Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets
Figure 1
Chronic high glucose induces apoptosis and proteasome activities decrease in INS-1E cells.
INS-1E cells were cultured for 48 hours at increasing concentrations of glucose ranging from 10 mM (G10) to 33 mM (G33). A: Protein levels of cleaved caspase-3, cleaved PARP and actin were analyzed by Western blotting in INS-1E cells exposed to different glucose concentrations. Actin was used as a loading control. Immunoblots presented are representative of 5 independent experiments. B: Quantitative analysis of bands densities of Western blot (as presented in A) for cleaved caspase-3 and cleaved PARP were normalized to actin. Results are presented as means ± SEM of 5 independent experiments and expressed as fold increase compared to the G10 value. C: Total cell death was measured in the culture supernatants of INS-1E cells after 48 hours. Results are presented as means ± SEM of 4 independent experiments and expressed as percentage of the G10 value. D: Chymotrypsin-like, caspase-like, and trypsin-like activities were measured in lysates from G10- or G33-exposed INS-1E cells. Results are presented as means ± SEM of 6 independent experiments and expressed as percentage of the G10 value. E: Levels of polyubiquitinated proteins, CHOP protein -an endoplasmatic reticulum stress marker-, 20S-β5 protein -a proteasome subunit-, and actin were analyzed by Western blottin in INS-1E cells after 48 hours of culture either in 10 mM or 33 mM glucose. Actin was used as a loading control. Immunoblots presented are representative of 4 independent experiments. F: Quantitative analysis of bands densities of Western blots (as presented in E) were normalized to actin. Results are presented as means ± SEM of 4 independent experiments and expressed in arbitrary unit (AU). *P<0.05, **P<0.01, and ***P<0.001.