Elucidation of the mechanism of impaired insulin release in diabetes mellitus by the molecular cell biological techniques

为了阐明糖尿病胰岛素释放障碍的机制，我们首先研究了SNAREs及其相关蛋白在胰腺β细胞中的表达。我们发现MIN6 β细胞表达syntaxin 1A、syntaxin 2（表imorphin）、突触体相关蛋白25kda （SNAP25）、囊泡相关膜蛋白-2 （VAMP2或synaptobrevin）、synaptotagmin I、III、IV。为了研究这些蛋白如何参与糖尿病的病因和进展，我们检测了syntaxin 1A和SNAP25在2型糖尿病动物模型GK大鼠胰腺β细胞中的表达水平。这些蛋白质水平下降到对照组的一半左右。然后，我们试图利用重组腺病毒系统将下降的表达水平恢复到正常水平。GK大鼠胰岛感染Adex 1CA-syntaxin 1A和Adex 1CA-SNAP25后，这些蛋白水平恢复，因此受损的胰岛素释放部分正常化。因此，我们的数据强烈表明t-SNAREs表达参与2型糖尿病的病因和进展。然后我们研究了融合后胰岛素分泌颗粒的内吞作用与胰岛素释放的关系。为此，我们克隆了定位于早期核内体的新型纤维素蛋白相关肽（NGRP）。为了确定NCRP（内啡肽）在胰岛素分泌中的功能作用，我们利用腺病毒系统过表达野生型和显性阴性内啡肽。数据显示，内啡肽通过内吞囊泡和内核体之间的相互作用，在胰岛素的外胞吞作用中发挥生理作用。最后，我们开发了一种新的策略，通过胰岛素与ph敏感GFP突变体（pHluorin）之间的融合蛋白来检测胰岛素融合本身。

In order to elucidate the mechanism of impaired insulin release in diabetes mellitus, we firstly investigated the expression of SNAREs and its related proteins in pancreatic β cells. We revealed that MIN6 β cells expressed syntaxin 1A, syntaxin 2 (epimorphin), synaptosomal associated protein of 25 kDa (SNAP25), vesicle associated membrane protein-2 (VAMP2 or synaptobrevin), synaptotagmin I, III, IV. To examine how these proteins are involved in the etiology and progression of diabetes mellitus, we examined the expression levels of syntaxin 1A and SNAP25 in pancreatic β cells of GK rats, type 2 DM animal model. These protein levels were decreased to about a half-fold that of control. Then, we tried to recover the decreased expression levels to the normal levels using recombinant adenovirus system. Infection of Adex 1CA-syntaxin 1A and Adex 1CA-SNAP25 with GK rat islets recovered these protein levels, thereby the impaired insulin release was partly normalized. Thus, our data strongly indicated the involvement of t-SNAREs expression in the etiology and progression of type 2-DM. We then investigated how endocytosis insulin secretory granules post fusion is associated with insulin release. For this purpose, we have cloned the novel-cellubrevin related peptide (NGRP), which was localized in the early endosome. To determine the functional role of NCRP (endobrevin) in the exocytosis of insulin, wild-type and dominant-negative endobrevin were overexpressed using adenovirus system. The data revealed that endobrevin plays a physiological role in the exo-endocytosis of insulin via an interaction between endocytic vesicles and the endosome. Finally, we developed the new strategy to detect the insulin fusion per se by fusion protein between insulin and pH-sensitive GFP mutant (pHluorin).