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Keywords:

  • 3T3-L1;
  • 18β-glycyrrhetinic acid;
  • Cannabinoid receptor type 1;
  • Licorice;
  • Obesity

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

Scope

Previous reports suggest that licorice extract has various metabolically beneficial effects and may help to alleviate adiposity and hyperlipidemia. However, underlying anti-obesity mechanisms still remain elusive. Moreover, it is unknown which single ingredient in licorice extract would mediate such effects. We aimed to demonstrate that licorice extract and its active ingredients can inhibit adipocyte differentiation and fat accumulation.

Methods and results

18β-glycyrrhetinic acid (18β-GA) alleviated the effects of CB1R agonist, anandamide (AEA) on CB1R signaling in a concentration-dependent manner. Consistently, 18β-GA suppressed AEA-induced adipocyte differentiation in 3T3-L1 cells through the downregulation of AEA-induced MAPK activation and expression of adipogenic genes including C/EBP-α and PPAR-γ. The protein levels of fatty acid synthase and stearoyl-CoA desaturase 1 were also decreased and the phosphorylation of acetyl-CoA carboxylase was increased in 18β-GA pretreated cells. The supplementation of 18β-GA significantly lowered body weight, fat weight, and plasma lipids levels in obese animal models.

Conclusion

These results may provide a novel insight into the molecular mechanism involved in anti-adipogenic and anti-obesity effects of 18β-GA by suppressing the activation of CB1R induced by AEA. Thus, 18β-GA may exert beneficial effects against obesity-related metabolic disorders.

Abbreviations
18β-GA

18β-glycyrrhetinic acid

ACC

acetyl-CoA carboxylase

AEA

anandamide/N-arachidonoylethanolamine

BBB

blood–brain barrier

CB1R

cannabinoid receptor type 1

ERK

extracellular signal-regulated kinase

FAS

fatty acid synthase

FBS

fetal bovine serum

FER

food efficiency ratio

GL

glycyrrhizin

HFD

high-fat diet

MAPK

mitogen-activated protein kinase

PPAR-γ

peroxisome proliferator-activated receptor gamma

STD

standard diet

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

The endocannabinoid system is considered to be an important regulator of energy homeostasis at central and peripheral levels [1]. It is composed of the cannabinoid receptors type 1 (CB1R) and 2, and their natural ligands called endocannabinoids, among which anandamide (N-arachidonoylethanolamine, AEA) is the most extensively studied. Central actions of CB1R agonists on the control of food intake are well established, but increasing attention is being directed to its peripheral effects [2, 3]. In adipocytes, the expression of CB1R increases during differentiation, and CB1R stimulation accelerates the differentiation of pre-adipocytes through transcriptional activation of peroxisome proliferator-activated receptor gamma (PPAR-γ) [4-6]. CB1R activation also enhances the biosynthesis of fatty acid and triglyceride by upregulation of fatty acid synthase (FAS), while fatty acid oxidation is suppressed by inhibition of adenylate cyclase and activation of mitogen-activated protein kinases (MAPKs) [6-8]. In contrast, CB1R blockade in human and rodent adipocytes inhibits adipocyte differentiation and fat accumulation [9, 10]. Consistently, CB1R knockout mice (CB1R−/−) are lean and resistant to high-fat diet (HFD) induced obesity [11, 12] and a selective CB1R antagonist, SR141716 (rimonabant) reduces body weight in obese animals even if the suppressive effect on food intake diminishes within 1 or 2 weeks of dosing, suggesting that CB1R blockade may improve peripheral lipid profiles, leading to reduced body fat content [13-15].

Fat content is determined by the balance between lipogenesis and fatty acid oxidation. Lipogenesis encompasses the processes of fatty acid synthesis and subsequent triglyceride production that take place in both adipose tissue and liver. Many hormones, nutrients, and transcriptional factors modulate fat accumulation [16]. Two adipogenic transcription factors, CCAAT enhancer binding protein-α (C/EBP-α) and PPAR-γ are key components of cellular orchestrating fat accumulation in adipocytes. Cross-regulation of C/EBP-α and PPAR-γ controls the transcriptional pathway of both adipogenesis and lipogenesis. In particular, the in vitro conversion of 3T3-L1 adipoblasts to adipocytes has provided a useful system to study the adipogenesis and lipogenesis. The 3T3-L1 cell line was originally developed by clonal expansion from murine Swiss 3T3 cells. These cells exhibit a fibroblast-like morphology and proliferate continuously when maintained in growth medium. Upon appropriate hormonal stimulation, 3T3-L1 cells terminate mitotic growth and acquire morphological and enzymatic properties characteristic of adipocytes [17, 18]. Meanwhile, lipids deposited in adipocytes are broken down through lipolysis, releasing fatty acids via hydrolysis of triglycerides. Fatty acid oxidation which generates acetyl coenzyme A and ATP within mitochondria, is increased in the catabolic state of the adipocytes [19].

Glycyrrhiza glabra L. (licorice) root and its ingredients are widely used as a natural sweetener, flavoring additive and in herbal medicines for antiviral, anticancer, and anti-inflammatory effects [20, 21]. The major active ingredients of licorice root are triterpenoid saponins such as glycyrrhizin (GL), 18β-glycyrrhetinic acid (18β-GA) and its diastereomer 18α-GA, and a flavonoid, liquiritin [22, 23]. Prenylflavonoids such as glycycoumarin, glycyrin, dehydroglyasperin C, and dehydroglyasperin D in licorice were also found to possess various therapeutic properties [24]. In this study, we have investigated whether 18β-GA, a key chemical of major triterpenoid saponins from licorice extract, might regulate CB1R activity, leading to anti-adipogenesis in 3T3-L1 cells and anti-obesity in diet-induced obese animals. Although it has been previously suggested that licorice extract show metabolically beneficial effects including anti-atherosclerosis and body weight loss, their underlying mechanisms are not clearly understood [25-29]. In addition, it is unknown which single chemical in licorice extract would mediate such effects. We found that 18β-GA decreases adiposity and ameliorates lipid dysregulation as well as body weight gain. More importantly, we investigated that 18β-GA could potently attenuate the effects of AEA on CB1R activation. Taken together, these results suggest that 18β-GA may be useful for the treatment of obesity-related metabolic diseases.

