Figure 4

Pioglitazone ameliorated hepatic steatosis through enhancing lipolysis, β-oxidation, and autophagy dependent on PPARα and PPARγ activation. (A) AML12 cells treated with (a,e) control (b,f) 400 µM palmitic acid (PA), (c,g) 400 µM PA and 10 µM pioglitazone (PioG), (d,h) 400 µM PA and 30 µM pioglitazone. Cells with bright field and Oil-red O/hematoxylin stain to show lipid droplet. Magnification: 400X. (B,C) AML12 cells, transfected with siRNA against PPARα, PPARγ, and non-targeting negative siRNA, were treated with palmitic acid (PA, 400 μM) with or without pioglitazone (PioG) for 3 days. Protein expressions of the PPARα, PPARγ, ATGL, HSL, LC3, and CPT-1A were analyzed to control with GAPDH and β-actin. Data are expressed as fold change of the negative siRNA. P < 0.05 indicated as significant, *vs Control (con), ^Δvs PA, ^$vs PA + PioG 10 μM, ^#vs PA + PioG 30 μM. (D) Autophagic flux was determined by treating lysosomal inhibitor, leupeptin, for 4 hours to compare the LC3 expression with loading control of β-actin. Net LC3 flux was indicated by the difference of LC3II/β-actin ratio between leupeptin treated and untreated cells. Con: (lane 5-lane 1), PA: (lane 6-lane 2), PA + PioG10 μM: (lane 7-lane 3), PA + PioG 30 μM: (lane 8-lane 4). (E) AML12 cells, incubated with PA and pioglitazone as indicated for 48 hours, were then treated with leupeptin (100 µM) for 6 and 12 hours. The lipid content was determined by Oil-red O stain and quantified by reading absorbance at 492 nm. Data was expressed as fold or percentage change in mean + SEM. P < 0.05 indicated as significant, *vs Con, ^Δvs PA.