Role and regulation of GLP-1 receptor trafficking in pancreatic beta cells

Type 2 diabetes and obesity are two interconnected major public health problems, due to their epidemic proportions and association with adverse consequences such as cardiovascular disease and cancer. Patients with type 2 diabetes often experience pancreatic beta cell failure leading to reduced production and secretion of insulin. Glucagon-like peptide-1 receptor (GLP-1R) agonists are a new class of drugs for the treatment of type 2 diabetes that activate the GLP-1R, which in turn stimulates the release of insulin from beta cells in the pancreas, with better control of blood glucose levels than other therapies, while at the same time increasing the total number of beta cells and inducing a reduction in appetite leading to weight loss.
GLP-1R belongs to a family of receptors known as G protein-coupled receptors (GPCRs). These receptors exist in many different types of cells, and are normally localised at the plasma membrane of cells were they can detect signals in the form of agonists from outside the cell and get activated, sending these signals inside the cell and generating a cellular response. Once activated, these receptors are relocated to different organelles in the cell in a phenomenon called trafficking, where they are sorted to either be degraded so the signal is terminated, recycled for another round of activation, or sent to specific locations where they can signal from inside the cell. Receptor trafficking has therefore the potential to control the activity of GLP-1R, but the trafficking of GLP-1R in pancreatic beta cells has not been studied in depth.
I therefore propose to study the trafficking of GLP-1R in pancreatic beta cells using state-of-the-art high-resolution microscopy techniques. I will identify the main players that regulate this trafficking by manipulating the levels of candidate proteins in the cell and analysing any effects on GLP-1R trafficking. I will also investigate the presence of trafficking defects in mouse models of the human disease. Additionally, I will measure the activation of signals in the cell by the receptor, and I will establish whether this can be modified by the manipulation of GLP-1R trafficking. I will also evaluate any differences in the response of beta cells by measuring cell survival and proliferation levels as well as the amount of insulin they secrete. Finally, I will assess the type of trafficking elicited by a number of GLP-1R agonists which are currently being developed as new diabetes therapies, and which are known to have differences in the type of signals that they generate in pancreatic beta cells.
These studies will identify fundamental new mechanisms capable of regulating GLP-1R trafficking in the beta cell, and how these affect the type of signals being generated by the receptor. They will also establish the importance of trafficking in the regulation of the known effects of the receptor on insulin secretion, survival and proliferation of beta cells, and might open new avenues for the development of improved therapy approaches for type 2 diabetes that might also be of use for the treatment of obesity.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that induces insulin secretion from pancreatic beta cells via the glucose-regulated activation of the G protein-coupled receptor (GPCR) GLP-1 receptor (GLP-1R), resulting in adenylate cyclase activation, cAMP generation and downstream signalling. As well as promoting insulin release, GLP-1R increases beta cell mass, suppresses glucagon secretion, slows gastric emptying and induces satiety. Various GLP-1R agonists have been successfully developed as therapies for type 2 diabetes.
Endocytic sorting and trafficking of GPCRs is a well-known mechanism for the modulation of receptor activities in a wide variety of cell types. GLP-1R trafficking is however a relatively understudied area whose deeper understanding may provide new ways to improve the efficacy of GLP-1R agonists.
I will use confocal, super-resolution and electron microscopy, as well as biochemical approaches to analyse the trafficking pathways followed by GLP-1R in beta cell lines and in physiologically relevant primary beta cells. The molecular machinery regulating GLP-1R traffic will be identified by si/shRNA depletion of candidate proteins, an siRNA library screen of membrane trafficking factors and analysis of GLP-1R-interacting proteins by co-immunoprecipitation and mass spectrometry, with defects on GLP-1R traffic probed in murine models of diabetes. The effect of manipulating GLP-1R trafficking on downstream signalling will be assessed with FRET-based cAMP sensors and other biochemical readouts, as well as measures of beta cell insulin secretion, survival and proliferation. I will also characterise the trafficking patterns elicited by a panel of GLP-1R agonists with known differences in GLP-1R signalling.
These studies will identify fundamental mechanisms regulating GLP-1R trafficking after exposure to GLP-1 and other agonists, and will reveal novel molecular links between agonist-induced GLP-1R trafficking, signalling and beta cell behaviour.

The proposed research is likely to benefit the general population of the UK, as well as the UK pharmaceutical industry and the UK science base.

1. The general population of the UK. Type 2 diabetes affects ~3m people in the UK (https://www.diabetes.org.uk/About_us/What-we-say/Statistics/Diabetes-prevalence-2012/). These values are predicted to grow further to epidemic proportions driven by increasingly sedentary lifestyles and obesity. The complications of diabetes include stroke, retinopathy, neuropathy, renal failure, cardiovascular disease and cancer. Treatment of diabetes is estimated to cost ~£8000 per year per patient, or £24 billion in total. Diabetic patients are 3.5 times more likely to be admitted for hospital treatment than the rest of the population (http://www.physorg.com/news151077389.html). These direct economic costs account for 7-13% of health care costs in most developed societies (IDF Diabetes Atlas, 2003).
Pancreatic beta cell dysfunction plays a central role in the pathophysiology of type 2 diabetes, and therefore strategies to improve beta cell function and survival are at the forefront of diabetes research. The GLP-1 receptor (GLP-1R) has become an important target for such therapy strategies due to its capacity to improve beta cell survival and insulin secretion while offering both a positive impact on obesity and cardiovascular protection, and as a consequence, a number of GLP-1R agonists are now regularly prescribed to diabetic patients. The proposed project is based on the analysis of the endocytic trafficking of this prominent drug target and the implications of its trafficking on the modulation of receptor activity in beta cells, and will therefore contribute to broaden our understanding of the mechanism of action this key G protein-coupled receptor (GPCR) while providing novel approaches to the treatment of diabetes.

2. The UK Pharmaceutical Industry. GPCRs represent ~50% of the current drug targets. This family of membrane receptors plays a crucial role in drug discovery today. GPCRs are prominent components of pipelines in drug companies, and many GPCR agonist drugs have been developed for indications such as cardiovascular, metabolic, neurodegenerative, psychiatric, and oncologic diseases (http://pubs.acs.org/subscribe/archive/mdd/v07/i11/html/1104feature_filmore.html).
The global market for anti-diabetes drugs is estimated to be worth ~$30 billion and new drug targets and leads are desperately needed for the pharmaceutical industry to produce novel approaches to diabetes treatment. Major companies such as Novo Nordisk, AstraZeneca and GSK have developed and are continuing to develop different formulations for GLP-1R agonists. However, there is still much to be learned about how GPCRs work and how they can be selectively regulated. Studying the role of GLP-1R trafficking in fine-tuning of GLP-1R activity is potentially of major interest for the UK pharmaceutical industry and could lead to novel ways of controlling receptor activity in diabetic patients. Pharmaceutical interest in GLP-1R traffic is evidenced by the recent publication of a study analysing GLP-1R trafficking in real-time by a Novo Nordisk research group (Mol Cell Endocrinol. 2014 Feb 15;382(2):938-49).

3. The UK science base. This project involves the application of super-resolution and electron microscopy (EM) techniques to the field of GPCR trafficking, pancreatic beta cell and diabetes research. It will therefore be of interest to a wide range of researchers from different scientific areas that will benefit from the extension of the scope of these powerful technologies. Additionally, it will allow for a research technician working in the project to be trained in the challenging techniques associated with EM, and will therefore expand the UK-based EM work pool, while allowing me to enhance my professional management skills and my training in super-resolution microscopy techniques and thus develop my academic skills.