Anti-obesity effects of Lysimachia foenum-graecum characterized by decreased adipogenesis and regulated lipid metabolism

Abstract

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 μg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 μg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Figure 1

LFE inhibits adipocyte differentiation. (A) Mouse 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of various concentrations of LFE for 6 days. Intracellular lipids were stained with Nile Red and fluorescence intensity was measured. Relative lipid accumulation was calculated as follows: (fluorescence intensity of treated cells / fluorescence intensity of DMSO-treated control cells) ×100. (B) After staining with Nile Red, cells were counter-stained with Hoechst 33342 and photographed with IN Cell Analyzer 1000 (GE Healthcare). (C) Cells were harvested at day 6 and the expression of PPARγ and C/EBPα was analyzed by Western blotting. (D) Total RNA was extracted at day 6 and the expression level of PPARγ, FAS, ACC1, SCD1, aP2, and adiponectin mRNA was analyzed by qPCR. Each bar represents mean ± SD of triplicate PCR reactions. Similar results were obtained from two independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Figure 2

LFE suppresses the expression and activation of PPARγ. (A) Differentiated 3T3-L1 adipocytes were treated with DMSO or LFE (2 or 10 µg/ml) for 24 h. Total protein was extracted and the expression of PPARγ and C/EBPα was analyzed by Western blotting. Experiment was done in duplicate. (B) HEK 293 cells were transfected with DR-1-luciferase reporter along with PPARγ and RXRα expression vectors. After 6 h, cells were treated with DMSO, rosiglitazone (1 µM), BADGE (100 µM), or LFE (10 µg/ml) and incubated for 24 h. Experiments were done in quadruple and luciferase activity was measured and expressed as mean ± SD. Similar results were obtained from two independent experiments. ^*P < 0.05 vs. lane 2, ^#P < 0.05 vs. lane 3.

Figure 3

LFE modulates the expression of genes involved in lipid metabolism. Differentiated 3T3-L1 adipocytes (A) or C2C12 cells (B) were treated with 10 µg/ml LFE for 24 h. Total RNA was isolated and analyzed by qPCR for the expression of lipogenic genes (ADD1/SREBP1c, FAS, ACC1 and SCD1) or lipolytic genes (ACO1, CPT1, PPARα and PPARδ). Each bar represents mean ± SD of triplicate PCR reactions. Similar results were obtained in two independent experiments. ^*P < 0.05, ^**P < 0.01.

Figure 4

LFE stimulates fatty acid oxidation by activating AMPK. (A) Differentiated 3T3-L1 adipocytes were treated with DMSO, LFE (2 and 10 µg/ml) or berberine (30 µg/ml) for 30 min after starving cells in DMEM containing 0.1% BSA for 16 h. Berberine was used as a positive control for activating AMPK. Total cell lysates were subjected to Western blot analysis using antibodies specific for phospho-AMPK, phospho-ACC, total AMPK, total ACC, and β-actin. (B) Differentiated C2C12 cells were treated with or without LFE (10 µg/ml) or compound C (10 µM) and fatty acid oxidation assays were performed. Each bar represents mean ± SD from three independent experiments. *P < 0.05 vs. DMSO, ^#P < 0.05 vs. LFE.

Figure 5

LFE ameliorates obesity induced by HFD. Mice were fed either a ND or HFD for 6 weeks in the presence (30, 100, and 300 mg/kg/day) or absence of LFE (n = 8). (A) Changes in body weight. (B) Absolute amount of food intake per mouse. (C) Comparison of fat pad weight from abdominal subcutaneous, epididymal, and perirenal WATs. (D) Histological analysis of the epididymal WATs after staining with hematoxylin and eosin. (E) Effect of LFE on the expression of genes involved in lipid metabolism in the epididymal WAT. (F) Effect of LFE on the expression of C/EBPα and PPARγ proteins in the epididymal WAT. Epididymal WATs were obtained from two animals of each group and each lane represents the protein level of individual WAT. (G) Effect of LFE on the phosphorylation of AMPK in the epididymal WAT. Each bar represents mean ± SD from eight mice. ^*P < 0.05 vs. ND+Vehicle, ^***P <0.001 vs. ND + Vehicle, ^#P < 0.05 vs. HFD + Vehicle, ^##P < 0.01 vs. HFD + Vehicle, ^###P < 0.001 vs. HFD + Vehicle.

Figure 6

LFE improves plasma lipid parameters. Plasma was obtained after fasting for 16 h from ND or HFD mice treated with either vehicle or 100 mg/kg LFE as described in Figure 5. (A) Concentration of triglyceride and glucose in plasma. (B) Plasma concentration of leptin and resistin. (C) Plasma adiponectin levels adjusted to epididymal fat mass. Each bar represents mean ± SD from eight individual animals. ^***P < 0.001 vs. ND + Vehicle, ^#P < 0.05 vs. HFD+Vehicle, ^##P < 0.01 vs. HFD + Vehicle, ^###P < 0.001 vs. HFD+Vehicle.