Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. affects proteasome activity and ubiquitination In INS-1E cells, increasing glucose from an optimal (10 mM) to a supra-physiologic (33 mM) level during 48 h is deleterious and leads to dose-dependent increases in cleaved-caspase-3, cleaved-PARP (Figures 1A and 1B), and total cell death (Figure 1C). Besides, this chronic exposure to high-glucose significantly decreases the 3 proteasome activities, with a 20C25% loss of the chymotrypsin-like, caspase-like, and trypsin-like activities (Figure 1D). In parallel, the polyubiquitinated proteins level is increased by 26% in the presence of high glucose, whereas the 20S-5 proteasome subunit level is not significantly altered (Figures 1E and 1F). Finally, we confirm that endoplasmic Rabbit Polyclonal to TPD54 reticulum (ER) stress, as evidenced by the two fold increase in CHOP expression (Figures 1E and 1F), is involved in the increased apoptosis observed in beta cells submitted to high glucose. Open in a separate window 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, MTX-211 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-5 protein -a proteasome subunit-, and actin were MTX-211 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. Impaired proteasome activities in hyperglycemic GK rat islets We assess the impact of a hyperglycemic environment on beta cell proteasome function using the GK rat diabetic model [32], [33]. Pancreatic islets from 5 GK rats exhibiting mild hyperglycemia (around 9.0 mM) are compared to islets from 9 Wistar control rats exhibiting normoglycemia (around 5.0 mM). GK rats islets show a slight increase in apoptosis, as revealed by PARP MTX-211 cleavage (Figures 2A and 2B). More importantly, GK rat islets display a 25% reduction in caspase-like activity (p<0.01), a 40% reduction in trypsin-like activity (p<0.01), whereas chymotrypsin-like activity was not significantly decreased (?10%, p?=?0.20) (Figure 2C). This suggests that the hyperglycemic environment could be linked to decreased proteasome activities a deleterious impact on beta cell survival. Noteworthily, this pro-apoptotic effect of PIs exists even for a slight ?20%- reduction of proteasome activity, the same percentage of inhibition induced by high-glucose culture or 50 nM MG-132. Our results are in accordance MTX-211 with previous studies showing that high doses of PIs reduce viability of clonal MIN6 and INS-1E beta cells [13], [17]. For entire islets, the literature data were controversial, as a decrease in viability was observed in human islets cultured with epoxomycin [17], whereas lactacystin had no impact on beta cell viability of young rats [13]. We confirm here that immortalized cell lines are more sensitive to the pro-apoptotic effect of PIs than primary cells, even if the latter can still be impacted by higher dose of PIs [37]. We show that inhibition of proteasome activity in beta cells could be a new link between glucotoxicity and apoptosis. This phenomenon -via genetic predisposition or epigenetic regulation- may thus exist in diabetic patients, participating in beta cell dysfunction. Indeed, Bugliani transgenic mice model that ER stress could.