Sodium butyrate stimulates expression of fibroblast growth factor 21 in liver by inhibition of histone deacetylase 3

Abstract

Fibroblast growth factor 21 (FGF21) stimulates fatty acid oxidation and ketone body production in animals. In this study, we investigated the role of FGF21 in the metabolic activity of sodium butyrate, a dietary histone deacetylase (HDAC) inhibitor. FGF21 expression was examined in serum and liver after injection of sodium butyrate into dietary obese C57BL/6J mice. The role of FGF21 was determined using antibody neutralization or knockout mice. FGF21 transcription was investigated in liver and HepG2 hepatocytes. Trichostatin A (TSA) was used in the control as an HDAC inhibitor. Butyrate was compared with bezafibrate and fenofibrate in the induction of FGF21 expression. Butyrate induced FGF21 in the serum, enhanced fatty acid oxidation in mice, and stimulated ketone body production in liver. The butyrate activity was significantly reduced by the FGF21 antibody or gene knockout. Butyrate induced FGF21 gene expression in liver and hepatocytes by inhibiting HDAC3, which suppresses peroxisome proliferator-activated receptor-α function. Butyrate enhanced bezafibrate activity in the induction of FGF21. TSA exhibited a similar set of activities to butyrate. FGF21 mediates the butyrate activity to increase fatty acid use and ketogenesis. Butyrate induces FGF21 transcription by inhibition of HDAC3.

FIG. 1.

Butyrate increases FGF21 mRNA and protein in HepG2 cells. A: Increase of FGF21 mRNA expression by butyrate. The cells were serum starved in DMEM supplemented with 0.25% BSA overnight and treated with bezafibrate (100 μmol/L), fenofibrate (500 μmol/L), butyrate (1 mmol/L), BOH (1 mmol/L), and hexanoate (1 mmol/L) for 2 h. The total RNA was extracted and subjected to qRT-PCR analysis for FGF21 mRNA. B: Increase of FGF21 protein by butyrate. The cells were serum starved in DMEM supplemented with 0.25% BSA overnight and treated with bezafibrate (100 μmol/L), fenofibrate (500 μmol/L), butyrate (1 mmol/L), BOH (1 mmol/L), and hexanoate (1 mmol/L) for 6 h. The FGF21 protein was determined in the whole-cell lysate by enzyme-linked immunosorbent assay. C: Butyrate increases FGF21 mRNA in a dose-dependent manner. D: Butyrate induces FGF21 protein in a dose-dependent manner. E: TSA increases FGF21 mRNA in a dose-dependent manner. F: TSA induces FGF21 protein in a dose-dependent manner. Data are mean ± SEM, n = 4. *P < 0.05 vs. control.

FIG. 2.

Butyrate administration increases serum and hepatic FGF21 in obese mice. A: Serum FGF21 concentrations at basal level and 7 h after butyrate injection in obese mice (n = 8). B: Serum FGF21 concentrations at basal levels and 7 h after TSA injection in obese mice (n = 5–6). C: Serum FGF21 concentrations at basal levels and 7 h after hexanoate injection in obese mice (n = 5–6). D: FGF21 mRNA expressions in the liver of obese mice at 2 h after butyrate injection (n = 4). E: FGF21 protein levels in the liver of obese mice at 2 h after butyrate injection (n = 4). Data are mean ± SEM. In A and B, *P < 0.05 vs. basal. In D and E, *P < 0.05 vs. PBS.

FIG. 3.

Butyrate administration enhances ketogenesis via induction of FGF21. A: Serum β-hydroxybutyrate concentrations at 7 h after butyrate injection in obese mice. B: Ketogenic gene (HMGCS2 and CPT1a) mRNA expressions in the liver of obese mice. *P < 0.05 vs. PBS. C: Correlations of increasing values of serum β-hydroxybutyrate with increasing values of serum FGF21 in obese mice treated with butyrate. D: Correlations of increasing values of serum β-hydroxybutyrate with increasing values of serum FGF21 in obese mice treated with TSA. Data are mean ± SEM (n = 6–8). E: Serum β-hydroxybutyrate concentrations at basal levels and 7 h after butyrate injection in the FGF21 KO and WT mice. F: Ketogenic gene (HMGCS2 and CPT1a) mRNA expressions after butyrate injection compared with baseline in the liver of FGF21 KO and WT mice. Data are mean ± SEM (n = 5). *P < 0.05 vs. WT.

