Research articleIndole-3-carbinol prevents diet-induced obesity through modulation of multiple genes related to adipogenesis, thermogenesis or inflammation in the visceral adipose tissue of mice☆
Introduction
The adipose tissue is an endocrine organ which plays a fundamental role in metabolism and homeostasis regulation through the secretion of several biologically active adipokines including cytokines and chemoattractant proteins along with molecules involved in glucose and lipid metabolism, adipogenesis and thermogenesis [1]. For this reason, obesity, especially increased visceral adiposity, leads to increased production of several inflammatory cytokines that play a critical role in obesity-related inflammation and metabolic pathologies [2]. Tumor necrosis factor-alpha (TNFα) is a potent cytokine that induces the production of interleukin-6 (IL-6), which is the major determinant of the acute phase response, and also has effects on glucose transport, lipid metabolism and insulin action [2]. It has been reported that, in obese individuals and animal models, the levels of TNFα and IL-6 are persistently elevated, whereas a reduction of adipose tissue mass leads to a decrease in these expression levels [2]. There is increasing evidence for the fundamental role of toll-like receptors (TLRs) in linking nutrition, adipose tissue and innate immunity. TLRs play an important role in regulating innate immune responses to both infectious and sterile inflammatory stimuli, and free fatty acids (FFAs) are important ligands for TLR2 and TLR4. TLRs therefore represent a direct molecular link between hyperlipidemia, a central clinical feature of obesity, and activation of the innate immune system. TLR2 and TLR4 are expressed in a wide range of cells, including macrophages and adipocytes, and upon binding FFAs, activate NFκB, upregulate inflammatory cytokine expression and induce insulin resistance [3].
Adipose tissue growth, such as that observed during normal development and obesity, is the result of both hypertrophy (increase in size) and hyperplasia (increase in number) of adipocyte [4]. Adipogenesis is mainly regulated by peroxisome proliferator activated receptor gamma (PPARγ), a member of the nuclear receptor family that serves as the master transcriptional regulator. Downstream targets for PPARγ include a host of genes involved in lipid accumulation and metabolism, such as adipocyte lipid binding protein (aP2), cluster of differentiation 36 (CD36), lipoprotein lipase and phosphoenolpyruvate carboxykinase [5]. Increased expression and/or activation of uncoupling proteins (UCPs) in the adipose tissue uncouples oxidative phosphorylation, resulting in the conversion of energy to heat [6]. Therefore, it is possible that drugs activating or increasing the expression of UCP1 and UCP3 would have important effects on energy expenditure or on the rate of nutrient oxidation.
Indole-3-carbinol (I3C, 1H-indol-3-methanol, C9H9NO) is a naturally occurring compound found in cruciferous vegetables of the genus Brassica, which includes cabbage, broccoli, cauliflower, brussel sprouts, turnip, kale and kohlrabi. I3C is produced when these vegetables are macerated, cut or cooked via the myrosinase-catalyzed hydrolysis of glucobrassicin, an indole glucosinolate [7]. Anderton et al. reported that after oral administration of I3C (250 mg/kg) to mice, I3C was rapidly absorbed and had already reached an apparent peak concentration of 4.1 μg/ml at the earliest sampling time point of 15 min in plasma. Furthermore, maximal plasma I3C concentrations observed were considerably higher than those for its acid condensation products such as diindolylmethane (DIM) (I3C, 4.1 μg/ml vs. DIM, 0.2 μg/ml) [8]. At the concentration range of 50–100 μM, I3C has been shown to suppress the proliferation of various cancer cells, including those of breast [9], colon [10], prostate [11] and endometrium [12] cancers, by targeting a wide spectrum of signaling pathways governing hormonal homeostasis, cell-cycle progression, and cell proliferation and survival [13]. Although I3C has been shown to possess anticancer properties, its antiobesity effect has not been studied yet. The main objective of this study was to investigate the weight-lowering effect of I3C supplementation in mice maintained on the high-fat diet (HFD) and to study the potential molecular mechanisms of this effect, focusing on the expression of genes involved in adipogenesis, thermogenesis, and inflammation in the adipose tissue.
Section snippets
Materials
3T3-L1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). I3C (MW 147.17 g/mol, 96% purity) and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Bovine calf serum (BCS) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS), penicillin/streptomycin and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Isopropanol was purchased from Amresco (Solon, OH, USA).
I3C inhibits 3T3-L1 cell differentiation
I3C decreased the cell population growth in a dose-dependent manner with an IC50 value of 35.2 mM against the 3T3-L1 preadipocytes (data not shown). To study the potential inhibitory effects of I3C on adipogenesis, various concentrations of I3C were added to the differentiation medium for 10 days during the culture of 3T3-L1 cells. Macroscopic observations of oil red O staining are shown in Fig. 1A. With increasing concentrations of I3C in culture media, a significant inhibition of
Discussion
The effect of I3C supplementation on body weight loss did not depend on decreased food or energy intake because there were no significant differences in food intake among groups. In the present study, I3C supplemented to the HFD significantly suppressed the expressions of GalR and CTSS in the epididymal adipose tissue of mice. Galanin is one of the most inducible neuropeptides that is known to stimulate the intake of a fat-rich diet [14]. The conventional galanin-mediated signaling involves the
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This work was performed with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ006696),” Rural Development Administration, Republic of Korea and the Korea Health 21 R & D Project (Project No. A110532), Ministry of Health & Welfare, Republic of Korea.