Application of pharmacological, gene silencing and cell biological techniques to explore the effects of the gut hormone GLP-1 on glucagon release
Glucagon is also known as a counter regulatory hormone which opposes the action of insulin by promoting glucose production in the liver, thereby increasing blood glucose concentrations and preventing hypoglycaemia1. Thus, glucose homeostasis is tightly regulated by the joint actions of both insulin and glucagon, secreted from the pancreatic beta- and alpha-cells respectively. Despite the importance of glucagon, very little is known about the precise regulatory machinery that controls its secretion from alpha-cells2. It has been established that, in addition to glucose, a number of both endocrine and paracrine hormones influence glucagon secretion.
For example, insulin, somatostatin and γ-aminobutyric (GABA) all decrease glucagon exocytosis while the gut hormone, gastric inhibitory polypeptide (GIP), increases its release3 . However, the actions of the mature form of GLP-1, released from the intestinal L-cells in response to food intake, are far from clear, since its ability to inhibit glucagon release in vivo contrasts with its effects in single alpha-cells. Likewise, our preliminary data indicate that GLP-1 (9-36) amide, the inactivated form of GLP-1 generated following enzymatic degradation, can also reduce secretion of glucagon, with high potency, in a cAMP-independent manner. In contrast, exenatide, a synthetic and stable analogue of GLP-1 (7-36) amide has been separately documented to both increase and decrease glucagon secretion from mouse alpha-cells4 . Interestingly, expression levels of both the glucagon receptor (GCGR) and the GLP-1 receptor (GLP-1R) found within alpha-cells are between 30-100 fold lower than those detected in pancreatic beta-cells5. Recent work from our laboratory has shown that GLP-1 (7-36) amide as well as GLP-1 (9-36) can strongly supress glucagon secretion from pancreatic islets isolated from GLP-1 receptor - / - 'knockout' mice; This implies that GLP-1 utilizes a novel mechanism for modulating glucagon secretion. Given these seemingly conflicting reports, and a lack of a clear definitive mechanism, the aim of this project is to determine how GLP-1 regulates the secretion of glucagon in a GLP-1R-independent manner. The complex nature of islets means that many of the components for modulating glucagon secretion are also found in beta-cells and delta-cells. Therefore simpler model systems to enable pharmacological characterization of candidate receptors for GLP-1 binding are required. Our initial receptors of choice are the GCGR and the GIPR since recent reports suggest that both exhibit a preference for binding GLP-1 (7-36) amide over exenatide. Further preliminary data suggests that administration of a GCGR antagonist blocks GLP-1 (9-36) amide-mediated glucagon secretion from human islets. GPCR pharmacology will be studied as part of an ongoing collaboration with Dr Graham Ladds at the University of Cambridge and islet physiological evaluation will be undertaken at the Oxford Centre for Diabetes, Endocrinology and Metabolism.
The specific objectives of the proposed work are as follows:
- Pharmacological characterization of the GCGR and GIPR as targets for GLP-1 activity;
- Evaluation of other G-protein coupled receptors (GPCRs) in modulating glucagon secretion;
- Analysis of the GPCR-based signaling components found in alpha-cells – including GPCRs, G-proteins and receptor activity modifying proteins (RAMPs).
- Evaluation of the role of somatostatin in modulating glucagon secretion. This work is essential since somatostatin receptors are predominately Gαi-coupled in alpha-cells.
- Assessment of receptor physiology in islets by genetic knockdown of GCGR, GIPR and GLP-1R to pharmacologically and physiologically characterise their responses to both GLP-1 (7-36) amide and GLP-1 (9-36) amide.
These experiments will establish the mechanisms by which both GLP-1 (7-36) amide and GLP-1 (9-36) amide modulate glucagon secretion. In the long-term, the insights gained here will also facilitate the design of more efficacious drugs aimed at regulating glycaemia.
This is a collaborative project between two internationally renowned dynamic groups with diverse expertise – pharmacology and cell signalling and cell biology and physiology, linked by GPCR signalling. Both groups comprise world-renowned enthusiastic scientists working in well-equipped laboratories. Also, each group has extensive collaborative networks. Therefore the project offers a unique opportunity to learn and evelop an extensive repertoire of multi-disciplinary techniques to tackle important questions on the role of GLP-1 and GPCRs on the regulation of glucagon secretion. Various techniques will be employed including islet isolation, tissue culture, radioimmunoassay, RNA interference and gene silencing and microscopy techniques- EM and confocal, western blotting, immunoprecipitation and quantitative PCR. There will also be the opportunity to use more complex methodologies including ligand binding, β-arrestin recruitment and internalization and signaling bias assays.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
|1||Unger RH. 1971. Glucagon physiology and pathophysiology.N. Engl. J. Med., 285 (8), pp. 443-9. - http://www.ncbi.nlm.nih.gov/pubmed/4997492|
|2||Ramracheya R, Ward C, Amisten S, Shigeto M, Walker JN, Zhang Q, Johnson PR, Rorsman P, Braun M. Membrane potential-dependent inactivation of voltage-gated ion channels in α-cells inhibits glucagon secretion from human islet. Diabetes 59: 2198-208, 2010. https://www.ncbi.nlm.nih.gov/pubmed/20547976|
|3||Degn KB, Brock B, Juhl CB, Djurhuus CB, Grubert J, Kim D, Han J, Taylor K, Fineman M, Schmitz O. 2004. Effect of intravenous infusion of exenatide (synthetic exendin-4) on glucose-dependent insulin secretion and counterregulation during hypoglycemia.Diabetes, 53 (9), pp. 2397-403. - http://www.ncbi.nlm.nih.gov/pubmed/15331551|
|4||Ma X, Zhang Y, Gromada J, Sewing S, Berggren PO, Buschard K, Salehi A, Vikman J, Rorsman P, Eliasson L. 2005. Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors.Mol. Endocrinol., 19 (1), pp. 198-212. - http://www.ncbi.nlm.nih.gov/pubmed/15459251|
|5||Tornehave D, Kristensen P, Rømer J, Knudsen LB, Heller RS. Expression of the GLP-1 receptor in mouse, rat, and human pancreas. J Histochem Cytochem. 2008 Sep;56(9):841-51. https://www.ncbi.nlm.nih.gov/pubmed/18541709|