Anti-Obesity and Lipid Lowering Activity of Bauhiniastatin-1 is Mediated Through PPAR-γ/AMPK Expressions in Diet-Induced Obese Rat Model

Abstract

Objective: To evaluate the therapeutic efficacy and underlying molecular mechanisms of Bauhiniastatin-1 (BSTN1) to alleviate adiposity in diet-induced obese rodent model and in 3T3-L1 cells. Methods: BSTN1 was purified and confirmed through HPLC. In-vitro experiments such as MTT assay, Oil Red-O (ORO) stain, cellular lipid content, glycerol release and RT-PCR analysis were performed in 3T3-L1 cells in the presence and absence of BSTN1. In animal experiments, rats were divided into Group-I: normal pellet diet-fed, Group-II: HFD-fed, Groups-III, IV and V: HFD-fed BSTN1 (1.25, 2.5, and 5 mg/kg.b.wt./day/rat)-treated and Group-VI: HFD-fed Orlistat-treated. The rats were fed either normal diet or high fat diet (HFD) for 18 weeks and water ad-libitum. BSTN1 was orally administered from 13th week onwards to the selected HFD-fed groups. Body composition parameters, biochemical assays, histopathology examination and western blot analysis were performed to identify the predicted targets related to obesity. Molecular docking studies threw light on the binding interactions of BSTN1 against PPAR-γ, FAS and AMPK. Results: BSTN1 at 20 μM significantly (p < 0.001) inhibited adipocyte differentiation and lipid accumulation in 3T3-L1 cells. A conspicuous down-regulation in the mRNA expression levels of PPAR-γ, FAS and SREBP1 was observed but AMPK expression remained unchanged in BSTN1 treated 3T3-L1 cells. A substantial decrease in body weight gain, fat percent, total body fat, serum and liver lipid profile (except high-density lipoprotein), glucose, insulin and insulin resistance in BSTN1 treated rats was noticed in a dose dependent manner. In BSTN1 (5 mg/kg.b.wt.)-treated groups significantly (p < 0.01) elevated plasma adiponectin level but reduced leptin level as well as fall in serum AST and ALT were noticed. Further, the disturbed structural integrity and architecture of adipose and hepatic tissues due to high fat diet feeding were considerably recovered with BSTN1 treatment. Down-regulation in the protein expression level of PPAR-γ and activation of AMPK through phosphorylation was observed in BSTN1 treated rats than the untreated. Molecular docking studies revealed strong binding interactions of BSTN1 against PPAR-γ and AMPK and thus supported the experimental results. Conclusion: Taken together, the results suggest that BSTN1 could be a promising pharmacological molecule in the treatment of obesity and dyslipidemia.

Keywords: 3T3-L1 cells; AMPK; PPAR-γ; adipogenesis; bauhiniastatin-1; diet-induced obesity; insulin resistance; molecular docking.

FIGURE 1

Molecular characterization of isolated compound/s from the chloroform fraction of B. purpurea ethanolic extract. (A) and (B) LC-MS analysis showing TOF MS ES + BPI and Diode Array (Retention Time) (C) HPLC peak representing structure and retention time (5.942) of BSTN1.

FIGURE 2

Effect of BSTN1 on lipid levels in 3T3-L1 cells by (A) Oil Red O staining of 3T3-L1 cells (Magnification 40×). (i) Undifferentiated, untreated pre-adipocytes (ii) Differentiated, untreated adipocytes (control) (iii) DMSO (0.01%) treated adipocytes (vehicle control). (iv), (v), and (vi) BSTN1 treated adipocytes (5, 10, and 20 μM) showing decreased adipogenesis in dose dependent manner. (B) Quantification of lipid content (%) by Oil Red O stain extraction in BSTN1-treated and other groups. (C) Lipolysis assay indicating the amount of glycerol content released from 3T3-L1 adipocytes into the media. (D) Cytotoxicity assay results on the percentage of cell viability of untreated, DMSO and BSTN1 treated pre-adipocytes determined after 48 h using MTT assay. Data are presented as mean ± SD of triplicate. *, **, and *** indicate significant difference between control and treated 3T3-L1 cells at p < 0.05, p < 0.01, and p < 0.001 respectively.

FIGURE 3

Effect of BSTN1 on mRNA expression of target genes in 3T3-L1 cells by RT-PCR. Quantitation of fold change of key adipogenic and lipogenic markers (A) FAS mRNA (B) SREBP1 mRNA, (C) AMPK mRNA and (D) PPAR-γ mRNA. Data are presented as mean ± SD of triplicate. * and ** indicate significant difference between control and treated 3T3-L1 cells at p < 0.05 and p < 0.01, respectively. FAS, fatty acid synthase; SREBP1, sterol regulatory element-binding protein 1; AMPK, AMP-activated protein kinase and PPAR-γ, peroxisome proliferator-activated receptor-γ.

FIGURE 4

Western blot analysis: Protein expression of control and treated rats in (A) adipose tissue and (E) liver tissue. β-actin is used as a housekeeping gene. Histograms showing quantification of expressed proteins: (B) Adipose-p-AMPK (C) adipose-AMPK (D) adipose-PPAR-γ, (F) Hepatic-p-AMPK (G) Hepatic-AMPK and (H) Hepatic-PPAR-γ. Histograms represent mean ± SD, n = 6. * indicates significant difference (p < 0.01) between HFD control and treated groups. AMPK, AMP-activated protein kinase and PPAR-γ, peroxisome proliferator-activated receptor-γ.

FIGURE 5

Effect of BSTN1 on adipokines and histology of white adipose tissue (40x magnification). (A) Plasma adipokines levels represented as mean ± SD, n = 6. ^# denotes significant (p < 0.001) difference between normal control and HFD control. * and ** denote significant difference between HFD control and treated groups at p < 0.05 and p < 0.01, respectively. (B) Histology of normal control adipose tissue (C) HFD-fed group showing enlarged and lipid laden adipocytes (D) HFD fed BSTN1 treated group showing reduction in adipocytes’ size and lipid content (E) HFD fed orlistat treated group. AC, Adipocyte Cell and AV, Adipocyte Volume. Scale bar: 100 µm.

FIGURE 6

Effect of BSTN1 on liver enzymes (AST and ALT) levels and liver tissue histology (40x magnification). (A) Serum AST and ALT levels represented as mean ± SD, n = 6. ^# denotes significant (p < 0.001) difference between normal control and HFD control. * and ** denote significant difference between HFD control and treated groups at p < 0.05 and p < 0.01, respectively. (B) Histology of normal control hepatic tissue (C) HFD fed group showing disrupted tissue structure with enlarged central vein (D) HFD fed BSTN1 treated group showing improvement in hepatic structure (E) HFD fed orlistat treated group. CV, Central Vein; FD, Fat Droplets and Ep, Epithelial cells. Scale bar: 100 µm.

FIGURE 7

BSTN1 docking to FAS, AMPK and PPAR-γ. (A−C) Computational docking of BSTN1 (red) with the FAS (6NNA). (D−F) Computational docking of BSTN1 (red) with the amino acids of targeting AMPK (6C9F). (G−I) Computational docking reveals that BSTN1 (red) strongly interacts with hydroxyl and amino groups of the tyrosine, serine and histidine residues of PPAR-γ (3WMH). FAS, fatty acid synthase; AMPK, AMP-activated protein kinase and PPAR-γ, peroxisome proliferator-activated receptor-γ.