Standardized Ethanol Extract of Cassia mimosoides var. nomame Makino Ameliorates Obesity via Regulation of Adipogenesis and Lipogenesis in 3T3-L1 Cells and High-Fat Diet-Induced Obese Mice

Figures

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  Figure 1. (A) UPLC of the ethanol extract of C. mimosoides var. nomame Makino (EECM) detected at 254 nm. (B) High-resolution mass spectrometry (HR/MS) spectrum of [M + H] ions (m/z 499) of quercitrin (Rt: 4.033 min). High-performance liquid chromatography (HPLC) profiles of (C) EECM and (D) standard quercitrin.

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  Figure 2. Inhibitory effects of EECM on intracellular lipid accumulation, adipogenesis, and lipogenesis in 3T3-L1 cells. The 3T3-L1 cells were cultured in the growth medium (GM) for four days. After the cells reached confluency, they were cultured with or without methylisobutylxanthine, dexamethasone, and insulin (MDI) and various concentrations of EECM. (A) Relative microscopic images of Oil-Red-O-stained 3T3-L1 cells. (B) Quantitative lipid accumulation ratio. After differentiation, total protein was extracted and detected by via Western blotting analysis. Expression levels of (C) AMPKα pathway proteins and (D) lipogenic and (E) adipogenic transcription factors. Values are represented as the mean ± standard deviation (SD). # p < 0.05 vs. the GM group; *** p < 0.001 vs. the differentiation medium (DM) group.

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  Figure 3. Regulatory effect of EECM on the MCE in 3T3-L1 cells. Differentiated cells were treated with EECM (6.25, 12.5, and 25 μg/mL) for 24 h. (A) Cell cycle phase ratio and (B) cell-cycle-related proteins expression were measured via propidium iodide (PI) staining and Western blotting, respectively.

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  Figure 4. Repressive effect of EECM on body weight gain in HFD (60%)-induced obese mice. Mice of EECM treatment were provided the indicated dose of EECM (100 and 300 mg/kg) daily during the experiment. (A) Body weight changes, (B) total body weight at the endpoint of the experiment, and (C) total body weight gain for 10 weeks. Values are represented as the mean ± SEM of eight mice per group. # p < 0.05 vs. the control (CON) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the HFD (60%)-induced obese group.

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  Figure 5. Regulatory effects of EECM on body composition and fat mass in HFD (60%)-induced obese mice. (A) Radiography images of the fat tissues are presented. (B) Fat weight in each group was measured via DEXA analysis. Fat was obtained from each tissue after the sacrifice of mice, and the weight was measured. (C–F) Subcutaneous fat, mesenteric fat, gonadal fat, and perirenal fat weights. Values are represented as the mean ± SEM (DEXA analysis; n = 3/group, fat in tissue weight: n = 8/group). # p < 0.05 vs. the CON group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the HFD (60%)-induced obese group.

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  Figure 6. Regulatory effect of EECM on the production of adipocyte-released hormones in HFD (60%)-induced obese mice. Plasma levels of (A) insulin, (B) leptin, and (C) adiponectin were detected using ELISA kit. Values are represented as the mean ± SEM (n = 8/group). # p < 0.05 vs. the CON group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the HFD (60%)-induced obese group.

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  Figure 7. Effects of EECM on adipocyte size, adipogenesis, and lipogenesis in the subcutaneous fat tissues of HFD (60%)-induced obese mice. (A) Histological analysis of subcutaneous fat tissue via H&E staining at ×20 magnification. (B) Representative diameter of adipocytes in the tissue. Expression levels of (C) p-AMPKα, (D) lipogenic proteins, and (E) adipogenic transcription factors were determined via Western blotting analysis. Values are represented as the mean ± SEM (n = 8/group). # p < 0.05 vs. the CON group; *** p < 0.001 vs. the HFD (60%)-induced obese group.

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  Figure 8. Effect of EECM on lipid accumulation in the liver tissues of HFD (60%)-induced obese mice. (A) The images of H&E stained the liver tissues at ×40 magnification. (B) Comparison of liver weights of each group. Expression levels of (C) p-AMPKα, (D) lipogenic proteins, and (E) adipogenic transcription factors were determined via Western blotting analysis. Values are represented as the mean ± SEM (n = 8/group). # p < 0.05 vs. the CON group; *** p < 0.001 vs. the HFD (60%)-induced obese group.

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  Figure 9. Stimulating effects of EECM on thermogenesis in the BAT. (A) Weights of BAT in each group. (B) Expression levels of thermogenesis-related proteins PGC-1α, PPARα, SIRT1, UCP-1, and COX-IV were determined via Western blotting analysis. Values are represented as the mean ± SEM (n = 8/group). # p < 0.05 vs. the CON group.

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  Figure 10. Regulatory effects of EECM on microbiota composition and colonization. (A) β-diversity between groups was analyzed using the principal coordinate analysis (PCoA) plot. (B) Ratio of phylum level composition ratio of each group. (C–F) Relative ratio of Bacteroidetes, Firmicutes, and Proteobacteria and the Firmicutes/Bacteroidetes (F/B) ratio. Values are represented as the mean ± SEM of five mice per group. # p < 0.05 vs. the CON group; * p < 0.05 and ** p < 0.01 vs. the HFD (60%)-induced obese group.