Protective Effects of Licochalcone A Ameliorates Obesity and Non-Alcoholic Fatty Liver Disease Via Promotion of the Sirt-1/AMPK Pathway in Mice Fed a High-Fat Diet

Abstract

Licochalcone A is a chalcone isolated from Glycyrrhiza uralensis. It showed anti-tumor and anti-inflammatory properties in mice with acute lung injuries and regulated lipid metabolism through the activation of AMP-activated protein kinase (AMPK) in hepatocytes. However, the effects of licochalcone A on reducing weight gain and improving nonalcoholic fatty liver disease (NAFLD) are unclear. Thus, the present study investigated whether licochalcone A ameliorated weight loss and lipid metabolism in the liver of high-fat diet (HFD)-induced obese mice. Male C57BL/6 mice were fed an HFD to induce obesity and NAFLD, and then were injected intraperitoneally with licochalcone A. In another experiment, a fatty liver cell model was established by incubating HepG2 hepatocytes with oleic acid and treating the cells with licochalcone A to evaluate lipid metabolism. Our results demonstrated that HFD-induced obese mice treated with licochalcone A had decreased body weight as well as inguinal and epididymal adipose tissue weights compared with HFD-treated mice. Licochalcone A also ameliorated hepatocyte steatosis and decreased liver tissue weight and lipid droplet accumulation in liver tissue. We also found that licochalcone A significantly regulated serum triglycerides, low-density lipoprotein, and free fatty acids, and decreased the fasting blood glucose value. Furthermore, in vivo and in vitro, licochalcone A significantly decreased expression of the transcription factor of lipogenesis and fatty acid synthase. Licochalcone A activated the sirt-1/AMPK pathway to reduce fatty acid chain synthesis and increased lipolysis and β-oxidation in hepatocytes. Licochalcone A can potentially ameliorate obesity and NAFLD in mice via activation of the sirt1/AMPK pathway.

Keywords: AMPK; HepG2; licochalcone A; lipolysis; nonalcoholic fatty liver disease; obesity.

Figure 1

Licochalcone A (LA) reduced body weight in high-fat diet (HFD)-induced obese mice. (A) Male mice were fed an HFD (containing 60% fat) for 16 weeks, and administered DMSO, 5 mg/kg licochalcone A (LA5), or 10 mg/kg licochalcone A (LA10) by intraperitoneal injection (I.P.) twice a week from week 4 to 16. (B) Weight gain was measured for 16 weeks. (C) The appearance of the animal and (D) weight gain measured in the last week. (E) Food intake monitored each day. Data are presented as the mean ± SEM; n = 12. * p < 0.05, ** p < 0.01 compared with mice with HFD-induced obesity.

Figure 2

Licochalcone A (LA) reduced the epididymal and inguinal adipose tissue weights in HFD-induced obese mice. (A) The appearance and (B) weight of epididymal adipose tissue. (C) HE staining of epididymal adipose tissue (200× magnification). (D) The adipocyte size in epididymal adipose tissue. (E) The appearance and (F) weight of inguinal adipose tissue. (G) HE staining of inguinal adipose tissue (200× magnification). (H) The adipocyte size in inguinal adipose tissue. Data are presented as the mean ± SEM; n = 12. * p < 0.05, ** p < 0.01 compared with mice with HFD-induced obesity.

Figure 3

Licochalcone A (LA) ameliorated hepatic steatosis in HFD-induced obese mice. (A) The appearance of the liver, (B) liver weight, and (C) HE staining of liver tissues (200× magnification). (D) The calculated number of lipid droplets in liver tissue. (E) PAS staining demonstrating the glycogen distribution in the liver (200× magnification). (F) TG and (G) TC levels in the liver. (H) NAFLD scores based on the evaluation of HE staining of liver tissues. Data are presented as the mean ± SEM; n = 12. * p < 0.05, ** p < 0.01 compared with mice with HFD-induced obesity.

Figure 4

Effects of licochalcone A (LA) on lipid metabolism in mouse liver tissue. (A) Expression of transcription factors associated with adipogenesis and lipogenesis, (B) lipolysis, (C) β-oxidation, and (D) the sirt-1/AMPK pathway were detected by Western blot. (E,F) The fold expression levels were measured relative to the expression of β-actin. Three independent experiments were analyzed using β-actin as an internal control. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 compared to the HFD group.

Figure 5

Licochalcone A (LA) modulated lipogenesis and lipolysis gene expression in liver tissue. Gene expression levels were determined using real-time PCR. * p < 0.05, ** p < 0.01 compared with mice with HFD-induced obesity.

Figure 6

Licochalcone A (LA) modulated FAS, sirt-1, and CPT-1 expression in the liver. Expression levels of CPT-1, sirt1, and FAS were analyzed by immunohistochemistry and labeled as a brown color drop.

Figure 7

Licochalcone A (LA) reduced lipid accumulation in HepG2 cells. HepG2 cells were treated with 0.5 mM oleic acid (OA) at 37 °C for 48 h to induce lipid accumulation in hepatocytes, followed by treatment with licochalcone A (1.5–12 μM) for 24 h. (A) The fluorescent dye BODIPY 493/503 (green) was used to detect hepatic lipid droplets using a fluorescent microscope. Three independent experiments were analyzed. Nuclei were stained with DAPI (blue), and (B) Fluorescent images were quantified. (C) Oil red O staining showed lipid accumulation. (D) HepG2 cells treated with isopropanol and lipid accumulation measured using the absorbance at OD 490 nm. Three independent experiments were analyzed. The data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01 compared with oleic acid-induced HepG2 cells.

Figure 8

Licochalcone A (LA) reduced lipoperoxidation and fatty acid uptake into HepG2 cells. (A) HepG2 cells were treated with 0.5 mM oleic acid (OA) at 37 °C for 48 h to induce lipid accumulation in hepatocytes, followed by treatment with licochalcone A (1.5–12 μM) for 24 h. The fluorescent dye BODIPY 581/591 C11 (red) was used to detect hepatic lipoperoxidation under a fluorescent microscope. Three independent experiments were analyzed. Nuclei were stained with DAPI (blue). (B) Fluorescent images were quantified and compared with oleic acid-induced HepG2 cells. (C) Oleic acid-induced HepG2 cells were treated with licochalcone A for 24 h before staining with the fluorescent probe BODIPY FL C12 (green). Nuclei were stained with DAPI (blue). (D) Fluorescent images were quantified and data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01 compared with oleic acid-induced HepG2 cells. Three independent experiments were analyzed.

Figure 9

Effects of licochalcone A (LA) on lipid metabolism in HepG2cells. HepG2 cells were treated with 0.5 mM oleic acid (OA) for 48 h to induce lipid accumulation in hepatocytes, followed by treatment with licochalcone A (1.5–12 μM) for 24 h. (A) The expression of transcription factors associated with adipogenesis and lipogenesis proteins, (B) lipolysis, (C) β-oxidation, and (D) the AMPK/Sirt-1 pathway were detected by Western blot. (E,F) The fold expression levels were measured relative to the expression of β-actin. Three independent experiments were analyzed, and data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 compared to oleic acid-induced HepG2 cells.

Figure 10

Effects of licochalcone A (LA) on AMPK/Sirt-1 pathway in HepG2 cells. HepG2 cells were treated with 0.5 mM oleic acid (OA) for 48 h, followed by licochalcone A (12 μM) with or without (A,B) an AMPK inhibitor (compound C) or (C,D) an AMPK activator (AICAR) for 24 h. Three independent experiments were analyzed using β-actin as an internal control. The images were quantified and data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01 compared with oleic acid-induced HepG2 cells.