Glucose decreases β cell I-κB expression and induces IL-1β–meditated NF-κB activation, Fas expression, and DNA fragmentation. (a) Relative NF-κB activity. Human islets were cultured in suspension for 44 hours in 5.5 or 33.3 mM glucose alone or in the presence of IL-1β, IL-1Ra, or both. HeLa cells stimulated with 5 ng/ml IL-1α were used as positive control. Each bar represents the mean ± SEM of three experiments from three separate donors. *P < 0.05 relative to islets cultured in 5.5 mM glucose alone. (b) Immunoblotting of NF-κB (p65), Fas, and actin. Human islets cultured in suspension at 5.5 or 33.3 mM glucose with and without IL-1β or IL-1Ra were analyzed after 44 hours of incubation. The antibodies were blotted on the same membrane after stripping. One of three experiments from three donors is shown. Each experiment displayed similar results. (c) Double immunostaining for I-κB in red (boxes 1 and 3) and insulin in green (boxes 2 and 4) in sections of cultured human islets exposed for 44 hours to media containing 5.5 mM (boxes 1 and 2) or 33.3 mM glucose (boxes 3 and 4). (d) Double immunostaining for NF-κB (p65) in red (boxes 1 and 3) and insulin in green (boxes 2 and 4) in human islets exposed for 44 hours to media containing 5.5 mM (boxes 1 and 2) or 33.3 mM glucose (boxes 3 and 4). The arrows mark β cell nuclei stained positive for NF-κB. Magnification: ×750. (e) RT-PCR detection and quantification of Fas mRNA expression. Total RNA was isolated from human islets cultured for 44 hours in medium containing 5.5 or 33.3 mM glucose alone or in the presence of IL-1Ra. In the LightCycler quantitative PCR system, the level of Fas expression was normalized against GAPDH, and the results were expressed as mRNA levels relative to control incubations at 5.5 mM. Results are shown as mean ± SEM of six independent experiments from six donors. *P < 0.05 compared with islets cultured in 5.5 mM glucose alone. **P < 0.05 compared with islets cultured in 33.3 mM glucose. (f) Double immunostaining for Fas in red (boxes 1, 3, 5, 7, 9, and 11) and insulin in green (boxes 2, 4, 6, 8, 10, and 12) in human islets exposed for 4 days to media containing 5.5 mM glucose without IL-1β (boxes 1 and 2), with IL-1β alone (boxes 3 and 4), or with IL-1Ra (boxes 5 and 6) or 33.3 mM glucose without (boxes 7 and 8) and with IL-1Ra (boxes 9 and 10) or IL-1β (boxes 11 and 12). Magnification: ×250. (g) Human islets were cultured for 4 days in 5.5 and 33.3 mM glucose alone or in the presence of IL-1β and/or IL-1Ra, or (h) with and without PDTC. Results are mean ± SEM of the percentage of TUNEL-positive β cells. The mean number of islets scored from each donor was 49 (range 35–63) for each treatment condition. Islets were isolated from five organ donors. *P < 0.01 relative to islets cultured in 5.5 mM glucose. **P < 0.01 relative to islets cultured in 33.3 mM glucose. ⁺P < 0.01 relative to islets cultured in 5.5 mM glucose plus IL-1β.