2 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

2.1 Materials

GL, 18α-GA, 18β-GA, and AEA were purchased from Sigma (Sigma, MO, USA). Liquiritin was from Chromadex (Irvine, CA, USA). SR141716 was synthesized in the Amorepacific R&D center (Yongin, Korea) with a purity of > 95%. The optimum extraction condition of licorice root was established: the use of ethanol/water (30:70, v/v) as an extraction solvent, and 60 min dipping time at 50°C. Compounds were dissolved in DMSO as a 10 mM stock solution and then diluted with phosphate buffered saline.

2.2 Ca2+ flux assay in CB1-expressing Chem-1 cell

For measurement of the antagonist activity to CB1 receptor, we used the Chemicon's cloned human CB1-expressing cell line (Chemicon Inc., HTS019C) and Flu-4 NW (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The Chemicon's cloned human CB1-expressing cell line was made in the Chem-1 host, which supports high levels of recombinant CB1 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Cells were seeded at 5 × 104 cells per well in a 96-well assay plate in growth media (DMEM containing 4.5 g/L glucose, 10% FBS, 10 mM nonessential amino acids, 10 mM HEPES, 100 U/mL each penicillin and streptomycin) lacking Genetecin/G418, and incubated overnight. The following day, the growth media were removed and 100 μL of the dye loading solution containing 2.5 mM probenecid for the inhibition of organic–anion transporters located in the cell membrane was added to each well. Cells were further incubated at 37°C, 5% CO2 for 30 min and then at room temperature for an additional 30 min. These cells were treated with or without compounds in DMSO (final 0.5%) for 10 min at room temperature and then 3 μM anandamide in DMSO (final 0.5%) was added to cells. The fluorescence was measured at excitation 485 nm and emission 525 nm using a Flex station III microplate reader (Molecular Devices, Sunnyvale, CA, USA). All assays were executed in triplicate and results were presented as mean ± SD of three separate experiments.

2.3 Cell culture and adipocyte differentiation

Mouse 3T3-L1 preadipocytes (ATCC CL-173, Manassas, VA, USA) were maintained in DMEM (Gibco, Carlsbad, CA, USA) containing 10% v/v calf serum and antibiotics. For differentiation, cells at 2 days after reaching confluence were cultured in the medium comprising DMEM supplemented with 10% v/v fetal bovine serum (FBS), 10 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone. After 2 days, the culture medium was changed to DMEM containing 10% FBS and 10 μg/mL insulin. The medium was replaced again with fresh DMEM containing 10% FBS after another 2 days. The adipocytes were used at 6∼8 days after the initiation of differentiation. To investigate effects of AEA or 18β-GA on adipogenic differentiation, AEA with or without 18β-GA was added throughout differentiation at given concentrations mentioned in figures.

2.4 Oil Red O staining and quantification of lipid content

Accumulation of lipid droplets was monitored by microscopic analysis and confirmed by Oil Red O staining (Sigma). For Oil red O staining, 3T3-L1 cells were washed twice with PBS, fixed in 3.7% formaldehyde for 1 h, and stained for 30 min with 1% w/v Oil Red O solution in 60% v/v isopropanol. After being washed with distilled water and evaporated completely, the cells were observed under a microscope. Stained oil droplets were extracted using isopropanol, and the absorbance was measured at 520 nm using a microplate reader (Dibitech Ltd., Korea).

2.5 Protein extraction and immunoblotting

The 3T3-L1 cells were washed with ice-cold PBS, and harvested pellets were homogenized in 1× RIPA buffer (Cell Signaling, Beverly, MA, USA) with protease inhibitor mixture and 1 mM PMSF (Sigma) followed by centrifugation at 14 000 × g for 10 min at 4°C. The resulting supernatants were collected and concentrations were determined by BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein extracts (50 μg/lane) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membranes were then probed with a 1:1000 dilution of primary antibody. Antibodies to the following proteins were purchased from the indicated sources: rabbit polyclonal anti-mouse extracellular signal-regulated kinase (ERK), p-ERK (Thr202/Tyr204), p38, p-p38 (Thr180/Tyr182), and β-actin were obtained from Cell Signaling Technology (Cell signaling); rabbit polyclonal anti-mouse FAS and mouse monoclonal anti-mouse SCD-1 were obtained from Abcam (Abcam, Cambridge, MA, USA). After incubation with horseradish peroxidase conjugated goat anti-rabbit or anti-mouse IgG (Cell signaling) as secondary antibody for 1 h at room temperature, signals were detected and quantified using a chemiluminescent detection system, LAS-3000 (Fujifilm).

