42-OR: Hub Cells Orchestrate 3-Dimensional Pancreatic Beta-Cell Ca2+ Dynamics In Vivo

Abstract

Aim: In-vitro studies reveal a subpopulation of β cells which co-ordinate insulin release from pancreatic islets. This connectivity has not been established in 3 dimensions across the islet or in the living animal. We investigated this in the living zebrafish D. rario or in mouse islets engrafted in the anterior eye chamber (ACE), using the recombinant Ca2+ sensor GCaMP6 under the insulin promoter/C57Bl6, Ins1Cre:GCaM6ff/f. Methods: Whole islet live imaging of D. rario primary islets in situ at an acquisition rate of 0.8Hz, covering 700μm, was achieved using resonant scanner technology with a Leica inverted laser scanning confocal system, using a 25X/N.A. 0.95 water lens. Fully vascularized islets in the murine ACE were imaged under isofluorane anaesthesia on a modified Nikon Ti-E spinning disc confocal microscope (20x 0.75 NA water immersion objective, 3 Hz frame rate). Results: 3-dimensional β cell connectivity (Pearson R statistic) significantly rose to 88% across the zebrafish islet in response to glucose stimulation. Focused ablation of the co-ordinating “hub” (but not follower) β cells significantly attenuated the subsequent response to a glucose challenge. In mouse islets, high circulating glucose levels were also associated with a significant rise in 3D beta cell connectivity from 65 to 86% (n=3; p=0.02). Granger causality analysis on these islets revealed that even on prolonged (>10 minute) glucose stimulation, pan-islet connectivity was sustained and the most highly connected cells were those in the region of the islet from which Ca2+ waves emanated. Binarized signal analysis (n=6) separately confirmed the presence of a highly-connected β cell subpopulation (8.7 ± 3.6% of cells). Conclusions: These studies evidence beta cell “hubs” which are responsible for the initiation and sustained coordination of the Ca2+ response to glucose across fish and mouse islets in vivo. Disclosure V. Salem: None. L.F. Delgadillo Silva: None. K. Suba: None. A. Martin Alonso: None. W. Kim: None. V. Kalogianni: None. N. Akhtar: None. N. Mousavy Gharavy: None. E. Georgiadou: None. I. Leclerc: None. L. Briant: None. D. Hodson: None. W. Distaso: None. N.N. Ninov: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding Diabetes UK; UK Wellcome Trust