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受损的机制，我们首先研究了SNARE及其相关蛋白在胰岛β细胞中的表达。我们发现MIN 6 β细胞表达syntaxin 1A、syntaxin 2（epimorphin）、突触体相关蛋白25（SNAP 25）、囊泡相关膜蛋白2（VAMP 2或synaptobrevin）、突触结合蛋白I、III、IV。为了研究这些蛋白质如何参与糖尿病的病因和进展，我们检测了2型糖尿病动物模型GK大鼠胰腺β细胞中syntaxin 1A和SNAP 25的表达水平。这些蛋白质水平降低至对照的约一半。然后，我们尝试使用重组腺病毒系统将降低的表达水平恢复到正常水平。Adex 1CA-syntaxin 1A和Adex 1CA-SNAP 25感染GK大鼠胰岛可恢复这些蛋白水平，从而使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-SNARE表达参与2型糖尿病的病因和进展。然后，我们研究了融合后的内吞胰岛素分泌颗粒如何与胰岛素释放相关。为此，我们克隆了一种新的小泡纤维素相关肽（NGRP），它定位于早期内体。为了确定NCRP（内泡蛋白）在胰岛素胞吐中的功能作用，使用腺病毒系统过表达野生型和显性负性内泡蛋白。这些数据表明，endobrevin通过内吞囊泡和内体之间的相互作用在胰岛素的外吞-内吞中发挥生理作用。最后，我们开发了一种新的策略，通过胰岛素和pH敏感的GFP突变体（pHluorin）之间的融合蛋白来检测胰岛素本身的融合。

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).