2.6 RNA extraction and real-time quantitative PCR analysis

Total RNA was isolated from 3T3-L1 cells or mouse liver tissue using the RNeasy mini kit (Qiagen, UK) according to the manufacturer's instructions. Reverse transcription of total RNA (1.2 μg) was performed using SuperScript TM RT (Invitrogen) and aliquots were stored at −20°C. Expression studies were carried out using gene specific primers for mouse C/EBP-α, PPAR-γ, acetyl-CoA carboxylase (ACC), FAS, SCD-1, and glyceraldehydes 3-phosphate dehydrogenase that were obtained from Applied Biosystems (assay identifications are as follows: Mm00514283-s1, Mm01184322-m1, Mm01304257-m1, Mm00433237-m1, Mm01197142-m1, and Mm99999915-g1). Quantitative mRNA expression levels of target genes were measured by real-time PCR using the 7500 Fast real-time PCR system (Applied Biosystems). All quantifications were performed in duplicate and the experiments were independently repeated three times.

2.7 Animal care and experimental protocol

Seven-week-old male C57BL/6J mice were obtained from Orient Bio (Gyunggi-do, Korea) and housed one per cage in the specific pathogen-free facility in 12-h light/12-h dark cycles. All of the mice consumed a normal chow diet and tap water ad libitum for 1 week prior to their division into three weight-matched groups (n = 8 per group). The mice were fed a standard diet (STD; AIN-76A, Research Diets Inc., New Brunswick, NJ) or a HFD (60% of kcal derived from fat, Research Diets Inc.) with either vehicle (1% methylcellulose; 1 mL/kg, p.o.) or 18β-GA (30 mg/kg, per oral (p.o.)) once a day for 8 weeks. Body weight gain followed once a week, and 24-h food intake per cage was measured twice a week by weighing the food bottles and taking spillages into account. Food efficiency ratio (FER) was calculated as the ratio of body weight gain (g) per food intake (g). At the end of the experimental period, all mice were euthanized and dissected tissue specimens were immediately stored at −80°C until analysis. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Pacificpharma Corporation.

2.8 In silico evaluation of 18β-GA blood–brain barrier (BBB) permeability

The propensity of BBB passage by 18β-GA has been preliminarily evaluated on the basis of the following well established parameters: the rate of brain penetration (log PS), and the ratio of the steady-state concentrations of the drug molecule in the brain and in the blood, usually expressed as log(Cbrain/Cblood) or, more simply, log BB. Negative values of log BB, particularly ‹−1.0, has been determined for sparingly BBB permeable compounds. 18β-GA log PS and log BB values were calculated in silico by using algorithms based on putative physicochemical properties of the compound, predicted from its formula with the PhysChem ADME-Tox prediction modules on the ACD/Percepta platform. To compare 18β-GA with reference CB1 antagonist, log PS, and log BB values were also determined for SR141716A.

2.9 Statistical analysis

Statistical analyses were made using Minitab v. 14.0 software (Minitab Inc. PA. USA). Bivariate analyses were made using the unpaired two-tailed Student's t-test and one-way ANOVA. Post hoc comparisons were made using the Dunnett's multiple comparison test. A p value of < 0.05 was considered to indicate statistical significance. Unless otherwise indicated, data are given as mean ± SD of at least three independent experiments.

3 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

3.1 Effect of licorice extract and 18β-GA on AEA induced- CB1R activation

To investigate whether the extract of licorice root and its main active ingredients may mitigate the effects of AEA on CB1R signaling, we examined the inhibitory activity in human CB1R-expressing Chem-1 cells. An endogenous cannabinoid receptor ligand, anandamide (AEA) activated Ca2+ flux with an EC50 value of 0.91 ± 0.08 μM in CB1R-expressing Chem-1 cells (Fig. 1A). However, whole extract of licorice root inhibited the Ca2+ flux in a concentration-dependent manner, with IC50 value of 9.17 ± 1.62 μg/mL against 3 μM AEA (Fig. 1B). Major active ingredients of licorice root that include liquirtin, GL, 18α-GA, and 18β-GA also exhibited inhibitory activity at 30 μM of each of the test compounds against Ca2+ flux induced by 3 μM AEA, where 18β-GA exhibited the strongest potency exceeding 90% inhibition in responses to CB1R agonist (Fig. 1C). 18β-GA elicited dose dependent decrease in intracellular Ca2+ levels with IC50 value of 1.96 ± 0.05 μM (Fig. 1D). These results suggest that 18β-GA may be the major active principle for AEA induced-CB1R downregulatory effect of licorice root extract.

image

Figure 1. Licorice and its ingredients suppress AEA activated Ca2+ flux in CB1R-expressing Chem-1 cells. Full-length human CB1R-transfected Chem-1 cells were incubated for 1 h in loading media with Fluo-4 and then washed with assay buffer. Anandamide (AEA) evoked increases in Ca2+ flux at the indicated concentrations (0.1, 0.3, 1, 3, and 10 μM) (A). Whole extract (0.1, 1, 3, 10, 30, and 100 μg/mL) from licorice root (B), various active compounds (30 μM) (C), or 18β-GA (0.1, 0.3, 1, 3, 10, and 30 μM) (D) were added to the cells, and incubated for 10 min at 37°C. Ca2+ in response to AEA (3 μM) was determined in triplicate on a Flex station.

