Short Communication | Published:

Indole-3-carbinol directly targets SIRT1 to inhibit adipocyte differentiation

International Journal of Obesity volume 37, pages 881884 (2013) | Download Citation

Abstract

Indole-3-carbinol (I3C), a natural product of Brassica vegetables such as broccoli and cabbage, inhibits proliferation and induces apoptosis in various cancer cells. I3C has recently received attention as a possible anti-obesity agent. However, how I3C interacts with specific targets in the pathways involved in obesity and metabolic disorders is unknown. Silent mating type information regulation 2 homolog 1 (SIRT1), a NAD+-dependent deacetylase sirtuin, has recently emerged as a novel therapeutic target for metabolic diseases. Herein, we report that I3C is a potent, specific SIRT1 activator efficacious in cultured 3T3-L1 cell lines. A pull-down assay showed that I3C binds to SIRT1. To assess the significance of this binding, we determined whether I3C could activate SIRT1 deacetylase activity in a cell-free system. We found that I3C binds to SIRT1 and activates SIRT1 deacetylase activity in 3T3-L1 cells. In addition, I3C did not inhibit adipocyte differentiation in 3T3-L1 cells in which SIRT1 was knockdowned. Further, reverse transcriptase polymerase chain reaction analysis showed that I3C treatment reduced mRNA levels of adipogenic genes that encode for C/EBPα, PPARγ2, FAS, and aP2 in 3T3-L1 cells but not in SIRT1 knockdown cells. Overall, these results suggested that I3C ameliorates adipogenesis by activating SIRT1 in 3T3-L1 cells.

Introduction

Indole-3-carbinol (I3C), a natural product found in broccoli and cabbage, has chemotherapeutic properties, including antiproliferative and proapoptotic activities, against various cancers.1 In vitro, I3C reportedly targets a broad range of signaling molecules involved in cell-cycle regulation and survival, including serine/threonine protein kinase Akt, NF-κB, B-cell leukemia/lymphoma 2, mitogen-activated protein kinases, cyclin-dependent kinases, and cyclin D1.2 Recently, I3C received special attention as a possible anti-obesity agent. I3C improved hyperglycemia and hyperinsulinemia in mice fed the HFD3 and decreased the expression of inflammatory cytokines, such as IL-6, in the co-cultured adipocytes and macrophages.4 Furthermore, we previously found that I3C efficiently blocked adipocyte differentiation in a cultured 3T3-L1 cell line.5 Our in vivo results demonstrated that 0.1% I3C supplementation (100 mg/kg/day) significantly reduced body weight and visceral fat-pad weight gains in HFD-fed mice.5 The daily I3C intake in our mice was equivalent to an intake of about 8.1 mg/kg human body weight when calculated based on normalization to body surface area under the recommendations of the U.S. Food and Drug Administration (http://www.fda.gov/cder/cancer/animalframe.htm) and Reagen-Shaw et al.6 In the meantime, Reed et al.7 reported that I3C has been well tolerated by individuals with a daily dose ranges between 7 and 14 mg/kg human body weight as a nutritional supplement. In a clinical trial, consumption of large amounts of I3C -usually more than 14 mg/kg human body weight per day- increased the risk for developing liver cancer.8 The mechanism of action of I3C may involve multiple mechanisms, including decreased adipogenesis and inflammation as well as activated thermogenesis.5 However, it remains unclear how I3C interacts with specific targets in the pathways involved in obesity and metabolic disorders.

Sirtuins, a family of NAD+-dependent deacetylases, recently emerged as novel therapeutic targets for metabolic diseases. Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) is thought to influence these diverse biological processes through direct deacetylation of peroxisome proliferator-activated receptor γ coactivator-1α, forkhead box O1, and p53, as well as indirect activation of AMP-activated protein kinase signaling, stimulating fat utilization to prevent diet-induced obesity, and associated disorders.9 Herein, we report that I3C is a potent and specific SIRT1 activator efficacious in a cultured 3T3-L1 cell line. Combined with previous work on I3C (in press), the current study provides complementary in vitro evidence validating that SIRT1 is a target of I3C for the treatment of metabolic disorders.

