Fig 2: Role of FFAs and Que on the regulation of ACACA activity. (A) HepG2 cells were incubated in low glucose (LG) or in high glucose (HG) medium, in the presence or in the absence of 0.75 mM FFAs and 5 µM Que for 24 h. Total proteins were extracted from the cells and separated by SDS/PAGE. After incubation with antibodies against phosphorylated ACACA, the protein content was quantified by densitometric analysis, and results were expressed as a percentage of control cells (LG). Results are representative of three different experiments. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). Values are means ± S.D. (B) Cells were treated as reported in A. ACACA activity was determined in cell lysates as in Material and Methods. The activity in each sample was expressed as a percentage of control cells (LG). Values are means ± S.D., n = 3. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). (C) After the separation of cell lysates by SDS/PAGE, proteins were transferred onto nitrocellulose membrane and blotted against phosphorylated AMPK. The protein content was quantified by densitometric analysis and expressed as a percentage of control cells (LG). Results are representative of three different experiments. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). (D) PP2A activity was determined in cell lysates as reported in Material and Methods. The activity in each sample was expressed as a percentage of control cells (LG). Values are means ± S.D., n = 3. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG).

Fig 3: PFKP promotes mitochondrial recruitment of AMPK and enhances the phosphorylation of ACC2.a PFKP IP in H1299 cells under control or GS. b Immunofluorescence analysis of mito-GFP and PLA puncta (interacted PFKP and AMPK) in H1299 cells under control (Ctrl) or GS for 4 h. The gray values of green and red signals between two the arrows were plotted. Scale bar, 10 µm. c, d Mitochondria fraction analysis of H1703 (c) and H1299 (d) cells under control or GS. e IP of ACC1 and ACC2 in H1703 cells under control or GS. f ACC2 IP in H1299 cells under control or treated with GS for 4 h or 8 h. Data are shown as means ± SD with three biological replicates. ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.

Fig 4: Effects of EGCG on the gene expression and protein in FFAs-induced hepatocytes. A TG levels in hepatocytes; B effects of EGCG on protein expression of ACC1, FASn, PPAR–FGF21–AMPK; C quantification of Fig. B; D levels of FGFR2,FGFR3 and FGFR4 in hepatocytes; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs BSA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs FFA