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Journal of Ethnopharmacology 155 (2014) 1342–1352
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Research Paper
Ginseng treatment reverses obesity and related disorders by inhibiting angiogenesis in female db/db mice Hyunghee Lee a, Mina Kim a, Soon Shik Shin b, Michung Yoon a,n a b
Department of Biomedical Engineering, Mokwon University, Daejeon 302-729, Republic of Korea Department of Formula Sciences, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 27 March 2014 Received in revised form 24 June 2014 Accepted 16 July 2014 Available online 27 July 2014
Ethnopharmacological relevance: Korean red ginseng (ginseng, Panax ginseng C.A. Meyer) has traditionally been used in the treatment of most ageing-related diseases, such as obesity, diabetes, and dyslipidemia, but the mechanism of the effects is unclear. The aim of this study was to determine the effects of ginseng on obesity in a mouse model of female obesity (obese female db/db mouse) and to investigate the mechanism of anti-obesity effects. Materials and methods: After female db/db (B6.Cg-m Leprdb/þ þ/J) mice were treated with 5% (w/w) ginseng for 13 weeks, variables and parameters of obesity and disorders related to obesity were examined. Blood vessel density and the expression of genes involved in angiogenesis were also measured. Results: Mice treated with ginseng for 13 weeks had less body weight and lower adipose tissue mass compared to control, untreated mice. The size of adipocytes was smaller in visceral adipose tissues of ginseng-treated mice. Obesity-related complications, such as hepatic steatosis, hypertriglyceridemia, and hyperglycemia, were markedly improved in treated mice. Blood vessel density was lower in visceral adipose tissue sections from treated mice than those from control mice. Concomitantly, mRNA levels for VEGF-A and FGF-2 were lower in both visceral adipose tissue from treated mice and treated 3T3-L1 cells compared to those from untreated controls. Protein levels for VEGF were also lower in visceral adipose tissue from treated mice. In contrast, ginseng increased mRNA expression of genes responsible for energy expenditure and fatty acid β-oxidation in visceral adipose tissue during ginseng-induced weight reduction. Conclusions: These results suggest that ginseng may effectively treat female obesity and related disorders in part by inhibition of angiogenesis. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Korean red ginseng Obesity Angiogenesis Female db/db Mouse Adipose tissue growth
1. Introduction Angiogenesis, the process of new blood vessel formation, is required for adipose tissue growth in adults, similar to its necessity for tumor growth (Crandall et al., 1997; Rupnick et al., 2002). Most tissues do not normally grow throughout adulthood, and their supporting vasculature is quiescent, whereas adult adipose tissue is probably one of the most plastic tissues in the body, and it expands and shrinks throughout adulthood (Cao, 2007). Adipose tissue is highly vascularized, and each adipocyte is nourished by an extensive capillary network (Bouloumié et al., 2002; Cao, 2007). It has therefore been suggested that adipose tissue growth can be regulated by the adipose tissue vasculature and is angiogenesis-dependent (Rupnick et al., 2002; Bråkenhielm et al., 2004; Kim et al., 2010). Thus,
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Corresponding author. Tel.: þ 8242 829 7581; fax: þ 8242 829 7580. E-mail address: [email protected] (M. Yoon).
http://dx.doi.org/10.1016/j.jep.2014.07.034 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
modifying or blocking angiogenesis may help manage or eliminate obesity. Growing adipocytes produce angiogenic factors, such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2, contributing to the formation of new blood vessels inside the fat pad (Claffey et al., 1992; Voros et al., 2005; Cao, 2007). VEGF-A and FGF-2 stimulate proliferation and migration of endothelial cells and enhance adipocyte differentiation (Carmeliet et al., 1996; Bikfalvi et al., 1997). Anti-angiogenic drugs, such as those targeting VEGF-A or FGF-2 pathways, may be key components of therapy for obesity and obesity-related metabolic diseases. Ginseng, the root of Panax ginseng C.A. Mayer (Araliaceae), has historically been used as one of the most popular herbal medicines to treat various diseases in Korea, China and Japan for thousands of years. Ginseng has been traditionally used in the treatment of most ageing-related diseases, such as diabetes, obesity, and dyslipidemia (Yin et al., 2008). Ginseng and its active components ginsenosides inhibit obesity in several obese animal models, such
H. Lee et al. / Journal of Ethnopharmacology 155 (2014) 1342–1352
as high fat-fed Sprague-Dawley rats, C57BL/6J mice, and Balb/c mice, and genetically obese ob/ob mice (Kim et al., 2005; Karu et al., 2007; Mollah et al., 2009; Lee et al., 2012; Lee et al., 2013), but the mechanism of the effects is unclear. Accumulating evidence also shows that ginseng has been reported to inhibit angiogenesis, which prevents tumor growth (Sato et al., 1994; Ho et al., 2012). Ginsenosides exhibit potential as potent cancer chemopreventive agents because of this anti-angiogenic activity (Yue et al., 2006; Jeong et al., 2010). Based on the aforementioned literature that ginseng modulates angiogenesis (Sato et al., 1994; Liu et al., 2009) and adipose tissue growth is regulated by angiogenesis (Cao, 2010), we hypothesized that the anti-angiogenic properties of Korean red ginseng (ginseng) could treat obesity and its metabolic diseases. It is, moreover, known that males and females differ in the regulation of body weight and body fat (Brown and Clegg, 2010). Men and post-menopausal women accumulate more fat in intraabdominal depots than pre-menopausal women, resulting in a greater risk of developing complications associated with obesity (Geer and Shen, 2009). Our previous results demonstrated that ginseng effectively prevents male obesity in high fat diet-fed C57BL/6J mice (Lee et al., 2013). Thus, we examined the effects of ginseng on female obesity using female homozygous leptin receptor-deficient db/db mice, which exhibit an obesity syndrome characterized by severe adiposity, dyslipidemia, and insulin resistance (Clement et al., 1998; Muoio and Lynis Dohm, 2002). These mice have body weights three times greater than normal and exhibit a five-fold increase in body fat content (Friedman and Halaas, 1998). We show that ginseng suppresses female obesity by angiogenesis inhibition. Ginseng-treated mice showed less body weight and adipose tissue mass compared with untreated mice. Blood vessel density was concomitantly lower in visceral adipose tissues of treated mice compared to obese mice. Ginseng also modulated the mRNA and protein expression of angiogenic factors. We also observed that ginseng increased the expression of genes involved in energy expenditure and fatty acid oxidation in visceral adipose tissues during ginseng-induced weight loss. In addition, ginseng treatment is a reversal protocol since ginseng was given to obese mice. This protocol mimics the intervention in obese patients rather than the prevention of obesity by testing in lean animals fed an obesogenic diet simultaneously with the intervention. Therefore, these results suggest that the anti-angiogenic actions of ginseng may inhibit adipose tissue growth and reverse obesity and related disorders in female db/db mice.
2. Materials and methods 2.1. Ginseng preparation Ginseng extract powder was commercially prepared from ginseng cultivated with care in well-fertilized fields for 6 years (KT&G, Daejeon, Korea). Total ginsenosides (TGSs) were obtained by extraction from the ginseng extract powder (Yoon et al., 2003). Briefly, ginseng extract powder (100 g) was placed into a 1-L flask with a refluxing condenser and extracted twice with 500 mL of water-saturated 1-butanol for 1 h at 80 1C. The extracted solution was passed through Whatman filter paper (No. 41) after cooling. The process was repeated twice. The residue and filter paper were washed with 100 mL of water-saturated 1-butanol, and then the filtrate was washed twice with 100 mL of water in a 2-L separating funnel. The butanol layer was then evaporated to dryness. The concentrate was extracted to remove any traces of fat with 100 mL of diethyl ether for 30 min at 36 1C in a flask with a refluxing condenser, after which the ether solution was decanted.
