1723-P: Dual-Label Kinetic Assay to Monitor ß-Cell Death and Proliferation

Abstract

A deficit of β-cell mass is common to all types of diabetes. Thus, many research efforts are dedicated to developing therapies to preserve and expand β-cell mass. Current methods assessing β-cell proliferation and cell death are limited due to reliance on a single time point, and usually cannot measure proliferation and death simultaneously. Here we have developed and optimized a novel method, based on the single-cell and population-level analyses using real-time kinetic labeling (SPARKL), to study kinetics of cell proliferation and death in β cells. SPARKL uses two fluorescent dyes - SYTO21 labels all cells in green, and YOYO3, a red cell-impermeable viability dye that stains dead cells. We continuously imaged cells every few hours over numerous days and have optimized growth conditions and dye concentrations to monitor INS1 or primary rodent or human islet cells on single cell and population levels. As a first step, we compared growth under 6, 11 and 20 mM glucose and glucolipotoxicity (20 mM glucose and 1 mM palmitate). We found a doubling time of 52±2, 48±4, 44±3 and >120 h, respectively, in INS1 cells. At 48 h, fewer than 16% of the cells are dead in varying glucose concentrations compared to 69±5% dead cells in the glucolipotoxic condition (N=8). Harmine, a known mitogen of β-cells, enhanced proliferation in all glucose concentrations by 5-15% and protected from glucolipotoxic cell death (N=3). In primary islets, as expected, the proliferation rates were extremely low and often masked by cell death in varying glucose concentrations. Glucolipotoxicity led to >80% cell death in mouse and human islets (N=3). Finally, we screened a library of 73 epigenetic modifiers to screen for compounds that rescued INS1 cells from glucolipotoxic-like stress (overexpression of ChREBPβ). Our screen yielded four epigenetic modifiers that reduced cell death to less than 2% at 48 h. In conclusion, SPARKL is a novel and useful method that is accurate, unbiased, and requires minimal handling to simultaneously measure β-cell proliferation and death. Disclosure L.S.Katz: None. P.Wang: None. H.U.Kaniskan: None. J.D.Gelles: None. J.Chipuk: None. A.F.Stewart: None. D.Scott: None. Funding National Institutes of Health (R01DK130300)