Table 1.
List of human gene primers used for quantitative PCR.
Fig 1.
Palmitic acid stimulates lipid accumulation in hepatocytes.
(A) HepG2 cells were treated with 200–500 μM palmitic acid (PA) for 24 hours. Triglycerides were extracted from cultured cells and quantitated by enzymatic assays. Triglyceride levels were normalized to total cellular protein contents. (B) HepG2 cells were treated with 400 μM PA for 24 hours. The mRNA expression levels of perilipin 1 (PLIN1), perilipin 2 (PLIN2) and perilipin 3 (PLIN3) were normalized to the level of β-actin. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with control cells.
Fig 2.
Exendin-4 inhibits palmitic acid-induced hepatic lipogenesis and TG synthesis.
HepG2 cells were treated with palmitic acid (PA; 400 μM) either with or without exendin-4 (Ex-4; 100 nM) for 24 hours. The mRNA expression levels of SREBP-1c, PPARγ, SCD1, FAS, ACC, DGAT1, and DGAT2 were normalized to the level of β-actin. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with control cells; # p<0.05, ## p<0.01 compared with PA-treated cells.
Fig 3.
Dose-dependent and time-dependent effects of exendin-4 on β-catenin expression in hepatocytes.
(A) HepG2, Huh7 and AML12 cells were treated with three different concentrations (50–500 nM) of exendin-4 for 24 hours. (B) HepG2, Huh7 and AML12 cells were treated with 100 nM exendin-4 for different lengths of time up to 24 hours. β-catenin expression was measured using quantitative (real-time) PCR and normalized to β-actin as a control. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with control cells.
Fig 4.
Exendin-4 induces active β-catenin signaling in an in vitro model of steatosis.
Cytosolic and nuclear extracts were prepared from HepG2 cells treated with palmitic acid (PA; 400 μM) either with or without exendin-4 (Ex-4; 100 nM) for 24 hours. The levels of (A) cytosolic phosphorylated GSK-3β, (B) nuclear β-catenin and TCF4 were analyzed by western blotting. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with control cells; # p<0.05, ## p<0.01 compared with PA-treated cells.
Fig 5.
β-catenin downregulates the expression of lipogenic transcription factors.
HepG2 cells were transfected with 10 nM siRNA directed against β-catenin for 24 hours. The protein levels of nuclear β-catenin, SREBP-1, and PPARγ were detected by western blot analysis. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with negative control (NC) siRNA-transfected cells.
Fig 6.
Exendin-4 reduces PA-induced lipid accumulation via β-catenin signaling in HepG2 cells.
Cells treated with 400 μM PA were stimulated with 100 nM exendin-4 in the absence or presence of 10 μM IWR-1 for 24 hours. (A) Cells were stained with Oil Red O and images were captured under a light microscope (magnification, 400x). Scale bars = 100 μm. The absorbance of the extracted dye was measured at 540 nm. (B) Triglycerides were extracted from cultured cells and quantitated by enzymatic assays. Triglyceride levels were normalized to total cellular protein contents. All values are expressed as the mean ± SE (n = 6). * p<0.05, ** p<0.01 compared with control cells.
Fig 7.
Schematic diagram illustrating a possible mechanism by which exendin-4-driven β-catenin signaling alleviates PA-induced hepatic steatosis.