Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, 530 45th Street, Pittsburgh, PA 15201, USA.
Diabetologia. 2011 Oct;54(10):2702-12. doi: 10.1007/s00125-011-2251-0. Epub 2011 Jul 20.
AIMS/HYPOTHESIS: Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosis
Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence or absence of C-peptide and tested for protein quantity and activity of caspase-3 and other apoptosis markers by ELISA, TUNEL and immunoblotting. Intracellular ROS were measured by flow cytometry using the ROS sensitive dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)-DCDFA). NAD(P)H oxidase activation was assayed by lucigenin. Membrane and cytoplasmic levels of the NAD(P)H subunit ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC-1) and its GTPase activity were studied by immunoblotting and ELISA. RAC-1 (also known as RAC1) gene expression was investigated by quantitative real-time PCR.
C-peptide significantly decreased caspase-3 levels and activity and upregulated production of the anti-apoptotic factor B cell CLL/lymphoma 2 (BCL-2). Glucose-induced ROS production was quenched by C-peptide and this was associated with a decreased NAD(P)H oxidase activity and reduced RAC-1 membrane production and GTPase activity.
CONCLUSIONS/INTERPRETATION: In glucose-exposed endothelial cells, C-peptide acts as an endogenous antioxidant molecule by reducing RAC-1 translocation to membrane and NAD(P)H oxidase activation. By preventing oxidative stress, C-peptide protects endothelial cells from glucose-induced apoptosis.
目的/假设：高血糖产生的活性氧（ROS）被认为与糖尿病血管并发症的发展有关。高葡萄糖增加内皮细胞的氧化应激并诱导细胞凋亡。在暴露于葡萄糖的内皮细胞中，ROS 的主要来源是 NAD（P）H 氧化酶。几项研究表明，胰岛素原在胰腺β细胞内切割产生的 C 肽在某些血管功能障碍模型中具有抗炎作用。然而，这种作用的分子机制尚不清楚。我们假设 C 肽通过减少 NAD（P）H 氧化酶的激活来减少葡萄糖诱导的 ROS 生成，并防止细胞凋亡。
将人主动脉内皮细胞（HAEC）暴露于 25mmol/l 葡萄糖中，存在或不存在 C 肽，并通过 ELISA、TUNEL 和免疫印迹法检测半胱天冬酶-3 和其他凋亡标志物的蛋白含量和活性。通过使用 ROS 敏感染料氯甲基-2'，7'-二氯二氢荧光素二乙酸酯（CM-H（2）-DCDFA）通过流式细胞术测量细胞内 ROS。通过荧光素酶测定法测定 NAD（P）H 氧化酶的激活。通过免疫印迹法和 ELISA 研究 NAD（P）H 亚基 ras 相关 C3 肉毒杆菌毒素底物 1（rho 家族，小 GTP 结合蛋白 Rac1）（RAC-1）及其 GTP 酶活性的膜和细胞质水平。通过定量实时 PCR 研究 RAC-1（也称为 RAC1）基因表达。
C 肽显著降低了半胱天冬酶-3 水平和活性，并上调了抗凋亡因子 B 细胞 CLL/淋巴瘤 2（BCL-2）的产生。C 肽抑制了葡萄糖诱导的 ROS 产生，这与 NAD（P）H 氧化酶活性降低以及 RAC-1 膜产生和 GTP 酶活性降低有关。
结论/解释：在暴露于葡萄糖的内皮细胞中，C 肽通过减少 RAC-1 向膜的易位和 NAD（P）H 氧化酶的激活来充当内源性抗氧化分子。通过防止氧化应激，C 肽保护内皮细胞免受葡萄糖诱导的细胞凋亡。
