Corni Fructus Containing Formulation Attenuates Weight Gain in Mice with Diet-Induced Obesity and Regulates Adipogenesis through AMPK

Abstract

Obesity is a metabolic disorder characterized by chronic inflammation and dyslipidemia and is a strong predictor for the development of hypertension, diabetes mellitus, and cardiovascular disease. This study examined the antiobesity effect of an ethanol extract of Corni Fructus containing formulation (CDAP), which is a combination of four natural components: Corni Fructus, Dioscoreae Rhizoma, Aurantii Fructus Immaturus, and Platycodonis Radix. The cellular lipid content in 3T3-L1 adipocytes was assessed by Oil Red O staining. Expressions of peroxisome proliferator-activated receptor- γ (PPAR- γ ), CCAAT/enhancer-binding protein- α (C/EBP- α ), and lipin-1 were determined by real-time RT-PCR. Western blot was used to determine the protein levels of PPAR- γ , C/EBP- α , and AMP-activated protein kinase- α (AMPK- α ). The CDAP extract suppressed the differentiation of 3T3-L1 adipocytes by downregulating cellular induction of PPAR- γ , C/EBP- α , and lipin-1. The CDAP extract also significantly upregulated phosphorylation of AMPK- α . An in vivo study showed that CDAP induced weight loss in mice with high-fat-diet-induced obesity. These results indicate that CDAP has a potent anti-obesity effect due to the inhibition of adipocyte differentiation and adipogenesis.

Figure 1

Effect of CDAP on cell viability and lipid accumulation in 3T3-L1 cells. (a) Cells were incubated with CDAP at the indicated concentration for 48 h. Cell viability was assessed by MTS assay. (b) Postconfluent 3T3-L1 cells were differentiated in the absence or in the presence of CDAP (0, 10, 50, and 100 μg/mL) for 6 days. Lipid droplets were measured by Oil Red O staining. EGCG was used as a positive control. All values are mean ± S.D. ^# P < 0.05 versus undifferentiated control cells; *P < 0.05 versus differentiated control cells.

Figure 2

Effect of CDAP on the expression of transcription factors and adipocyte-specific genes in differentiation of 3T3-L1 cells. Postconfluent 3T3-L1 cells were differentiated in the absence or presence of CDAP (0, 10, 50, and 100 μg/mL) for 6 days. The mRNA of PPAR-γ (a), C/EBP-α (b), and lipin-1 (c) was analyzed by real-time RT-PCR. Results were expressed relative to untreated cells after normalization to GAPDH mRNA. EGCG was used as a positive control. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. ^# P < 0.05 versus undifferentiated control cells; *P < 0.05 versus differentiated control cells.

Figure 3

Effect of CDAP on the protein expressions of transcription factors in differentiation of 3T3-L1 cells. Postconfluent 3T3-L1 cells were differentiated in the absence or presence of CDAP (0, 10, 50, and 100 μg/mL) for 6 days. PPARγ (a), C/EBPα (b), and lipin-1 (c) protein expressions were analyzed by western blot analysis. EGCG was used as a positive control. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. ^# P < 0.05 versus undifferentiated control cells; *P < 0.05 and **P < 0.01 versus differentiated control cells.

Figure 4

Effect of CDAP and its constituents on phosphorylation of AMPK during 3T3-L1 differentiation. Postconfluent 3T3-L1 cells were differentiated in the presence or absence of Corni Fructus, Dioscoreae Rhizoma, Aurantii Fructus Immaturus, and Platycodonis Radix for 6 days. (a) Lipid accumulation was measured by an Oil Red O staining assay. (b) The cytotoxicity of each constituent, naringin and platycodin D, which are more effective herb's constituents on lipid accumulation, was examined using an MTS assay. (c) Effect of CDAP on AMPK activation was compared with naringin and platycodin D, a major constituent of Aurantii Fructus Immaturus and Platycodonis Radix, respectively. Protein levels of phosphorylated AMPK (pAMPK) and total AMPK were determined by western blot analysis. The protein expression differences are normalized to AMPK-α. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. ^# P < 0.05 versus undifferentiated control cells; *P < 0.05 versus differentiated control cells.

Figure 5

Effects of CDAP in HF diet-induced obesity mice. Mice (n = 5 per group) were administrated CDAP extract (100 mg/kg/day) with their HF diet for 16 weeks. Normal diet (blank) fed mice were administrated with vehicle. Slinti (5 mg/kg/day) was administrated as a positive control. Changes in body weight (a), the weight difference between the start weight and end weight of each group (b), LDL cholesterol (c), HDL cholesterol (d), total cholesterol (e), and fructosamine (f) of the mice were measured. All values are mean ± S.D. ^# P < 0.05 significantly different from blank (normal diet group); *P < 0.05 significantly different from HF diet group (60%).

Figure 6

HPLC chromatogram of CDAP. The peaks were assigned based on the isolation of each compound. (a) Corni Fructus: logarnin; (b) Dioscoreae Rhizoma: allantoin; (c) Aurantii Fructus Immaturus: naringin; (d) Platycodonis Radix: platycodin D. The herbs were extracted with 80% ethanol (80% EtOH).