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

Choi OM, Cho YH, Choi S, Lee SH, Seo SH, Kim HY, Han G, Min DS, Park T, Choi KY - Int J Obes (Lond) (2013)

Bottom Line: 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. 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea [2] Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

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.

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Related in: MedlinePlus

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).
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fig2: 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).

Mentions: Treatment of differentiation-induced preadipocytes with I3O followed by ORO staining confirmed that I3O decreased the accumulation of lipid droplets in 3T3-L1 cells in a dose-dependent manner (Figure 2a). Quantitative analysis of the ORO staining showed that lipid accumulation was reduced by 38% at 4 μM and 80% at 20 μM, respectively (Figure 2b). Treatment with I3O also revealed an 82% reduction of triglyceride accumulation by 20 μM I3O (Figure 2c). The mRNA levels of both C/EBPα and PPARγ, the two major adipogenic transcription factors induced during adipocyte differentiation,23 were significantly reduced in differentiation-induced preadipocytes treated with I3O (Figures 2d and e). mRNA levels of the PPARγ target genes, which include adipocyte protein 2 (aP2), adiponectin and lipoprotein lipase (LPL), were also significantly reduced (Supplementary Figure 2). At the protein level, the expressions of both C/EBPα and PPARγ were also reduced by I3O; concomitantly, there was an increase in β-catenin and a decrease of p-GSK3β (Figure 2f). Cell viabilities of 3T3-L1 cells were not changed by treatment of 4 μM I3O, and decreased slightly by treatment of 20 μM I3O (Supplementary Figure 3). Overall, I3O inhibits adipocyte differentiation by activating the Wnt/β-catenin pathway, resulting in the reduced expression of factors involved in adipogenesis, such as C/EBPα and PPARγ.


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

Choi OM, Cho YH, Choi S, Lee SH, Seo SH, Kim HY, Han G, Min DS, Park T, Choi KY - Int J Obes (Lond) (2013)

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).
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Related In: Results  -  Collection

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fig2: 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).
Mentions: Treatment of differentiation-induced preadipocytes with I3O followed by ORO staining confirmed that I3O decreased the accumulation of lipid droplets in 3T3-L1 cells in a dose-dependent manner (Figure 2a). Quantitative analysis of the ORO staining showed that lipid accumulation was reduced by 38% at 4 μM and 80% at 20 μM, respectively (Figure 2b). Treatment with I3O also revealed an 82% reduction of triglyceride accumulation by 20 μM I3O (Figure 2c). The mRNA levels of both C/EBPα and PPARγ, the two major adipogenic transcription factors induced during adipocyte differentiation,23 were significantly reduced in differentiation-induced preadipocytes treated with I3O (Figures 2d and e). mRNA levels of the PPARγ target genes, which include adipocyte protein 2 (aP2), adiponectin and lipoprotein lipase (LPL), were also significantly reduced (Supplementary Figure 2). At the protein level, the expressions of both C/EBPα and PPARγ were also reduced by I3O; concomitantly, there was an increase in β-catenin and a decrease of p-GSK3β (Figure 2f). Cell viabilities of 3T3-L1 cells were not changed by treatment of 4 μM I3O, and decreased slightly by treatment of 20 μM I3O (Supplementary Figure 3). Overall, I3O inhibits adipocyte differentiation by activating the Wnt/β-catenin pathway, resulting in the reduced expression of factors involved in adipogenesis, such as C/EBPα and PPARγ.

Bottom Line: 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. 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea [2] Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

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.

Show MeSH
Related in: MedlinePlus