Department of Physiology, Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, ON, M5S 1A8, Canada.
Diabetologia. 2014 Apr;57(4):809-18. doi: 10.1007/s00125-013-3143-2. Epub 2013 Dec 20.
AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1), secreted by the enteroendocrine L cell, is an incretin hormone that potently stimulates insulin secretion. Although signalling pathways promoting GLP-1 release are well characterised, the mechanisms by which GLP-1-containing granules fuse to the L cell membrane are unknown. As soluble NSF attachment proteins (SNAREs) are known to mediate granule-membrane fusion, the role of vesicle-associated membrane proteins (VAMPs) in GLP-1 exocytosis was examined.
SNARE expression was determined in murine GLUTag L cells by RT-PCR and immunoblot and in primary murine L cells by immunofluorescence. Co-immunoprecipitation was used to examine SNARE interactions, while tetanus toxin (TetX)-mediated cleavage of VAMP was used with a GLP-1 secretion assay and total internal reflection fluorescence microscopy to determine the role of VAMP2 in exocytosis.
VAMP2 was expressed in murine L cells and localised to secretory granules in GLUTag cells. VAMP1/3 and the core membrane proteins syntaxin1a and synaptosomal-associated protein 25 kDa (SNAP25) were also detected. TetX cleaved VAMPs in GLUTag cells. However, only VAMP2 interacted with syntaxin1a, as did SNAP25 and Munc18-1. TetX treatment of GLUTag cells prevented glucose-dependent insulinotrophic peptide- and oleic-acid-stimulated GLP-1 secretion (p < 0.05-0.01), as well as K(+)-stimulated single-cell exocytosis (p < 0.05-0.001), while TetX-resistant VAMP2 expression rescued GLP-1 secretion (p < 0.01-0.001).
CONCLUSIONS/INTERPRETATION: Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag L cell in response to a variety of established secretagogues. An improved understanding of the mechanisms governing the release of GLP-1 may lead to new therapeutic approaches to enhance the levels of this incretin hormone in patients with type 2 diabetes.
目的/假设：胰高血糖素样肽-1（GLP-1）由肠内分泌 L 细胞分泌，是一种强烈刺激胰岛素分泌的肠促胰岛素激素。虽然促进 GLP-1 释放的信号通路已经得到很好的描述，但 GLP-1 包含的颗粒与 L 细胞膜融合的机制尚不清楚。由于已知可溶性 NSF 附着蛋白（SNAREs）介导颗粒-膜融合，因此研究了囊泡相关膜蛋白（VAMPs）在 GLP-1 胞吐作用中的作用。
通过 RT-PCR 和免疫印迹法测定小鼠 GLUTag L 细胞中的 SNARE 表达，并通过免疫荧光法测定原代小鼠 L 细胞中的 SNARE 表达。共免疫沉淀用于检测 SNARE 相互作用，而破伤风毒素（TetX）介导的 VAMP 切割与 GLP-1 分泌测定和全内反射荧光显微镜一起用于确定 VAMP2 在胞吐作用中的作用。
VAMP2 在小鼠 L 细胞中表达，并在 GLUTag 细胞中的分泌颗粒中定位。还检测到 VAMP1/3 和核心膜蛋白 syntaxin1a 和突触相关蛋白 25 kDa（SNAP25）。TetX 在 GLUTag 细胞中切割 VAMPs。然而，只有 VAMP2 与 syntaxin1a 相互作用，SNAP25 和 Munc18-1 也是如此。TetX 处理 GLUTag 细胞可防止葡萄糖依赖性胰岛素促分泌肽和油酸刺激的 GLP-1 分泌（p <0.05-0.01），以及 K+刺激的单细胞胞吐作用（p <0.05-0.001），而 TetX 抗性 VAMP2 表达可恢复 GLP-1 分泌（p <0.01-0.001）。
结论/解释：综上所述，这些发现表明 VAMP2 在各种已建立的分泌刺激物作用下从 GLUTag L 细胞中 GLP-1 胞吐作用中起重要作用。对调节 GLP-1 释放的机制的更好理解可能会导致新的治疗方法来提高 2 型糖尿病患者中这种肠促胰岛素激素的水平。
