Fig 1.
Methylation status and leptin expression.
A, B: Leptin (A) and adiponectin (B) mRNA expression in epididymal (eWAT) and subcutaneous white adipose tissue (sWAT) from mice at age 12 weeks, and in 3T3-L1 adipocytes. C: Percentage of CpGs methylated in the leptin promoter in eWAT, sWAT, and 3T3-L1 cells. In A, B, C, data are mean ± SEM and *; p < 0.05, **; p < 0.01 versus eWAT.
Fig 2.
Effects of demethylation on leptin expression in 3T3-L1 cells.
A, B: Promoter methylation (A) and leptin expression (B) in 3T3-L1 pre-adipocytes treated with 0.5 or 5 μM 5-azacytidine for 7 days. C, D: Promoter methylation (C) and leptin expression (D) in adipocytes derived from 3T3-L1 precursors treated with or without 5-azacytidine, measured 6 days after differentiation. Products from real-time qPCR were also separated on agarose. E, F: Expression (E) and secretion (F) of leptin in adipocytes derived from precursors treated with 5-azacytidine, as measured by western blotting and ELISA, respectively. Data were normalized to total protein concentration. G: Activity of methylated and unmethylated leptin promoter in adipocytes differentiated from 3T3-L1 cells. H: Leptin expression in serum-starved cells treated with 100 nM insulin (Ins), 1 μM troglitazone (Tro), or 1 μM dexamethasone (Dex). Cells are adipocytes derived from 3T3-L1 precursors treated with 5-azacytidine. Results are mean ± SEM. *, p < 0.05; **, p < 0.01 versus untreated cells in A-F, versus methylated promoter in G, and versus vehicle in H.
Fig 3.
Effects of 5-azacytidine on adipogenesis in 3T3-L1 precursors treated with or without 5-azacytidine.
A: Lipid accumulation, as visualized with Oil Red O in cells fixed in 3.7% formaldehyde. B: Cell proliferation. C-G: Expression of the adipocyte-specific genes PPARγ (C), C/ EBPα (D), aP2 (E), GLUT4 (F), and adiponectin (G). Results are mean ± SEM. *, p < 0.05; **, p < 0.01.
Fig 4.
Effects of DNMT1 knockdown in 3T3-L1 pre-adipocytes.
A, B: DNMT1 mRNA (A) and protein (B). C, D: Promoter methylation (C) and leptin expression (D). E: Expression of the adipogenic genes, PPARγ, C/EBPα, aP2, GLUT4, adiponectin, and leptin. F: Cell proliferation. Results are mean ± SEM. *, p < 0.05; **, p < 0.01 versus non-targeting siRNA in A-D, and versus 0 h in F.
Fig 5.
Effects of adipocyte enlargement.
A: CpG methylation in eWAT, liver, skeletal muscle, and brain tissue from mice 12 weeks of age. B: CpG methylation in adipocytes and stromal vascular fraction of whole adipose tissue. C: Methylation in adipocytes isolated from eWAT of mice on normal chow (NC) or high-fat (HF) diet. D: Time course of leptin expression in adipocytes differentiated from untreated or 5-azacytidine-treated 3T3-L1 precursors. E, F: Leptin promoter methylation at day 6 and 20 in adipocytes derived from untreated (E) and 5-azacytidine-treated (F) 3T3- L1 pre-adipocytes. Data are mean ± SEM. *, p < 0.05; **, p < 0.01 versus eWAT (A), adipocytes (B), normal chow (C), untreated cells (D), and day 6 (E-F).