Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-Activated Protein Kinase Alpha In vivo and In vitro

Abstract

Chrysophanic acid (CA) is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD)-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK) inhibitor, the action of CA on AMPKα was nullified in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases, we suggest the new potential of CA as an anti-obese pharmacotherapy.

Keywords: AMP-activated protein kinase alpha; adipogenesis; chrysophanic acid; obesity; thermogenesis.

FIGURE 1

Chrysophanic acid suppresses weight gain in HFD-induced obese C57BL/6J mice. (A) Weight changes for 16 weeks, (B) iWAT, eWAT, liver, and BAT tissue weights were measured. (C) Total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, glucose, (D) AST, and AST of blood serum were measured. Data represent means ± SD. ^#p < 0.05 compared with NC, ^∗p < 0.05 compared with HFD. NC, normal control group; HFD, high-fat diet group; HFD + CA, high-fat diet plus CA group (n = 5 per group).

FIGURE 2

Chrysophanic acid shows inhibitory effects on adipogenesis-related factors in tissues of C57BL/6J mice. (A) Visual comparisons and (B) H&E stainings of iWAT (magnification 200×, scale bar 100 μm), liver (magnification 200×, scale bar 100 μm), and BAT (magnification 400×, scale bar 100 μm) were performed. Real-time RT-PCR analyses of (C) Pparg, Cebpa, and Sirt1 in iWAT, (D) Pparg, Cebpa, and Lipin1 in liver, and (E) Ucp1, Pgc1a, and Sirt3 in BAT were performed. GAPDH was used as endogenous control. Data represent means ± SD of three independent experiments. ^#p < 0.05 compared with NC, ^∗p < 0.05 compared with HFD. NC, normal control group; HFD, high-fat diet group; HFD + CA, high-fat diet plus CA group (n = 5 per group).

FIGURE 3

Chrysophanic acid impairs adipocyte development in zebrafish. Zebrafish larvae treated with either DMSO (10 μM) or CA (10 μM) were shown in lateral views with anterior to the right. Both bright-field (Left) and fluorescent images (Right) were paired to visualize the location of adipocytes detected by Nile red. The quantification of the signal intensity from Nile red-positive adipocytes was measured by making multi-point selections of adipocytes using ImageJ. ^∗p < 0.05 compared with DMSO-treated control. DMSO, DMSO-treated control group; CA, CA-treated group (n = 15 per group).

FIGURE 4

Chrysophanic acid suppresses adipogenic factors in 3T3-L1 adipocytes. (A) An Oil Red O analysis was performed in order to measure the lipid accumulation (magnification 200×, scale bar 100 μm). (B) Relative optic density was measured at 500 nm. Real-time RT-PCR analyses of (C) Pparg, Cebpa, (D) Glut4, Lipin1, aP2, and (E) Sirt1 were performed. EGCG (10 μM) and GW (10 μM) were used as positive controls. Trog (10 μM) was used as a negative control. GAPDH was used as endogenous control. Data represent means ± SD of three independent experiments. ^#p < 0.05 compared with MDI-uninduced preadipocytes, ^∗p < 0.05 compared with MDI-induced adipocytes. MDI, differentiation medium.

FIGURE 5

Chrysophanic acid suppresses PPARγ in competition against the PPARγ agonist troglitazone in 3T3-L1 adipocytes. (A) Western blot analyses of PPARγ and C/EBPα were performed and (B) quantification of the protein bands was measured using ImageJ. (C) Western blot analysis of PPARγ was performed under co-treatment of CA with GW or Trog, and (D) quantification of the protein bands was measured using ImageJ. EGCG (10 μM) and GW (10 μM) were used as positive controls. Trog (10 μM) was used as a negative control. GAPDH was used as endogenous control. Data represent means ± SD of three independent experiments. ^#p < 0.05 compared with MDI-uninduced preadipocytes, ^∗p < 0.05 compared with MDI-induced adipocytes. MDI, differentiation medium.

FIGURE 6

Chrysophanic acid upregulates brown-fat-specific factors at both mRNA and protein levels in primary cultured brown adipocytes. (A) An MTS assay was performed to measure the survival rate of primary cultured brown adipocytes after treatment with CA. (B) Mitochondrial abundance in primary brown adipocytes was analyzed by MitoTracker Red staining (magnification 200×, scale bar 100 μm). (C) Real-time RT-PCR analyses of Ucp1, Pgc1a, and Prdm16 were performed. (D) Western blot analyses of UCP1 and PGC1α were performed. (E) Quantification of the protein bands was measured using ImageJ. RSV (500 nM) was used as a positive control. GAPDH was used as endogenous control. Data represent means ± SD of three independent experiments. ^#p < 0.05 compared with DM-uninduced preadipocytes, ^∗p < 0.05 compared with DM-induced adipocytes. DM, differentiation medium.

FIGURE 7

Chrysophanic acid induces AMPK phosphorylation resulting in inhibition of adipogenesis in 3T3-L1 adipocytes and activation of thermogenesis in primary cultured brown adipocytes. (A) Western blot analyses of p-AMPKα and AMPKα were performed in CA-treated 3T3-L1 adipocytes. (B) Western blot analyses of p-AMPKα and AMPKα were performed under co-treatment of CA and CC in 3T3-L1 adipocytes. (C) Western blot analyses of p-AMPKα and AMPKα were performed under co-treatment of CA and CC in primary cultured brown adipocytes. The phosphorylation level of AMPKα was measured by p-AMPKα/AMPKα rate. GAPDH was used as endogenous control. Data represent means ± SD of three independent experiments. ^#p < 0.05 compared with MDI- or DM-uninduced preadipocytes, ^∗p < 0.05 compared with MDI- or DM-induced adipocytes. MDI and DM, differentiation medium.