Molecular mechanism of the novel oxidative stress in diabetes

据报道，糖尿病患者和动物血液中的α-生育酚(维生素E)浓度降低，可能通过降低抗氧化活性而引发另一种氧化应激。已证实肝脏通过α-生育酚转移蛋白(a-TTP)将食物来源的α-生育酚分泌到血液循环中起主要作用，而α-TTP是血浆α-生育酚水平的主要决定因素。我们发现链脲佐菌素诱导的糖尿病大鼠肝脏α-TTP水平降低，低浓度胰岛素(&lt；10^&lt；-10&gt；M)培养的肝细胞α-TTP水平也降低。因此，本研究旨在探讨胰岛素耗竭状态下肝脏α-TTP浓度降低的分子机制。原代培养的大鼠肝细胞分别与环己胺(蛋白质合成抑制剂)和不同浓度的胰岛素孵育不同时间，用Western印迹法检测α-TTP水平。结果表明，低浓度胰岛素(10^~(-10)-10&gt；M，0M)孵育12、24小时后，α-TTP水平下降，而高浓度胰岛素(10^~(-8)~(-8)M，10^~(-6)&gt；M)对α-TTP水平无明显影响。此外，α-TTP的减少通过阻止蛋白质合成而消失。因此，α-TTP的降低可能是由于低浓度胰岛素引起的蛋白酶活性降解所致。接下来，我们研究了溶酶体或蛋白酶体抑制剂对α-TTP浓度的影响。结果表明，加入溶酶体抑制剂后，α-TTP浓度显著降低，但不受蛋白酶体抑制剂的影响。综上所述，肝脏α-TTP在低浓度胰岛素作用下可能被降解，α-TTP蛋白酶可能被溶酶体快速降解。

It has bee reported that a-tocopherol (vitamin E) concentration in blood was reduced in diabetic patients and animals that could induce another oxidative stress by reducing anti-oxidative activity. It has been established that liver has the main role to secrete food-derived a-tocopherol to circulation through function of a-tocopherol transfer protein (a-TTP) and a-TTP was a major determinant of plasma a-tocopherol level by this mechanism. We have found that hepatic a-TTP level was reduced in streptozotocin-induced diabetic rats and reduction of a-TTP concentration was also observed in hepatocytes cultured with low concentration of insulin (<10^<-10>M). Therefore, the aim of this study was to investigate the molecular mechanism of the reduction of hepatic a-TTP concentration under insulin depleted condition. Primary cultured rat hepatocyte was incubated with/without cyclohexymide (inhibitor of protein synthesis) and with various concentration of insulin for various periods and a-TTP level was measured by Western blot analysis. The results showed that a-TTP level decreased with low concentrations of insulin (10^<-10>M, 0M) while a-TTP level was not changed with high concentrations of insulin (10^<-8>M, 10^<-6>M) for 12 and 24 hours incubation. Furthermore, the decrease of a-TTP disappeared by preventing protein synthesis. Therefore, the decrease of a-TTP might be due to degradation by protease activity induced by low insulin concentration. Next, we investigated whether concentration of a-TTP were influenced by lysosome or proteasome inhibitor. The results showed that a-TTP concentration was greatly reduced by addition of lysosome inhibitors but not influened by a proteasome inhibitor. In conclusion, hepatic a-TTP may be degraded under low concentration of insulin, and a-TTP protease may be rapidly procecced by lysosome.