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

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.