Figure 1.
Cereulide causes apoptotic cell death in 3 beta-cell models, but not in other mammalian cell lines after 24 h exposure.
Cell death rate was determined with Hoechst-PI staining for MIN6 cells (A, n = 6), mouse islets (B, n = 3), INS-1E (C, n = 3), COS-1 (D, n = 3) and HepG2 (E, n = 3), as well as caspase 3/7 activation (F, n = 3) in MIN6 cells after 24 h exposure to cereulide. Data are presented as mean ± SEM. * P<0.05, ** P<0.01 *** P<0.001 vs control.
Figure 2.
Cereulide impairs glucose-stimulated insulin secretion.
MIN6 cells (A, n = 11) and whole mouse islets (B, n = 3) were exposed to cereulide for 24 h, and incubated with KREBS containing low (white bars) or high (black bars) concentrations of glucose. Insulin secretion is expressed as a percentage of total insulin content. Data are presented as mean ± SEM. * P<0.05, *** P<0.001 vs control (2 way ANOVA, followed by Bonferroni test for MIN6; student t test for islets).
Figure 3.
mRNA levels of intrinsic mitochondrial apoptosis and ER stress mediators are upregulated in MIN6 cells after 24 h of exposure to cereulide.
Death protein 5 (Dp5, n = 4, A) and p53 upregulated modulator of apoptosis (Puma, n = 8, B) are dose dependently upregulated, as are activating transcription factor 4 (Atf4, n = 8, C) and CCAAT/-enhancer-binding protein homologous protein (Chop, n = 8, D), while binding immunoglobulin protein (Bip, n = 8, E) and spliced X-box binding protein are not (Xbp1s, n = 8, F). Data are presented as mean ± SEM (n 4–8). * P<0.05 vs control, *** P<0.001 vs control.
Figure 4.
Cereulide disrupts normal mitochondrial structure in MIN6 cells.
Electron microscopic evaluation of MIN6 cells in control condition (A) or after 24 h exposure to 0.5 ng/ml cereulide (B) (magnification 2500x). Arrows indicate normal mitochondria in control condition (A) or enlarged mitochondria with less cristae after cereulide exposure (B). Morphometric analysis confirmed fewer mitochondria (D) and swelling of the remaining mitochondria (E) in the exposed cells, as is calculated by dividing the mitochondrial area by the number of mitochondria. In the cereulide exposed condition, we noted cells with chromatin condensation (indicated by CC in panel 4C), compared to normal nuclei (indicated by NC in panel A). After cereulide exposure there were more autophagic vacuoles (F, indicated by white arrow in panel C) Data are presented as mean ± SEM. * P<0.05 vs control; *** P<0.001 vs control (student t test).
Figure 5.
Cereulide negatively affects mitochondrial function in MIN6 cells after 24 h exposure.
Oxygen consumption rate in basal conditions was reduced (A, n = 7). Cereulide caused a rise in reactive oxygen species, as shown by the increase in dichlorofluorescindiacetate fluorescence (B, n = 8). Cereulide exposure caused cytochrome c release into the cytoplasm (C, n = 4). Data are presented as mean ± SEM. * P<0.05 vs control, **P<0.01 vs control.