All-optical deconstruction of the islet wiring patterns underlying insulin secretion in health and disease

Lead Research Organisation: University of Birmingham
Department Name: Clinical and Experimental Medicine

Abstract

The NHS spends 9% of its annual budget on treating diabetes and its associated complications, a figure widely projected to double by 2035. Diabetes prevalence is currently 5.1% of the UK adult population, with T2DM accounting for the majority (90%) of cases. This life-threatening disease state is typified by a failure of the insulin-secreting beta cell mass to adapt to increased peripheral resistance. The resulting metabolic dysregulation increases the incidence of a wide range of complications including cardiovascular disease, renal and liver failure, retinal degeneration, and cancer. Despite considerable research investment over the last two decades, disease prevalence is increasing. To halt or stop this trend, new avenues of investigation are urgently required to identify therapeutic targets. Recently, we have uncovered a new pathway by which glucose and other secretagogues regulate insulin secretion from the islets of Langerhans. By combining sophisticated in situ imaging approaches with circuit neuroscience methodology to map cell-cell connectivity, we and others have shown that beta cells wire themselves within islets as glucose- and incretin-responsive subnetworks which support synchronised activity. These subnetworks orchestrate a gain-of-function in insulin release that is lost when beta cells are dissociated or electrically uncoupled. A striking feature of beta cell connectivity is the existence of superconnected hubs that dominate islet dynamics by coordinating and pacemaking responses between distant islet regions. Since hub cells are rare events (dozens per islet), they are robust in the face of random insult but are prone to collapse following targeted insult, a scenario that would be catastrophic for islet function. Thus, the intraislet regulation of insulin release, and in particular hub cells, may be key components of the pancreatic infrastructure which fails during T2DM. Using optogenetics and photopharmacology to yield precise millisecond optical control over single cells, the aims of the present proposal are to: 1) interrogate the wiring patterns underlying generation of islet dynamics; 2) define the first blueprint for a functional role of hubs in insulin release; 3) understand what is special about such cells; and 4) show how the islet circuitry and hub cell function may be targeted during T2DM to impact insulin secretion. It is anticipated that these studies will lead to the characterisation of a novel route of insulin release, which may eventually allow the reversal of T2DM through restoration of the functional beta cell mass.

Technical Summary

Beta cell connectivity describes the phenomenom whereby the islet context improves insulin secretion by providing a three-dimensional platform for intercellular signaling processes. Thus, the precise flow of information through homotypically interconnected beta cells leads to the large-scale organization of hormone-releasing activities, producing a gain-of-function in hormone release. Importantly, a growing number of studies suggest that such cell-cell communication may be targeted by both environmental and genetic factors in T2DM to perturb normal beta cell function and insulin release. Using rapid Nipkow imaging combined with mathematical graph theory, we have shown that beta cells wire themselves as glucose- and incretin-responsive subnetworks, which support synchronised activity. Such subnetworks display small-world properties and their topology is dominated by a few cells that host the majority of the functional connections, linking and pacemaking distant regions of the islet. These superconnected cells, or "hubs", may provide an important route for the intra-islet regulation of insulin secretion, and may represent a novel target for T2DM insults. By melding cutting-edge optogenetic and photopharmaceutical approaches, we now aim to: 1) remote control the function of single beta cells within islets using halorhodopsin and a photoswitchable sulfonylurea; 2) optically deconstruct the islet circuitry underlying insulin secretion; 3) gain a handle on hub cell function using online analysis of islet dynamics and hormone output; and 4) determine the susceptibility of islet wiring patterns and hub cell function to T2DM insults. The outcome of these studies will be the characterisation of a novel mode of regulation of insulin secretion, which may be targeted to improve the functional beta cell mass during T2DM.

