Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

Abstract

Aim: To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.

Methods: HepG2 and C2C12 cells were used. Cell viability was determined using MTT assay. Real-time PCR was performed to measure the gene expression. Western blotting assay was applied to investigate the protein phosphorylation level. Enzymatic assay kits were used to detect the total cholesterol (TC), triglyceride (TG) and glucose contents.

Results: Danthron (0.1, 1, and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in both HepG2 and C2C12 cells. Meanwhile, danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions, and the TC and TG levels. In addition, danthron treatment efficiently increased glucose consumption. The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.

Conclusion: Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

Figure 1

Regulation of danthron on AMPK activation and lipid accumulation in HepG2 cells. (A) Chemical structure of danthron. (B) Concentration-dependent effects of danthron on AMPK and ACC phosphorylation detected by Western blotting. (C) Effect of danthron on cell viability examined using the MTT assay. (D and E) The mRNA levels of SREBP1c and FAS determined by RT-PCR. (F and G) The levels of intracellular TG and TC measured by the enzymatic colorimetry method. GS (40 μmol/L) was used as a positive control. Data are presented as the mean±SEM of three independent experiments. ^bP<0.05, ^cP<0.01 vs DMSO.

Figure 2

Increases in ABCA1 and ABCG1 promoter activities and gene expressions by danthron treatment. (A and B) The effects of danthron on ABCA1 and ABCG1 promoter activities were determined by luciferase reporter gene-based assay. TO90 (TO901317, 2 μmol/L) was used as a positive control. (C and D) The gene expressions of ABCA1 and ABCG1 were detected in HepG2 cells. (E and F) The mRNA levels of ABCA1 and ABCG1 were tested in C2C12 cells. Data are presented as the mean±SEM of three independent experiments. ^bP<0.05, ^cP<0.01 vs DMSO.

Figure 3

Effects of danthron on AMPK activation and lipid accumulation in mature C2C12 cells. (A) Danthron efficiently increased the phosphorylation of AMPK and ACC. (B and C) Concentration-dependent effects of danthron on SREBP1c and FAS gene expressions were determined. (D and E) Danthron attenuated the intracellular TG and TC contents. The concentrations of danthron were indicated. Data are presented as the mean±SEM of three independent experiments. ^bP<0.05, ^cP<0.01 vs DMSO.

Figure 4

The regulation of the lipid metabolism-related genes by danthron was AMPK-dependent. The phosphorylation of both AMPK and ACC was increased after treatment with danthron and abolished by co-incubation with compound C in both HepG2 (A) and C2C12 (D) cells. Meanwhile, decreases in SREBP1c and FAS mRNA levels induced by danthron were reversed after co-incubation with compound C in HepG2 (B and C) and C2C12 (E and F) cells. Met (10 mmol/L) was used as a positive control. The concentrations of both danthron and compound C were 10 μmol/L. Data are presented as the mean±SEM of three independent experiments. ^bP<0.05, ^cP<0.01 vs DMSO or as indicated.

Figure 5

Enhancements by danthron on glucose consumption in HepG2 and C2C12 cells. The glucose uptake was concentration-dependently increased with danthron administration in both HepG2 (A) and C2C12 (C) cells. Such increases were attenuated by compound C in HepG2 (B) and C2C12 (D) cells, respectively. Key proteins of the insulin signaling pathway (E) were detected in HepG2 cells by Western blotting. The concentrations of compounds were indicated. Data are presented as the mean±SEM of three independent experiments. ^bP<0.05, ^cP<0.01 vs DMSO or as indicated.