Molecular analysis of the protein machinery underlying insulin secretion

Our increasingly sedentary lives, combined with the widespread availability of high energy foods, has resulted in a dramatic increase in obesity-linked diseases. Under normal conditions we consume food irregularly, throughout the day, and our body compensates by storing energy, in the form of the sugar glucose, when it is plentiful and releasing this energy, when required, between meals. The storage of glucose is controlled by insulin, which is released from the pancreas to stimulate excess glucose uptake from the blood and storage, primarily in the liver and muscles. However, under conditions of obesity, this system is put under pressure to store the extremely high levels of ingested energy. Initially the body adapts and more insulin is released from the pancreas, stimulating the storage of more glucose. However, eventually this compensation mechanism begins to fail and the body is no longer able to control the levels of glucose in the blood. Through undefined mechanisms, these high blood glucose concentrations are toxic to the pancreatic cells responsible for insulin release, decreasing insulin secretion even further, compounding the problem, and resulting in the development of Type 2 diabetes.

The highly specialised cells in the pancreas that release insulin are called beta-cells. This process of insulin secretion is highly regulated, utilising proteins as molecular machines to release insulin only when required. While we know a great deal about the proteins involved, to date we have been unable to observe them directly in beta-cells. Recently a new type of technology has been developed that allows for tens of thousands of individual proteins to be observed. This allows for both their precise location and movement to be analysed with an accuracy of one-thousandth the thickness of a human hair. Using these new approaches it is now possible to answer the key questions of how insulin release is regulated and how this changes during Type 2 diabetes.

In this research project, we aim to understand the organisation of the secretory protein machinery under normal physiological conditions and how this changes under the conditions experienced in Type 2 diabetes. To achieve this we will use cutting edge molecular microscopy techniques to examine the spatial arrangements of the proteins, their movements and their interactions with other proteins. Once we understand the normal physiological organisation of the secretory protein machinery we will examine how this is altered under Type 2 diabetic conditions. This study will provide answers to three key questions. First, how is the secretory protein machinery organised. Second, how is this organisation maintained, Third, how is this changed under conditions where high glucose concentrations begin to decrease insulin secretion in Type 2 diabetes. This research will greatly improve our understanding of how the protein machinery is organised to coordinate insulin release. Ultimately this could lead to new methods of diagnosis, prevention of transition from compensation to loss of beta-cell function, and therapeutic treatments for Type 2 diabetes.

Regulated exocytosis is a highly orchestrated cascade of protein-protein interactions culminating in the fusion of cargo containing vesicles with the plasma membrane. In beta-cells the primary cargo is insulin, which is released as part of glucose homeostasis. In common with other highly specialised secretory cells the secretory apparatus is composed of the SNARE proteins present on the plasma membrane (tSNARE) and the cargo-containing vesicle (vSNARE). It is clear that the SNARE proteins act in a highly coordinated manner and that disruption of these proteins results in a marked decrease in insulin release. However, how these proteins are organised spatially, how they move on the plasma membrane and where they interact relative to secretory vesicles in beta cells remains unknown. Recently it was reported that the SNARE proteins in beta-cells can be directly affected by the high extracellular glucose concentrations encountered during Type 2 diabetes. This project will examine at the molecular level the organisation and interactions of the plasma membrane tSNAREs and how this is altered under chronic elevated glucose concentrations. Specifically we will utilise advanced super-resolution single molecule approaches to observe the spatial organisation, molecular dynamics and interactions of large cohorts of tSNAREs in intact beta-cells. We will examine the underlying molecular basis for the observed organisation and analyse how different tSNARE isoforms, known to be involved in different phases of insulin secretion are organised. Finally we will investigate how this organisation is altered under chronic elevated extracellular glucose concentrations. Our extensive preliminary data, demonstrating all of the required techniques are in place in the laboratory, shows the feasibility of the project. Ultimately this could lead to new methods of diagnosis and therapeutic treatments for Type 2 diabetes.

This research will benefit:

1. The academic researchers directly involved in the project by developing their research, professional and transferable skills. This is a varied and ambitious project and the PDRA will be encouraged to develop public engagement, communication and other skills benefitting from the wide range of courses and opportunities in place at Heriot-Watt University.

2. Academic researchers worldwide, including those interested in regulated exocytosis, membrane fusion, beta-cell function, insulin release and glucose homeostasis, membrane protein organisation, super-resolution microscopy and the wider signal processing community. They will gain from new data, scientific knowledge, methodologies and ideas produced from this research. Our findings will be communicated to the scientific community by means of publication in peer-reviewed journals and presentation at national and international meetings. In addition we will generate and make available new tools and reagents to assist in other research projects. Finally we will make available all raw image data, post-publication on an open access or collaborative basis.

3. Exploitation of discoveries and interaction with commercial private companies. While this proposed project is concerned with the elucidation of the fundamental principles governing the organisation of the secretory machinery and how this is altered during Type 2 diabetic conditions, this project may lead to development of diagnosis or therapeutic applications in the longer term. This could involve detection of changes to protein distributions detected using GSDIM (see section 3.3.2 of the 'Case for Support' for details of abbreviations) in patient biopsies or therapeutic treatment to correct any abnormal protein organisation. The Heriot-Watt Research and Enterprise Service is on hand to advise on knowledge transfer and exploitation, including advising on IP issues, assisting with patent and licensing and connecting with industry. They have a proven track record of translating academic research into successful start-ups or licensing to commercial entities. They will be fully briefed on progress made to this project on an annual basis.

4. The general public. I will increase the public's awareness and understanding of science in my area of research. I will actively participate in public engagement activities and publicise existing findings and outcomes of our research beyond academic circles. I will continue to engage the local public and schoolchildren through public outreach activities including the twice-yearly Royal Society of Edinburgh Science Master Class. In the longer term, I aim to generate improvements in economic, health and welfare in the UK by contributing research data, understanding of regulated insulin secretion and how this is altered during Type 2 diabetes. To enable this I would interact with companies and other academic groups during or after this project as detailed above.