Wang Yao, Lin Haiyan, Hao Nana, Zhu Zhengqiu, Wang Dong, Li Yuan, Chen Hong, Zhu Yunxia, Han Xiao
Department of Endocrinology, Zhongda Hospital, Institute of Diabetes, Southeast University, Nanjing, People's Republic of China.
Diabetologia. 2015 Jun;58(6):1272-81. doi: 10.1007/s00125-015-3561-4. Epub 2015 Mar 22.
AIMS/HYPOTHESIS: The transcription factor forkhead box O1 (FOXO1) induces pancreatic islet beta cell endoplasmic reticulum stress and is involved in fatty-acid-induced insulin-secretion defects. Cask is a downstream target gene of FOXO1. Using INS-1 cells with palmitate-induced insulin-release defects, we investigated the relationship between FOXO1 and Cask.
The expression levels and location of calcium/calmodulin-dependent serine protein kinase (CASK) and FOXO1 were evaluated by real-time PCR, western blotting and immunofluorescence. The regulation of Cask by FOXO1 was examined using chromatin immunoprecipitation (ChIP) and luciferase assays. Potassium-stimulated insulin-secretion assays were used to verify the function of INS-1 cells and islets. Electron microscopy was used to establish the anchoring process of the insulin granules after CASK knockdown in islets.
Palmitic acid reduced CASK levels and increased FOXO1 levels. ChIP and luciferase assays demonstrated FOXO1 binding with the Cask promoter, which was enhanced by palmitate treatment. CASK knockdown reduced insulin release in INS-1 cells and primary islets, and Cask overexpression reversed the palmitate-induced insulin reduction. CASK knockdown attenuated forskolin-enhanced insulin release, but Cask overexpression did not change the insulin-secretion suppression induced by nifedipine. In pancreatic islet beta cells, CASK knockdown reduced the anchoring of insulin vesicles to cell membranes.
CONCLUSIONS/INTERPRETATION: The induction of beta cell insulin-secretion defects by fatty acids is mediated, at least in part, by FOXO1 via downregulation of Cask expression. It is characterised mainly as an obstruction of the anchoring of insulin granules to beta cell membranes.
目的/假设：转录因子叉头框O1（FOXO1）可诱导胰岛β细胞内质网应激，并参与脂肪酸诱导的胰岛素分泌缺陷。Cask是FOXO1的下游靶基因。我们使用具有棕榈酸诱导的胰岛素释放缺陷的INS-1细胞，研究了FOXO1与Cask之间的关系。
通过实时PCR、蛋白质印迹法和免疫荧光评估钙/钙调蛋白依赖性丝氨酸蛋白激酶（CASK）和FOXO1的表达水平及定位。使用染色质免疫沉淀（ChIP）和荧光素酶测定法检测FOXO1对Cask的调控。采用钾刺激胰岛素分泌试验验证INS-1细胞和胰岛的功能。利用电子显微镜观察胰岛中CASK敲低后胰岛素颗粒的锚定过程。
棕榈酸降低了CASK水平，提高了FOXO1水平。ChIP和荧光素酶测定表明FOXO1与Cask启动子结合，棕榈酸处理可增强这种结合。CASK敲低降低了INS-1细胞和原代胰岛中的胰岛素释放，而Cask过表达可逆转棕榈酸诱导的胰岛素减少。CASK敲低减弱了福斯可林增强的胰岛素释放，但Cask过表达并未改变硝苯地平诱导的胰岛素分泌抑制。在胰岛β细胞中，CASK敲低减少了胰岛素囊泡与细胞膜的锚定。
结论/解读：脂肪酸诱导的β细胞胰岛素分泌缺陷至少部分是由FOXO1通过下调Cask表达介导的。其主要特征是胰岛素颗粒与β细胞膜的锚定受阻。
