Glucose-Dependent Insulin Secretion from ß Cell Spheroids Is Enhanced by Embedding into Softer Alginate Hydrogels Functionalised with RGD Peptide.

Type 1 diabetes results from the loss of pancreatic ß cells, reduced insulin secretion and dysregulated blood glucose levels. Replacement of these lost ß cells with stem cell-derived ß cells, and protecting these cells within macro-device implants is a promising approach to restore glucose homeostasis. However, to achieve this goal of restoration of glucose balance requires work to optimise ß cell function within implants. We know that native ß cell function is enhanced by cell-cell and cell-extracellular matrix interactions within the islets of Langerhans. Reproducing these interactions in 2D, such as culture on matrix proteins, does enhance insulin secretion. However, the impact of matrix proteins on the 3D organoids that would be in implants has not been widely studied. Here, we use native ß cells that are dispersed from islets and reaggregated into small spheroids. We show these ß cell spheroids have enhanced glucose-dependent insulin secretion when embedded into softer alginate hydrogels conjugated with RGD peptide (a common motif in extracellular matrix proteins). Embedding into alginate-RGD causes activation of integrin responses and repositioning of liprin, a protein that controls insulin secretion. We conclude that insulin secretion from ß cell spheroids can be enhanced through manipulation of the surrounding environment.

Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion.

A developing understanding suggests that spatial compartmentalisation in pancreatic ß cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact ß-cell structure, and enhances glucose-dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating ß cells. Integrins and FAK are exclusively activated at the ß-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where ß-cell contact the islet capillaries.

Presynaptic-like mechanisms and the control of insulin secretion in pancreatic ß-cells.

Exocytotic release of hormones from endocrine cells must encompass mechanisms that direct the hormone into the blood stream. Increasing evidence indicates an intimate link between pancreatic ß-cells and the capillary bed of islets of Langerhans in both mouse and human. Integrins are exclusively activated at the region where ß-cells contact extracellular matrix proteins that surround the islet capillaries; furthermore, insulin granule exocytosis is targeted to this same region, therefore delivering hormone directly into the blood stream. In this review we discuss evidence suggesting that the capillary interface of ß-cells forms a specialised domain that is analogous to the presynaptic active zone of neurones. Pancreatic ß-cells possess many of the same proteins as found in the neuronal active zone, including several key presynaptic scaffold proteins. These scaffold proteins are enriched at the capillary interface of ß-cells and some have also been shown to control insulin secretion. We present a model that suggests this active zone-like domain in ß-cells may anchor key components of the stimulus secretion cascade, to not only target granule exocytosis to this region but also function as a significant regulator of insulin secretion.

Machine Learning Algorithms, Applied to Intact Islets of Langerhans, Demonstrate Significantly Enhanced Insulin Staining at the Capillary Interface of Human Pancreatic ß Cells.

Pancreatic ß cells secrete the hormone insulin into the bloodstream and are critical in the control of blood glucose concentrations. ß cells are clustered in the micro-organs of the islets of Langerhans, which have a rich capillary network. Recent work has highlighted the intimate spatial connections between ß cells and these capillaries, which lead to the targeting of insulin secretion to the region where the ß cells contact the capillary basement membrane. In addition, ß cells orientate with respect to the capillary contact point and many proteins are differentially distributed at the capillary interface compared with the rest of the cell. Here, we set out to develop an automated image analysis approach to identify individual ß cells within intact islets and to determine if the distribution of insulin across the cells was polarised. Our results show that a U-Net machine learning algorithm correctly identified ß cells and their orientation with respect to the capillaries. Using this information, we then quantified insulin distribution across the ß cells to show enrichment at the capillary interface. We conclude that machine learning is a useful analytical tool to interrogate large image datasets and analyse sub-cellular organisation.