Abstract
Aims/hypothesisWe 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.MethodsHuman 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.ResultsAssessment 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/interpretationStructural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.Graphical abstract
Highlights
Understanding the functions of human pancreatic islets underpins future approaches to treating diabetes [1]
Localisation of the apical polarity determinant, partitioning defective 3 homologue (Par3), shows significant enrichment in regions away from the vasculature (Fig. 2g–i, comparing vascular with avascular fluorescence intensity, Par3 was enriched p < 0.001, n = 45 cells). Immunostaining of these polarity determinants in isolated human beta cells shows that these proteins are present in beta cells (ESM Fig. 2). These results demonstrate that human beta cells are polarised and this polarisation was consistent for all beta cells in the islet
Our work reveals that individual beta cells in human islets are polarised, as shown by classical polarity determinants, with a basal pole at the capillary interface and apical pole opposite
Summary
Understanding the functions of human pancreatic islets underpins future approaches to treating diabetes [1]. In terms of islet architecture, some studies indicate that human islets have an alpha cell mantle and beta cell core, similar to rodent islets [5], with a multi-lobular morphology in larger islets [3, 6]. Other reports suggest either a random arrangement of different endocrine cells [7] or a laminar, folded structure [4]. Another important aspect of cell organisation is the endocrine cell contacts with the islet capillary bed [8, 9]. In human islets, one study suggests a subpopulation of beta cells may not touch the capillaries [14], raising the possibility that some beta cells behave differently, consistent with current ideas that beta cell heterogeneity is important to islet function [15, 16]
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