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3.2 Inhibitory effect of 18β-GA on adipocyte differentiation of 3T3-L1 preadipocytes

Previous studies have demonstrated that AEA stimulates adipogenesis through the CB1R activation and CB1R blockade in adipocyte inhibits the differentiation of preadipocytes into mature adipocytes [30, 31]. Therefore, we investigated the effects of AEA and 18β-GA on the conversion of 3T3-L1 preadipocytes to adipocytes by Oil Red O staining. Preadipocytes with AEA at various concentrations (0.1, 1, and 10 μM) showed increased lipid accumulation when assessed after 8 days (Fig. 2A). However, when 18β-GA was present together with 10 μM of AEA during differentiation, the stimulatory effects of AEA were abolished in a concentration dependent manner (Fig. 2B). The optical densities of Oil Red O eluted solutions showed 63% decrease in lipid accumulation by 30 μM 18β-GA compared with the vehicle control (Fig. 2C). Treatment with SR141716, a potent and selective CB1 receptor antagonist, significantly inhibited the adipocyte differentiation at a concentration of 1 μM (Fig. 2B and C). Moreover, treatment of 3T3-L1 preadipocytes with 18β-GA alone also decreased the adipocyte differentiation in a dose-dependent manner (Supporting Information Fig. 1).

image

Figure 2. 18β-GA inhibits lipid accumulation in AEA-treated 3T3-L1 preadipocytes. Effect of AEA on lipid accumulation and adipogenesis in 3T3-L1 cells (A). Preadipocytes were induced to differentiate with AEA in increasing concentrations (0.1, 1, and 10 μM) for 8 days. Anti-adipogenic effect of 18β-GA in AEA-treated 3T3-L1 cells (B). Preadipocytes were induced to differentiate with AEA (10 μM) and 18β-GA in increasing concentrations (3, 10, and 30 μM) or SR141716 (1 μM) for 8 days. The cellular lipid content was assessed by Oil Red O staining, and quantified by isopropanol (C). Each experiment was independently performed at least three times. Each bar represents mean ± SD. **p < 0.01 versus AEA-treated control. Con, vehicle control; GA, 18β-GA; SR, SR141716.

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3.3 Inhibitory effect of 18β-GA on the expression of adipogenic markers by AEA-induced MAPK inactivation in 3T3-L1 cells

Since the inhibition of ERK and p38 MAPK activity may play a role in the inhibition of adipogenesis by CB1R antagonist [32, 33], we investigated the effects of 18β-GA on ERK and p38 MAPK activation during 3T3-L1 adipogenesis. AEA triggered ERK and p38 MAPK activation in 3T3-L1 cells and the appearance of phospho-ERK and phospho-p38 became evident at 5 min after treatment (Fig. 3A). In contrast, 30 μM 18β-GA or 1 μM SR141716 pretreatment during adipogenesis strongly attenuated the 10 μM AEA-stimulated phosphorylations of both ERK and p38 MAPK (Fig. 3B). To further elucidate the mechanism underlying the blockade of adipocyte differentiation by 18β-GA, we used quantitative real-time PCR to examine the expressions of C/EBP-α and PPAR-γ. Thirty micromolars 18β-GA or 1 μM SR141716 suppressed significantly AEA-induced C/EBP-α and PPAR-γ expression (Fig. 3C). Moreover, we measured the protein activities of key lipogenic enzymes including ACC, FAS, and stearoyl-CoA desaturase 1 (SCD-1), which are regulated by C/EBP-α or PPAR-γ [32]. 18β-GA or SR141716 promoted phosphorylation of ACC and downregulated the expression levels of FAS and SCD-1 (Fig. 3D) whose patterns were also well correlated with the downregulation of C/EBP-α and PPAR-γ. Interestingly, the effect of 18β-GA on the ACC phosphorylation was significantly higher than that of the SR141716. In agreement with the changes in morphology, 18β-GA reversed the gene and protein expressions upregulated by AEA.

image

Figure 3. 18β-GA regulates expression of adipogenic and lipogenic markers in AEA-related 3T3-L1 cells. Fully differentiated 3T3-L1 adipocytes were exposed to serum-free DMEM for overnight and then, cells were incubated for the indicated time periods with 10 μM AEA (A). Preadipocyte 3T3-L1 cells were grown to confluence and induced to differentiate into mature adipocytes in the presence 30 μM 18β-GA, 1 μM SR141716, or vehicle. On day 8 after differentiation, culture medium was changed and cells were cultured in serum-free DMEM for overnight with 18β-GA, SR141716 or vehicle. The levels of ERK and p38 MAPK phosphorylation in serum starved cells were measured by immunoblotting after 5 min incubation with 10 μM AEA (B). Preadipocytes were induced to differentiate with 18β-GA, SR141716, or vehicle in the absence or presence of 10 μM AEA for 8 days. Adipogenesis-related genes were determined by qRT-PCR (C). Lipogenic proteins in total cell extracts were analyzed by immunoblotting (D). These data shown are representative of three independent experiments. Each bar represents mean ± SD. ##p < 0.01 versus the AEA-untreated control; *p < 0.05, ** p < 0.01 versus AEA-treated control. Con, vehicle control; GA, 18β-GA; SR, SR141716.

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3.4 Effect of 18β-GA on body weight gain and the plasma levels of lipid parameters in obese mice

To investigate whether the inhibitory activity of 18β-GA on the CB1R could ameliorate body weight, mice were fed a HFD for 8 weeks in the presence of 18β-GA and body weight gain was monitored once a week. Oral administration of 18β-GA (30 mg/kg/day for 8 weeks) significantly reduced body weight gain by HFD and the FER of the 18β-GA-fed mice was 31% lower than that of the HFD-fed mice (Fig. 4A and B). Furthermore, in the presence of 18β-GA, the levels of plasma triglycerides, total cholesterol markedly decreased (Fig. 4C), which seemed to be associated with reduced adiposity and lipid metabolism.

image

Figure 4. 18β-GA ameliorates obesity induced by HFD. Mice were fed either a standard diet (STD) or high-fat diet (HFD) for 8 weeks in the presence (30 mg/kg/day) or absence of 18β-GA (n = 8 per group). Body weight changes (A), Body weight gain and FER (FER, food efficiency ratio (%) = Body weight gain for the experimental period (g)/Food intake for the experimental period (g) X 100) (B) in vehicle- or 18β-GA-treated mice. Plasma levels of triglyceride, total cholesterol were measured (C). Each bar represents mean ± SD. of eight mice. #p < 0.05, ##p < 0.01 versus STD; *p < 0.05, ** p < 0.01 versus HFD. STD, standard diet; HFD, high-fat diet; HFD + GA, HFD and 30 mg/kg/day 18β-GA.

