Fig. 2.

NF-κB is required for cytokine-mediated decreases in insulin secretion and cellular production of nitric oxide. A–F: 832/13 cells were transduced with adenoviruses expressing either AdCMV-β-Gal [control (C)] or increasing doses of AdCMV-IκBα^SR (low and high). Eight hours after adenoviral transduction, cells were then NT or stimulated for an additional 18 h with 1 ng/ml IL-1β. A: immunoblot analysis of whole cell lysates probed with antibodies against iNOS, IκBα, and tubulin. Solid arrow, adenovirally overexpressed IκBα; dashed arrow, endogenous IκBα. B and C: densitometric analysis of iNOS (B) and IκBα (C) protein abundance from the immunoblot shown in A. D: nitric oxide determination by electron paramagnetic resonance (EPR) spectroscopy combined with N-methyl-d-glucamine dithiocarbamate (MGD)₂Fe²⁺ spin trap. E: quantification of the EPR spectroscopy data shown in D. F: nitrite accumulation in the media was measured by Griess assay. G and H: 832/13 cells were cultured with the indicated adenoviruses for 8 h, followed by exposure to 1 ng/ml IL-1β for either 6 (G) or 18 h (H). Insulin secretion was measured in response to basal (2 mM) and stimulatory (16 mM) glucose concentrations, with data shown as %stimulated control. The high dose of AdCMV-IκBα^SR was used in H. *P < 0.05 vs. β-Gal-IL-1β; **P < 0.01 vs. β-Gal-IL-1β; #P < 0.1 vs. β-Gal-IL-1β. Data represent means ± SE of 3 independent experiments.