Figure 3. Transcriptional regulation of adiponectin by iron.

(A) Media adiponectin levels in 3T3-L1 cells 12 hours following 12-hour pretreatment. P < 0.0001. (B) RT-PCR quantification of adiponectin mRNA levels in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO₄ for 24 hours, normalized to cyclophilin A. *P = 0.02. (C) Adiponectin promoter-driven luciferase activity in the presence or absence of 100 μM FeSO₄. ^‡P = 0.0025. (D) Western blot for acetylated FOXO1 (Ac-FOXO1), phosphorylated FOXO1 (P-FOXO1), total FOXO1 (Tot-FOXO1), and β-actin in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO₄ for 8 hours. (E) Quantitation of Western blots (total n = 6 independent determinations) normalized to β-actin. *P < 0.05. (F) Quantitation of Western blots for phosphorylated AKT in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO₄ for 8 hours and insulin (10 nM) for 1 hour. (G) ChIP showing FOXO1 occupancy of adiponectin promoter FOXO1 sites and PPRE and PPARγ occupancy of PPRE in 3T3-L1 adipocytes (n = 3 experiments each assayed in duplicate, *P < 0.05). (H) Immunoprecipitation of 3T3-L1 adipocyte extracts, treated overnight in the presence or absence of 100 μM FeSO₄, by antibodies to FOXO1, followed by immunoblotting for FOXO1 (t-FOXO1) and C/EBPα (0.58 ± 0.15 density units for control, 0.61 ± 0. 26 density units for iron-treated extracts, P = 0.93).