Ascochlorin Attenuates the Early Stage of Adipogenesis via the Wnt/β-Catenin Pathway and Inhibits High-Fat-Diet-Induced Obesity in Mice

Abstract

This study investigated the effects of ascochlorin (ASC), a natural compound derived from the fungus Ascochyta viciae, on adipogenesis and obesity. We determined the effects of ASC on 3T3-L1 preadipocytes and whether it ameliorated to mitigate high-fat diet (HFD)-induced obesity in C57BL/6J mice. We found that ASC significantly inhibited the differentiation of preadipocytes by modulating the Wnt/β-catenin signaling pathway, a key regulator of adipogenic processes. Treatment with ASC not only reduced the mRNA and protein expression of key adipogenic transcription factors such as C/EBPα and PPARγ, but also reduced lipid accumulation both in vitro and in vivo. In addition, treatment HFD-fed mice with ASC significantly reduced their weight gain and adiposity vs. control mice. These results suggest that ASC has considerable potential as a therapeutic agent for obesity, owing to its dual action of inhibiting adipocyte differentiation and reducing lipid accumulation. Thus, ASC represents a promising candidate as a natural anti-obesity agent.

Keywords: adipogenesis; ascochlorin; high-fat diet; obesity; β-catenin.

Figure 1

Effect of various compounds on lipid accumulation, TG content, and cell viability in 3T3-L1 adipocytes. (A) Two-day post-confluency preadipocytes were incubated with differentiation medium in the presence of various compounds (0, 1, 10, 20, and 30 µM) for 48 h. Cell viability was detected by MTT. (B,D) Accumulation of intracellular lipid droplets in Oil Red O staining of the undifferentiated control (UC) and differentiated control (DC) or compound-treated cells on day 8 (D8) after differentiation induction. Metformin (Met 10 mM) was used as a positive control. 3T3-L1 cells were imaged using a microscope (×200). (C,E) Quantification of intracellular lipid content (O.D.₅₁₀). (F) Measurement of intracellular triglyceride (TG) content on D8. Rosiglitazone (Rosi 10 µM) was used as a negative control. Data represent the mean ± SD of three independent experiments. *** p < 0.001 compared to the DC.

Figure 2

Effect of ASC on lipid accumulation and lipogenic gene expression in 3T3-L1 cells. (A) Representative image of BODIPY-stained lipid droplets in DC- or ASC (10 µM)-treated mature 3T3-L1 adipocytes. (B) Densitometric analysis of each BODIPY fluorescence ratio was performed using ImageJ (version 2018). (C) Two-day post-confluency preadipocytes were incubated with differentiation medium in the presence of ASC (0, 1, 5, and 10 µM). Western blotting of the cells treated with a series of doses of ASC as indicated for PPARγ, C/EBPα, FASN, SREBP1, FABP4, and adiponectin. Protein expression was quantified using ImageJ software and normalized against β-actin as a loading control. (D) The expression of PPARγ, C/EBPα, and FASN was evaluated by qRT-PCR with specific primer pairs on D8. (E) The expression of KLF2, Pref1, and GATA2 was evaluated by qRT-PCR on D2. (F) The expression of Perillipin, FAS, HSL, and SREBP1c was evaluated by qRT-PCR on D8. The relative qRT-PCR values were corrected to expression levels and normalized with respect to the control. Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DC.

Figure 3

Effect of ASC on MCE and adipogenic differentiation in 3T3-L1 cells. Cells were differentiated according to the standard protocol. (A) Flow cytometry analysis of cells treated with MDI and 10 µM ASC for 18 h. (B) Cellular extracts were isolated and analyzed for the expression of the proteins indicated by Western blotting. Protein expression was quantified using ImageJ software and normalized against β-actin as a loading control. (C) Two-day post-confluency preadipocytes were incubated with differentiation medium in the presence of 10 µM ASC for 48 h. 3T3-L1 cells were subjected to adipocyte differentiation and harvested on day 0, 2, 4, 6, and 8 for qRT-PCR. mRNA levels of anti-adipogenesis-associated genes (Pref1, GATA2, and KLF2). (D) Protein levels of pre-adipogenesis-associated genes (PPARγ, c/EBPα, FASN, and FABP4). (E) mRNA levels of pre-adipogenesis-associated genes (PPARγ, c/EBPα, and FAS). Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DC.

Figure 4

Activation of Wnt/β-catenin signaling by ASC blocked adipogenesis. (A) mRNA expression levels of β-catenin in 3T3-L1 adipocytes treated with ASC (10 µM) at days 2 and 8. (B) The expression of β-catenin, KLF2, Pref1, and GATA2 was evaluated by qRT-PCR with specific primer pairs on D8. (C) Representative Western blot images and quantification of β-catenin and p-GSK3β protein levels in fully matured 3T3-L1 adipocytes. Protein expression was quantified using ImageJ software and normalized against β-actin as a loading control. (D) The expression of Wnt10b, Wnt16, and id2 was evaluated by qRT-PCR with specific primer pairs on D8. (E) 3T3-L1 cells were transfected with control siRNA or β-catenin siRNA, and then treated with ASC (10 µM) and harvested on day 8. Cells were analyzed by Western blotting for the β-catenin, PPARγ, and c/EBPα. β-actin was used as a loading control. (F) Representative images of 3T3-L1 cells were transfected with control siRNA or β-catenin siRNA, and then treated with ASC (10 µM) and stained with Oil Red O on day 8. (G) Quantification of intracellular lipid content. Data represent the mean ± SD of three independent experiments. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DC.

Figure 5

Effect of ASC on HFD-induced obesity. (A) Weekly change in total body weight and (B) representative appearance of mice fed with normal diet (ND, n = 5), high-fat diet (HFD, n = 5), ASC (5 mg/kg, n = 5), and metformin (Met, 200 mg/kg, n = 3). (C) Representative appearance of liver, inguinal subcutaneous (iWAT), and epididymal fat tissue (eWAT). (D) Changes in liver and (E) eWAT and (F) iWAT weight. (G–K) Plasma level of TC, TG, HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), and alanine aminotransferase (ALT). (L) Representative Western blot images and quantification of β-catenin protein levels in eWAT are shown. Protein expression was quantified using ImageJ software and normalized against β-actin as a loading control. (M,N) The expression of Wnt10b and id2 was evaluated in eWAT by qRT-PCR using specific primer pairs. (O) H&E-stained images of liver tissue. (P) β-catenin expression of the liver tissues by immunohistochemistry, 20×. Data represent the mean ± SD of three independent experiments. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the HFD.