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3.5 Inhibitory effect of 18β-GA on adiposity and expression of genes involved in lipid metabolism in obese mice

To examine if the reduced body weight gain in 18β-GA treated group is related to decreased fat accumulation, the weight of the epididymal white adipose tissue and the sizes of adipocytes were examined. In obese mice, 18β-GA greatly decreased the weight of epididymal adipose tissue (Fig. 5A) and adipocyte fat cell size was significantly reduced (Fig. 5B and C). Next, to better understand the molecular mechanism by which 18β-GA mediates the beneficial effects on obese animals described above, the expression of genes involved in energy metabolism was investigated. Consistent with the reduced plasma and adipocyte lipid content upon 18β-GA administration in obese animals, expression of lipogenic genes, ACC, FAS, and SCD-1, in adipose tissue was downregulated by 18β-GA (Fig. 6A). Moreover, in the epididymal white adipose tissue of mice chronically treated with 18β-GA we also observed a down-regulation of metabolic enzymes of lipid and endocannabinoid system such as CB1R and fatty acid amide hydrolase (Fig. 6B). These data indicate that 18β-GA might alleviate HFD-induced metabolic abnormalities, at least in part, through regulating the expression of a certain set of metabolic genes.

image

Figure 5. 18β-GA decreases adiposity in obese mice. Fat pad weight from epididymal white adipose tissue (WAT) (A), Histological analysis of the epididymal WAT after staining with hematoxylin and eosin (B) from vehicle- or 18β-GA-treated obese mice and adipocyte size was quantified (C). Each bar represents mean ± SD. of eight mice. ##p < 0.01 versus STD; ** p < 0.01 versus HFD. STD, standard diet; HFD, high-fat diet; HFD + GA, HFD and 30 mg/kg/day 18β-GA.

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image

Figure 6. 18β-GA regulates expression of genes involved in lipid metabolism. Total RNA was isolated from epididymal WAT of vehicle- or 18β-GA-treated obese mice. Relative mRNA levels of metabolic enzymes of lipid (A) and endocannabinoid system (B) were determined using qRT-PCR. The gene expression levels were normalized by the level of glyceraldehydes 3-phosphate dehydrogenase mRNA. Experiments were independently performed at least three times. Data represent mean ± SD. #p < 0.05, ##p < 0.01 versus STD; *p < 0.05, ** p < 0.01 versus HFD. STD, standard diet; HFD, high-fat diet; HFD + GA, HFD and 30 mg/kg/day 18β-GA.

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4 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

In the current study, we demonstrated that licorice extract and its active ingredients, liquiritin, GL, 18α-GA, and 18β-GA were able to attenuate the effects of AEA on CB1R signaling. More importantly, we showed that 18β-GA, the most potent ingredient among tested, displayed anti-adipogenesis and anti-obesity effects, which may ultimately induce weight loss and have a positive effect on lipid metabolism. Although many synthetic drug candidates including SR141716 (rimonabant) have been developed and studied, little is known about phytochemicals or natural products exhibiting CB1R inhibition. With this study, we believe that a new insight has been provided into the mechanism underlying anti-obesity effects of licorice and its active ingredients.

CB1R is implicated in the modulation of energy metabolism in peripheral tissues such as adipose tissue, skeletal muscle, pancreas and liver [19]. Adipose tissue is the most-frequently studied tissue in the context of anti-obesity effects of CB1R antagonists at peripheral levels. Endocannabinoids including AEA have several effects on adipocytes that include the promotion of adipocyte differentiation, triglyceride synthesis and glucose uptake [1, 3, 35]. In contrast, CB1R blockade induces lipolysis, thereby increasing the availability of free fatty acids for oxidation and possibly manifesting anti-obesity effects overall [36]. In our study, 18β-GA plays an inhibitory role in Ca2+ flux by AEA-induced CB1R activation and dose-dependently inhibited adipocyte differentiation. Furthermore, the pretreatment with 18β-GA significantly reduced lipid accumulation, which had been prompted by AEA-induced CB1R activation (Fig. 1, 2).

Suppression of AEA induced-CB1R activation by 18β-GA was further demonstrated by the blockade of MAPK activation and C/EBP-α and PPAR-γ upregulation (Fig. 3B, C). MAPK, C/EBP-α, and PPAR-γ activation are the major events underlying the CB1R mediated-adipocyte differentiation [37]. The endocannabinoids directly bind to PPAR-γ, activate CB1R on the cell surface or initiate intracellular signaling that may lead to adipogenic transcription factors such as C/EBP-α or PPAR-γ expression through the activation of ERK and p38 MAPK [3, 32, 38]. In our results, AEA activated the ERK and p38 MAPK in 3T3-L1 cells, and the effect was abolished by 18β-GA. Furthermore, AEA-induced upregulation of C/EBP-α and PPAR-γ expression was attenuated by co-incubation with 18β-GA, indicating that inhibitory effects of 18β-GA against adipocyte differentiation may be mediated by blockade of CB1R-induced C/EBP-α and PPAR-γ upregulation via the suppression of MAPK activation at least in part. Here we also showed that 18β-GA treatment caused induction of ACC phosphorylation and reduction of FAS and SCD-1 (Fig. 3D). Eventually, it leads to the suppression of unnecessary lipid accumulation in adipocytes. Although details of the mechanism by which 18β-GA influences expression of these lipogenic proteins remain to be elucidated, previous reports have shown that CB1R inactivation upon treatment with SR141716 also controls expression of those proteins [39]. The chronic effects by CB1R inactivation involve changes in gene expression, which likely contribute to reduced fat cell differentiation and again contributing to lipid lowering, reduced fat mass, and improved insulin sensitivity [40, 41]. Recently, several reports have shed light on the connection between CB1R and PPAR-γ in the regulation of energy homeostasis [15, 33, 42-45]. For instance, activation of CB1R induces transcription factor PPAR-γ and its target lipogenic enzymes leading to regulated lipid metabolism [4, 34, 45].