Planned Impact

Pharmaceutical industry: Sulfonylureas are still widely used for the treatment of T2DM, but are associated with an increased risk of cardiovascular disease, weight gain and persistent hypoglycaemia. Our photoswitchable sulfonylurea, JB253, could in the future provide a novel treatment for T2DM by allowing the specific targeting of drug activity to the islets, reducing side effects associated with extra-pancreatic effects. More generally, we expect the proposed research to provide new mechanistic insights into insulin release, namely by characterising hub cells. This may lead to the identification of drug targets for the normalisation of glucose homeostasis in man. Indeed, treatment regimens are based on single cell characteristics, yet increasing evidence suggests that we should be aiming to reverse "networkopathies" (i.e. defects in the functional pattern of cell activation). Since the diabetes drugs market is worth billions per year, the licensing or spin-off of any molecules/novel targets may contribute to UK economic competitiveness. G.A.R. interacts with several major Pharma companies as part of IMIDIA (EU FP7), and these contacts will be exploited where appropriate.

Life Sciences suppliers: JB253 is a useful research tool and could equally be licensed as a reagent with which to optically manipulate cell electrical activity in KATP channel-expressing tissue without the need for genetic introduction of optogenes. The timescale to institute a licensing agreement would not depend on granting of a patent, as we can control supply.

Patients living with diabetes: JB253 may be of interest to patients living with T2DM, since it would allow better control over blood glucose levels by allowing insulin secretion to be entrained to peak demand using light (i.e. food intake). This would lead to an enhanced quality of life ('healthy ageing'), whilst reducing burden on the healthcare system due to complications arising from hyperglycemia. Similarly, the characterisation of novel mechanisms governing insulin secretion from islets may eventually lead to therapies which allow better or ancillary control of blood glucose levels (e.g. by preventing hub cell failure or targeting hub cells specifically with therapy).

Researchers: Optogenetics has allowed neuroscientists to gain a better handle on the neural circuits which dictate many brain functions. Although optochemicals are less broad in their application, they possess distinct advantages as a research tool. For example, they are exogenously applied and so can be used in a range of systems, they act upon endogenous ion channels, and finally they are process-specific. Since KATP channels are expressed in the brain, vasculature, pancreas and muscle, we anticipate that JB253 may be a popular tool for a number of research fields. Furthermore, the use of optogenetics in endocrine cells is still in its infancy and we expect that characterisation of halorhodopsin-mediated cell silencing will allow this technique to be rolled out in the pancreas and other endocrine tissues (e.g. the pituitary gland).

Staff: The PDRA and technician will be recruited into a Section of Cell Biology (~ 25 researchers), headed by G.A.R., providing excellent opportunities for multi-disciplinary training. The present project proposal seeks to apply cutting-edge technologies and mathematical modelling to functionally dissect the processes underlying the intraislet regulation of insulin secretion. This would equip the PDRA with a broad and interdisciplinary skills-set, which would open up the possibility to work in a number of other academic fields, thus increasing their future career prospects. In addition, such a background is desirable for the private sector. For example, PhD candidates with strong numeracy and modelling skills are attractive not only to the pharmaceutical industry, but also to the financial sector. As a further demonstration of commitment to s

Publications

10 25 50
 
Description European Research Council Starting Grant
Amount € 1,700,000 (EUR)
Funding ID 715884 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 06/2017 
End 06/2022
 
Description Research Grant
Amount £187,000 (GBP)
Funding ID 17/0005681 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2017 
End 04/2020
 
Title A light activated agonist for optical control of GPR40 
Description A light activated agonist (GW9508), which allows the optical control of GPR40 in cells and tissue. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact The tool was used to show that the effects of lipids and free fatty acid receptors (i.e. GPR40) on insulin release stem from actions on Ca2+ and K+ channels as well as diacylglycerols. 
URL http://pubs.rsc.org/en/content/articlelanding/2017/sc/c7sc01475a#!divAbstract
 
Title A light-activated incretin for the functional dissection of GLP-1R signaling 
Description Incretin mimetics are set to become a mainstay of type 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer-biased GLP-1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact First demonstration of optical control of a class B GPCR using a peptidomimetic 
URL http://www.ncbi.nlm.nih.gov/pubmed/26585495
 
