Diabetes, a disease affecting >300 million people worldwide, is characterized by uncontrolled elevated blood glucose levels. A failure of pancreatic islet β-cells to secrete sufficient insulin for regulating blood glucose homeostasis is the common cause of all diabetes. Clinical treatments to treat and cure diabetes broadly aim to restore or replace the dysfunctional pancreatic islets to provide a well regulated source of insulin secretion. Examples include pharmacological treatments for elevating insulin secretion, artificial pancreas (glucose monitoring/insulin pump), islet transplantation or β-cell regeneration. Our lab's research is involved with understanding how the pancreatic islet functions and its disruption during the development of diabetes, as well as improving clinical diagnostics and treatments for both type 1 and type 2 diabetes.
Multi-cellular interactions and dynamics of islet function:
We know well how insulin secretion is regulated from individual β-cells, but we do not have a good understanding of how the β-cell functions in the context of the islet: β-cells in the islet secrete insulin ~10 times more effectively than equivalent isolated β-cells. We are focusing on understanding precisely how communication between cells within the islet enhances the regulation of insulin secretion. We are also investigating how the disruption of these mechanisms early in the development of type 1 and type 2 diabetes contributes to islet dysfunction. This research will assist in the development of pharmacological treatments to restore normal insulin secretion and will also assist in improving the survival and function of cell-replacement therapies (see below).
eft: Pancreatic islet where a defined percentage of constituent cells express a KATP channel mutation which alteres excitability (GFP+, green). Right: In the presence of this perturbation, we measure how cell-cell communication modulates the overall islet response to glucose. In this case glucose-stimulated calcium elevation is suppressed throughout the islet via gap junction channels; thereby supressing insulin release and causing diabetes.
In studying how different cellular populations within the islet interact, we follow an approach whereby precise perturbations in signalling activity are introduced into a well-defined population of cells in the islet, utilizing transgenic mouse models, microfluidics and optogenetics. Quantitative confocal and two-photon microscopy, together with biochemical and physiological approaches, are applied to measure the effect of these perturbations across the islet and how they manifest in the overall islet dynamical response. Predictive mathematical models are then used to describe these results.
Left: Phase map showing propagation of calcium waves across the islet, from areas of red to areas of blue. Middle: This can be reproduced in a multicellular islet model. Right: Representing the islet as a coupled resistor network also allows connectivity analysis to predict properties of calcium wave propagation and oscillation synchronization.
Optimizing pancreatic islet transplant function:
Transplantation of human islets obtained from cadaveric donors can cure type-1 diabetes. Currently, transplant success is temporary, with a duration of insulin independence varying from ~weeks to ~years (average ~6 months). Harvested human islet tissue widely varies in quality and there is a reduction in transplant function and survival over time. We are building upon our understanding of islet function to asses and improve the function of islet transplant tissue. We are developing methods to make faster and more sensitive functional assessments of harvested human islet tissue, based on quantitative fluorescence. Furthermore we are integrating this with microfluidic technology to allow high-throughput islet screening and sorting, to enable more efficient and better functioning transplant tissue. Similarly, we are also developing engineered 'pseudo-islets', utilizing our knowledge of islet cell-cell communication. These islets contain additional cells that release protective and stimulatory factors to increase insulin secretion and promote β-cell survival. Better functioning islet tissue will prolong transplant success and insulin independence.
Left: Human islet with highlighted coordination domains. Right: The dynamics of Ca2+ activity can be measured to assess whether the islet batch is functioning normally and be suitable for transplantation. Measurements are similarly performed for other signalling mechanisms underlying insulin secretion.
Quantitative fluorescence microscopy development:
We are also developing and applying new quantitative fluorescence microscopy and spectroscopy approaches for biological applications. By using optical-switchable fluorophores and fluorescent proteins, we can modulate the fluorescence with a defined phase and frequency and apply lock-in based detection to eliminate background signal. This can be combined with Förster Resonance Energy Transfer for measuring bio-molecular interactions which can enable the resolution of interacting and non-interacting populations with unprecedented sensitivity (termed photo-chromic FRET, or pcFRET). We are applying these techniques to study membrane-bound protein-protein interactions and organization in pancreatic β-cells which underlie the regulation of insulin secretion.
Left: Time course of fluorescence from a reversibly switchable fluorescent protein rsTagFFP (red) upon repeated switching between a fluorescent and non-fluorescent state with a defined phase and frequency. A SYFP in close proximity undergoes FRET with the rsTagFFP leading to a modulation in YFP fluorescence (yellow), the relative strength of which is a measure of the strength of the interaction. Right: This modulation strength can be spatially mapped to reveal areas where an interaction occurs (membrane bound, high FRET efficiency) and areas where no interaction occurs (cytosolic, low FRET efficiency).