Figure 1.

Free fatty acids (FFAs) treatment induces the expression of ATP-citrate lyase (ACLY). (A) HepG2 cells were incubated in the presence of a mixture of free oleic and palmitic fatty acids (FFAs) in the molar ratio of 2:1 for the indicated times. Cells were then harvested, and total RNA was extracted. ACLY and Sterol Regulatory Element-Binding Protein-1 (SREBP-1) mRNA levels were determined by using RT-qPCR and normalized with 18S rRNA. Values were reported in histograms as fold change relative to the untreated control cells. Values are means ± S.D., n = 5 (For ACLY 0 h vs. 24 h * p ≤ 0.05; 0 h vs. 48 h ** p ≤ 0.01; For SREBP-1, 0 h vs. each time of treatment, * p ≤ 0.05). (B) Proteins (60 μg) were prepared from FFAs-treated cells, separated by SDS/PAGE and immunoblotted with antibody against ACLY or SREBP-1. The content of ACLY, precursor SREBP-1 (pSREBP-1) and mature SREBP-1 (nSREBP-1) in FFAs-treated cells was analyzed by Western blotting, quantified by densitometric analysis, and expressed as fold change relative to untreated control cells. Values are means ± S.D., n = 4 (For ACLY, 0 h vs. 6 h and 24 h * p ≤ 0.05; 0 h vs. 48 h ** p ≤ 0.01; For pSREBP-1 and nSREBP-1, 0 h vs. 6 h * p ≤ 0.05; 0 h vs. 12 h, or 24 h, or 48 h ** p ≤ 0.01). (C) HepG2 cells, incubated for 6h or 48h in DMEM with either vehicle (BSA) or 0.75 mM FFAs, were then treated with 2 μg/mL puromycin, inhibitor of protein synthesis. At different times, cells were harvested and the content of ACLY protein was measured by Western blot analysis. The semilog plot represents the decay curve of ACLY protein in control (filled circle), 6 h FFAs-treated (open square), and 48h FFAs-treated (open circle) HepG2 cells. The results are from a representative experiment, with similar results being obtained in three independent experiments.