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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 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).
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fig5: 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).

Mentions: The reduction of epididymal white adipose tissue in I3O-supplemented HFD mice was primarily due to significantly smaller adipocytes, as shown by 59 and 72% reductions in adipocyte size in 25 and 100 mg kg−1 mice, respectively (Figures 5a and b). We further confirmed the decrease in visceral white adipose tissue by examining the effect of I3O on subcutaneous fat (Figures 5c and d). The thickness of fat between the skin and muscle layers decreased significantly in the I3O-supplemented HFD mice compared with HFD mice. We confirmed the reduction in expression levels of PPARγ and C/EBPα mRNA (Figures 5e and f). Additionally, the mRNA levels of PPARγ target genes, aP2, adiponectin and LPL, were also significantly reduced (Supplementary Figures 8A–C). To monitor the glucose homeostasis, we determined the concentrations of insulin and adipokines in the blood of I3O-fed and non-fed mice (Figures 5g and h and Supplementary Figures 8D–F). The higher insulin concentration in the HFD-fed mice decreased in a dose-dependent manner by the treatment of I3O (Figure 5g). Moreover, the insulin concentration in the blood of mice fed with 100 mg kg−1 I3O was lower than the mice fed a chow diet (Figure 5g). The concentrations of resistin, IL-6, TNFα and leptin, adipokines known to increase insulin resistance,30 were elevated by the HFD, and in contrast, were reduced by I3O in a dose-dependent manner (Figure 5h and Supplementary Figures 8D–F). Immunohistochemical analyses for β-catenin in the epidermal fat of the I3O-fed mice showed a significant increase of nuclear β-catenin in the adipocytes (Figure 5i). We further confirmed the I3O-mediated reduction of PPARγ and C/EBPα by immunoblotting analyses (Figure 5j). Collectively, these findings verify that I3O reduces both subcutaneous and visceral fat by decreasing adipocyte size through the inhibition of PPARγ and C/EBPα via activation of Wnt/β-catenin signaling.


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

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

fig5: 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).
Mentions: The reduction of epididymal white adipose tissue in I3O-supplemented HFD mice was primarily due to significantly smaller adipocytes, as shown by 59 and 72% reductions in adipocyte size in 25 and 100 mg kg−1 mice, respectively (Figures 5a and b). We further confirmed the decrease in visceral white adipose tissue by examining the effect of I3O on subcutaneous fat (Figures 5c and d). The thickness of fat between the skin and muscle layers decreased significantly in the I3O-supplemented HFD mice compared with HFD mice. We confirmed the reduction in expression levels of PPARγ and C/EBPα mRNA (Figures 5e and f). Additionally, the mRNA levels of PPARγ target genes, aP2, adiponectin and LPL, were also significantly reduced (Supplementary Figures 8A–C). To monitor the glucose homeostasis, we determined the concentrations of insulin and adipokines in the blood of I3O-fed and non-fed mice (Figures 5g and h and Supplementary Figures 8D–F). The higher insulin concentration in the HFD-fed mice decreased in a dose-dependent manner by the treatment of I3O (Figure 5g). Moreover, the insulin concentration in the blood of mice fed with 100 mg kg−1 I3O was lower than the mice fed a chow diet (Figure 5g). The concentrations of resistin, IL-6, TNFα and leptin, adipokines known to increase insulin resistance,30 were elevated by the HFD, and in contrast, were reduced by I3O in a dose-dependent manner (Figure 5h and Supplementary Figures 8D–F). Immunohistochemical analyses for β-catenin in the epidermal fat of the I3O-fed mice showed a significant increase of nuclear β-catenin in the adipocytes (Figure 5i). We further confirmed the I3O-mediated reduction of PPARγ and C/EBPα by immunoblotting analyses (Figure 5j). Collectively, these findings verify that I3O reduces both subcutaneous and visceral fat by decreasing adipocyte size through the inhibition of PPARγ and C/EBPα via activation of Wnt/β-catenin signaling.

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