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The residue was dried at 50 1C and weighed. Individual GSs were analyzed by the HPLC/ELSD system (Fig. 1). The HPLC/ELSD analytical conditions are as follows: (i) Column, Lichrosorb NH column (25 0.4 cm, 5 μm, Merck). (ii) Mobile phase, mixtures of solvent A (acetonitrile/water/isopropanol ¼80:5:15) and solvent B (acetonitrile/water/isopropanol¼ 80:20:15). (iii) Gradient profile of solvent A to solvent B, from 70:30 to 0:100 for 0–20 min; 0:100 for 20–55 min; from 0:100 to 70:30 for 55–65 min; flow rate, 1.0 ml/min. (iv) Detector, ELSD (ELSD 2000, Alltech, dearfield, IL, USA): detection temperature 92 1C; neutralizing gas, nitrogen, 2.0 l/min. 2.2. Animal study Female, eight-week-old, genetically obese B6.Cg-m Leprdb/þ þ/J (db/db) mice and lean B6.Cg-m Leprdb/þ/J (db/þ) mice were housed at Mokwon University under pathogen-free conditions with a standard 12-h light/dark cycle. Mice were divided into three groups (n¼ 8/group): a lean group given a standard diet (normal mice), a genetically obese group given a standard diet (obese control mice), and a genetically obese group given a standard diet supplemented with 5% (w/w) ginseng (ginseng-treated mice). For ginsengsupplemented high fat diet, 50 g ginseng extract powder was mixed with 1 kg high fat diet. Mice received approximately 4.5 g ginseng extract/kg BW/day. Ginseng extract at these doses did not show any toxic effects. Body weights were measured daily by a person blinded to each treatment group. Food intake was determined by estimating the amount of food consumed by the mice throughout the treatment period. The small amount of food spillage that was typically noted when the cages were inspected was collected to ensure accurate measurement of food intake. After a 12-h fast on the last day of the study, the animals were sacrificed by cervical dislocation. Blood was collected from the retro-orbital sinus into tubes, and serum was separated and stored at 80 1C until analysis. Visceral adipose tissues were removed, weighed, snap-frozen in liquid nitrogen, and stored at 80 1C until use. Portions of the liver and visceral adipose tissues were prepared for histology. All animal experiments were approved by the Institutional Animal Care and Use Committees of Mokwon University, following National Research Council Guidelines. 2.3. 3T3-L1 differentiation Mouse 3T3-L1 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (Invitrogen, Carlsbad, CA, USA). After cells were maintained at confluence for 2 days, they were incubated in an MDI induction medium (day 0) containing 0.5 mM 1-methyl-3isobutyl-xanthine, 1 μM dexamethasone, and 1 μg/ml insulin in DMEM containing 10% fetal bovine serum (FBS, Invitrogen). The cultures were continued for 2 days to induce adipocyte differentiation. Thereafter, cells were cultured in DMEM containing 10% FBS for the remainder of the differentiation process. All other treatments including ginseng, TGSs, and individual GSs were administered on days 0–2 only, and the medium was changed every other day. 2.4. Blood analysis Levels of triglycerides and free fatty acids were measured using an automatic blood chemical analyzer (CIBA Corning, Oberlin, OH, USA) and a SICDIA NEFAZYME kit (Shinyang Chemical, Seoul, Korea), respectively. Levels of serum glucose and insulin were measured using an Accu-Chek Performa System (Roche, Germany) and an insulin radioimmunoassay kit (Linco, St. Charles, MO, USA), respectively.
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Fig. 1. The HPLC profile of ginseng extract. Individual ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, and Rg3 are analyzed by the HPLC/ELSD system. The most abundant ginsenoside is Rb1. Peak numbers denote retention times.
2.5. Histological analysis Liver and visceral adipose tissue samples were fixed in 10% phosphate-buffered formalin (PBF) for 1 day and processed in a routine manner for paraffin sections. Tissue sections (5 μm) were cut and stained with hematoxylin and eosin (H and E) for microscopic examination. To quantify adipocyte size, the H and E-stained sections were analyzed using the Image-Pro Plus analysis system (Media Cybernetics, Bethesda, MD, USA). Blood vessel staining was performed using a blood vessel staining kit (Chemicon, Billerica, MA, USA). Visceral adipose tissue samples were fixed in 10% PBF for 1 day and processed for paraffin sectioning using standard methods. Sections of 3-μm thickness were cut and irradiated in a microwave oven for epitope retrieval. Sections were sequentially incubated with rabbit anti-von Willebrand factor (vWF) antibody as a primary antibody, goat antirabbit antibody as a secondary antibody, and streptavidin-alkaline phosphatase solution. A freshly prepared chromogen reagent was added to the sections to visualize blood vessels. Blood vessel density was analyzed using the Image area analyzer program.
2.6. Quantitative real time PCR Total cellular RNA from visceral adipose tissues and 3T3-L1 adipocytes was prepared using the Trizol reagent (Gibco-BRL, Grand Island, NY, USA). Reverse transcription of 2 μg of total cellular RNA using Moloney murine leukemia virus reverse transcriptase resulted in the generation of an antisense primer cDNA. Synthesized cDNA fragments were amplified using AccuPowers GreenStarTM qPCR PreMix (Bioneer, Deajeon, Korea) on a
ExcyclerTM 96 Real Time Quantitative Thermal Block machine (Bioneer). The PCR sequences for VEGF-A are as follows (GeneBank number NM_001287056): VEGF-A forward primer 50 -gcagcttgagttaaacgaacg- 30 and reverse primer 50 -ggttcccgaaaccctgag-30 . The PCR sequences for FGF-2 are as follows (GeneBank number NM_008006): FGF-2 forward primer 50 -ccaaccggtaccttgctatga-30 and reverse primer 50 -ttcgttcagtgccacatacca-30 . The PCR sequences for peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) are as follows (GeneBank number BC066868): PGC-1α forward primer 50 -ccctgccatgttaagacc-30 and reverse primer 50 tgctgctgttcctgttttc-30 . The PCR sequences for uncoupling protein 1 (UCP1) are as follows (GeneBank number NM_009463): UCP1 forward primer 50 -ggcattcagaggcaaatcagct-30 and reverse primer 50 -caatgaacactgccacacctc-30 . The PCR sequences for UCP2 are as follows (GeneBank number NM_011671): UCP2 forward primer 50 ctggtcgccggcctgcagcgc-30 and reverse primer 50 -gatcccttcctctcgtgcaat-30 . The PCR sequences for carnitine palmitoyltransferase 1 (CPT-1) are as follows (GeneBank number NM_009948): CPT-1 forward primer 50 -ttcaacactacacgcatccc-3'and reverse primer 50 gccctcatagagccagacc-30 . The PCR sequences for medium-chain acyl coenzyme A dehydrogenase (MCAD) are as follows (GeneBank number NM_007382): MCAD forward primer 50 -ccgaagagttggcgtatggg-30 and reverse primer 50 -50 -gggctctgtcacacagtaagc-30 . The PCR sequences for β-actin are as follows (GeneBank number BC138614): β-actin forward primer 50 -taccacaggcattgtgatgg-30 and reverse primer 50 -tttgatgtcacgcacgattt-3´. The conditions of PCR were a denaturing step at 95 1C for 5 min followed by 50 cycles at 95 1C for 10 s, 60 1C for 40 s, and 72 1C for 10 s. Their concentrations were calculated as copies per ml using the standard curve and the relative expression levels were presented as a ratio of target gene cDNA versus β-actin cDNA.
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2.7. Western blot analysis Visceral adipose tissues were lysed in ice-cold lysis buffer [50 mM Tris–Cl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, and 1% Triton X-100] containing protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin). Lysates were centrifuged at 12,000 rpm for 20 min at 4 1C, and the resulting supernatants were subjected to electrophoresis on 10% polyacrylamide gels. The separated proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 10% nonfat milk (DIFCO, Detroit, MI, USA) in Tris-buffered saline (TBS). The membrane was washed three times with TBS containing 0.1% Tween-20 (TBS-T), followed by incubation with an anti-VEGF antibody (catalog number sc-507, Santa Cruz, CA, USA) (1:200 dilution). The membrane was washed three times with TBS-T and then incubated with the HRP-conjugated goat anti-rabbit IgG (catalog number sc-2004, Santa Cruz) diluted in TBS (1:2500 dilution) as a secondary antibody. The membrane was washed three times with TBS-T and visualized by an enhanced chemiluminescence western blot detection system (WEST-ZOL plus, Intron, Daejeon, Korea).
2.8. Statistics All values are expressed as mean 7standard deviation (SD). Statistical analysis was performed by ANOVA followed by Post-Hoc analysis using the SPSS software. Statistical significance was defined as a value of p o0.05.