Title An azosulfonylurea for the optical control of insulin release 
Description In collaboration with Dirk Trauner (LMU, Munich), we have designed, developed, synthesised and tested a fourth generation sulfonylurea for the optical control of KATP channel activity and insulin secretion. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Our azosulfonylurea, JB253, allows the optical interrogation of beta cell activity using an exogenously applied chemical. This avoids the use of optogenes, which require introduction using viral approaches or transgenic mouse models. In addition, JB253 can be used to reversibly and repeatedly stimulate cell activity, unlike caged compounds. Lastly, using JB253, we have provided the first evidence that photopharmacology (the targeting and manipulation of drug activity using light) is applicable to the treatment of metabolic disease states such as type 2 diabetes. The technology is currently the source of an invention disclosure. We have also used this tool directly to uncover a role for beta cell hubs (https://www.ncbi.nlm.nih.gov/pubmed/27452146) 
URL http://www.ncbi.nlm.nih.gov/pubmed/25311795
 
Title CRIPSR GLP1R-/- mice 
Description We used CRISPR to introduce a single nucleotide deletion into exon 2 of the Glp1r. This resulted in a frameshift, nonsense mediated mRNA decay and complete loss of the protein. These mice are useful for pre-clinical testing of drugs targeted against the GLP1R. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact We have provided the mice to labs in the UK and Germany under a standard MTA provision. We are likely to get further requests once the model is published. 
URL https://www.nature.com/articles/s41467-020-14309-w
 
Title Cell impermeable SNAP tag labels 
Description SNAP tag labels are usually cell impermeable due to the fluorophore which they use. This precludes use of a number of super-resolution imaging-compatible fluorophores, including SiR, JF549 and JF646. We have synthesised novel SNAP tag labels which incorporate a charged sulfonate, thus rendering any fluorophore cell impermeable. This allows surface proteins to be specifically interrogated. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact The novel SNAP tag labels increase resolution of super-resolution microscopy. They also allow the specific interrogation of surface proteins for the first time using nanoscopy approaches. 
URL https://pubs.rsc.org/en/content/articlelanding/2020/sc/d0sc02794d#!divAbstract
 
Title Far red antagonist peptide labels for visualising GLP1R 
Description Far red antagonist peptide labels for visualising GLP1R. The probes are wash free and allow detection of endogenous GLP1R in vitro and in vivo. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact We have disseminated probes to multiple labs in the UK, EU and North America. The probes are being used for a number of projects related to GLP1R function, with relevance for the treatment of diabetes. 
URL https://www.nature.com/articles/s41467-020-14309-w
 
Title SNAP_GLP1R mice for visualization of GLP1R in the living system 
Description We used CRISPR to knock-in a SNAP self-labelling tag onto the N-terminus of the GLP1R. We can now label GLP1R in vivo in a mammalian species using a range of fluorophores. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2020 
Provided To Others? No  
Impact SNAP_GLP1R mice will help us understand GLP1R distribution and function, as well as test novel drugs. This will impact potentially impact how we treat diabetes, since many drugs target the GLP1R. 
 
Title Second generation potent conformationally locked positive allosteric modulators (PAMs) of the GLP-1R 
Description Novel PAMs that allow biased signallng at the GLP-1R to be explored. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The GLP-1R is a blockbuster target for type 2 diabetes therapy. Despite this, the mechanisms underlying GLP-1R are poorly understood. The abovementioned coformationally-locked PAMs will allow biased signalling to be explored. Moreover, they may provide more specific and selective therapies for type 2 diabetes. 
 
Title Tethered pharmacology compounds 
Description Conditional targeting of pharmacophores to specific cells using enzyme self-labeling technology. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact The technology is still at an early stage, but we have already received a number of requests from other labs for reagent. Moreover, we have used the technology backbone to produce a range of bright fluorescent labels that allow visualization of GPCRs in live tissue. 
URL https://www.ncbi.nlm.nih.gov/pubmed/29532016
 
