Limits...
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.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125748&req=5

fig1: 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).

Mentions: To identify small molecular activators of the Wnt/β-catenin pathway, we adopted a cell-based approach using HEK293 stable cells harboring the Wnt/β-catenin TOPflash reporter to screen a synthetic library of pharmacologically active compounds. A total of 1280 small molecules were tested at a final concentration of 20 μM and assayed for luciferase activity 24 h after treatment (Figure 1a). VPA (500 μM)18, 19 or LiCl (20 mM)20 was used as a positive control for the activation of Wnt/β-catenin signaling. Confirmation screening identified two compounds, I3O and SB-415286, as potential candidates that activated the Wnt/β-catenin pathway; these compounds were selected for further testing (Supplementary Figure 1). Both I3O and SB-415286 dose-dependently activated TOPflash reporter activity without showing any significant cytotoxicity by the cell viability assay (Figures 1b and c). ORO staining showed that treatment with these compounds at a concentration of 20 μM markedly inhibited the adipocyte differentiation of 3T3-L1 preadipocytes without inducing abnormal cell morphologies (Figure 1d). Although both compounds showed anti-adipogenic effects, we chose I3O (Figure 1e) for further study because the anti-adipogenic effects of SB-415286 had been previously described.21 The half-maximal effective concentration (EC50) of I3O for the activation of the TOPflash reporter was 4 μM (Figure 1f). The role of I3O in the activation of the Wnt/β-catenin signaling pathway was further confirmed by the significant increase in expression and nuclear localization of β-catenin in 3T3-L1 preadipocytes (Figure 1g). The levels of β-catenin and the active form of GSK3β (p-Tyr216)22 increased and decreased, respectively, by I3O treatment in a dose-dependent manner (Figure 1h).


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)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125748&req=5

fig1: 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).
Mentions: To identify small molecular activators of the Wnt/β-catenin pathway, we adopted a cell-based approach using HEK293 stable cells harboring the Wnt/β-catenin TOPflash reporter to screen a synthetic library of pharmacologically active compounds. A total of 1280 small molecules were tested at a final concentration of 20 μM and assayed for luciferase activity 24 h after treatment (Figure 1a). VPA (500 μM)18, 19 or LiCl (20 mM)20 was used as a positive control for the activation of Wnt/β-catenin signaling. Confirmation screening identified two compounds, I3O and SB-415286, as potential candidates that activated the Wnt/β-catenin pathway; these compounds were selected for further testing (Supplementary Figure 1). Both I3O and SB-415286 dose-dependently activated TOPflash reporter activity without showing any significant cytotoxicity by the cell viability assay (Figures 1b and c). ORO staining showed that treatment with these compounds at a concentration of 20 μM markedly inhibited the adipocyte differentiation of 3T3-L1 preadipocytes without inducing abnormal cell morphologies (Figure 1d). Although both compounds showed anti-adipogenic effects, we chose I3O (Figure 1e) for further study because the anti-adipogenic effects of SB-415286 had been previously described.21 The half-maximal effective concentration (EC50) of I3O for the activation of the TOPflash reporter was 4 μM (Figure 1f). The role of I3O in the activation of the Wnt/β-catenin signaling pathway was further confirmed by the significant increase in expression and nuclear localization of β-catenin in 3T3-L1 preadipocytes (Figure 1g). The levels of β-catenin and the active form of GSK3β (p-Tyr216)22 increased and decreased, respectively, by I3O treatment in a dose-dependent manner (Figure 1h).

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