3. Results 3.1. Body weight, adipose tissue mass, and food intake Obese (db/db) control mice showed markedly higher body weights compared with lean (db/þ ) mice after 13 weeks. However, mice fed a diet supplemented with ginseng had 21% lower body weights compared with obese control mice (Fig. 2A). Consistent with the decreases in body weight, total adipose tissue mass was also decreased in ginseng-treated mice compared with obese control mice. Total adipose tissue weights were decreased by 30% in ginseng-treated mice (Fig. 2B). The amount of food intake was not significantly changed by adding ginseng to the diet of obese control mice (Fig. 2C).
3.2. Adipocyte size Analysis of H and E-stained visceral adipose tissue sections revealed that adipocytes of ginseng-treated mice were much smaller than those of obese control mice (Fig. 3A). The average size of adipocytes in ginseng-treated mice (11,589 73630 μm2) was 49% smaller than that in obese control mice (22,539 7 1453 μm2, Fig. 3B).
3.3. Hepatic lipid accumulation The accumulation of hepatic lipids was determined after ginseng treatment in mice by H and E-staining and light microscopy. As shown in Fig. 4, obese control mice exhibited considerable hepatic lipid accumulation compared with lean normal mice. When obese mice were treated with ginseng, there was a complete inhibition of hepatic lipid accumulation, showing that ginseng inhibits hepatic steatosis.
Fig. 2. Effects of ginseng on body weight, adipose tissue mass, and food intake in female db/db mice. Mice (n¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Body weights at the end of the treatment period are significantly different between the normal and control groups (po0.05) and between the control and ginseng groups (po0.05). (B) Adipose tissue weights. (C) Food intake. All values are expressed as mean7SD. npo0.05 compared with normal group. nnpo0.05 compared with control group.
3.4. Blood lipids, glucose, and insulin levels Compared with normal mice, obese control mice exhibited much higher levels of serum triglycerides, free fatty acids, glucose,
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Fig. 3. Light microscopic analysis of visceral adipocytes. Mice (n¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Representative hematoxylin and eosin-stained sections (5-μm thickness) of visceral adipose tissue. Adipocyte size from the ginseng groups is smaller than that from control groups. (B) The size of adipocytes in a fixed area (1,000,000 μm2) is measured. All values are expressed as the mean7SD. npo0.05 compared with normal group. nnpo0.05 compared with control group.
and insulin. In contrast, ginseng-treated mice had significantly lower serum levels of triglycerides and free fatty acids (29% and 44% reductions, respectively) than untreated obese controls (Fig. 5A and B). Serum insulin and glucose levels were also lower (72% and 14%, respectively) in ginseng-treated mice compared with those in untreated controls (Fig. 5C and D). 3.5. Angiogenesis in adipose tissues Staining of visceral adipose tissue sections with an antibody against vWF, an endothelial cell marker, showed that blood vessel density was lower in ginseng-treated mice than in obese control mice. Ginseng-treated mice had 53% lower blood vessel density compared with controls (Fig. 6). 3.6. Expression of genes involved in angiogenesis in adipose tissues and 3T3-L1 adipocytes The expression patterns of genes involved in angiogenesis were investigated in visceral adipose tissues and 3T3L1 adipocytes. The mRNA expression of angiogenic factors was down-regulated in ginseng-treated mice compared with those in control mice. The mRNA levels of VEGF-A and FGF-2 were 80% and 85% lower, respectively, in ginseng-treated mice compared with those in control mice (Fig. 7A). The protein levels of VEGF were 54% lower
in mesenteric adipose tissues of ginseng-treated mice compared with those in obese control mice (Fig. 7B). Treatment of 3T3-L1 adipocytes with ginseng, TGSs, and individual GSs decreased the mRNA levels of VEFG-A and FGF-2 compared with their expression in untreated adipocytes. Ginseng treatment reduced VEFG-A and FGF-2 mRNA levels by 4% and 16%, respectively, and TGSs decreased their mRNA levels by 45% and 9%, respectively (Fig. 7C). In particular, Rb1 effectively decreased the mRNA levels of VEFG-A and FGF-2 by 42% and 34%, respectively.
3.7. Expression of genes involved in energy expenditure and fatty acid β-oxidation in adipose tissues The expression patterns of genes involved in energy expenditure and fatty acid β-oxidation were investigated in visceral adipose tissues. Their expression was up-regulated in ginsengtreated mice compared with those in control mice although UCP1 expression was down-regulated. The mRNA levels of PGC-1α, UCP2, CPT-1, and MCAD were 20%, 309%, 64%, and 110% higher, respectively, in ginseng-treated mice compared with those in control mice (Fig. 8). In contrast, UCP1 mRNA levels were 85% lower in ginseng-treated mice compared with those in control mice.
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Fig. 4. Inhibition of hepatic lipid accumulation by ginseng in female db/db mice. Mice (n ¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Representative H and E-stained sections of livers are shown (original magnification 100). Arrows indicate the fatty changes in hepatocytes. (B) Histological analyses of hepatic lipid accumulation. 0, no lesion; 1, mild; 2, moderate; 3, severe; 4, very severe. np o 0.05 compared with normal group. nnp o0.05 compared with control group.
4. Discussion Ginseng has been used as a traditional medicine in oriental societies and is now a popular and worldwide used natural medicine. Numerous studies have reported that ginseng has many pharmacological effects on the central nervous system, as well as on the endocrine, immune, and cardiovascular systems (Gillis, 1997; Attele et al., 1999; Yin et al., 2008; Lu et al., 2009). Ginseng has also been used as a traditional medicine in the treatment of most ageing-related diseases, such as obesity, diabetes, and dyslipidemia (Yin et al., 2008), but the mechanism of the effects is not clear. Based on reports showing that adipose tissue mass can be regulated by angiogenesis (Rupnick et al., 2002; Cao, 2007) and that ginseng inhibits angiogenesis (Sato et al., 1994; Ho et al., 2012; Park and Yoon, 2012), we examined the effects of ginseng on obesity and to investigate the mechanism of anti-obesity effects in obese db/db mice. It is, moreover, known that males and females have important differences in the regulation of body weight and body fat (Brown and Clegg, 2010). Since our previous results demonstrated that ginseng effectively prevents male obesity by inhibiting angiogenesis (Lee et al., 2013), we examined the effects of ginseng on female obesity and found that female db/db mice treated with ginseng showed alleviation of obesity and its related disorders, such as hepatic steatosis, hyperlipidemia, and hyperglycemia and that this process may be mediated, at least in part, through inhibiting angiogenesis. In addition, administration of
ginseng is a reversal protocol as ginseng was given to obese mice. This protocol mimics the intervention in obese patients rather than the prevention of obesity by testing in lean animals fed an obesogenic diet simultaneously with the intervention. Body weights and adipose tissue mass of ginseng-treated mice were much lower than those of untreated control db/db mice. Similarly, our previous results showed that ginseng-treated, high fat diet-fed obese mice had lower body weights and adipose tissue mass compared with male mice fed a high fat diet alone (Lee et al., 2013), indicating that ginseng regulates both genetically and nutritionally induced obesity. Interestingly, inhibitory effects of ginseng on obesity were more prominent in female mice than in male mice. It is thought that this is may be due to the presence of the ovarian steroid hormone estrogen (Brown and Clegg, 2010). We also observed that the magnitude of reduction in weight loss is greater in db/db mice than in high fat diet-fed obese mice because of the greater fat content in db/db mice. Newly formed adipose tissue depends on continued angiogenesis for further growth (Rupnick et al., 2002; Bråkenhielm et al., 2004), and different angiogenesis inhibitors have been shown to significantly reduce body weight and adipose tissue mass (Cao, 2007). Our results are supported by other reports in the literature showing that body weight gain and adipose tissue mass in obese animals are significantly reduced by several kinds of angiogenesis inhibitors, such as angiostatin, endostatin, TNP-470, TNP-470 analog CKD732, and VEGF receptor 2-specific inhibitors (Rupnick et al., 2002;
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Fig. 5. Changes in circulating levels of free fatty acids, triglycerides, glucose, and insulin. Mice (n¼ 8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). Serum (A) free fatty acids, (B) triglycerides, (C) glucose, and (D) insulin are determined after 13 weeks of ginseng treatment. npo0.05 compared with normal group. nnpo0.05 compared with control group.
