 2在人类基因组起源中的假定作用。 糖尿病的慢性并发症，以及越来越多的证据表明AR 2 参与生理性神经调节并受其调节。 的 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）部分地由 或通过AR 2从葡萄糖生产山梨糖醇的间接后果， 单体NADPH依赖性醛酮还原酶家族成员。 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。 AR 2基因表达的调控。 初步研究表明， 实验室检测了培养的RPE细胞中AR 2 mRNA的变化 经受高渗介质。 暴露于300 mM后24小时内 葡萄糖，3-O-甲基葡萄糖或甘露醇，AR 2 mRNA增加40倍， 山梨醇含量随后增加。 在四种RPE细胞系中 经过广泛的研究，三个人证明了这种“正常”的AR 2诱导， 响应渗透胁迫，但第四个（RPE 91）表现出组成性 AR 2 mRNA高水平表达， 山梨醇的积累。 AR 2的异常表达，例如 如果存在于糖尿病患者中， 可能导致糖尿病并发症的发生。 基于这些考虑，本申请提出了四个具体的 目的： 1)定量RPE中稳态AR 2 mRNA水平的变化 细胞对暴露于葡萄糖、3-O-甲基葡萄糖和甘露醇的反应 并将mRNA水平与AR 2蛋白的变化相关联， 山梨醇。 2)为了确定AR 2基因转录的改变，和/或 mRNA的稳定性引起稳态AR 2 mRNA水平的变化。 3)为了表征AR 2基因的启动子，并鉴定AR 2基因的启动子。 调节渗透诱导的顺式作用调节序列 转录。 4)目的探讨RPE 91细胞中AR 2表达异常的机制。

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.