Materials and methods

In vitro pull-down assay

Recombinant SIRT1 (2 μg) was incubated with I3C-Sepharose 4B beads (or Sepharose 4B beads alone as a control) (50 μl; 50% slurry) in a reaction buffer (50 mM tris(hydroxymethyl)aminomethane, pH 7.5, 5 mM EDTA, 150 mM sodium chloride (NaCl), 1 mM dithiothreitol, 0.01% NP-40, and 2 μg ml−1 bovine serum albumin). After incubation with gentle rocking overnight at 4 °C, the beads were washed five times with buffer (50 mM tris(hydroxymethyl)aminomethane, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% NP-40, and 0.02 mM phenylmethylsulfonyl fluoride), and proteins bound to the beads were analyzed by western blotting.

Animal care and SIRT1deacetylase assay

All mice were housed in the specific pathogen-free facility of the Yonsei University, Seoul, Republic of Korea, and this study was approved by the Institutional Animal Care and Use Committee of Yonsei University. Thirty male C57BL/6N mice (age, 5 weeks) were purchased from Orient Bio (Gyeonggi-do, Republic of Korea) and conditioned in the animal facility for 1 week. The normal diet was a purified diet based on the AIN-76 rodent diet composition. The HFD (20% fat, wt/wt) was formulated to provide 40% of the total energy generated by the diet from fat by replacing carbohydrates with lard and corn oil. The I3C-supplemented diet was identical to HFD and contained 0.1% (wt/wt) I3C. Diets were provided in the form of pellets for 10 weeks. Nuclear extracts from epididymal adipose tissue were isolated using a separate nuclear extraction kit (Active Motif, Carlsbad, CA, USA), with minor modifications. SIRT1 activity was determined with a SIRT1 fluorometric kit (BIOMOL International, Plymouth Meeting, PA, USA) according to the manufacturer’s instructions.

Cell culture and semiquantitative reverse transcriptase-PCR

Lentiviral infection was performed as described previously.10 I3C (molecular weight 147.17 g mol−1, 96% purity) was obtained from Sigma-Aldrich (St Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). 3T3-L1 cells were treated with varying doses of I3C (0.1, 1, 10, 50 or 100 μM) for 10 days during differentiation. Fat accumulation was visualized by staining the lipids with Oil Red O. Intracellular triglyceride levels were measured in cell lysates using an enzymatic method with a reagent kit from Boehringer Mannheim (Irvine, CA, USA). Total RNA was isolated from cultured cells using Trizol (Invitrogen, Carlsbad, CA, USA) and was then reverse-transcribed using the Superscript II kit (Invitrogen) according to the manufacturer’s recommendations. The forward (F) and reverse (R) primers for each mouse gene are shown in Supplementary Table 1. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard for measuring mRNA levels of genes related to adipogenesis in the 3T3-L1 cells.

Statistical analysis

Statistical analyses were conducted by the Student t-test or one-way analysis of variance using the Statistical Package for the Social Sciences (SPSS) software. A P value of less than 0.05 was considered significant.

Results

I3C binds to SIRT1 and activates SIRT1 deacetylase activity

Identification of I3C-binding proteins is crucial for delineating the mechanism of the anti-obesity effects of I3C. Here, with I3C-Sepharose 4B, we found that I3C binds to commercially available, active SIRT1 (Figure 1A). The results showed that active SIRT1 bound to I3C-Sepharose 4B but not Sepharose 4B. To assess the significance of this binding, we determined whether I3C activated SIRT1 deacetylase activity in a cell-free system. Dose–response experiments showed that I3C doubled the rate of deacetylation catalyzed by SIRT1 at an I3C concentration of approximately 10 μM; the rate was maximal at I3C concentrations of 100–200 μM (Figure 1B). Although I3C did not significantly affect the 2 Vmax determinations when either substrate or NAD+ was varied (Figures 1C and D), I3C had a pronounced influence on the 2 apparent Michaelis constants (Km). Furthermore, I3C supplemented to mice fed a HFD significantly restored the HFD-induced down-regulation of SIRT1 deacetylase activity in their epididymal adipose tissue (Figure 1E).

