The small molecule indirubin-3'-oxime activates Wnt/β-catenin signaling and inhibits adipocyte differentiation and obesity

Abstract

Objectives: Activation of the Wnt/β-catenin signaling pathway inhibits adipogenesis by maintaining preadipocytes in an undifferentiated state. We investigated the effect of indirubin-3'-oxime (I3O), which was screened as an activator of the Wnt/β-catenin signaling, on inhibiting the preadipocyte differentiation in vitro and in vivo.

Methods: 3T3L1 preadipocytes were differentiated with 0, 4 or 20 μM of I3O. The I3O effect on adipocyte differentiation was observed by Oil-red-O staining. Activation of Wnt/β-catenin signaling in I3O-treated 3T3L1 cells was shown using immunocytochemical and immunoblotting analyses for β-catenin. The regulation of adipogenic markers was analyzed via real-time reverse transcription-PCR (RT-PCR) and immunoblotting analyses. For the in vivo study, mice were divided into five different dietary groups: chow diet, high-fat diet (HFD), HFD supplemented with I3O at 5, 25 and 100 mg kg(-1). After 8 weeks, adipose and liver tissues were excised from the mice and subject to morphometry, real-time RT-PCR, immunoblotting and histological or immunohistochemical analyses. In addition, adipokine and insulin concentrations in serum of the mice were accessed by enzyme-linked immunosorbent assay.

Results: Using a cell-based approach to screen a library of pharmacologically active small molecules, we identified I3O as a Wnt/β-catenin pathway activator. I3O inhibited the differentiation of 3T3-L1 cells into mature adipocytes and decreased the expression of adipocyte markers, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ, at both mRNA and protein levels. In vivo, I3O inhibited the development of obesity in HFD-fed mice by attenuating HFD-induced body weight gain and visceral fat accumulation without showing any significant toxicity. Factors associated with metabolic disorders such as hyperlipidemia and hyperglycemia were also improved by treatment of I3O.

Conclusion: Activation of the Wnt/β-catenin signaling pathway can be used as a therapeutic strategy for the treatment of obesity and metabolic syndrome and implicates I3O as a candidate anti-obesity agent.

Figure 1

Screening a chemical library to identify a small molecule activator of the Wnt/β-catenin signaling pathway. (a) TOPflash reporter activity of HEK293-TOP cells treated with individual small molecules in DMEM at a concentration of 20 μM. After 24 h firefly luciferase activities of whole-cell lysates were measured. (b) Dose-dependency of I3O and SB-415286 on the activation of Wnt/β-catenin pathway in HEK293-TOP cells. VPA (500 μM) was used as a positive control (black colored bar). (c) Cell viability of HEK293-TOP cells was measured after treatment with either DMSO, I3O, or SB-415286 at a concentration of 20 μM. (d) ORO staining of 3T3-L1 preadipocytes that were induced to differentiate using MDI and treated with either DMSO, I3O or SB-415286 at a concentration of 20 μM. (e) Chemical structure of I3O. (f) Activation curve for TOPflash reporter activity induced by I3O. (g) 3T3-L1 preadipocytes were treated with 20 μM of I3O and immunocytochemical analysis was performed to detect β-catenin (red). Nuclei were counterstained with DAPI (blue). (h) 3T3-L1 preadipocytes were treated with 0, 2 and 40 μM of I3O, and immunoblotting analysis was performed to detect β-catenin, p-GSK3b (pY216) and α-tubulin. Values are mean±standard error of the mean (s.e.m., n=3). **P<0.01, ***P<0.001 (Student's t-test).

Figure 2

Effects of I3O on MDI-induced adipogenesis in 3T3-L1 cells. (a) 3T3-L1 preadipocytes were induced to differentiation with DMSO, 4, 20 μM I3O or 20 mM LiCl. Lipid droplets were stained using ORO 14 days post-differentiation. (b) ORO staining was quantified using a spectrophotometer at 590 nm. Values were normalized to the differentiated control. (c) 3T3-L1 preadipocytes were treated with I3O and triglyceride content was measured. Real time RT-PCR analysis of adipocyte markers PPARγ (d) and C/EBPα (e) after treatment with I3O. (f) Immunoblot analysis of protein expression of adipocyte markers, β-catenin, and p-GSK3β in 3T3-L1 cells that were induced to differentiation and treated with I3O. (g) 3T3-L1 preadipocytes were transfected with β-catenin or control siRNA. After 16 h, cells were induced to differentiate and the effects of β-catenin siRNA on the intracellular accumulation of lipid were observed by ORO staining. (h) Lipid accumulation was quantified using a spectrophotometer. Data are presented as mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).

Figure 3

Effects of I3O on HFD-induced obesity in C57BL/6N mice. Fifty male C57BL/6N mice were divided into groups (n=10 per group) and fed a chow diet, HFD, or HFD supplemented with I3O at 5, 25 and 100 mg kg⁻¹. (a) Whole-body images of Representative control and I3O-treated mice. (b) Total body weight. (c) Representative images of visceral white adipose tissue and liver of mice. Plasma levels of triglyceride (d), total cholesterol (e), free fatty acid (f), and glucose levels (g) in mice after 8 weeks of feeding with chow, HFD, or I3O-supplemented HFD. Values are expressed as means±s.e.m. (n=10). *P<0.05, **P<0.01, ***P<0.001 (Kruskal-Wallis test).

Figure 4

Effects of I3O on the liver of HFD-induced mice. Plasma levels of triglyceride (a), total cholesterol (b), and free fatty acid (c) in the liver of mice after 8 weeks. (d) Immunohistochemical analysis of markers in liver. Values are expressed as means±s.e.m. (n=10). *P<0.05, **P<0.01, ***P<0.001 (Kruskal-Wallis test).

Figure 5

Effects of I3O on adipose tissue. (a) Histological analysis of epididymal white adipose tissue of mice fed with chow, HFD, or I3O-supplemented HFD. Tissues were stained with hematoxylin and eosin (H&E). (b) Adipocyte size was measured using Image J Software. (c) H&E staining of subcutaneous fat of mice. (d) Thickness of fat between skin and muscle region was measured using Image J Software. RT-PCR analysis of adipocyte markers C/EBPα (e) and PPARγ (f). Concentrations of insulin (g) and resistin (h) in the bloods of mice fed with chow, HFD, or I3O-supplemented HFD were measured using ELISA. (i) Immunohistochemistrical analyses were performed to detect β-catenin in epidermis fat tissues of mice fed with chow, HFD, or I3O-supplemented HFD. (j) Immunoblot analyses to detect expression levels of β-catenin, PPARγ, C/EBPα and ERK. Data are presented as mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001 (Kruskal-Wallis test).