In this study, we also observed that 18β-GA could alleviate body weight, visceral adiposity, and hyperlipidemia in high fat induced obese animals. 18β-GA efficiently reduces body weight, adipose mass, adipocyte hypertrophy, and the expression of lipogenic genes was potently suppressed in adipose tissue of 18β-GA-treated obese mice (Fig. 4-6A). These are further supported by our findings showing a downregulation of CB1R and fatty acid amide hydrolase mRNA in the fat tissue of high fat induced obese mice treated with 18β-GA (Fig. 6B). It is very likely that 18β-GA reduce adipogenesis and lipogenesis in 3T3-L1 cells, via CB1R inactivation, which might contribute to reduced adipose tissue mass and adipocyte hypertrophy. Thus, it is plausible that 18β-GA alters the expression of genes involved in lipid metabolism, which might chronically decrease lipid dysregulation in obese animals.

It is commonly recognized that SR141716A has been withdrawn from the market for reported side effects [46, 47]. Despite its clinically ascertained effect in human body weight reduction and in cardiovascular risk factor improvement, serious CNS side effects such as anxiety, depression, and suicidal ideation have been in fact evidenced [48]. The CNS side effects have been considered to be related with the ability of the compounds to cross the BBB and access CNS CB1R [49-51]. Therefore, we identified the ratio of the steady state distribution of molecules between the brain and the plasma usually expressed as log BB by the in silico comparison of 18β-GA with SR141716 (Table 1). Interestingly, log BB calculated value of 18β-GA is in particular the negative and it approaches to −1.0 that is considered the limit to avoid BBB crossing. However, this could not assure the total lack of 18β-GA in the brain and also should be proved through the determination of the pharmacokinetic parameters of 18β-GA in the further studies.

Table 1. The propensity of BBB passage by 18β-GA
Compoundlog PSlog BB
  1. The following has been preliminarily evaluated on the basis of the parameters: the rate of brain penetration (log PS), and the ratio of the steady-state concentrations of the drug molecule in the brain and in the blood, usually expressed as log(Cbrain/Cblood) or, more simply, log BB. The data analysis was performed with the PhysChem ADME-Tox prediction modules on the ACD/Percepta platform.

SR141716−1.20.07
18β-GA−2.9−0.18

Another safety concern of licorice is its potential effect on blood pressure. The active compound of licorice, glycyrrhizic acid (GA), is hydrolyzed in the body to form glycyrrhetinic acid, which inhibits renal 11β-HSD2 and by that mechanism increases access of cortisol to its receptors to produce renal sodium retention and potassium loss [52]. Considering that a regular intake of 100 mg GA/day is the lowest-observed-adverse-effect level and using a safety factor of 10, a daily intake of 10 mg GA would represent a safe dose for most healthy adults [53].

In conclusion, licorice extract and its active ingredient 18β-GA inhibit adiposity and metabolic disorders in obese animals. These effects of 18β-GA are produced through strong and sustained changes in lipid homeostasis by alleviating the effects of AEA on CB1R signaling, implying that 18β-GA would be a new strategy in the prevention and treatment of obesity and metabolic disorders.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

This study was financially supported by the grant from National Research Foundation, Korea (No. 2012R1A2A1A03006092).

The authors have declared no conflict of interest.

