Optical interrogation of glucose-regulated beta cell connectivity
N.R. Johnston1, R.K. Mitchell1, J. Ferrer1, B. Thorens2, G.A. Rutter1, D.J. Hodson1; 1Department of Medicine, Imperial College London, UK, 2Center for Integrative Genomics, University of Lausanne, Switzerland.
Background and aims:
The multicellular organization of beta cell dynamics produces a gain-of-function in insulin release through the generation of rhythmic and synchronous activity patterns. Here, we describe an optical silencing strategy to allow the precise and specific interrogation of the functional wiring patterns which orchestrate and pace beta cell interactivity.
Materials and methods: Functional multicellular calcium (Ca2+) imaging (fMCI) was used in combination with online Monte Carlo-based correlation analyses to construct the wiring patterns underlying cell-cell connectivity and hormone release. Rapid Nipkow disk microscopy allowed video rate recording at speeds in excess of 5 Hz. Beta cell-specific expression of the light-activated inward chloride (Cl-) channel, halorhopsin, was directed by crossing the Ins1Cre deletor strain with animals engineered to express eNpHR3.0-EYFP following excision of a loxP-flanked STOP cassette. To allow user-directed single cell silencing within the field of view, a diffraction-limited 585nm laser was coupled via a fibre optic to a custom-manufactured dichroic array.
Results: Beta cells form a scale-free network which supports the synchronous propagation of glucose-stimulated Ca2+ waves by efficiently connecting distant regions of the intact islet (n = 12 recordings from 5 animals) (power law fit; R² = 0.7247). A typical feature of such a topology was the non-random appearance of superconnected hub cells whose firing activity repetitively preceded that of the remainder of the population. Online mapping of islet functional architecture using MATLAB routines coupled directly to the imaging setup revealed the presence of a statistically-stable hub cell population. Silencing of individual identified hubs using a pinpointing laser had catastrophic consequences for coordinated islet responses to glucose, and this could be reversed simply by ceasing illumination (11.5 ± 1.8 versus 3.1 ± 0.8 % correlated cell pairs, laser OFF versus laser ON, respectively; P<0.01) (n = 8 recordings from 4 animals). By contrast, specific silencing of non-hub or follower cells was unable to significantly perturb islet dynamics (7.3 ± 1.8 versus 9.9 ± 2.0 % correlated cell pairs, laser OFF versus laser ON, respectively; P>0.05) (n = 7 recordings from 4 animals). Further supporting a role for distinct wiring patterns in glucose-regulated islet connectivity, low-grade cytokine (IL-1β and IL-6) treatment resulted in a dramatic and rapid collapse in correlated cell-cell activity due to impaired hub function (9.6 ± 0.9 versus 5.1 ± 0.6 % correlated cell pairs, 0 versus 4 hrs cytokine exposure, respectively; P< 0.01) (n = 7 recordings from 3 animals).
Conclusion: The intra-islet circuitry is dominated by superconnected hub cells which dictate population responses to glucose. Notably, these hubs are vulnerable to pro-inflammatory T2D insults and may contribute to the reduced functional beta cell mass that accompanies glucose intolerance.
Supported by: Diabetes UK, Wellcome Trust, MRC and the Royal Society.