Pathogenesis and Impact of Islet Amyloid

项目摘要/摘要 胰岛淀粉样蛋白是2型糖尿病的一种病理特征，导致β细胞丢失和分泌 这种疾病的特征是功能障碍。这些淀粉样蛋白沉积的独特多肽成分是β- 细胞分泌产物胰岛淀粉样多肽，聚集形成淀粉样蛋白，导致β-细胞 细胞凋亡。人IAPP(HIAPP)聚集与线粒体功能障碍和氧化应激有关， 但胰岛淀粉样蛋白的形成导致线粒体功能障碍的机制尚不清楚。 β细胞中胆固醇的积累也与细胞丢失和分泌功能障碍有关；然而， 胆固醇积累有害的机制也仍然难以捉摸。 类固醇合成急性调节蛋白(STAR)将胆固醇从外向内转运 随后代谢的线粒体膜。在经典的类固醇生成组织中，胆固醇 裂解酶(Cyps)代谢导致糖皮质激素、盐皮质激素和性激素的产生 类固醇。在非经典的类固醇生成组织中，STAR对胆汁酸、糖皮质激素的产生至关重要， 保护或伤害细胞的神经类固醇和氧化类固醇。 我们已经建立了在β细胞中存在STAR，并在淀粉样蛋白条件下特异性上调 队形。这种β-细胞中STAR的上调导致线粒体胆固醇增加，降低 线粒体功能和细胞活力下降。我们还发现，参与氧固醇的细胞色素P27A1 产量随着胰岛淀粉样蛋白的形成而下调，而Cyp11a1和其他下游的Cyps 负责类固醇产生的基因在胰岛中不存在。最后，虽然已知STAR的表达式是 在转录因子CREB(cAMP反应元件结合蛋白)的强烈调控下，我们发现了CREB 在淀粉样蛋白形成的条件下，活性随着恒星的出现而逐渐增加。 基于我们的初步数据，我们假设胰岛淀粉样蛋白的形成诱导了 CREB依赖的方式，导致胆固醇向线粒体的运输增加，进而导致 线粒体功能障碍、β细胞功能障碍和β细胞丢失。 这一假设将在以下三个具体目标中得到解决，每个目标都旨在回答 重要的问题。 特异性目标1：确定STAR在介导β-细胞胆固醇升高的毒性效应中的作用 体外培养。这一目标将回答以下问题：a)在胆固醇条件下，STAR基因是否被击倒 负载保护hIAPP胰岛免受淀粉样蛋白诱导的毒性？B)STAR过度表达是否会损害线粒体 功能和结果β-细胞功能障碍和丢失？C)过表达的细胞色素P27A1可以保护胰岛免受 淀粉样蛋白形成条件下STAR对线粒体功能的不利影响？ 特定目标2：确定体内STAR表达的长期减少是否改善了 胰岛淀粉样蛋白沉积对β细胞质量和功能的不利影响。这个目标将回答以下问题 问：选择性β细胞STAR缺陷是否能减少淀粉样蛋白形成下的β细胞丢失和功能障碍 在体内的条件？ 具体目标3：确定胰岛淀粉样蛋白是否需要CREB和CRTC2的持续激活。 诱导了STAR的上调。这一目标将回答以下问题：a)淀粉样蛋白是否需要CREB- 诱导STAR上调？B)CRTC2对于CREB增加STAR的行动是必要的和/或充分的吗 在小岛上的表情？C)在淀粉样蛋白形成条件下，CREB是否直接与STAR启动子结合？ 这些研究将提供关于STAR在淀粉样蛋白诱导的线粒体中的作用的新数据 功能障碍和β细胞毒性，从而可能确定用于治疗2型糖尿病的新的β细胞靶点。

PROJECT SUMMARY/ABSTRACT Islet amyloid is a pathological characteristic of type 2 diabetes, contributing to the β-cell loss and secretory dysfunction that characterize the disease. The unique peptide constituent of these amyloid deposits is the β- cell secretory product islet amyloid polypeptide (IAPP), which aggregates to form amyloid resulting in β-cell apoptosis. Human IAPP (hIAPP) aggregation is associated with mitochondrial dysfunction and oxidative stress, but the mechanism by which formation of islet amyloid results in mitochondrial dysfunction remains unclear. Cholesterol accumulation in the β-cell is also associated with cellular loss and secretory dysfunction; however, the mechanism by which cholesterol accumulation is deleterious also remains elusive. Steroidogenic Acute Regulatory Protein (StAR) transports cholesterol from the outer to the inner mitochondrial membrane for subsequent metabolism. In classical steroidogenic tissues, cholesterol metabolism by cleavage enzymes (CYPs) results in production of glucocorticoids, mineralocorticoids and sex steroids. In non-classical steroidogenic tissues, StAR is vital to the production of bile acids, glucocorticoids, neurosteroids and oxysterols that either protect or harm the cell. We have established StAR to be present in β-cells and specifically upregulated under conditions of amyloid formation. This upregulation of StAR in β-cells resulted in increased mitochondrial cholesterol, decreased mitochondrial function and reduced cell viability. We also found that CYP27A1, which is involved in oxysterol production, was down regulated with islet amyloid formation, while CYP11A1 and other downstream CYPs responsible for steroid production were not present in islets. Finally, while expression of StAR is known to be regulated acutely by the transcription factor CREB (cAMP response element binding protein), we found CREB activity to be increased chronically along with StAR under amyloid forming conditions. Based on our preliminary data, we hypothesize that islet amyloid formation induces StAR expression in a CREB-dependent manner, resulting in increased transport of cholesterol into mitochondria that in turn leads to mitochondrial dysfunction, β-cell dysfunction and β-cell loss. This hypothesis will be addressed in the following three specific aims, each of which is intended to answer important questions. Specific Aim 1: To determine the role of StAR in mediating the toxic effects of increased β-cell cholesterol in vitro. This aim will answer the following questions: a) Does StAR knockdown under conditions of cholesterol loading protect hIAPP islets from amyloid-induced toxicity? b) Does StAR overexpression impair mitochondrial function and result in β-cell dysfunction and loss? c) Can overexpression of CYP27A1 protect islets from the detrimental effects of StAR on mitochondrial function under amyloid-forming conditions? Specific Aim 2: To determine whether long-term reduction of StAR expression in vivo ameliorates the detrimental effects of islet amyloid deposition on β-cell mass and function. This aim will answer the following question: Does selective β-cell StAR deficiency decrease β-cell loss and dysfunction under amyloid forming conditions in vivo? Specific Aim 3: To determine whether sustained activation of CREB and CRTC2 is required for islet amyloid- induced upregulation of StAR. This aim will answer the following questions: a) Is CREB required for amyloid- induced upregulation of StAR? b) Is CRTC2 necessary and/or sufficient for CREB’s action to increase StAR expression in islets? c) Does CREB directly bind the StAR promoter under amyloid forming conditions? These studies will provide novel data regarding the role of StAR in amyloid-induced mitochondrial dysfunction and β-cell toxicity, and thereby potentially identify new β-cell targets for treating type 2 diabetes.