Bråkenhielm et al., 2004; Kim et al., 2007; Lijnen et al., 2007). The size of adipocytes was considerably smaller in ginseng-treated mice than in untreated control mice, eventually resulting in decreased adipose tissue mass and body weights. Adipose tissue is highly vascularized and each adipocyte is nourished by an extensive capillary network (Bouloumié et al., 2002; Cao, 2007). Thus, inhibition of adipose tissue angiogenesis can be therapeutic targets of obesity and obesity-related metabolic diseases. Actually, we have observed that liver lipid accumulation in ginseng-treated mice was markedly inhibited, and serum levels of triglycerides, glucose, and insulin were lower in treated mice relative to untreated control mice. These results suggest that ginseng may alleviate metabolic diseases, such as liver steatosis, hypertriglyceridemia, and insulin resistance as consequences of anti-angiogenicinduced weight reduction. Ginseng-induced weight loss occurred without appetite changes, as evidenced by a similar food consumption profile between ginseng-treated and untreated control obese mice. It has been reported that treatment with angiogenesis inhibitors endostatin and low doses of TNP-470 or angiostatin, as well as antiangiogenic herbal composition Ob-X, did not suppress appetite (Rupnick et al., 2002; Kim et al., 2010). Rupnick et al. (2002) proposed that adipose tissue vessel may be maintained in a relatively immature state relative to that of other organs that are mass stable. Because anti-angiogenic agents target growing or newly formed vessels, adipose tissue vessels may be susceptible to angiogenesis inhibitors. Thus, anti-angiogenic ginseng may selectively target adipose tissue and cause weight reduction. Similar
results were observed in several obese animal models, such as fatty Zucker rats, high fat-fed Sprague-Dawley rats, and Otsuka Long-Evans Tokushima fatty rats. When these animals were treated with ginseng root extract, there was no significant difference in food intake between ginseng-treated and untreated groups (Banz et al., 2007; Lee et al., 2009, 2012). In contrast, food consumption was reduced by ginseng berry extract in ob/ob mice and by crude saponin in Sprague-Dawley rats fed a high fat diet (Attele et al., 2002; Kim et al., 2005). These conflicting data may be due to differences in the source of ginseng, such as root versus berry, the total ginseng versus active components of ginseng, and the animals used for analysis. Weight reduction without appetite suppression may be due to the changes in the expression of genes responsible for energy expenditure and fatty acid β-oxidation. First, we measured the mRNA levels of UCP1, the hallmark of brown adipocytes, in visceral adipose tissues in order to examine the involvement of the white adipose tissue browning and increased energy expenditure. The conversion of white adipose tissue to the thermogenically active beige adipose tissue has been suggested as a potential strategy to treat obesity (Wu et al., 2012). In the present study, UCP1 mRNA levels were not increased in ginseng-treated mice compared with untreated control mice, showing that weight reduction was not due to the browning of white adipose tissue. Next, we measured mRNA levels of energy expenditure and fatty acid β-oxidation genes, such as PGC-1α, UCP2, CPT-1, and MCAD in visceral adipose tissues (Boss et al., 2000; Spiegelman, 2007; Yoon, 2009). Ginseng significantly increased the mRNA levels of all the above genes in
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Fig. 6. Histological analysis of the blood vessels in visceral adipose tissues stained with an antibody against vWF. Female db/db mice (n¼ 8/group) are either untreated (control) or treated with ginseng for 13 weeks (ginseng). (A) Representative immunostained sections (original magnification 100). (B) Quantification of blood vessel density. All values are expressed as mean7 SD. np o 0.05 compared with control group.
visceral adipose tissues of obese mice. These results suggest that treated mice may exhibit the increased energy expenditure and preferentially use fatty acids as energy substrates during ginsenginduced weight loss. The endothelial cell marker vWF-positive cells were lower in adipose tissue sections from ginseng-treated mice than in those from control mice, indicating that ginseng can inhibit angiogenesis in growing adipose tissues. Rupnick et al. (2002) reported that weight loss from TNP-470-treated ob/ob mice was associated with adipose tissue endothelial cell apoptosis. Proliferating endothelial cells were decreased and endothelial apoptosis was significantly increased in epididymal fat sections from TNP-470-treated ob/ob mice. This coordinated growth may be mediated by interaction of endothelial cells and adipocytes. Evidence has shown that ginseng acts as a potential cancer chemopreventive agent by exerting antiangiogenic activities (Sato et al., 1994). For examples, the active ginsenosides Rb1 and Rb3 suppress tumor metastasis in part due to inhibition of angiogenesis, and the ginsenoside metabolite compound K inhibits the migration and tube formation of endothelial cells (Sengupta et al., 2004; Yue et al., 2006; Xu et al., 2008;
Jeong et al., 2010). These results suggest that ginseng can inhibit obesity through its angiosuppressive activities. To adapt to changes in the size of adipocytes and adipose tissue mass, adipose vasculature requires proper modulation by angiogenic regulators. Adipose tissue-derived angiogenic factors, such as VEGF-A and FGF-2, can synergistically induce angiogenesis (Cao et al., 2001). It is generally well known that the VEGF system accounts for most of the angiogenic actions in adipose tissue (Hausman and Richardson, 2004). VEGF plays a central role in most growing or developing tissue tissues. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, and VEGF-D. Among the several VEGFs, VEGF-A is a major angiogenic factor that stimulates proliferation and migration of endothelial cells. Loss of a single VEGF allele causes abnormal blood vessel development and lethality of embryos (Carmeliet et al., 1996). Overexpression of VEGF increases blood vessel size and number in both white adipose tissue and brown adipose tissue (Elias et al., 2012). Various strategies for inhibiting VEGF have been investigated as targets of anti-angiogenic agents. These include neutralizing antibodies to VEGF, VEGF receptor inhibitors, and soluble VEGF constructs
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Fig. 7. Expression of angiogenic factors in visceral adipose tissues of female db/db mice and 3T3-L1 cells. (A) Analysis of the VEGF-A and FGF-2 mRNA levels in visceral adipose tissues. (B) Analysis of the VEGF protein levels in visceral adipose tissues. Representative protein bands (n¼ 5/group) are shown. (C) Analysis of VEGF-A and FGF-2 mRNA levels in 3T3-L1 cells. 3T3-L1 cells are treated with MDI induction medium (control), MDI plus ginseng (10 μg/ml), TGSs (10 μg/ml), or individual GSs [i.e., Rb1, Rb2, Rc, Rd, Rf, Rg1, Rg2, Rg3 (10 μM each), and Re (0.1 μM)]. All values are expressed as the mean7 SD of R.D.U. using β-actin as a reference. np o 0.05 compared with control group.
(Murukesh et al., 2010). FGF-2 is also a potent stimulator of proliferation, differentiation, and migration of endothelial cells, and increases adipocyte differentiation in vivo (Kawaguchi et al., 1998). During angiogenesis, FGF-2 induces VEGF expression in endothelial cells and stromal cells, which is required for angiogenic activities of FGF-2 (Seghezzi et al., 1998; Claffey et al., 2001; Tsunoda et al., 2007), suggesting that FGF-induced angiogenesis
requires activation of VEGF system (Presta et al., 2005). FGF-2 increases the expression of proteinases, such as collagenase and urokinase-type plasminogen activator, and integrin to form new capillaries (Okamura et al., 1991; Tienari et al., 1991). In the present study, mRNA levels of VEGF-A and FGF-2 as well as protein expression of VEGF were significantly lower in treated mice. Moreover, ginseng decreased VEGF-A and FGF-2 mRNA
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Fig. 8. Expression of genes involved in energy expenditure and fatty acid β-oxidation in visceral adipose tissues of female db/db mice. All values are expressed as the mean 7 SD of R.D.U. using β-actin as a reference. npo 0.05 compared with control group.
levels in 3T3-L1 adipocytes compared with control. Their mRNA levels were also reduced by TGSs and individual GSs including Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Rg3. In particular, Rb1, which is the most abundant GS in ginseng root in different sources and species (Washida and Kitanaka, 2003), was the most effective among the GSs. Our data indicate that ginseng exerts an inhibitory effect on VEGF and FGF-2 expression in visceral adipose tissues and downregulation of angiogenic factors in adipose tissues may lead to treatment of obesity and related disorders. In conclusion, these studies suggest that ginseng may treat female obesity and related metabolic diseases in genetically obese db/db mice and that this process may be mediated in part through the inhibition of angiogenesis. Thus, ginseng provides a promising therapeutic approach for controlling human female obesity and its related disorders.
