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

Abstract

SummaryProgrammed b-cell death plays an important role in both type 1 and type 2 diabetes. Most of what is known about the mechanisms of bcell 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 b-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 b-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 b-cells. Taken together, our data define the kinetic progression of b-cell death mechanisms under different conditions and illustrate the heterogeneity and plasticity of cell death modes in b-cells. We conclude that apoptosis is not the primary mode of adult primary b-cell death.

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress

“…Our Bax-Bak double knockout model allowed us to identify both apoptotic and non-apoptotic βcell death during STS treatment and glucolipotoxicity. This agrees with the finding that stressed primary β-cells often die without all the morphological features of apoptosis (45). Moreover, death of Pdx-1-deficient β-cells happens by apoptosis, as well as necrosis involving the mitochondrial permeability transition pore (mPTP) (46)(47)(48).…”

Types of cell death and apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman spectroscopy

“…Thus, when the cell shown in Figure a becomes PI positive, the Hoechst signal has faded. However, image analysis software is still able to qualitatively identify most of these apoptotic cells as Hoechst positive, as is shown in the final panel in Figure b that does not show a decrease of Hoechst‐positive apoptotic cells (Yang & Johnson, ).…”

Differential effects of voclosporin and tacrolimus on insulin secretion from human islets

“…Pancreatic islets from human cadaveric donors were dispersed and seeded into 384-well plates and cultured in similar manner as previously described (20)(21)(22)(23). Cells were stained with 50 ng/mL Hoechst 33342 (Invitrogen) and 0.5 μg/mL propidium iodide (PI) (Sigma-Aldrich) in RPMI 1640 medium (Invitrogen) with 5 mM glucose (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Invitrogen), 10% vol/vol fetal bovine serum (FBS) (Invitrogen) with or without a cytokine cocktail (25 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ; R&D Systems).…”