Sensing And Signalling Of Extracellular Ca2+ In The Islet Of Langerhans

该项目旨在开发一种新的传感平台来解决一个长期存在的难题：细胞外Ca2+如何协调胰岛β细胞进行搏动胰岛素分泌。后者是维持血糖稳态的关键。自由离子Ca2+现在被认为是在几乎所有哺乳动物细胞生命的几乎每个方面介导细胞活动不可或缺的信使。尽管细胞内Ca2+在不同生物功能中的作用已经得到了很好的表征，但由于缺乏适当的传感方法，无法区分高基础水平Ca2+的细微变化（例如，毫摩尔级基线的微摩尔尺度差异），对细胞外Ca2+的认识受到了限制。对于一种主流的传感器-荧光指示器，它们特别受到背景自身荧光的限制（由于使用较短的波长激发，即使在没有感兴趣的离子的情况下，指示器本身也会发出较长波长的荧光）。为了克服这一限制，拟议的研究将引入最先进的上转换发光，其特点是能够将980 nm红外光激发转化为可见发射，并与传统的荧光指示器相结合。这种混合传感器将代表世界上第一个能够定量表征细胞外Ca2+的传感器。上转换协同混合传感器将应用于解决细胞外Ca2+如何参与协调胰岛β细胞的脉动胰岛素分泌-一个与葡萄糖代谢耦合的关键代谢过程。这种传感器的出现，与定制的微流体装置相结合，将提供一个机会来量化细胞外Ca2+和脉冲胰岛素分泌之间的联系，从小鼠分离的胰岛在不同的代谢状态（健康，前驱糖尿病和2型糖尿病）。这些分析将导致基础知识的获得，可以提高目前对2型糖尿病病理生理学的理解，并可能为这一主要慢性疾病的管理提出新的治疗策略，从而使2型糖尿病患者受益（到2021年，英国有490万人，全球有5.366亿人）。

This project aims to develop a new sensing platform to resolve a long-standing puzzle: how extracellular Ca2+ coordinates pancreatic islet beta-cells for pulsatile insulin secretion. The latter is key to maintain the blood glucose homeostasis.Free ionic Ca2+ is now recognised as an indispensable messenger in mediating cellular activities in nearly every aspect of cellular life for almost all mammalian cells. Although the roles of intracellular Ca2+ in relation to different biological functions have been well characterised, insights into extracellular Ca2+ are constrained by the lack of appropriate sensing methodology capable of distinguishing the subtle changes of Ca2+ over the high basal level (e.g., micromolar scale variances in millimolar order baseline). For one sensor mainstream - fluorescent indicators, they are particularly limited by the background autofluorescence (due to the use of a shorter wavelength excitation, indicators themselves fluoresce with longer wavelength emissions even in the absence of ions of interest). To overcome this limit, the proposed research will introduce state-of-the-art upconversion luminescence, which features the capacity of converting 980 nm infrared light excitation into visible emissions, to combine with conventional fluorescent indicators. Such a hybrid sensor will represent the world-first one that enables characterising extracellular Ca2+ quantitatively.The upconversion-synergised hybrid sensor will then be applied to address how extracellular Ca2+ participates in coordinating pulsatile insulin secretion from pancreatic islet beta-cells - a key metabolic process coupled to glucose metabolism. The advent of such a sensor, in conjunction with a bespoke microfluidic device, will provide an opportunity to quantify the link between extracellular Ca2+ and pulsatile insulin secretion in isolated islets from mice at different metabolic states (health, prediabetes and type 2 diabetes). These analyses will lead to fundamental knowledge gains which can improve the current understanding on the pathophysiology of type 2 diabetes and may suggest new therapeutic strategies for the management of this major chronic disorder, thereby benefiting people living with type 2 diabetes (in 2021, 4.9 million in the UK and 536.6 million worldwide).