FIG. 4.

Butyrate administration promotes fatty acid oxidation via FGF21 in obese mice. Substrate use, O₂ consumption, and spontaneous physical activity were examined using the metabolic chamber in obese mice (n = 8). Butyrate was injected at 500 mg/kg body wt at 12 A.M., and data were recorded during 7 P.P. to 7 A.M. after the injection. A: Substrate use in mice is expressed by RER. B: Serum FGF21 was determined after injection of 3 μg rabbit–anti-mouse FGF21 antibody (Ab) or normal IgG. Data are mean ± SEM. *P < 0.05 vs. basal.

FIG. 5.

Butyrate activates FGF21 gene transcription by targeting HDAC3. Transient transfection was conducted with the FGF21 luciferase reporters in HEK 293 cells. In cotransfection, the expression plasmids for PSG5–PPAR-α, retinoid X receptor, siRNA for HDACs (siHDAC1, siHDAC2, and siHDAC3), and scrambled siRNA were used. The cells were treated with bezafibrate, butyrate, or TSA at 24 h after transfection and harvested 16 h later for luciferase assay. In all of the transient transfection experiments, the internal control was 0.1 μg/well of simian virus 40 (SV40) R. luciferase reporter plasmid, and the total DNA concentration was corrected in each well with a control plasmid. A: Butyrate response element. Cells were transfected with the FGF21 luciferase reporter plasmids in the presence of SV40. Cells were treated with 100 μmol/L bezafibrate or 0.5 mmol/L butyrate for 16 h. B: Dose-dependent activity of butyrate. Cells were transfected with the long promoter (−1,497/5) reporter in the presence of PPAR-α expression plasmid or control plasmid. Cells were treated with bezafibrate (100 μmol/L), butyrate (0.5, 1.5, and 2.0 mmol/L), or TSA (100, 200, and 400 nmol/L) for 16 h. C: HDAC3 knockdown. In the FGF21 (−1,497/5) reporter system, HDACs were inhibited with siRNA of HDAC1, HDAC2, and HDAC3. Scrambled siRNA was used as a negative control. D: HDAC3 interaction with PPAR-α. In the FGF21 luciferase reporter system, the reporter activity was induced by cotransfection of PPAR-α expression vector. HDAC3 was knocked down by siRNA. Data are mean ± SEM. *P < 0.05 vs. control; #P < 0.05 vs. shorter promoters. TATA, TATA box.

FIG. 6.

HDAC3 interacts with PPAR-α in the FGF21 promoter. ChIP assays were performed using liver tissues collected after bezafibrate and butyrate injection. Immunoprecipitation was performed with antibodies to PPAR-α (A), polymerase II (B), and HDAC3 (C). Rabbit IgG was used in the negative control. The specific chromatin DNA was quantified in qRT-PCR with the primer against the distal PPRE (−1,119/−1,044) in the mouse FGF21 gene promoter. Data are mean ± SEM, n = 4. *P < 0.05 vs. IgG control.

FIG. 7.

Butyrate enhances PPAR-α agonist activity in the stimulation of FGF21 expression. A: FGF21 protein expression in HepG2 cells. FGF21 protein was determined in the whole-cell lysate by enzyme-linked immunosorbent assay (ELISA) after HepG2 cells were treated with bezafibrate and butyrate. *P < 0.05 vs. control; #P < 0.05 vs. bezafibrate. B: Serum FGF21 concentration. Serum FGF21 was determined in obese mice at 7 h after treatment with bezafibrate (100 mg/kg body wt) and butyrate (500 mg/kg body wt) (n = 5–6). *P < 0.05 vs. basal; #P < 0.05 vs. bezafibrate. C: FGF21 mRNA in the liver of obese mice. The liver tissue was collected at 2 h after the mice were treated with bezafibrate and butyrate (n = 4). The mRNA expression was determined in qRT-PCR. D: Protein level in the liver. FGF21 protein was determined in the liver tissue by ELISA. E: Serum β-hydroxybutyrate concentration at 7 h after butyrate and bezafibrate injection in obese mice (n = 5–6). F: Genes associated with ketogenesis and fatty acid oxidation (HMGCS2, CPT1a, ACADVL, ACSL1, ACO, and L-FABP) mRNA expressions in the liver. Data are mean ± SEM. *P < 0.05 vs. PBS; #P < 0.05 vs. bezafibrate.