Background
Environmental factors play a major role in the rising prevalence of type 1 and type 2 diabetes mellitus. Cereulide is a lipophilic peptide that is often found at low concentrations in starchy food. It is a culprit to consider in this era of prepackaged meals.
Materials and methods
Mouse and rat insulin producing beta cell lines, MIN6 and INS-1E respectively, as well as whole mouse islets, isolated from 2 week old C57Bl/6J mice, were exposed to cereulide concentrations ranging from 0.05ng/ml to 5ng/ml for 24 and 72h. Cell death was evaluated by a Hoechst/Propidium Iodide assay, and compared to cell death in human hepatocellular HepG2 and monkey fibroblast-like COS-1 cells. Subsequently, MIN6 cells were exposed to low concentrations of cereulide (0.15 - 0.5 ng/ml) for 24h and glucose-stimulated insulin secretion was evaluated as well as mechanisms of toxicity by mRNA profiling, electron microscopy and caspase activation and cytochrome c release assay.
Results
Cereulide exposure caused cell death in MIN6, INS-1E and pancreatic islets, but not in HepG2 or COS-1E cells (Table 1). Caspase 3/7 activation confirmed the apoptotic cell death process. Glucose-stimulated insulin secretion decreased from 10.48 ± 3.33 fold to 2.01 ± 0.51 (P < 0.05) in MIN6 cells after 24h exposure with 0.25 ng/ml cereulide. Exposure to 0.25ng/ml cereulide induced markers of mitochondrial stress, including PUMA (p53 upregulated modulator of apoptosis; 271 ± 77 % of control; P < 0.05) but also markers of ER stress, such as CHOP (CCAAT/-enhancer-binding protein homologous protein; 641 ± 190 % of control; P < 0.01). EM revealed swelling and loss of mitochondria, and cytoplasmic cytochrome c release confirmed mitochondrial cell death signalling (360 ± 83 % of control after exposure to 0.5 ng/ml for 24h (P < 0.05).
Table 1.
Apoptosis induced after 24h exposure to cereulide (mean percentage ± SEM).
| MIN6 (n=5) | INS-1E (n=4) | HepG2 (n=3) | COS (n=3) | Islets (n=3) | |
|---|---|---|---|---|---|
| Medium | 7.3 ± 1.3 | 2.5 ± 0.3 | 5.8 ± 0.6 | 1.2 ± 0.6 | 3.1 ± 1.2 |
| 0.05 ng/ml cereulide | 5.9 ± 1.0 | 3.2 ± 0.5 | 6.6 ± 2.1 | 1.6 ± 0.4 | 3.9 ± 1.5 |
| 0.25 ng/ml cereulide | 31.6 ± 5.8 * | 58.1 ± 11.4 * | 6.9 ± 1.5 | 2.9 ± 0.7 | 8.6 ± 2.4 |
| 0.5 ng/ml cereulide | 43.6 ± 6.1 * | 100.0 ± 0.0 * | 11.9 ± 2.5 | 2.6 ± 0.6 | 49.2 ± 9.0 |
| 5 ng/ml cereulide | 100.0 ± 0.0 * | 100.0 ± 0.0 * | 7.7 ± 2.3 | 4.3 ± 0.9 | 96.4 ± 3.5* |
* p ≤ 0.05 vs control
Conclusion
Cereulide, a toxin frequently found in prepackaged or prepared starchy meals, increases levels of mitochondrial and ER stress markers in beta cells of rats and mice, even at low doses. In a dose dependent way, it also leads to impaired beta cell function and apoptosis. Cereulide might thus be involved in the current diabetes.