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Research Paper
Ginseng treatment reverses obesity and related disorders by inhibiting angiogenesis in female db/db mice Hyunghee Lee a, Mina Kim a, Soon Shik Shin b, Michung Yoon a,n a b
Department of Biomedical Engineering, Mokwon University, Daejeon 302-729, Republic of Korea Department of Formula Sciences, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 27 March 2014 Received in revised form 24 June 2014 Accepted 16 July 2014 Available online 27 July 2014
Ethnopharmacological relevance: Korean red ginseng (ginseng, Panax ginseng C.A. Meyer) has traditionally been used in the treatment of most ageing-related diseases, such as obesity, diabetes, and dyslipidemia, but the mechanism of the effects is unclear. The aim of this study was to determine the effects of ginseng on obesity in a mouse model of female obesity (obese female db/db mouse) and to investigate the mechanism of anti-obesity effects. Materials and methods: After female db/db (B6.Cg-m Leprdb/þ þ/J) mice were treated with 5% (w/w) ginseng for 13 weeks, variables and parameters of obesity and disorders related to obesity were examined. Blood vessel density and the expression of genes involved in angiogenesis were also measured. Results: Mice treated with ginseng for 13 weeks had less body weight and lower adipose tissue mass compared to control, untreated mice. The size of adipocytes was smaller in visceral adipose tissues of ginseng-treated mice. Obesity-related complications, such as hepatic steatosis, hypertriglyceridemia, and hyperglycemia, were markedly improved in treated mice. Blood vessel density was lower in visceral adipose tissue sections from treated mice than those from control mice. Concomitantly, mRNA levels for VEGF-A and FGF-2 were lower in both visceral adipose tissue from treated mice and treated 3T3-L1 cells compared to those from untreated controls. Protein levels for VEGF were also lower in visceral adipose tissue from treated mice. In contrast, ginseng increased mRNA expression of genes responsible for energy expenditure and fatty acid β-oxidation in visceral adipose tissue during ginseng-induced weight reduction. Conclusions: These results suggest that ginseng may effectively treat female obesity and related disorders in part by inhibition of angiogenesis. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Korean red ginseng Obesity Angiogenesis Female db/db Mouse Adipose tissue growth
1. Introduction Angiogenesis, the process of new blood vessel formation, is required for adipose tissue growth in adults, similar to its necessity for tumor growth (Crandall et al., 1997; Rupnick et al., 2002). Most tissues do not normally grow throughout adulthood, and their supporting vasculature is quiescent, whereas adult adipose tissue is probably one of the most plastic tissues in the body, and it expands and shrinks throughout adulthood (Cao, 2007). Adipose tissue is highly vascularized, and each adipocyte is nourished by an extensive capillary network (Bouloumié et al., 2002; Cao, 2007). It has therefore been suggested that adipose tissue growth can be regulated by the adipose tissue vasculature and is angiogenesis-dependent (Rupnick et al., 2002; Bråkenhielm et al., 2004; Kim et al., 2010). Thus,
n
Corresponding author. Tel.: þ 8242 829 7581; fax: þ 8242 829 7580. E-mail address: [email protected] (M. Yoon).
http://dx.doi.org/10.1016/j.jep.2014.07.034 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
modifying or blocking angiogenesis may help manage or eliminate obesity. Growing adipocytes produce angiogenic factors, such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2, contributing to the formation of new blood vessels inside the fat pad (Claffey et al., 1992; Voros et al., 2005; Cao, 2007). VEGF-A and FGF-2 stimulate proliferation and migration of endothelial cells and enhance adipocyte differentiation (Carmeliet et al., 1996; Bikfalvi et al., 1997). Anti-angiogenic drugs, such as those targeting VEGF-A or FGF-2 pathways, may be key components of therapy for obesity and obesity-related metabolic diseases. Ginseng, the root of Panax ginseng C.A. Mayer (Araliaceae), has historically been used as one of the most popular herbal medicines to treat various diseases in Korea, China and Japan for thousands of years. Ginseng has been traditionally used in the treatment of most ageing-related diseases, such as diabetes, obesity, and dyslipidemia (Yin et al., 2008). Ginseng and its active components ginsenosides inhibit obesity in several obese animal models, such
H. Lee et al. / Journal of Ethnopharmacology 155 (2014) 1342–1352
as high fat-fed Sprague-Dawley rats, C57BL/6J mice, and Balb/c mice, and genetically obese ob/ob mice (Kim et al., 2005; Karu et al., 2007; Mollah et al., 2009; Lee et al., 2012; Lee et al., 2013), but the mechanism of the effects is unclear. Accumulating evidence also shows that ginseng has been reported to inhibit angiogenesis, which prevents tumor growth (Sato et al., 1994; Ho et al., 2012). Ginsenosides exhibit potential as potent cancer chemopreventive agents because of this anti-angiogenic activity (Yue et al., 2006; Jeong et al., 2010). Based on the aforementioned literature that ginseng modulates angiogenesis (Sato et al., 1994; Liu et al., 2009) and adipose tissue growth is regulated by angiogenesis (Cao, 2010), we hypothesized that the anti-angiogenic properties of Korean red ginseng (ginseng) could treat obesity and its metabolic diseases. It is, moreover, known that males and females differ in the regulation of body weight and body fat (Brown and Clegg, 2010). Men and post-menopausal women accumulate more fat in intraabdominal depots than pre-menopausal women, resulting in a greater risk of developing complications associated with obesity (Geer and Shen, 2009). Our previous results demonstrated that ginseng effectively prevents male obesity in high fat diet-fed C57BL/6J mice (Lee et al., 2013). Thus, we examined the effects of ginseng on female obesity using female homozygous leptin receptor-deficient db/db mice, which exhibit an obesity syndrome characterized by severe adiposity, dyslipidemia, and insulin resistance (Clement et al., 1998; Muoio and Lynis Dohm, 2002). These mice have body weights three times greater than normal and exhibit a five-fold increase in body fat content (Friedman and Halaas, 1998). We show that ginseng suppresses female obesity by angiogenesis inhibition. Ginseng-treated mice showed less body weight and adipose tissue mass compared with untreated mice. Blood vessel density was concomitantly lower in visceral adipose tissues of treated mice compared to obese mice. Ginseng also modulated the mRNA and protein expression of angiogenic factors. We also observed that ginseng increased the expression of genes involved in energy expenditure and fatty acid oxidation in visceral adipose tissues during ginseng-induced weight loss. In addition, ginseng treatment is a reversal protocol since ginseng was given to obese mice. This protocol mimics the intervention in obese patients rather than the prevention of obesity by testing in lean animals fed an obesogenic diet simultaneously with the intervention. Therefore, these results suggest that the anti-angiogenic actions of ginseng may inhibit adipose tissue growth and reverse obesity and related disorders in female db/db mice.
2. Materials and methods 2.1. Ginseng preparation Ginseng extract powder was commercially prepared from ginseng cultivated with care in well-fertilized fields for 6 years (KT&G, Daejeon, Korea). Total ginsenosides (TGSs) were obtained by extraction from the ginseng extract powder (Yoon et al., 2003). Briefly, ginseng extract powder (100 g) was placed into a 1-L flask with a refluxing condenser and extracted twice with 500 mL of water-saturated 1-butanol for 1 h at 80 1C. The extracted solution was passed through Whatman filter paper (No. 41) after cooling. The process was repeated twice. The residue and filter paper were washed with 100 mL of water-saturated 1-butanol, and then the filtrate was washed twice with 100 mL of water in a 2-L separating funnel. The butanol layer was then evaporated to dryness. The concentrate was extracted to remove any traces of fat with 100 mL of diethyl ether for 30 min at 36 1C in a flask with a refluxing condenser, after which the ether solution was decanted.
