Multi-parameter single-cell kinetic analysis reveals multiple modes of cell death in primary pancreatic β-cells

Programmed β-cell death plays an important role in both type 1 and type 2 diabetes. Most of what is known about the mechanisms of β-cell death comes from single time-point, single parameter measurements of bulk populations of mixed cells. Such approaches are inadequate for determining the true extent of the heterogeneity in death mechanisms. Here, we characterized the timing and order of molecular events associated with cell death in single β-cells under multiple diabetic stress conditions, including hyperglycemia, cytokine exposure, nutrient deprivation and endoplasmic reticulum (ER) stress. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with brightfield imaging. We identified several cell death modes where the order of events that usually define apoptosis were not observed. This we termed 'partial apoptosis'. Remarkably, complete classical apoptosis, defined as cells with plasma membrane blebbing, caspase-3 activity, nuclear condensation and membrane annexin V labeling prior to loss of plasma membrane integrity, was found in only half of the cytokine-treated primary β-cells and never in cells stressed by serum removal. By contrast, in the MIN6 cell line, death occurred almost exclusively through complete classical apoptosis. Ambient glucose modulated the cell death mode and kinetics in primary β-cells. Taken together, our data define the kinetic progression of β-cell death mechanisms under different conditions and illustrate the heterogeneity and plasticity of cell death modes in β-cells. We conclude that apoptosis is not the primary mode of adult primary β-cell death.