FFAs通过影响hIAPP构象及内质网UPR在胰岛β细胞损伤中的作用及机制的研究

Damage effect of hIAPP on pancreatic islet beta cell is based on its unstable structure, which prone to form the α-helix, β-sheet and fibril conformation. So far, the protein conformation diseases, such as AD, PD and T2DM share the common characteristics including the production of insoluble protein and dysfunction of folded protein degradation, which caused the insoluble protein deposition. Additionally, accumulation of insoluble protein was repressed and eliminated by the unfolded protein response (UPR) initiated from the endoplasmic reticulum (ER).The pathologic development of type 2 diabetes mellitus (T2DM) and its complications dramatically are due to the high level of free fatty acids (FFAs). Indirectly, the evidence has been indicated that FFAs aggravated the amyloid deposition. Our previous study showed that serum hIAPP increased significantly in the patient suffered from the T2DM. Furthermore, we found beta cell injury induced by FFAs-treatment has been implicated in production of IAPP. However, it can be demonstrated without the direct evidence that alteration of hIAPP conformation and accumulation of amyloid under the high FFAs conditions. Based on the techniques of simulated reaction in vitro and chemical detection, effects of FFAs on hIAPP conformation have been studied; Also, we want to known what changes of the rIAPP/hIAPP conformation and UPR following the FFAs-treatment in the hIAPP-INS-1 cell which is characterized by the expression of hIAPP, endogenously. In the transgenic mouse which knocked in the pro-hIAPP gene, we will clarify whether the high fat accelerated the amyloid deposition, and how does the features of T2DM were induced by the hIAPP cooperated with FFAs. The aim of this proposal is to clarify the effects of FFAs on hIAPP conformation and UPR, to identify the process of amyloid deposition under high FFAs conditions, and to explore the mechanisms that the hIAPP cooperated with aberrant lipid metabolism on the pathologic development in T2DM.

hIAPP对胰岛细胞的损伤作用，与其结构不稳定，易发生α-螺旋、β-片层及纤维化有关，这种构象变化是已知蛋白构象性疾病的共有特征。内质网的UPR反应在细胞内形成有效的清除机制，防止构象异常蛋白过度聚集。T2DM及其并发症的产生和FFAs关系密切，间接证据表明FFAs可能和Amyloid沉积有关，我们前期工作发现T2DM患者血清hIAPP明显升高，高脂诱导的胰岛细胞损伤有IAPP参与，但尚无直接证据证明FFAs会导致异常构象的hIAPP聚集增多清除减少。本研究基于体外模拟技术研究FFAs对hIAPP构象的影响，针对内源性表达hIAPP的INS-1细胞株，研究FFAs干预如何导致rIAPP/hIAPP构象及UPR反应异常，在表达hIAPP的转基因小鼠上，研究高脂是否促进了Amyloid的沉积，二者的协同作用是如何诱导T2DM病理特征的，以揭示hIAPP构象异常和聚集在T2DM发病中的作用机制。

胰淀粉样蛋白沉积是二型糖尿病（T2DM）常见病理。高脂血症及脂类代谢异常是T2DM常见并发症。持续高血脂对胰淀粉样蛋白沉积的影响及其关键机制是本项目研究的重点。为阐明游离脂肪酸（FFAs）对胰淀粉样蛋白沉积及胰岛β细胞的损伤效应，本研究从三个水平开展了相对系统的研究。在分子模拟水平研究了FFAs对人源性胰岛淀粉样蛋白（hIAPP）构象的影响。在细胞水平研究了胰岛β细胞高表达hIAPP后，FFAs与内源性hIAPP的协同损伤效应,通过外源性给予人工合成的hIAPP联合FFAs对胰岛β细胞的损伤效应。在整体水平，研究了T2DM患者胰岛功能低下、血脂水平和血清IAPP水平的相关性；在hIAPP转基因小鼠上，通过高脂饮食喂养，研究了高脂饮食联合hIAPP高表达对胰岛β细胞的损伤效应，以及对中枢神经系统的损伤效应。我们的研究结果表明，常见的5种FFAs均可以加速hIAPP的纤维化，其中棕榈酸（PA）不但加速hIAPP纤维化，还能维持纤维化的稳定状态，促进hIAPP和质膜的相互作用，尤其阴离子脂质具有促进hIAPP纤维化的倾向。在细胞模型上，高表达hIAPP的胰岛β细胞对FFAs的损伤效应更加敏感，表现为内质网应激，细胞凋亡；合成的hIAPP联合FFAs处理胰岛β细胞，导致细胞膜通透性增加。在整体动物模型上，杂合子hIAPP转基因鼠给予高脂饮食喂养6个月、12月后均表现出胰岛损伤，胰岛内hIAPP聚集，在分离的胰岛组织中，观察到与内质网应激相关分子高表达，炎症信号增强。高脂喂养12个月的hIAPP转基因鼠，海马区老化神经细胞增加，海马区神经细胞葡萄糖利用率下降。以上结果表明，FFAs诱导hIAPP出现更多纤维状态，纤维化的hIAPP可以导致胰岛β细胞膜的通透性增加，同时诱导细胞内质网应激，加重细胞凋亡。高脂饲养诱导hIAPP高表达，并在胰岛组织内沉积，加剧了T2DM病理的形成。高脂诱导的hIAPP同时会影响脑的葡萄糖代谢，加速脑老化，动物表现为记忆功能下降。