5 References

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information
  • 1
    Di Marzo, V., Matias, I., Endocannabinoid control of food intake and energy balance. Nat. Neurosci. 2005, 8, 585589.
  • 2
    Quarta, C., Mazza, R., Obici, S., Pasquali, R., Pagotto, U., Energy balance regulation by endocannabinoids at central and peripheral levels. Trends Mol. Med. 2011, 17, 518526.
  • 3
    Matias, I., Petrosino, S., Racioppi, A., Capasso, R. et al., Dysregulation of peripheral endocannabinoid levels in hyperglycemia and obesity: effect of high fat diets. Mol. Cell. Endocrinol. 2008, 286, S66S78.
  • 4
    Matias, I., Gonthier, M. P., Orlando, P., Martiadis, V. et al., Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia. J. Clin. Endocrinol. Metab. 2006, 91, 31713180.
  • 5
    Gasperi, V., Fezza, F., Pasquariello, N., Bari, M. et al., Endocannabinoids in adipocytes during differentiation and their role in glucose uptake. Cell. Mol. Life Sci. 2007, 64, 219229.
  • 6
    Kim, J., Li, Y., Watkins, B. A., Endocannabinoid signaling and energy metabolism: a target for dietary intervention. Nutrition 2011, 27, 624632.
  • 7
    Bouaboula, M., Perrachon, S., Milligan, L., Canat, X. et al., A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. J. Biol. Chem. 1997, 272, 2233022339.
  • 8
    Watt, M. J., Adipose tissue-skeletal muscle crosstalk: are endocannabinoids an unwanted caller? Diabetologia 2009, 52, 571573.
  • 9
    Colombo, G., Agabio, R., Diaz, G., Lobina, C. et al., Appetite suppression and weight loss after the cannabinoid antagonist SR 141716. Life Sci. 1998, 63, PL113PL117.
  • 10
    Elmquist, J. K., Elias, C. F., Saper, C. B., From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 1999, 22, 221232.
  • 11
    Cota, D., Marsicano, G., Tschop, M., Grubler, Y. et al., The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. J. Clin. Invest. 2003, 112, 423431.
  • 12
    Ravinet, T. C., Delgorge, C., Menet, C., Arnone, M., Soubrie, P., CB1 cannabinoid receptor knockout in mice leads to leanness, resistance to diet-induced obesity and enhanced leptin sensitivity. Int. J. Obes. Relat. Metab. Disord. 2004, 28, 640648.
  • 13
    Poirier, B., Bidouard, J. P., Cadrouvele, C., Marniquet, X. et al., The anti-obesity effect of rimonabant is associated with an improved serum lipid profile. Diabetes Obes. Metab. 2005, 7, 6572.
  • 14
    Bensaid, M., Gary-Bobo, M., Esclangon, A., Maffrand, J. P. et al., The cannabinoid CB1 receptor antagonist SR141716 increases Acrp30 mRNA expression in adipose tissue of obese fa/fa rats and in cultured adipocyte cells. Mol. Pharmacol. 2003, 63, 908914.
  • 15
    Pagano, C., Rossato, M., Vettor, R., Endocannabinoids, adipose tissue and lipid metabolism. J. Neuroendocrinol. 2008, 20 (Suppl 1), 124129.
  • 16
    Girard, J., Perdereau, D., Foufelle, F., Prip-Buus, C., Ferre, P., Regulation of lipogenic enzyme gene expression by nutrients and hormones. FASEB J. 1994, 8, 3642.
  • 17
    Poulos, S. P., Dodson, M. V., Hausman, G. J., Cell line models for differentiation: preadipocytes and adipocytes. Exp. Biol. Med. 2010, 235, 118593.
  • 18
    Green, H., Kehinde, O., Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell 1976, 7, 105113.
  • 19
    Wang, S., Soni, K. G., Semache, M., Casavant, S. et al., Lipolysis and the integrated physiology of lipid energy metabolism. Mol. Genet. Metab. 2008, 95, 117126.
  • 20
    Baltina, L. A., Chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine. Curr. Med. Chem. 2003, 10, 15571.
  • 21
    Cho, H. J., Lim, S. S., Lee, Y. S., Kim, J.-S. et al., Hexane/ethanol extract of Glycyrrhiza uralensis licorice exerts potent anti-inflammatory effects in murine macrophages and in mouse skin. Food Chem. 2010, 121, 959966.
  • 22
    Rauchensteiner, F., Matsumura, Y., Yamamoto, Y., Yamaji, S., Tani, T., Analysis and comparison of Radix Glycyrrhizae (licorice) from Europe and China by capillary-zone electrophoresis (CZE). J. Pharm. Biomed. Anal. 2005, 38, 594600.
  • 23
    Montoro, P., Maldini, M., Russo, M., Postorino, S. et al., Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS. J. Pharm. Biomed. Anal. 2011, 54, 535544.
  • 24
    Kim, H. J., Lim, S. S., Park, I. S., Lim, J. S. et al., Neuroprotective effects of dehydroglyasperin C through activation of heme oxygenase-1 in mouse hippocampal cells. J. Agric. Food Chem. 2012, 60, 55835589.
  • 25
    Mae, T., Kishida, H., Nishiyama, T., Tsukagawa, M. et al., A licorice ethanolic extract with peroxisome proliferator-activated receptor-gamma ligand-binding activity affects diabetes in KK-Ay mice, abdominal obesity in diet-induced obese C57BL mice and hypertension in spontaneously hypertensive rats. J. Nutr. 2003, 133, 33693377.
  • 26
    Moon, M. H., Jeong, J. K., Lee, Y. J., Seol, J. W. et al., 18β-glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis. Biochem. Biophys. Res. Commun. 2012, 420, 805810.
  • 27
    Eisenbrand, G., Glycyrrhizin. Mol. Nutr. Food Res. 2006, 50, 10871088.
  • 28
    Visavadiya, N. P., Narasimhacharya, A. V., Hypocholesterolaemic and antioxidant effects of Glycyrrhiza glabra (Linn) in rats. Mol. Nutr. Food Res. 2006, 50, 10801086
  • 29
    Classen-Houben, D., Schuster, D., Da Cunha, T., Odermatt, A. et al., Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid. J. Steroid Biochem. Mol. Biol. 2009, 113, 248252.
  • 30
