Luppi Patrizia, Drain Peter
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.
Diabetes Metab Res Rev. 2014 Oct;30(7):599-609. doi: 10.1002/dmrr.2528.
Excessive generation of reactive oxygen species (ROS) causing oxidative stress plays a major role in the pathogenesis of diabetes by inducing beta cell secretory dysfunction and apoptosis. Recent evidence has shown that C-peptide, produced by beta cells and co-secreted with insulin in the circulation of healthy individuals, decreases ROS and prevents apoptosis in dysfunctional vascular endothelial cells. In this study, we tested the hypothesis that an autocrine activity of C-peptide similarly decreases ROS when INS1 beta cells are exposed to stressful conditions of diabetes.
Reactive oxygen species and apoptosis were induced in INS1 beta cells pretreated with C-peptide by either 22 mM glucose or 100 μM hydrogen peroxide (H2 O2 ). To test C-peptide's autocrine activity, endogenous C-peptide secretion was inhibited by the KATP channel opener diazoxide and H2 O2 -induced ROS assayed after addition of either exogenous C-peptide or the secretagogue glibenclamide. In similar experiments, extracellular potassium, which depolarizes the membrane otherwise hyperpolarized by diazoxide, was used to induce endogenous C-peptide secretion. ROS was measured using the cell-permeant dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2 -DCFDA). Insulin secretion and apoptosis were assayed by enzyme-linked immunosorbent assay.
C-peptide significantly decreased high glucose-induced and H2 O2 -induced ROS and prevented apoptosis of INS1 beta cells. Diazoxide significantly increased H2 O2 -induced ROS, which was reversed by exogenous C-peptide or glibenclamide or potassium chloride.
These findings demonstrate an autocrine C-peptide mechanism in which C-peptide is bioactive on INS1 beta cells exposed to stressful conditions and might function as a natural antioxidant to limit beta cell dysfunction and loss contributing to diabetes.
活性氧（ROS）过度生成导致氧化应激，通过诱导β细胞分泌功能障碍和凋亡，在糖尿病发病机制中起主要作用。最近的证据表明，在健康个体循环中由β细胞产生并与胰岛素共同分泌的C肽，可减少ROS并防止功能失调的血管内皮细胞凋亡。在本研究中，我们检验了这样一个假设，即当INS1β细胞暴露于糖尿病应激条件下时，C肽的自分泌活性同样会降低ROS。
用22 mM葡萄糖或100 μM过氧化氢（H2O2）对经C肽预处理的INS1β细胞诱导ROS生成和凋亡。为了测试C肽的自分泌活性，通过KATP通道开放剂二氮嗪抑制内源性C肽分泌，并在添加外源性C肽或促分泌剂格列本脲后检测H2O2诱导的ROS。在类似实验中，使用细胞外钾来诱导内源性C肽分泌，细胞外钾可使原本因二氮嗪而超极化的细胞膜去极化。使用细胞渗透性染料氯甲基-2',7'-二氯二氢荧光素二乙酸酯（CM-H2-DCFDA）测量ROS。通过酶联免疫吸附测定法检测胰岛素分泌和凋亡。
C肽显著降低高糖诱导和H2O2诱导的ROS，并防止INS1β细胞凋亡。二氮嗪显著增加H2O2诱导的ROS，而外源性C肽、格列本脲或氯化钾可使其逆转。
这些发现证明了一种C肽自分泌机制，其中C肽对暴露于应激条件下的INS1β细胞具有生物活性，并且可能作为一种天然抗氧化剂，限制导致糖尿病的β细胞功能障碍和损失。