Description Design, development and synthesis of azosulfonylureas 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution We tested a novel azosulfonylurea, JB253, for use in rodent and human islets as a research tool with which to optically control insulin release.
Collaborator Contribution Dirk Trauner's group (LMU, Munich) designed and synthesised JB253 and provided us with compound for testing. Johannes Broichhagen joined my lab for 6 months on an EASD Albert Reynolds Travelling Fellowship (providing only travel and living costs) to help conduct the lab work.
Impact A publication is currently in press: Broichhagen, J., Schönberger, M., Cork, S.C., Frank, J.A., Marchetti, P., Bugliani, M., Shapiro, A.M.J., Trapp, S., Rutter, G.A., Hodson, D.J.*, Trauner D* (2014). Optical control of insulin release using a photoswitchable sulfonylurea. Nature Communications. Accepted. *Corresponding authors. An invention disclosure has also been submitted to the LMU and Imperial College Patent Offices.
Start Year 2012
 
Description Design, synthesis and testing of allosteric modulators of the GLP-1R 
Organisation Max Planck Society
Department Max Planck Institute of Biophysics
Country Germany 
Sector Charity/Non Profit 
PI Contribution We helped design and test the abovementioned allosteric modulators of the GLP-1R
Collaborator Contribution Johannes Broichhagen (postdoc in the Johnsson group) performed helped design and synthesise the abovementioned allosteric modulators of the GLP-1R
Impact We are discussing the results with Pfizer, as these ligands are of potential interest as drug candidates.
Start Year 2016
 
Description Design, synthesis and testing of allosteric molecular photoswitches of the GLP-1R 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution We performed in vitro testing of the abovementioned allosteric photoswitches0
Collaborator Contribution The Trauner laboratory provided chemistry expertise.
Impact Allosteric Optical Control of a Class B G-Protein-Coupled Receptor. Broichhagen J, Johnston NR, von Ohlen Y, Meyer-Berg H, Jones BJ, Bloom SR, Rutter GA, Trauner D, Hodson DJ. Angew Chem Int Ed Engl. 2016 May 4;55(19):5865-8. doi: 10.1002/anie.201600957.
Start Year 2016
 
Description Design, synthesis and testing of light-activated DAGs for exploring cell signaling 
Organisation European Molecular Biology Laboratory
Department Cell Biology and Biophysics
Country Germany 
Sector Charity/Non Profit 
PI Contribution We performed in vitro testing of the abovementioned DAGs.
Collaborator Contribution The Schultz and Trauner labs provided chemistry expertise, as well as performed in vitro experiments.
Impact Photoswitchable diacylglycerols enable optical control of protein kinase C. Frank JA, Yushchenko DA, Hodson DJ, Lipstein N, Nagpal J, Rutter GA, Rhee JS, Gottschalk A, Brose N, Schultz C, Trauner D. Nat Chem Biol. 2016 Sep;12(9):755-62. doi: 10.1038/nchembio.2141. PMID: 27454932
Start Year 2016
 
Description Design, synthesis and testing of orthosteric peptide labels for visualizing GLP1R 
Organisation Max Planck Society
Country Germany 
Sector Charity/Non Profit 
PI Contribution We performed all the pharmacological and biological testing of next generation antagonist far red GLP1R peptide labels. To test specificity of the probes, we also generated GLP1R-/- mice using CRISPR technology.
Collaborator Contribution Kai Johnsson's and Johannes Broichhagen's group at the MPI for Medical Research designed and synthesised next generation antagonist far red peptide labels for detecting endogenous GLP1R. They also performed super-resolution microscopy (STED) using Stefan Hell's nanoscopy platform, located in Heidelberg, Germany.
Impact We have had numerous requests for probes and animals based upon conference presentations. So far, we have disseminated probes to muliple labs in UK, EU and North American. We have also distributed GLP1R-/- mice under MTA to Imperial College London and Helmholtz Munich. We are finalizing a licensing agreement with Spirochrome for supply of compound. We are also using these compounds in a number of other projects. The collaboration is highly multi-disciplinary spanning chemistry, chemical biology, structural biology, physiology and pharmacology.
Start Year 2018
 
