Figure 6

AMPK activation or inhibition of GSK3β diminishes development of endotoxin tolerance in LPS-treated macrophage. (A,B) Macrophages were first incubated with LPS (0 or 10 ng/mL) for 24 h. Next, cells were washed and treated with a second dose of LPS (0 or 10 ng/mL) for 4 h followed by measurement of TNF-α in culture media (ELISA). As indicated, cells were also treated with AMPK activators metformin (0 or 500 µmol/L) or GSK3β inhibitor SB216763 (0 or 30 µmol/L) for 60 min followed by inclusion of LPS (first dose). (C,D,E) AMPK activators or GSK3β inhibition prevented HIF-1α accumulation in LPS-treated macrophages. Peritoneal macrophages were treated with metformin (0 or 1 mmol/L) and LPS (30 ng/mL) for 0, 2, 4, 8 h followed by Western blot analysis of HIF-1α and β-actin. Cells were also treated with AICAR (0 or 300 µmol/L) or SB216763 (0 or 30 µmol/L) and then cultured with LPS for an additional 8 h (means ± SD, n = 3, *p < 0.05, **p < 0.01).