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

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.

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.

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