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The residue was dried at 50 1C and weighed. Individual GSs were analyzed by the HPLC/ELSD system (Fig. 1). The HPLC/ELSD analytical conditions are as follows: (i) Column, Lichrosorb NH column (25 0.4 cm, 5 μm, Merck). (ii) Mobile phase, mixtures of solvent A (acetonitrile/water/isopropanol ¼80:5:15) and solvent B (acetonitrile/water/isopropanol¼ 80:20:15). (iii) Gradient profile of solvent A to solvent B, from 70:30 to 0:100 for 0–20 min; 0:100 for 20–55 min; from 0:100 to 70:30 for 55–65 min; flow rate, 1.0 ml/min. (iv) Detector, ELSD (ELSD 2000, Alltech, dearfield, IL, USA): detection temperature 92 1C; neutralizing gas, nitrogen, 2.0 l/min. 2.2. Animal study Female, eight-week-old, genetically obese B6.Cg-m Leprdb/þ þ/J (db/db) mice and lean B6.Cg-m Leprdb/þ/J (db/þ) mice were housed at Mokwon University under pathogen-free conditions with a standard 12-h light/dark cycle. Mice were divided into three groups (n¼ 8/group): a lean group given a standard diet (normal mice), a genetically obese group given a standard diet (obese control mice), and a genetically obese group given a standard diet supplemented with 5% (w/w) ginseng (ginseng-treated mice). For ginsengsupplemented high fat diet, 50 g ginseng extract powder was mixed with 1 kg high fat diet. Mice received approximately 4.5 g ginseng extract/kg BW/day. Ginseng extract at these doses did not show any toxic effects. Body weights were measured daily by a person blinded to each treatment group. Food intake was determined by estimating the amount of food consumed by the mice throughout the treatment period. The small amount of food spillage that was typically noted when the cages were inspected was collected to ensure accurate measurement of food intake. After a 12-h fast on the last day of the study, the animals were sacrificed by cervical dislocation. Blood was collected from the retro-orbital sinus into tubes, and serum was separated and stored at 80 1C until analysis. Visceral adipose tissues were removed, weighed, snap-frozen in liquid nitrogen, and stored at 80 1C until use. Portions of the liver and visceral adipose tissues were prepared for histology. All animal experiments were approved by the Institutional Animal Care and Use Committees of Mokwon University, following National Research Council Guidelines. 2.3. 3T3-L1 differentiation Mouse 3T3-L1 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (Invitrogen, Carlsbad, CA, USA). After cells were maintained at confluence for 2 days, they were incubated in an MDI induction medium (day 0) containing 0.5 mM 1-methyl-3isobutyl-xanthine, 1 μM dexamethasone, and 1 μg/ml insulin in DMEM containing 10% fetal bovine serum (FBS, Invitrogen). The cultures were continued for 2 days to induce adipocyte differentiation. Thereafter, cells were cultured in DMEM containing 10% FBS for the remainder of the differentiation process. All other treatments including ginseng, TGSs, and individual GSs were administered on days 0–2 only, and the medium was changed every other day. 2.4. Blood analysis Levels of triglycerides and free fatty acids were measured using an automatic blood chemical analyzer (CIBA Corning, Oberlin, OH, USA) and a SICDIA NEFAZYME kit (Shinyang Chemical, Seoul, Korea), respectively. Levels of serum glucose and insulin were measured using an Accu-Chek Performa System (Roche, Germany) and an insulin radioimmunoassay kit (Linco, St. Charles, MO, USA), respectively.
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Fig. 1. The HPLC profile of ginseng extract. Individual ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, and Rg3 are analyzed by the HPLC/ELSD system. The most abundant ginsenoside is Rb1. Peak numbers denote retention times.
2.5. Histological analysis Liver and visceral adipose tissue samples were fixed in 10% phosphate-buffered formalin (PBF) for 1 day and processed in a routine manner for paraffin sections. Tissue sections (5 μm) were cut and stained with hematoxylin and eosin (H and E) for microscopic examination. To quantify adipocyte size, the H and E-stained sections were analyzed using the Image-Pro Plus analysis system (Media Cybernetics, Bethesda, MD, USA). Blood vessel staining was performed using a blood vessel staining kit (Chemicon, Billerica, MA, USA). Visceral adipose tissue samples were fixed in 10% PBF for 1 day and processed for paraffin sectioning using standard methods. Sections of 3-μm thickness were cut and irradiated in a microwave oven for epitope retrieval. Sections were sequentially incubated with rabbit anti-von Willebrand factor (vWF) antibody as a primary antibody, goat antirabbit antibody as a secondary antibody, and streptavidin-alkaline phosphatase solution. A freshly prepared chromogen reagent was added to the sections to visualize blood vessels. Blood vessel density was analyzed using the Image area analyzer program.
2.6. Quantitative real time PCR Total cellular RNA from visceral adipose tissues and 3T3-L1 adipocytes was prepared using the Trizol reagent (Gibco-BRL, Grand Island, NY, USA). Reverse transcription of 2 μg of total cellular RNA using Moloney murine leukemia virus reverse transcriptase resulted in the generation of an antisense primer cDNA. Synthesized cDNA fragments were amplified using AccuPowers GreenStarTM qPCR PreMix (Bioneer, Deajeon, Korea) on a
ExcyclerTM 96 Real Time Quantitative Thermal Block machine (Bioneer). The PCR sequences for VEGF-A are as follows (GeneBank number NM_001287056): VEGF-A forward primer 50 -gcagcttgagttaaacgaacg- 30 and reverse primer 50 -ggttcccgaaaccctgag-30 . The PCR sequences for FGF-2 are as follows (GeneBank number NM_008006): FGF-2 forward primer 50 -ccaaccggtaccttgctatga-30 and reverse primer 50 -ttcgttcagtgccacatacca-30 . The PCR sequences for peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) are as follows (GeneBank number BC066868): PGC-1α forward primer 50 -ccctgccatgttaagacc-30 and reverse primer 50 tgctgctgttcctgttttc-30 . The PCR sequences for uncoupling protein 1 (UCP1) are as follows (GeneBank number NM_009463): UCP1 forward primer 50 -ggcattcagaggcaaatcagct-30 and reverse primer 50 -caatgaacactgccacacctc-30 . The PCR sequences for UCP2 are as follows (GeneBank number NM_011671): UCP2 forward primer 50 ctggtcgccggcctgcagcgc-30 and reverse primer 50 -gatcccttcctctcgtgcaat-30 . The PCR sequences for carnitine palmitoyltransferase 1 (CPT-1) are as follows (GeneBank number NM_009948): CPT-1 forward primer 50 -ttcaacactacacgcatccc-3'and reverse primer 50 gccctcatagagccagacc-30 . The PCR sequences for medium-chain acyl coenzyme A dehydrogenase (MCAD) are as follows (GeneBank number NM_007382): MCAD forward primer 50 -ccgaagagttggcgtatggg-30 and reverse primer 50 -50 -gggctctgtcacacagtaagc-30 . The PCR sequences for β-actin are as follows (GeneBank number BC138614): β-actin forward primer 50 -taccacaggcattgtgatgg-30 and reverse primer 50 -tttgatgtcacgcacgattt-3´. The conditions of PCR were a denaturing step at 95 1C for 5 min followed by 50 cycles at 95 1C for 10 s, 60 1C for 40 s, and 72 1C for 10 s. Their concentrations were calculated as copies per ml using the standard curve and the relative expression levels were presented as a ratio of target gene cDNA versus β-actin cDNA.
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2.7. Western blot analysis Visceral adipose tissues were lysed in ice-cold lysis buffer [50 mM Tris–Cl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, and 1% Triton X-100] containing protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin). Lysates were centrifuged at 12,000 rpm for 20 min at 4 1C, and the resulting supernatants were subjected to electrophoresis on 10% polyacrylamide gels. The separated proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 10% nonfat milk (DIFCO, Detroit, MI, USA) in Tris-buffered saline (TBS). The membrane was washed three times with TBS containing 0.1% Tween-20 (TBS-T), followed by incubation with an anti-VEGF antibody (catalog number sc-507, Santa Cruz, CA, USA) (1:200 dilution). The membrane was washed three times with TBS-T and then incubated with the HRP-conjugated goat anti-rabbit IgG (catalog number sc-2004, Santa Cruz) diluted in TBS (1:2500 dilution) as a secondary antibody. The membrane was washed three times with TBS-T and visualized by an enhanced chemiluminescence western blot detection system (WEST-ZOL plus, Intron, Daejeon, Korea).
2.8. Statistics All values are expressed as mean 7standard deviation (SD). Statistical analysis was performed by ANOVA followed by Post-Hoc analysis using the SPSS software. Statistical significance was defined as a value of p o0.05.
