Shizukaol D isolated from Chloranthus japonicas inhibits AMPK-dependent lipid content in hepatic cells by inducing mitochondrial dysfunction

Abstract

This study is the first to demonstrate that shizukaol D, a natural compound isolated from Chloranthusjaponicus, can activate AMP- activated protein kinase (AMPK), a key sensor and regulator of intracellular energy metabolism, leading to a decrease in triglyceride and cholesterol levels in HepG2 cells. Furthermore, we found that shizukaol D induces mitochondrial dysfunction by depolarizing the mitochondrial membrane and suppressing energy production, which may result in AMPK activation. Our results provide a possible link between mitochondrial dysfunction and AMPK activation and suggest that shizukaol D might be used to treat metabolic syndrome.

Figure 1. Chemical structure of shizukaol D from Chloranthus japonicas .

Figure 2. Shizukaol D increases AMPK and ACC phosphorylation in HepG2 cells.

Western blotting analysis showing the levels of phosphorylated AMPK and ACC in HepG2 cells treated with shizukaol D. (A) HepG2 cells were treated with shizukaol D at the indicated concentrations for 1 h. Metformin (2 mM) was used as a positive control. (D) The cells were treated with 2 µM shizukaol D for the indicated time points. (B) (C), (E) and (F) the levels of phosphorylated AMPK and ACC were quantified from three independent experiments. *, p<0.05; **, p<0.01 compared to treatment with DMSO (one-way ANOVA).

Figure 3. Shizukaol D inhibits lipid accumulation in HepG2 cells.

HepG2 cells were starved in serum-free medium overnight and then treated with shizukaol D at the indicated concentrations for 24 h. Metformin (2 mM) was used as a positive control. Western blotting analysis showing phosphorylated AMPK and ACC (A). The triglyceride content (B) and cholesterol content (C) were measured (Results correspond to the mean ± SD of six independent experiments, statistical analysis was performed using one-way ANOVA followed by post- hoc test. *, p<0.05; **, p<0.01 versus the DMSO control). The cells were starved in serum-free medium overnight and then treated with shizukaol D at the indicated concentrations in the presence of 5.5 mM or 30 mM glucose for an additional 24 hours. The expression of AMPK and ACC was detected by western blotting (D), and the triglyceride content (E) and cholesterol content (F) were measured (graphics represent the mean ± SD from six independent experiments. *, p<0.05; **, p<0.01 versus the DMSO control).

Figure 4. Shizukaol D inhibits lipid accumulation in HepG2 cells in an AMPK-dependent manner.

HepG2 cells were transfected with AMPK siRNA or a control siRNA for 24 h followed by incubation with 2 µM shizukaol D or 2 mM metformin for an additional 24 h. AMPK and ACC phosphorylation was analyzed by western blotting (A), and the triglyceride content (B) and cholesterol content (C) were measured (n = 3). (D) The cells were pretreated with 20 µM compound C (an AMPK inhibitor) followed by treatment with 2 µM shizukaol D. AMPK and ACC phosphorylation was analyzed by western blotting (D), and the triglyceride content (E) and cholesterol content (F) were measured (n = 3). Statistical analysis was performed using two-way ANOVA followed by Tukey’ post-hoc test *, p<0.05; **, p<0.01.

Figure 5. Shizukaol D inhibits the mitochondrial membrane potential and increases the AMP/ATP ratio in HepG2 cells.

(A) HepG2 cells were incubated with shizukaol D for 10 min, and the mitochondrial membrane potential was measured. Treatment with CCCP was used as a positive control (n = 4). (B) HepG2 cells were treated with shizukaol D at the indicated concentrations for 1 h, and then the AMP/ATP ratio was measured (n = 3). (C) The cells were treated with 2 µM shizukaol D for the indicated time-points, and then the AMP/ATP ratio was measured (n = 3). *, p<0.05; **, p<0.01 compared to the DMSO control (one-way ANOVA).

Figure 6. Shizukaol D inhibits cellular respiration.

(A) Dose-dependent inhibition of HepG2 cell respiration by treatment with shizukaol D at the indicated concentrations (n = 4). (B) Effect of shizukaol D on the respiration of mitochondria isolated from HepG2 cells (n = 3). Shizukaol D did not inhibit mitochondrial respiration either in the presence of complex I (glutamate + malate) or complex II (succinate) substrates. (C) And (D) Lactate concentrations were measured in HepG2 cells treated with shizukaol D as indicated time (1 h and 4 h) (n = 3). *, p<0.05; **, p<0.01 compared to the DMSO control (one-way ANOVA).