Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.
Diabetes. 2010 Apr;59(4):808-18. doi: 10.2337/db09-1207. Epub 2010 Jan 27.
Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid-metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator-activated receptor (PPAR) delta was investigated.
Effects of 4-HDDE and PPARdelta on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions.
Using GW501516 (PPARdelta agonist) and GSK0660 (PPARdelta antagonist), we discovered that high-glucose-induced downregulation of the glucose transport system in VECs is mediated by PPARdelta. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARdelta in cells overexpressing human PPAR (hPPAR)delta but not hPPARalpha, -gamma1, or -gamma2. Moreover, silencing of PPARdelta prevented high-glucose-dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARdelta to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay.
Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARdelta.
血管内皮细胞（VECs）通过降低其典型葡萄糖转运体 GLUT-1 的表达来下调其葡萄糖摄取率，以响应高血糖。迄今为止，我们发现钙网织蛋白和花生四烯酸代谢酶 12-脂氧合酶在这个自身调节过程中起着关键作用。假设 12-脂氧合酶的过氧化产物 4-羟基十二碳-（2E，6Z）-二烯醛（4-HDDE）通过激活过氧化物酶体增殖物激活受体（PPAR）δ来介导这种下调机制，这一假说进行了研究。
通过药理学和分子干预，评估 4-HDDE 和 PPARδ 对原代牛主动脉内皮细胞葡萄糖转运系统和钙网织蛋白表达的影响。
使用 GW501516（PPARδ 激动剂）和 GSK0660（PPARδ 拮抗剂），我们发现高葡萄糖诱导的 VEC 葡萄糖转运系统下调是由 PPARδ 介导的。VEC 中的 PPAR 敏感荧光素酶报告基因测定显示，高葡萄糖显著增加荧光素酶活性，而 GSK0660 则使其丧失。高效液相色谱分析显示，高葡萄糖孵育可显著增加 VEC 中 4-HDDE 的生成。用 4-HDDE 处理暴露于正常葡萄糖的 VEC 可模拟高葡萄糖并下调葡萄糖转运系统并增加钙网织蛋白表达。与高葡萄糖一样，4-HDDE 显著激活细胞中过表达人 PPAR（hPPAR）δ而非 hPPARα、-γ1 或 -γ2 的 PPARδ。此外，高葡萄糖依赖性 GLUT-1 和钙网织蛋白表达的改变可被 PPARδ 沉默所阻止。最后，通过利用特定的染色质免疫沉淀测定，鉴定了 PPARδ 与钙网织蛋白基因启动子区域的 PPAR 反应元件的特异性结合。
总的来说，我们的数据表明，4-HDDE 通过激活 PPARδ 在 VEC 中下调葡萄糖摄取中起核心作用。
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