1747-P: Islet Ca2+ Dynamics, Heterogeneity, and Consistency in Three Dimensions with Activators of Pyruvate Kinase

Abstract

Pancreatic β-cell heterogeneity is important for Ca2+ triggering and insulin secretion and is affected in diabetes. Organization and consistency of β-cell subpopulations are poorly understood despite being key in understanding how individual cells contribute to islet function and diabetes. Existing studies analyze single 2D planes of the islet. However, subpopulation organization cannot be fully understood without examining β cell dynamics throughout the entire islet in 3D. Here, we imaged islets from β cell specific GCaMP6s Ca2+ reporter mice using high-speed light-sheet microscopy to analyze the 3D organization and consistency in high glucose and with pharmacologic activators of pyruvate kinase (PK), a glycolytic enzyme that controls βcell excitability. Phase analysis revealed that cells which initiated islet depolarization (leaders) are not highly consistent across oscillations, but the 3D region where the calcium wave originates is consistent, according to a metric based upon the KL-divergence (P<0.0001 over 11 oscillations). Using functional network analysis, β-cell hubs which drive islet synchrony are more consistent over an islet region than across individual cells (P<0.0001). The wave origin was more regionally dependent than the β-cell hubs (P<0.0001). We also found that network analysis is similar (P=0.04) and phase analysis is different between 2D and 3D (P<0.0001). Following quantification of heterogeneity in the 3D islet, certain sub-populations of β cells are similar throughout the islet, while others are not. Acute PK activation decreased the consistency of wave initiators (P<0.0001), but not the consistency of hub cells in the functional network (P=0.15), shedding new light onto the mechanism behind these subpopulations. Islet regions, rather than individual cells, are more consistent and thus likely responsible for initiating Ca2+ triggering and insulin secretion. These mechanisms help render the islet robust to single β cell failures. Disclosure J.Briggs: None. E.Jin: None. M.J.Merrins: None. R.K.Benninger: None. Funding William S. Middleton Memorial Veterans Hospital; U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01B005113); National Institutes of Health (R01DK113103, R01DK127637, R01DK102950, R01DK106412); National Science