Induction of HIF α-subunit protein levels by insulin via PI3K and by overexpression of activated PKB.
Cells treated with either insulin, wortmannin, or both, as well as cells transfected with the myrPKB expression vector, were cultured for 24 hours under arterial pO2. At 24 hours the medium was changed, and cells were further cultured for the next 24 hours under normoxic (16% O2) and hypoxic (8% O2) conditions. Three hours before harvesting the cells were treated with insulin (10 nM), wortmannin (20 nM), or in the controls with solvent (see “Materials and methods” for more information). (A) The HIF α-subunit mRNA levels were analyzed by Northern blotting. Representative Northern blot of 15 μg total RNA hybridized to digoxigenin-labeled HIF-1α, HIF-2α, HIF-3α, and β-actin antisense RNA probes (see “Materials and methods” for more information). (B,D) The HIF-α protein levels were measured by Western blotting. The protein level under normoxia (16% O2) was set equal to 100%. Values are expressed as means ± SEMs of 3 independent culture experiments. Studentt test for paired values: *significant difference 16% O2 versus 8% O2, **significant difference 16% O2 + insulin versus 16% O2 (control), 8% O2 + insulin versus 8% O2 (control),P ≤ .05; (D) **significant difference 16% O2 + myrPKB versus 16% O2 (control), 8% O2 + myrPKB versus 8% O2 (control),P ≤ .05. (C,E) Representative Western blots. Protein (50 μg) was subjected to Western analysis with an antibody against HIF-1α, HIF-2α, HIF-3α, GM, PKB-S473, PKB, or anti–EE-tag (see “Materials and methods” for more information). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry.
