Targeting GLP-1 receptor trafficking to improve therapies for type 2 diabetes

全球每年约有400万人死于2型糖尿病(T2D)，并导致更多人健康不佳。T2D的一个主要潜在问题是胰腺中被称为β细胞的特殊细胞开始衰竭，不能产生或分泌足够的胰岛素，而胰岛素是控制血糖所需的。通过激活一种被称为胰升糖素样肽-1受体(GLP-1R)的表面蛋白，可以刺激β细胞释放更多的胰岛素。GLP-1R治疗被称为GLP-1R激动剂，已经在T2D中使用。我们发现，一个被称为“受体转运”的过程限制了GLP-1R激动剂的有效性。当激动剂激活受体时，它也会将受体“内化”到细胞内，在那里它被隐藏起来，不再能被细胞外的激动剂激活。我们用目前在T2D中使用的GLP-1R激动剂的修改版本(exendin-4，也称为“Exenatide”)展示了这一点，我们设计这种药物来减少内化过程。这种名为“Ex-phe1”的新激动剂在患有T2D的小鼠身上比未经修饰的exendin-4有效得多，因为它允许受体在细胞表面停留更长时间并不断重新激活，导致更多的胰岛素释放。此外，尽管在β细胞中更有效，Ex-phe1并没有引起更多的恶心，这是GLP-1R激动剂众所周知的副作用。该项目有两个目标。首先是了解GLP-1R转运的变化如何改变β细胞的胰岛素释放。第二个是优化我们在初步工作中产生的化合物，以产生新的、更好的治疗T2D的方法，这将是并行进行的。我们的方法包括在β细胞和胰腺组织(“胰岛”)上进行实验，这让我们能够了解分子细节上的事件，以及在患有T2D的小鼠上，T2D告诉我们在整个身体水平上发生了什么。我们非常重视使用人类胰岛(在器官捐赠需求过剩的情况下提供研究)，因为我们认为这对于理解和治疗人类疾病是必不可少的。我们强调以下几个关键实验：1)我们将使用计算机建模和GLP-1R的基因修改的组合来确定受体与Ex-phe1或其他有偏见的GLP-1R激动剂之间的特定相互作用，这些相互作用支持它们不同的贩运和治疗效果。这一知识将对未来设计更有效的药物至关重要。2)我们将对人类胰岛进行基因改造，以降低“β-arrestin-2”的水平，我们认为β-arrestin-2是β细胞中的一种蛋白质，是GLP-1R激活、贩运和有益胰岛素释放之间的关键联系。我们还将在β细胞中培育缺乏β-arrestin-2的转基因小鼠。我们将使用这些系统来观察GLP-1R激动剂治疗后β细胞内蛋白质和基因激活的复杂模式。3)我们将研究我们新的、改进的GLP-1R激动剂Ex-phe1对移植到糖尿病小鼠体内的人胰岛的长期影响。重要的是，我们将使用一种新的方法，将胰岛实际移植到眼睛中，在显微镜下可以直接和重复地观察到胰岛形状、行为和整体健康的变化。这些小鼠将接受Exphe1或Exendin-4的长期配方治疗数月，我们将监测血糖的变化，使我们能够比较治疗对整体胰岛功能的影响。由于众所周知的小鼠和人类胰岛之间的差异，我们相信使用这种人源化的小鼠对于了解长期治疗效果至关重要。总而言之，我们已经确定了一种基于GLP-1R贩运的令人兴奋的新方法来改善T2D的治疗。在这个项目中，我们将确定这种方法为什么有效的分子细节，确定新的潜在药物靶点，并展示这种新疗法在独特的人源化小鼠身上的长期效果。

Type 2 diabetes (T2D) kills around 4 million people worldwide every year and causes ill health in many more. A major underlying problem in T2D is that specialised cells in the pancreas known as beta cells start to fail and do not produce or secrete enough insulin, which is needed to control blood sugar. Beta cells can be stimulated to release more insulin by activating a surface protein known as glucagon-like peptide-1 receptor (GLP-1R). GLP-1R treatments, known as GLP-1R agonists, are already used in T2D. We have found that a process known as "receptor trafficking" limits the effectiveness of GLP-1R agonists. When the agonist activates the receptor, it also "internalises" it into the cell where it is hidden and can no longer be activated by agonists outside the cell. We showed this using a modified version of a GLP-1R agonist currently used in T2D (exendin-4, also known as "Exenatide"), which we engineered to reduce the internalisation process. This new agonist called "Ex-phe1" was much more effective than unmodified exendin-4 in mice with T2D, as it allowed the receptor to remain longer at the cell surface and be continually reactivated, leading to more insulin release. Also, despite being more effective in beta cells, Ex-phe1 did not cause more nausea, a well-known side effect of GLP-1R agonists.This project has two goals. The first is to understand how changes in GLP-1R trafficking modify insulin release from beta cells. The second, to be performed in parallel, is to optimise the compounds we generated in our preliminary work to produce new, better, treatments for T2D. Our approach includes experiments in beta cells and pancreatic tissue ("islets"), which allow us to understand events in molecular detail, and in mice with T2D, which tell us what happens at the whole body level. We place a lot of emphasis on using human islets (provided for research when surplus to requirements for organ donation), as we believe this is essential to understand and treat human disease.We highlight below some key experiments:1) We will use a combination of computer modelling and genetic modification of the GLP-1R to identify specific interactions between the receptor and Ex-phe1 or other biased GLP-1R agonists which underpin their different trafficking and therapeutic effects. This knowledge will be critical for the design of even more effective drugs in the future.2) We will genetically modify human islets to reduce levels of "beta-arrestin-2", a protein in beta cells that we believe is a key link between GLP-1R activation, trafficking, and beneficial insulin release. We will also breed genetically modified mice lacking beta-arrestin-2 in beta cells. We will use these systems to look at complex patterns of protein and gene activation within beta cells in response to GLP-1R agonist treatment. From this we hope to identify molecules that could in the future be targeted as new T2D treatments.3) We will study the long-term effects of our new, improved GLP-1R agonist Ex-phe1 on human islets transplanted into diabetic mice. Importantly, we will use a new approach in which the islets are actually transplanted in the eye, where changes to islet shape, behaviour and overall health can be directly and repeatedly viewed in a microscope. The mice will be treated with a long-lasting formulation of Ex-phe1 or exendin-4 for several months, and we will monitor changes to blood sugar, allowing us to compare the treatments for their effects on overall islet function. Due to well-known differences between mouse and human islets, we believe that using this "humanised" mice is critical to understanding long-term treatment effects.In summary, we have identified an exciting new approach based on GLP-1R trafficking for improving the treatment of T2D. In this project we will establish the molecular details of why this approach works, identify new potential drug targets and demonstrate the long-term effects of this new treatment in unique humanised mice.