Chan Wen-Hsiung, Wu Hsin-Jung
Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan.
J Cell Biochem. 2008 Mar 1;103(4):1144-57. doi: 10.1002/jcb.21489.
Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.
高血糖和甲基乙二醛（MG）升高是糖尿病（DM）的症状。我们之前表明，高糖（HG；30 mM）或MG（50 - 400 microM）可诱导哺乳动物细胞凋亡，但这些剂量高于糖尿病患者血浆中葡萄糖和MG的生理浓度。正常血液循环中MG和葡萄糖的生理浓度分别约为1 microM和5 mM。在此，我们表明，与糖尿病患者血液循环中所见浓度相当的MG和葡萄糖（分别为5 microM和15 - 30 mM）联合处理可在体外导致人脐静脉内皮细胞（HUVECs）凋亡或坏死。HG/MG联合处理直接增加了HUVECs中的活性氧（ROS）含量，导致细胞内ATP水平升高，其可通过凋亡或坏死控制细胞死亡。用5 microM MG和20 mM葡萄糖联合处理HUVECs可显著增加细胞质游离钙水平、一氧化氮合酶（NOS）、半胱天冬酶 - 3和 - 9的激活、细胞色素c释放以及凋亡性细胞死亡。相反，在用5 microM MG和30 mM葡萄糖处理的HUVECs中未检测到这些凋亡生化变化，这些细胞似乎发生了坏死。用一氧化氮（NO）清除剂预处理可抑制5 microM MG/20 mM葡萄糖诱导的细胞色素c释放，降低半胱天冬酶 - 9和半胱天冬酶 - 3的激活，并增加已知参与凋亡信号传导的p53和p21的基因表达及蛋白水平。使用小干扰RNA（siRNA）抑制p53蛋白表达可阻断p21的激活以及5 microM MG/20 mM葡萄糖诱导的细胞凋亡。相反，用siRNA抑制p21蛋白表达可防止HUVECs凋亡，但对p53表达无影响。这些结果共同表明，MG和葡萄糖的处理剂量可决定HUVECs中的细胞死亡模式（凋亡与坏死），并且ROS和NO在MG/HG诱导的这些细胞凋亡中均起重要作用。
