Studies on the diabetic complications with regard to molecular mechanism of aldose reductase action

本研究旨在探讨糖尿病患者体内醛糖还原酶活性变化的分子机制及其代谢紊乱。总结如下：1.通过定点突变研究了His-42、His-188和Lys-263残基在人醛糖还原酶催化作用中的作用以及该酶的各种抑制剂。突变的人酶在杆状病毒-昆虫细胞系统中表达。结果表明，赖氨酸-263可能通过静电相互作用参与底物结合.研究了在包括肾脏和肝脏在内的几种组织中表达人醛糖还原酶的转基因小鼠。组织学检查见肾小球内有囊滴形成，肾血管内有血栓形成。因此，醛糖还原酶活性的增加可能在糖尿病逆转性并发症的发生中起重要作用.我们研究了高糖和高渗对培养的人脐静脉内皮细胞血小板源性生长因子（PDGF）的产生和PDGF-B链mRNA水平的影响。在过量的环境葡萄糖和高渗条件下，培养基中的PDGF浓度均显著增加。与蛋白分泌水平平行，PDGF-B链mRNA水平显示出显著增加。因此，高糖引起的血管内皮细胞PDGF释放增加可能在糖尿病血管病变的发病机制中起重要作用.基于这些研究，我们得出结论，高血糖和醛糖还原酶活性的升高在糖尿病并发症的发生和恶化中起着至关重要的作用。

We investigated in this study the molecular mechanism of aldose reductase activity and the metabolic derangements in the diabetic status. The summary is shown as follows.1. The role of His-42, His-188 and Lys-263 residues in the catalytic action of human aldose reductase was investigated in association with various inhibitors of this enzymes by site-directed mutagenesis. The mutated human enzymes were expressed in a faculovirus-insect cell system. The results are indicative of the possible role of Lys-263 in the substrate binding through electrostatic interaction.2. Transgenic mice expressing human aldose reductase in several tissues including kidney and liver were examined. Histologically, capsular drop formations in glomeruli and thrombi in renal vessels were founds. Therefore, acceleration of aldose reductase activity might have an important role in the development of reveral diabetic complications.3. We investigated the effects of high glucose and hyperosmolarity on platelet-derived growth factor (PDGF) production and PDGF-B chain mRNA levels in cultured human umbilical vein endothelial cells. Under an excess of ambient glucose and a hyperosmolar condition, PDGF concentrations in the culture medium were both significantly increased. Parallel to protein secretion levels, PDGF-B chain mRNA levels showed a significant increase. Thus, elevated PDGF released from endothelium by high glucose may play an important role the pathogenesis of diabetic angiopathy.4. Based on these studies, we conclude that hyperglycemia nad the elevation of aldose reductase activity have a crucial role in the initiation and deterioration of diabetic complications.