Palmitate induces fat accumulation by activating C/EBPβ-mediated G0S2 expression in HepG2 cells

Abstract

Aim: To determine the role of G0/G1 switch gene 2 (G0S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation.

Methods: HepG2 cells were treated with palmitate, or palmitate in combination with CCAAT/enhancer binding protein (C/EBP)β siRNA or G0S2 siRNA. The mRNA expression of C/EBPβ, peroxisome proliferator-activated receptor (PPAR)γ and PPARγ target genes (G0S2, GPR81, GPR109A and Adipoq) was examined by qPCR. The protein expression of C/EBPβ, PPARγ, and G0S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium.

Results: Palmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in HepG2 cells. In addition, palmitate increased the mRNA expression of C/EBPβ, PPARγ, and PPARγ target genes (G0S2, GPR81, GPR109A, and Adipoq) and the protein expression of C/EBPβ, PPARγ, and G0S2 in a dose-dependent manner. Knockdown of C/EBPβ decreased palmitate-induced PPARγ and its target genes (G0S2, GPR81, GPR109A, and Adipoq) mRNA expression and palmitate-induced PPARγ and G0S2 protein expression in HepG2 cells. Knockdown of C/EBPβ also attenuated lipid accumulation and augmented lipolysis in palmitate-treated HepG2 cells. G0S2 knockdown attenuated lipid accumulation and augmented lipolysis, while G0S2 knockdown had no effects on the mRNA expression of C/EBPβ, PPARγ, and PPARγ target genes (GPR81, GPR109A and Adipoq) in palmitate-treated HepG2 cells.

Conclusion: Palmitate can induce lipid accumulation in HepG2 cells by activating C/EBPβ-mediated G0S2 expression.

Keywords: Adipogenesis; CCAAT/enhancer binding protein β; G0/G1 switch gene 2; Lipolysis; Nonalcoholic fatty liver disease; Obesity; Proliferator-activated receptor γ; Saturated fatty acid.

Figure 1

Palmitate-induced lipid accumulation and inhibition of lipolysis in HepG2 cells. A and B: Dose response of lipid accumulation induced by palmitate. HepG2 cells were treated with various concentrations of palmitate for 24 h, and (A) stained with Oil Red O to visualize the intracellular lipid contents (original magnification, × 100); B: Lipid accumulation was quantified by absorbance value of the extracted Oil Red O dye at 500 nm. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^aP < 0.05 vs untreated control. C: Dose response of inhibition of lipolysis induced by palmitate. HepG2 cells were treated with various concentrations of palmitate for 24 h, and lipolysis was assessed by glycerol release into the medium. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^aP < 0.05 vs untreated control.

Figure 2

Palmitate-induced expression of C/EBPβ, PPARγ, and PPARγ target genes in HepG2 cells. A: Palmitate increased mRNA expression of C/EBPβ, PPARγ, and PPARγ target genes (G0S2, GPR81, GPR109A, and Adipoq) in a dose-dependent manner. mRNA was measured by qPCR. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^aP < 0.05 and ^bP < 0.01 vs untreated control. B: Palmitate increased protein expression of C/EBPβ, PPARγ, and G0S2 in a dose-dependent manner. Protein was examined by western blotting. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^aP < 0.05 and ^bP < 0.01 vs untreated control.

Figure 3

The effects of C/EBPβ knockdown on palmitate-induced PPARγ and its target gene expression in HepG2 cells. A: HepG2 cells were transfected with control siRNA or C/EBPβ siRNA, and C/EBPβ expression was measured by Western blotting. At least three independent experiments were conducted. Data are presented as means ± SE. ^dP < 0.01 vs control siRNA. B: C/EBPβ knockdown decreased palmitate-induced mRNA expression of PPARγ and its target genes (G0S2, GPR81, GPR109A, and Adipoq). mRNA was measured by qPCR. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^cP < 0.05 and ^dP < 0.01 vs control siRNA. C: C/EBPβ knockdown decreased palmitate-induced protein expression of PPARγ and G0S2. Protein was examined by Western blotting. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^dP < 0.01 vs control siRNA.

Figure 4

The effects of C/EBPβ knockdown on lipid accumulation and lipolysis in HepG2 cells treated with palmitate. A: C/EBPβ knockdown decreased palmitate-induced lipid accumulation. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye at 500 nm. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^cP < 0.05 vs control siRNA. B: C/EBPβ knockdown increased lipolysis in HepG2 cells treated with palmitate. Lipolysis was assessed by glycerol release into the medium. At least three independent experiments were conducted. Data are presented as means ± SE. ^dP < 0.01 vs control siRNA.

Figure 5

The effects of G0S2 knockdown on C/EBPβ, PPARγ and PPARγ target genes expression, lipid accumulation, and lipolysis in palmitate-treated HepG2 cells. A: HepG2 cells were transfected with control siRNA or G0S2 siRNA, and G0S2 expression was measured by Western blotting. At least three independent experiments were conducted. Data are presented as means ± SE. ^fP < 0.01 vs control siRNA. B: G0S2 knockdown decreased palmitate-induced lipid accumulation. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye at 500 nm. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. ^eP < 0.05 vs control siRNA. C: G0S2 knockdown increased lipolysis in HepG2 cells treated with palmitate. Lipolysis was assessed by glycerol release into the medium. At least three independent experiments were conducted. Data are presented as means ± SE. ^eP < 0.05 vs control siRNA. D: G0S2 knockdown did not affect palmitate-induced C/EBPβ, PPARγ, and other PPARγ target genes (GPR81, GPR109A, and Adipoq) mRNA expression. mRNA was measured by qPCR. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE. E: G0S2 knockdown did not affect palmitate-induced PPARγ and G0S2 protein expression. Protein was examined by Western blotting. At least three independent experiments were conducted for each measurement. Data are presented as means ± SE.