High glucose (28 mmol/l) does not induce NF-κB activation and DNA binding in β-cells, as assessed by immunofluorescence using
an antibody directed against the p65 NF-κB subunit. FACS-purified rat β-cells were exposed to 10 mmol/l (control, A) or 28 mmol/l glucose (B) for 12–24 h. The data at 12 h are shown here; similar observations were made at 24 h (not shown). As a positive control,
cells were exposed to IL-1β (30 units/ml, in medium containing 10 mmol/l glucose [C]) during the last 30 min of culture. NF-κB is located in the cytosol at 10 mmol/l (A) or 28 mmol/l (B) glucose, whereas it translocates to the nucleus after exposure to IL-1β, indicating activation (C). Subcellular NF-κB localization was counted in 200–400 cells using the same experimental conditions as above (D). □, Cytoplasmic NF-κB localization; ▪, nuclear localization. The results are means ± SE of three independent experiments.
*P < 0.001 vs. percent nuclear staining in the control by two-sided paired t test (D). Original magnification ×200.