3. Results 3.1. Body weight, adipose tissue mass, and food intake Obese (db/db) control mice showed markedly higher body weights compared with lean (db/þ ) mice after 13 weeks. However, mice fed a diet supplemented with ginseng had 21% lower body weights compared with obese control mice (Fig. 2A). Consistent with the decreases in body weight, total adipose tissue mass was also decreased in ginseng-treated mice compared with obese control mice. Total adipose tissue weights were decreased by 30% in ginseng-treated mice (Fig. 2B). The amount of food intake was not significantly changed by adding ginseng to the diet of obese control mice (Fig. 2C).
3.2. Adipocyte size Analysis of H and E-stained visceral adipose tissue sections revealed that adipocytes of ginseng-treated mice were much smaller than those of obese control mice (Fig. 3A). The average size of adipocytes in ginseng-treated mice (11,589 73630 μm2) was 49% smaller than that in obese control mice (22,539 7 1453 μm2, Fig. 3B).
3.3. Hepatic lipid accumulation The accumulation of hepatic lipids was determined after ginseng treatment in mice by H and E-staining and light microscopy. As shown in Fig. 4, obese control mice exhibited considerable hepatic lipid accumulation compared with lean normal mice. When obese mice were treated with ginseng, there was a complete inhibition of hepatic lipid accumulation, showing that ginseng inhibits hepatic steatosis.
Fig. 2. Effects of ginseng on body weight, adipose tissue mass, and food intake in female db/db mice. Mice (n¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Body weights at the end of the treatment period are significantly different between the normal and control groups (po0.05) and between the control and ginseng groups (po0.05). (B) Adipose tissue weights. (C) Food intake. All values are expressed as mean7SD. npo0.05 compared with normal group. nnpo0.05 compared with control group.
3.4. Blood lipids, glucose, and insulin levels Compared with normal mice, obese control mice exhibited much higher levels of serum triglycerides, free fatty acids, glucose,
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Fig. 3. Light microscopic analysis of visceral adipocytes. Mice (n¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Representative hematoxylin and eosin-stained sections (5-μm thickness) of visceral adipose tissue. Adipocyte size from the ginseng groups is smaller than that from control groups. (B) The size of adipocytes in a fixed area (1,000,000 μm2) is measured. All values are expressed as the mean7SD. npo0.05 compared with normal group. nnpo0.05 compared with control group.
and insulin. In contrast, ginseng-treated mice had significantly lower serum levels of triglycerides and free fatty acids (29% and 44% reductions, respectively) than untreated obese controls (Fig. 5A and B). Serum insulin and glucose levels were also lower (72% and 14%, respectively) in ginseng-treated mice compared with those in untreated controls (Fig. 5C and D). 3.5. Angiogenesis in adipose tissues Staining of visceral adipose tissue sections with an antibody against vWF, an endothelial cell marker, showed that blood vessel density was lower in ginseng-treated mice than in obese control mice. Ginseng-treated mice had 53% lower blood vessel density compared with controls (Fig. 6). 3.6. Expression of genes involved in angiogenesis in adipose tissues and 3T3-L1 adipocytes The expression patterns of genes involved in angiogenesis were investigated in visceral adipose tissues and 3T3L1 adipocytes. The mRNA expression of angiogenic factors was down-regulated in ginseng-treated mice compared with those in control mice. The mRNA levels of VEGF-A and FGF-2 were 80% and 85% lower, respectively, in ginseng-treated mice compared with those in control mice (Fig. 7A). The protein levels of VEGF were 54% lower
in mesenteric adipose tissues of ginseng-treated mice compared with those in obese control mice (Fig. 7B). Treatment of 3T3-L1 adipocytes with ginseng, TGSs, and individual GSs decreased the mRNA levels of VEFG-A and FGF-2 compared with their expression in untreated adipocytes. Ginseng treatment reduced VEFG-A and FGF-2 mRNA levels by 4% and 16%, respectively, and TGSs decreased their mRNA levels by 45% and 9%, respectively (Fig. 7C). In particular, Rb1 effectively decreased the mRNA levels of VEFG-A and FGF-2 by 42% and 34%, respectively.
3.7. Expression of genes involved in energy expenditure and fatty acid β-oxidation in adipose tissues The expression patterns of genes involved in energy expenditure and fatty acid β-oxidation were investigated in visceral adipose tissues. Their expression was up-regulated in ginsengtreated mice compared with those in control mice although UCP1 expression was down-regulated. The mRNA levels of PGC-1α, UCP2, CPT-1, and MCAD were 20%, 309%, 64%, and 110% higher, respectively, in ginseng-treated mice compared with those in control mice (Fig. 8). In contrast, UCP1 mRNA levels were 85% lower in ginseng-treated mice compared with those in control mice.
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Fig. 4. Inhibition of hepatic lipid accumulation by ginseng in female db/db mice. Mice (n ¼8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). (A) Representative H and E-stained sections of livers are shown (original magnification 100). Arrows indicate the fatty changes in hepatocytes. (B) Histological analyses of hepatic lipid accumulation. 0, no lesion; 1, mild; 2, moderate; 3, severe; 4, very severe. np o 0.05 compared with normal group. nnp o0.05 compared with control group.
4. Discussion Ginseng has been used as a traditional medicine in oriental societies and is now a popular and worldwide used natural medicine. Numerous studies have reported that ginseng has many pharmacological effects on the central nervous system, as well as on the endocrine, immune, and cardiovascular systems (Gillis, 1997; Attele et al., 1999; Yin et al., 2008; Lu et al., 2009). Ginseng has also been used as a traditional medicine in the treatment of most ageing-related diseases, such as obesity, diabetes, and dyslipidemia (Yin et al., 2008), but the mechanism of the effects is not clear. Based on reports showing that adipose tissue mass can be regulated by angiogenesis (Rupnick et al., 2002; Cao, 2007) and that ginseng inhibits angiogenesis (Sato et al., 1994; Ho et al., 2012; Park and Yoon, 2012), we examined the effects of ginseng on obesity and to investigate the mechanism of anti-obesity effects in obese db/db mice. It is, moreover, known that males and females have important differences in the regulation of body weight and body fat (Brown and Clegg, 2010). Since our previous results demonstrated that ginseng effectively prevents male obesity by inhibiting angiogenesis (Lee et al., 2013), we examined the effects of ginseng on female obesity and found that female db/db mice treated with ginseng showed alleviation of obesity and its related disorders, such as hepatic steatosis, hyperlipidemia, and hyperglycemia and that this process may be mediated, at least in part, through inhibiting angiogenesis. In addition, administration of
ginseng is a reversal protocol as ginseng was given to obese mice. This protocol mimics the intervention in obese patients rather than the prevention of obesity by testing in lean animals fed an obesogenic diet simultaneously with the intervention. Body weights and adipose tissue mass of ginseng-treated mice were much lower than those of untreated control db/db mice. Similarly, our previous results showed that ginseng-treated, high fat diet-fed obese mice had lower body weights and adipose tissue mass compared with male mice fed a high fat diet alone (Lee et al., 2013), indicating that ginseng regulates both genetically and nutritionally induced obesity. Interestingly, inhibitory effects of ginseng on obesity were more prominent in female mice than in male mice. It is thought that this is may be due to the presence of the ovarian steroid hormone estrogen (Brown and Clegg, 2010). We also observed that the magnitude of reduction in weight loss is greater in db/db mice than in high fat diet-fed obese mice because of the greater fat content in db/db mice. Newly formed adipose tissue depends on continued angiogenesis for further growth (Rupnick et al., 2002; Bråkenhielm et al., 2004), and different angiogenesis inhibitors have been shown to significantly reduce body weight and adipose tissue mass (Cao, 2007). Our results are supported by other reports in the literature showing that body weight gain and adipose tissue mass in obese animals are significantly reduced by several kinds of angiogenesis inhibitors, such as angiostatin, endostatin, TNP-470, TNP-470 analog CKD732, and VEGF receptor 2-specific inhibitors (Rupnick et al., 2002;
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Fig. 5. Changes in circulating levels of free fatty acids, triglycerides, glucose, and insulin. Mice (n¼ 8/group) are divided into three groups: lean db/þ mice (normal), obese db/db mice (control), and obese db/db mice treated with 5% ginseng (ginseng). Serum (A) free fatty acids, (B) triglycerides, (C) glucose, and (D) insulin are determined after 13 weeks of ginseng treatment. npo0.05 compared with normal group. nnpo0.05 compared with control group.