Description Design, synthesis and testing of orthosteric peptide photoswitches of the GLP-1R 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Experimental Vascular Medicine (EVM)
Country Germany 
Sector Academic/University 
PI Contribution We performed in vitro testing of the abovementioned orthosteric peptide photoswitches of the GLP-1R.
Collaborator Contribution The Trauner and Hoffmann-Röder provided chemistry expertise, as well as staff.
Impact Optical Control of Insulin Secretion Using an Incretin Switch. Broichhagen J, Podewin T, Meyer-Berg H, von Ohlen Y, Johnston NR, Jones BJ, Bloom SR, Rutter GA, Hoffmann-Röder A, Hodson DJ, Trauner D. Angew Chem Int Ed Engl. 2015 Dec 14;54(51):15565-9. doi: 10.1002/anie.201506384.
Start Year 2015
 
Description Understanding crosstalk between androgen and GLP1 signaling 
Organisation Tulane University
Country United States 
Sector Academic/University 
PI Contribution We have performed a number of FRET assays to study the effects of androgen on GLP1R signaling in mouse islets and cell lines.
Collaborator Contribution They developed the research area and are performing molecular and in vivo studies.
Impact We have published a paper in JCI Insight and are currently preparing another manuscript.
Start Year 2019
 
Description Diabetes UK student networking day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I was invited to attend the Diabetes UK student networking day where I judged poster presentation as well as hosted a media taster workshop, where I was interviewed by a press officer. The networking day sparked discussion concerning next steps and career options for second and final year PhD students.
Year(s) Of Engagement Activity 2018
 
Description Fellowship taster 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I made a YouTube video in which I was interviewed about 'a day in the life of a research fellow'. On the back of this, I was asked to attend a media training course to prepare me for more engagement activities.
Year(s) Of Engagement Activity 2016,2017
 
Description Interview by BBC's Alex Ritson on behalf of Diabetes UK 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact I was invited to Diabetes UK headquarters to be interviewed by the BBC's Alex Ritson in front of charitable donors.
Year(s) Of Engagement Activity 2018
 
Description LIfe Sciences in Six 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact Presentation of research findings in 6 slides (6 minutes) as part of the promotional activities for the University r.e. impact of life sciences research. All the presentations were filmed and placed on YouTube.
Year(s) Of Engagement Activity 2016
URL http://www.birmingham.ac.uk/university/colleges/mds/events/2016/11/life-sciences-6/index.aspx
 
Description Life Sciences In Six 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Our previous Life Sciences in Six gala proved so successful that we were asked to do it again by the University, but this time in the Birmingham Repertory Theatre.
Year(s) Of Engagement Activity 2017
URL https://www.birmingham.ac.uk/university/colleges/mds/events/2017/11/life-sciences-six-2017/index.asp...
 
Description Met the US Ambassador the UK, James 'Woody' Johnson 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact As part of the US Ambassador's visit to Birmingham, I was asked to present our MRC-funded research on diabetes. The US Ambassador was very interested because he is a major donor to the Juvenile Diabetes Research Foundation, having family affected by type 1 diabetes,
Year(s) Of Engagement Activity 2018
 
Description Optogenetics in Endocrinology workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I was invited by the British Society for Neuroendocrinology to present a workshop on Optogenetics in Endocrinology to early career fellows. As such, a number of people have started using this technique in endocrine tissues.
Year(s) Of Engagement Activity 2016
 
Description Pint of Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 50 members of the general public attended an informal presentation at a pub in central Birmingham. This sparked many questions and useful debate regarding drug therapy.
Year(s) Of Engagement Activity 2016
URL https://pintofscience.co.uk/event/how-does-our-environment-affect-our-wellbeing-
 
Description Presentation to local Diabetes UK Support Group (Heartlands Hospital, Sutton Coldfield) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact I presented research findings to the local Diabetes UK Support Group at the Heartlands Hospital in Sutton Coldfield. The presentation was well received and as a consequence, we will now arrange a visit to the lab.
Year(s) Of Engagement Activity 2017
 
Description Presented the labs work to patients with diabetes in Coventry 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact I presented the labs work to patients with type 1 and type 2 diabetes as part of a Diabetes UK networking event.
Year(s) Of Engagement Activity 2019