Exendin-4 Inhibits Hepatic Lipogenesis by Increasing β-Catenin Signaling

<div><p>The aim of this study is to investigate whether the beneficial effect of exendin-4 on hepatic steatosis is mediated by β-catenin signaling. After the HepG2 human hepatoma cells were treated with PA for 24 hours, total triglycerides levels were increased in a dose-dependent manner, and the expression levels of perilipin family members were upregulated in cells treated with 400 μM PA. For our in vitro model of hepatic steatosis, HepG2 cells were treated with 400 μM palmitic acid (PA) in the presence or absence of 100 nM exendin-4 for 24 hours. PA increased the expression of lipogenic genes, such as sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPARγ), stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) and triglyceride synthesis-involved genes, such as diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) in HepG2 cells, whereas exendin-4 treatment significantly prevented the upregulation of SREBP-1c, PPARγ, SCD1, FAS, ACC, DGAT1 and DGAT2. Moreover, exendin-4 treatment increased the expression of phosphorylated glycogen synthase kinase-3 beta (GSK-3β) in the cytosolic fraction and the expression of β-catenin and transcription factor 4 (TCF4) in the nuclear fraction. In addition, siRNA-mediated inhibition of β-catenin upregulated the expression of lipogenic transcription factors. The protective effects of exendin-4 on intracellular triglyceride content and total triglyceride levels were not observed in cells treated with the β-catenin inhibitor IWR-1. These data suggest that exendin-4 treatment improves hepatic steatosis by inhibiting lipogenesis via activation of Wnt/<a href="http://ko.wikipedia.org/wiki/%CE%92" target="_blank">β</a>-catenin signaling.</p></div

Exendin-4 reduces PA-induced lipid accumulation via β-catenin signaling in HepG2 cells.

<p>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.</p

List of human gene primers used for quantitative PCR.

<p>List of human gene primers used for quantitative PCR.</p

β-catenin downregulates the expression of lipogenic transcription factors.

<p>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.</p

Exendin-4 induces active β-catenin signaling in an in vitro model of steatosis.

<p>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.</p

Schematic diagram illustrating a possible mechanism by which exendin-4-driven β-catenin signaling alleviates PA-induced hepatic steatosis.

<p>Schematic diagram illustrating a possible mechanism by which exendin-4-driven β-catenin signaling alleviates PA-induced hepatic steatosis.</p

Exendin-4 inhibits palmitic acid-induced hepatic lipogenesis and TG synthesis.

<p>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.</p

Dose-dependent and time-dependent effects of exendin-4 on β-catenin expression in hepatocytes.

<p>(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.</p

Palmitic acid stimulates lipid accumulation in hepatocytes.

<p>(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.</p

Change of Sirt1 and AMPK pathway and GLP-1R expression by exendin-4 in mouse livers.

<p>Total RNA and protein were extracted from liver tissue and (A) <i>Nampt</i>, <i>Sirt1</i>, <i>Lkb1</i>, <i>AMPK</i>α1 and <i>AMPK</i>α1 mRNA and (B) GLP-1R, Sirt1, phospho-AMPKα, AMPK and β-actin protein expression were measured by quantitative real-time RT-PCR and western blot, respectively. Data were normalized to the β-actin of each sample. All values are expressed as the mean ± SE for n = 5–7 mice. * <i>p</i><0.05, ** <i>p</i><0.01 compared with a control and # <i>p</i><0.05, ## <i>p</i><0.01 compared with a HF.</p