Bråkenhielm et al., 2004; Kim et al., 2007; Lijnen et al., 2007). The size of adipocytes was considerably smaller in ginseng-treated mice than in untreated control mice, eventually resulting in decreased adipose tissue mass and body weights. Adipose tissue is highly vascularized and each adipocyte is nourished by an extensive capillary network (Bouloumié et al., 2002; Cao, 2007). Thus, inhibition of adipose tissue angiogenesis can be therapeutic targets of obesity and obesity-related metabolic diseases. Actually, we have observed that liver lipid accumulation in ginseng-treated mice was markedly inhibited, and serum levels of triglycerides, glucose, and insulin were lower in treated mice relative to untreated control mice. These results suggest that ginseng may alleviate metabolic diseases, such as liver steatosis, hypertriglyceridemia, and insulin resistance as consequences of anti-angiogenicinduced weight reduction. Ginseng-induced weight loss occurred without appetite changes, as evidenced by a similar food consumption profile between ginseng-treated and untreated control obese mice. It has been reported that treatment with angiogenesis inhibitors endostatin and low doses of TNP-470 or angiostatin, as well as antiangiogenic herbal composition Ob-X, did not suppress appetite (Rupnick et al., 2002; Kim et al., 2010). Rupnick et al. (2002) proposed that adipose tissue vessel may be maintained in a relatively immature state relative to that of other organs that are mass stable. Because anti-angiogenic agents target growing or newly formed vessels, adipose tissue vessels may be susceptible to angiogenesis inhibitors. Thus, anti-angiogenic ginseng may selectively target adipose tissue and cause weight reduction. Similar
results were observed in several obese animal models, such as fatty Zucker rats, high fat-fed Sprague-Dawley rats, and Otsuka Long-Evans Tokushima fatty rats. When these animals were treated with ginseng root extract, there was no significant difference in food intake between ginseng-treated and untreated groups (Banz et al., 2007; Lee et al., 2009, 2012). In contrast, food consumption was reduced by ginseng berry extract in ob/ob mice and by crude saponin in Sprague-Dawley rats fed a high fat diet (Attele et al., 2002; Kim et al., 2005). These conflicting data may be due to differences in the source of ginseng, such as root versus berry, the total ginseng versus active components of ginseng, and the animals used for analysis. Weight reduction without appetite suppression may be due to the changes in the expression of genes responsible for energy expenditure and fatty acid β-oxidation. First, we measured the mRNA levels of UCP1, the hallmark of brown adipocytes, in visceral adipose tissues in order to examine the involvement of the white adipose tissue browning and increased energy expenditure. The conversion of white adipose tissue to the thermogenically active beige adipose tissue has been suggested as a potential strategy to treat obesity (Wu et al., 2012). In the present study, UCP1 mRNA levels were not increased in ginseng-treated mice compared with untreated control mice, showing that weight reduction was not due to the browning of white adipose tissue. Next, we measured mRNA levels of energy expenditure and fatty acid β-oxidation genes, such as PGC-1α, UCP2, CPT-1, and MCAD in visceral adipose tissues (Boss et al., 2000; Spiegelman, 2007; Yoon, 2009). Ginseng significantly increased the mRNA levels of all the above genes in
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Fig. 6. Histological analysis of the blood vessels in visceral adipose tissues stained with an antibody against vWF. Female db/db mice (n¼ 8/group) are either untreated (control) or treated with ginseng for 13 weeks (ginseng). (A) Representative immunostained sections (original magnification 100). (B) Quantification of blood vessel density. All values are expressed as mean7 SD. np o 0.05 compared with control group.
visceral adipose tissues of obese mice. These results suggest that treated mice may exhibit the increased energy expenditure and preferentially use fatty acids as energy substrates during ginsenginduced weight loss. The endothelial cell marker vWF-positive cells were lower in adipose tissue sections from ginseng-treated mice than in those from control mice, indicating that ginseng can inhibit angiogenesis in growing adipose tissues. Rupnick et al. (2002) reported that weight loss from TNP-470-treated ob/ob mice was associated with adipose tissue endothelial cell apoptosis. Proliferating endothelial cells were decreased and endothelial apoptosis was significantly increased in epididymal fat sections from TNP-470-treated ob/ob mice. This coordinated growth may be mediated by interaction of endothelial cells and adipocytes. Evidence has shown that ginseng acts as a potential cancer chemopreventive agent by exerting antiangiogenic activities (Sato et al., 1994). For examples, the active ginsenosides Rb1 and Rb3 suppress tumor metastasis in part due to inhibition of angiogenesis, and the ginsenoside metabolite compound K inhibits the migration and tube formation of endothelial cells (Sengupta et al., 2004; Yue et al., 2006; Xu et al., 2008;
Jeong et al., 2010). These results suggest that ginseng can inhibit obesity through its angiosuppressive activities. To adapt to changes in the size of adipocytes and adipose tissue mass, adipose vasculature requires proper modulation by angiogenic regulators. Adipose tissue-derived angiogenic factors, such as VEGF-A and FGF-2, can synergistically induce angiogenesis (Cao et al., 2001). It is generally well known that the VEGF system accounts for most of the angiogenic actions in adipose tissue (Hausman and Richardson, 2004). VEGF plays a central role in most growing or developing tissue tissues. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, and VEGF-D. Among the several VEGFs, VEGF-A is a major angiogenic factor that stimulates proliferation and migration of endothelial cells. Loss of a single VEGF allele causes abnormal blood vessel development and lethality of embryos (Carmeliet et al., 1996). Overexpression of VEGF increases blood vessel size and number in both white adipose tissue and brown adipose tissue (Elias et al., 2012). Various strategies for inhibiting VEGF have been investigated as targets of anti-angiogenic agents. These include neutralizing antibodies to VEGF, VEGF receptor inhibitors, and soluble VEGF constructs
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Fig. 7. Expression of angiogenic factors in visceral adipose tissues of female db/db mice and 3T3-L1 cells. (A) Analysis of the VEGF-A and FGF-2 mRNA levels in visceral adipose tissues. (B) Analysis of the VEGF protein levels in visceral adipose tissues. Representative protein bands (n¼ 5/group) are shown. (C) Analysis of VEGF-A and FGF-2 mRNA levels in 3T3-L1 cells. 3T3-L1 cells are treated with MDI induction medium (control), MDI plus ginseng (10 μg/ml), TGSs (10 μg/ml), or individual GSs [i.e., Rb1, Rb2, Rc, Rd, Rf, Rg1, Rg2, Rg3 (10 μM each), and Re (0.1 μM)]. All values are expressed as the mean7 SD of R.D.U. using β-actin as a reference. np o 0.05 compared with control group.
(Murukesh et al., 2010). FGF-2 is also a potent stimulator of proliferation, differentiation, and migration of endothelial cells, and increases adipocyte differentiation in vivo (Kawaguchi et al., 1998). During angiogenesis, FGF-2 induces VEGF expression in endothelial cells and stromal cells, which is required for angiogenic activities of FGF-2 (Seghezzi et al., 1998; Claffey et al., 2001; Tsunoda et al., 2007), suggesting that FGF-induced angiogenesis
requires activation of VEGF system (Presta et al., 2005). FGF-2 increases the expression of proteinases, such as collagenase and urokinase-type plasminogen activator, and integrin to form new capillaries (Okamura et al., 1991; Tienari et al., 1991). In the present study, mRNA levels of VEGF-A and FGF-2 as well as protein expression of VEGF were significantly lower in treated mice. Moreover, ginseng decreased VEGF-A and FGF-2 mRNA
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Fig. 8. Expression of genes involved in energy expenditure and fatty acid β-oxidation in visceral adipose tissues of female db/db mice. All values are expressed as the mean 7 SD of R.D.U. using β-actin as a reference. npo 0.05 compared with control group.
levels in 3T3-L1 adipocytes compared with control. Their mRNA levels were also reduced by TGSs and individual GSs including Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Rg3. In particular, Rb1, which is the most abundant GS in ginseng root in different sources and species (Washida and Kitanaka, 2003), was the most effective among the GSs. Our data indicate that ginseng exerts an inhibitory effect on VEGF and FGF-2 expression in visceral adipose tissues and downregulation of angiogenic factors in adipose tissues may lead to treatment of obesity and related disorders. In conclusion, these studies suggest that ginseng may treat female obesity and related metabolic diseases in genetically obese db/db mice and that this process may be mediated in part through the inhibition of angiogenesis. Thus, ginseng provides a promising therapeutic approach for controlling human female obesity and its related disorders.
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