Figure 1
Figure 1

Effects of indole-3-carbinol on the kinetics of recombinant SIRT1. (A) Immunoblot analysis of Silent mating type information regulation 2 homolog 1 (SIRT1) following purification by indole-3-carbinol (I3C)-Sepharose 4B affinity column chromatography. (B) I3C dose–response of the SIRT1 catalytic rate. (C) SIRT1 initial rate with 3 mM NAD+ as a function of the p53-K382 acetylated peptide concentration in the presence or absence of 100 μM I3C. (D) SIRT1 initial rate with 1 mM p53-K382 acetylated peptide as a function of the NAD+ concentration in the presence or absence of 100 μM I3C. (E) Catalytic activity of SIRT1 in the adipose tissue mice fed experimental diets. The values are the means±s.e.m. of eight mice. Means not designated by a common superscript are different, abcP<0.05.

I3C-mediated inhibition of 3T3-L1 adipocyte differentiation did not affect SIRT1 knockdown cells

The I3C treatment significantly reduced intracellular fat accumulation in a dose-dependent manner relative to control cells treated with dimethyl sulfoxide (Figure 2A). These results were confirmed by measurement of the total amount of intracellular ORO (Figure 2B). The SIRT1 gene in 3T3-L1 cells was then downregulated by lentiviral infection with SIRT1 shRNA (Figure 2C). As shown in Figure 2D, no I3C-mediated fat reduction was observed in cells in which the SIRT1 level had been downregulated (SIRT1 knockdown cells). Downregulation of SIRT1 expression resulted in a significant increase in triglyceride accumulation after differentiation. The triglyceride content was reduced by I3C treatment in the control cells but not in the SIRT1 knockdown cells (Figure 2E). Treatment of 3T3-L1 cells with I3C (100 μM) significantly reduced the mRNA level of the CCAAT/enhancer binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid synthase (FAS), and adipocyte protein 2 (aP2). However, the mRNA levels of these genes in the SIRT1 knockdown cells were unaffected after I3C treatment (Figures 2F and G). There was no significant difference in the relative mRNA levels of GAPDH between I3C- and DMSO-treated conditions in the wild type 3T3-L1 (DMSO, 1 vs. I3C, 0.98) or SIRT1 knockdown cells (DMSO, 1 vs. I3C, 1.02) (Figure 2F).

Figure 2
Figure 2

Activation of SIRT1 by I3C decreases fat accumulation in differentiated adipocytes. (A) Ten days after the initiation of differentiation, the lipid accumulation in cells stained with Oil Red O was visualized after magnification ( × 200). (B) The lipid accumulation was measured through a spectrometer. The values with different superscripts are significantly different at P<0.05. (C) SIRT1 protein level in 3T3-L1 cells infected with SIRT1 shRNA vectors for downregulation of SIRT1. Effect of I3C on 3T3-L1 cells infected with the indicated viruses. The intracellular lipid content was evaluated by Oil Red O staining of whole cells (D) and in cell lysates (E). (F, G) I3C-mediated changes in the expression of adipogenic genes monitored by reverse transcriptase polymerase chain reaction with total RNA from 3T3-L1 cells and SIRT1 knockdown cells. Results are presented as the average±s.e.m. of at least 3 separate experiments, *P<0.05.