    D'Eon, T. M., Pierce, K. A., Roix, J. J., Tyler, A. et al., The role of adipocyte insulin resistance in the pathogenesis of obesity-related elevations in endocannabinoids. Diabetes 2008, 57, 12621268
  • 31
    Bouaboula, M., Hilairet, S., Marchand, J., Fajas, L. et al., Anandamide induced PPARgamma transcriptional activation and 3T3-L1 preadipocyte differentiation. Eur. J. Pharmacol. 2005, 517, 174181
  • 32
    Childers, S. R., Pacheco, M. A., Bennett, B. A., Edwards, T. A. et al., Cannabinoid receptors: G-protein-mediated signal transduction mechanisms. Biochem. Soc. Symp. 1993, 59, 2750.
  • 33
    Vettor, R., Pagano, C., The role of the endocannabinoid system in lipogenesis and fatty acid metabolism. Best Pract. Res. Clin. Endocrinol. Metab. 2009, 23, 5163.
  • 34
    Graugnard, D. E., Piantoni, P., Bionaz, M., Berger, L. L. et al., Adipogenic and energy metabolism gene networks in longissimus lumborum during rapid post-weaning growth in Angus and Angus × Simmental cattle fed high-starch or low-starch diets. BMC Genomics 2009, 10, 142.
  • 35
    Bellocchio, L., Cervino, C., Vicennati, V., Pasquali, R., Pagotto, U., Cannabinoid type 1 receptor: another arrow in the adipocytes’ bow. J. Neuroendocrinol. 2008, 20(Suppl 1), 130138.
  • 36
    Herling, A. W., Kilp, S., Elvert, R., Haschke, G., Kramer, W., Increased energy expenditure contributes more to the body weight-reducing effect of rimonabant than reduced food intake in candy-fed wistar rats. Endocrinology 2008, 149, 25572566.
  • 37
    Bosier, B., Muccioli, G. G., Hermans, E., Lambert, D. M., Functionally selective cannabinoid receptor signalling: therapeutic implications and opportunities. Biochem. Pharmacol. 2010, 80, 112.
  • 38
    Karaliota, S., Siafaka-Kapadai, A., Gontinou, C., Psarra, K. et al., Anandamide increases the differentiation of rat adipocytes and causes PPARgamma and CB1 receptor upregulation. Obesity 2009, 17, 18301838.
  • 39
    Jourdan, T., Djaouti, L., Demizieux, L., Gresti, J. et al., CB1 antagonism exerts specific molecular effects on visceral and subcutaneous fat and reverses liver steatosis in diet-induced obese mice. Diabetes 2010, 59, 926934
  • 40
    Eckardt, K., Sell, H., Taube, A., Koenen, M. et al., Cannabinoid type 1 receptors in human skeletal muscle cells participate in the negative crosstalk between fat and muscle. Diabetologia 2009, 52(4), 664674.
  • 41
    Tam, J., Vemuri, V. K., Liu, J., Batkai, S. et al., Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity. J. Clin. Invest. 2010, 120(8), 29532966
  • 42
    Osei-Hyiaman, D., DePetrillo, M., Pacher, P., Liu, J. et al., Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J. Clin. Invest. 2005, 115, 12981305.
  • 43
    Kyrou, I., Valsamakis, G., Tsigos, C., The endocannabinoid system as a target for the treatment of visceral obesity and metabolic syndrome. Ann. N. Y. Acad. Sci. 2006, 1083, 270305.
  • 44
    Kunos, G., Osei-Hyiaman, D., Liu, J., Godlewski, G., Batkai, S., Endocannabinoids and the control of energy homeostasis. J. Biol. Chem. 2008, 283, 3302133025.
  • 45
    Monsif, B., Sandrine, H., Jean, M., Lluis, F. et al., Anandamide induced PPARγ transcriptional activation and 3T3-L1 preadipocyte differentiation. Eur. J. Pharmacol. 2005, 517, 174181.
  • 46
    Christensen, R., Kristensen, P. K., Bartels, E. M., Bliddal, H., Astrup, Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials. Lancet 2007, 370, 17061713.
  • 47
    Van Gaal, L., Pi-Sunyer, X., Despres, J. P., McCarthy, C. et al., Efficacy and safety of rimonabant for improvement of multiple cardimetabolic risk factors in overweight/obese patients: pooled 1-year data from the Rimonabant in Obesity (RIO) program. Diabetes Care 2008, 31, S229S240.
  • 48
    Nathan, P. J., O'Neil, B. V., Napolitano, A., Bullmore, E. T., Neuropsychiatric adverse effects of centrally acting antiobesity drugs. CNS Neruosci. Ther. 2011, 17, 490505.
  • 49
    Clark, D. E., In silico prediction of blood-brain barrier permeation. Drug Discov. Today 2003, 8, 927933.
  • 50
    Christopoulou, F. D., Kiortsis, D. N., An overview of the metabolic effects of rimonabant in randomized controlled trials: Potential for other cannabinoid 1 receptor blockers in obesity. J. Clin. Pharm. Ther. 2011, 36, 1018.
  • 51
    Kirilly, E., Gonda, X., Bagdy, G., CB1 receptor antagonists: New discoveries leading to new perspectives. Acta Physiol. 2012, 205, 4160.
  • 52
    Serra, A., Uehlinger, D. E., Ferrari, P., Dick, B. et al., Glycyrrhetinic acid decreases plasma potassium concentrations in patients with anuria. J. Am. Soc. Nephrol. 2002, 13(1), 191196.
  • 53
    Stormer, F. C., Reistad, R., Alexander, J., Glycyrrhizic acid in liquorice-evaluation of health hazard. Food. Chem. Toxicol. 1993, 31(4), 303312.

Supporting Information

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgments
  8. 5 References
  9. Supporting Information

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FilenameFormatSizeDescription
mnfr2188-sup-0001-SuppMat.pdf345KFigure S1. 18β-GA inhibits adipogenesis of 3T3-L1 preadipocytes. Preadipocytes were induced to differentiate with 18β-GA in increasing concentrations (10 and 30 μM) for 8 days. Lipid droplets were stained with Oil Red O on day 8 of 3T3-L1 adipocyte differentiation and examined using light microscopy (A). And then cells were harvested, and the lipid accumulation was measured through a spectrophotometer (B)

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