Background and Aims: β cell connectivity is a feature of pancreatic islets in vitro but its existence in vivo, when innervated and continuously perfused with blood, has not yet been demonstrated. We imaged islets engrafted in the anterior chamber of the mouse eye (ACE) to explore this question.
Methods: Mouse (C57BL6, Ins1Cre::GCaM6mf/f) or human islets infected with adenovirus to express GCaMP6m, were engrafted and Ca2+ imaging performed under anaesthesia. Glucose or insulin were administered intravenously to achieve low glucose (46 mM) or high glucose (2530 mM) conditions Data were collected on a spinning disc confocal microscope using a 20×, 1.0 NA water immersion objective (3 Hz). Following movement correction, Ca2+ traces were analyzed with Image J. Connectivity analysis was performed with custom-built scripts in Matlab.
Results: Ca2+ waves spreading across the islet in 5/5 animals were observed. Even at low glucose concentrations, β cells form a highly connected syncytium. Increasing glucose concentrations augmented the proportion of connected β cells from 65 to 86% (n=5; P=0.02) and correlation strength (Pearson R with bootstrapping) from 0.34±0.07 to 0.46±0.08 (n=5; P=0.05). Granger causality analysis indicated that cells which responded first during Ca2+ pulses were causally linked to the activity of the largest number of other β cells in the islet. Moreover, the presence of a super-connected β cell subpopulation (8.7±3.6% of cells) was revealed by signal binarisation and Monte Carlo randomization. Pearson connectivity was increased from 58.3% to 63.9% (n=1 animal) in engrafted human islets
Conclusions: We demonstrate intercellular connectivity between β cells within the islet in vivo under conditions of normal islet perfusion and innervation. These findings are consistent with the existence of islet pacemaker cells which coordinate Ca2+ dynamics and possibly pulsatile insulin secretion in the physiological setting.