Discussion

Pharmacological programs that target transcriptional networks to regulate global gene expression favoring energy expenditure represent an attractive strategy to combat metabolic diseases. In this context, SIRT1 has emerged as an interesting target because the naturally occurring SIRT1 activator resveratrol protects cells from diet-induced metabolic disorders.11 At present, Sirtris Pharmaceuticals (now GlaxoSmithKline) is investigating a series of SIRT1 activators for the potential treatment of diabetes and obesity. We found that I3C upregulates SIRT1 activity by binding to SIRT1, lowering the Km value of the acetylated substrate. Because I3C influences only the Km value, it could be classified as a K system allosteric effector, perhaps indicating that only the substrate-binding affinity of the enzyme has been altered.

SIRT1 activation has been shown to stimulate several key cellular signaling pathways involved in regulating lipid metabolism, inflammation, and insulin resistance. SIRT1 physically interacts with PGC1α and catalyzes deacetylation reactions at multiple lysine sites, consequently increasing the activity of PGC1α leading to the induction of UCP1 and UCP3, which are involved in thermogenesis regulation.12 The SIRT1-mediated activation of PGC1α also induces the metabolic transcription network for mitochondrial fatty acid oxidation.13 Other studies have shown that SIRT1 physically interacts with NF-κB and inhibits the production of inflammatory cytokines.14 Additionally, recent studies demonstrated that SIRT1 interacts with IRS1, increasing the glucose uptake and reducing gluconeogenesis.15 Chang et al.4 reported that I3C decreased the expression of inflammatory cytokines, such as IL-6, in the co-cultured adipocytes and macrophages. Another study by the same author found that I3C decreased hyperglycemia and hyperinsulinemia in mice fed the HFD.3 Collectively, these results support the concept that I3C binds to SIRT1 and activates SIRT1 deacetylase activity in 3T3-L1 cells. Our previous study suggested that I3C may prevent obesity and metabolic disorders and that the in vivo action of I3C may involve multiple mechanisms such as decreased adipogenesis and inflammation as well as activated thermogenesis.5 Therefore, these results suggested that I3C may target SIRT1 because I3C appears to partially induce metabolic pathways that are activated by SIRT1.

In the current study, down-regulation of SIRT1 expression resulted in significantly increased triglyceride accumulation during adipocyte differentiation. Moreover, we observed that knockdown of SIRT1 caused increased C/EBPα, PPARγ2, FAS, and aP2 expression levels in 3T3-L1 cells. Furthermore, Picard et al.16 demonstrated that SIRT1 represses the action of PPARγ by docking to its cofactors: the nuclear receptor co-repressor and the silencing-mediator of retinoid and thyroid hormone receptors. This docking leads to increased fat mobilization and inhibition of adipogenesis. In summary, these results indicate that SIRT1 acts a negative modulator of adipogenesis in the 3T3-L1 model. We showed that the I3C-mediated inhibition of 3T3-L1 adipocyte differentiation was due to the activation of SIRT1 because there was no I3C-mediated fat reduction in cells in which the SIRT1 levels had been knocked down. Supporting these observations, the mRNA levels of adipogenic genes such as C/EBPα, PPARγ2, FAS, and aP2 in SIRT1 knockdown cells were unaffected after I3C treatment. These results suggested that I3C ameliorates adipogenesis by activating SIRT1 in 3T3-L1 cells. In conclusion, this is the first report showing that I3C specifically inhibits adipocyte differentiation by binding to SIRT1, thereby modulating the activity of SIRT1. These findings should be useful for developing drugs that target SIRT1; I3C appears to be a promising lead compound.

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Acknowledgements

This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare (#090282), Republic of Korea and by the SRC program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (#2012-0000643).

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Affiliations

  1. Department of Food and Nutrition, Yonsei University, Seoul, Republic of Korea

    • Y Choi
    •  & T Park
  2. Department of Bioscience and Biotechnology/Institute of Bioscience, BK21 Graduate Program, Sejong University, Seoul, Republic of Korea

    • S-J Um

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Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to T Park.

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DOI

https://doi.org/10.1038/ijo.2012.158

Supplementary Information accompanies the paper on International Journal of Obesity website (http://www.nature.com/ijo)