Department of Diabetes and Clinical Nutrition, Kyoto University, Japan.
Diabetes. 2011 Jan;60(1):218-26. doi: 10.2337/db10-0021. Epub 2010 Oct 26.
Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic β-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4.
Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells.
Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase-negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production.
Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.
活性氧（ROS）是破坏胰岛β细胞代谢分泌偶联的最重要因素之一。我们最近报道，Src 抑制可有效改善高糖诱导的糖尿病 Goto-Kakizaki（GK）大鼠胰岛 ROS 产生增加和 ATP 产生受损，提示 Src 活性上调。在本研究中，我们使用胰高血糖素样肽-1 类似物 exendin-4 研究了 GLP-1 信号是否调节 Src 活性并改善 GK 胰岛中的内源性 ROS 产生和 ATP 产生。
使用从 GK 和对照 Wistar 大鼠分离的胰岛进行免疫印迹分析以及 ROS 产生和 ATP 含量的测量。通过免疫沉淀胰岛裂解物然后进行免疫印迹检查Src 活性。使用分散胰岛细胞中的荧光探针测量 ROS 产生。
在 16.7mmol/l 葡萄糖暴露下，exendin-4 可显著降低 GK 胰岛中Src Tyr416 的磷酸化，这表明 Src 激活。GK 胰岛细胞中葡萄糖诱导的 ROS 产生（16.7mmol/l）通过 exendin-4 以及 Src 抑制剂 PP2 的共同暴露显著降低。GK 胰岛中 Src 激酶阴性突变体的表达显著降低了高葡萄糖诱导的 ROS 产生。exendin-4 以及 PP2 均显著增加了 GK 胰岛中高葡萄糖引起的 ATP 升高受损。exendin-4 降低 ROS 产生的作用不受 PKA 抑制剂 H-89 的影响，而 Epac 特异性 cAMP 类似物（8CPT-2Me-cAMP）显著降低了 Src Tyr416 磷酸化和 ROS 产生。
exendin-4 通过抑制 Src 激活降低糖尿病 GK 大鼠胰岛中的内源性 ROS 产生并增加 ATP 产生，这依赖于 Epac。
