Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes.

AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control 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.