Food and Chemical Toxicology 55 (2013) 100–105

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Food and Chemical Toxicology

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Anti-obesity effects of hot water extract and high hydrostatic pressure extractof garlic in rats fed a high-fat diet

Hyunjin Joo a, Chong-Tai Kim b, In-Hwan Kim c, Yangha Kim a,⇑a Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 120-750, Republic of Koreab Food Bio-Nano Research Group, Korea Food Research Institute, Seongnam, Gyeonggi, Republic of Koreac Department of Food and Nutrition, College of Health Sciences, Korea University, Seoul, Republic of Korea

a r t i c l e i n f o

Article history:Received 27 August 2012Accepted 27 December 2012Available online 7 January 2013

Keywords:ObesityHot water extractHigh hydrostatic pressure extractUCP2

0278-6915/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.12.044

⇑ Corresponding author. Tel.: +82 2 3277 3101; faxE-mail address: yhmoon@ewha.ac.kr (Y. Kim).

a b s t r a c t

The effects of hot water extract of garlic (WEG) and high hydrostatic pressure extract of garlic (HEG) onobesity were investigated in rats fed a high-fat (HF) diet. Supplementation with HEG significantlyreduced body weight gain and adipose tissue mass compared to those in the HF group, whereas WEGdid not. Serum levels of triglycerides, total cholesterol, and LDL-cholesterol were also decreased in theHEG supplemented group compared to those in the HF group. The level of fecal triglyceride in the HEGgroup was higher compared to that in the HF group. The mRNA levels of adipogenic genes, such as per-oxisome proliferator-activated receptor gamma (PPARc), sterol regulatory element-binding protein-1c(SREBP-1c), and fatty acid-binding protein (aP2) were significantly decreased in both the WEG andHEG groups. Additionally, uncoupling protein 2 (UCP2) mRNA were increased clearly in the HEG groupcompared to that in the HF group. These results suggested that HEG more efficiently reduced body weightgain than WEG, at least partially mediated by increase of the fecal triglyceride and downregulation of adi-pogenic genes expression together with upregulation of UCP2 gene expression in rats fed a high-fat diet.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Garlic (Allium sativum L.) has been widely used as a medicinalplant for preventing and treating many diseases (Agarwal, 1996).Garlic produces various biological benefits, including hypocholeste-rolemic (Stevinson et al., 2000), hypoglycemic (Jala et al., 2007),antioxidant (Banerjee et al., 2003), anticancer (Milner, 2001), andantiobesity effects (Lee et al., 2011b). The main components of garlicare water, carbohydrates, protein, fat, dietary fiber, essential aminoacids, vitamins, and minerals. Garlic also contains various organo-sulfur compounds such as alliin, allicin, ajoene, diallyl disulfide, dial-lyl trisulfide, allyl methanethiosulfinate, and S-allylcysteine(Lanzotti, 2006). The beneficial effects of garlic are supposedly de-rived from the diverse organosulfur compounds, and most bioactiveorganosulfur compounds are affected by cooking or processingmethods (Hemley, 2000; Ma et al., 2011). Therefore, the garlic pro-cessing technique may be very important to maintain the bioactivecompounds and increase the efficacy of garlic function.

The high hydrostatic pressure (HHP) extraction technique in thefood industry offers the chance to affect enzyme activity and foodfunctionality, as covalent bonds are less influenced by HHP com-pared to the thermal process (Butz et al., 2003; Tauscher, 2001).

ll rights reserved.

: +82 2 3277 2862.

An important application of the HHP extraction technique is to ex-tract bioactive constituents from plants or herbal materials atroom temperature (Lee et al., 2011a). When cell membranes aredestroyed by HHP, a greater number of bioactive compounds areextracted easily out of the cells. HHP technology affects the volatilecompound contents in garlic by decreasing alliinase activity, whichinfluences the formation of allicin (Ma et al., 2011; Iberl et al.,1990).

Obesity comes from excess energy intake over energy expendi-ture (Haslam and James, 2005). Obesity is a major public healthproblem throughout the world, because of its rising prevalenceand strong association with pathological disorders such as diabe-tes, cardiovascular diseases, and certain types of cancer (Ezzatiet al., 2002). In the field of food science, obesity studies have fo-cused on the search for food components that have potential tosuppress the accumulation of body fat. Dietary garlic reduces bodyweight and mass of various white adipose tissues (Lee et al.,2011b). A reduction in body weight by dietary garlic supplementa-tion is associated with an increase in uncoupling protein (UCP)mRNA and a decrease in expression of adipogenic genes in diet-induced obese mice (Lee et al., 2011b; Kim and Kim, 2011).

The aim of this study was to investigate the anti-obesity effectsof hot water extract of garlic (WEG) and high hydrostatic pressureextract of garlic (HEG) using high fat (HF) diet-induced obese rats.The mRNA expression of genes involved in adipogenesis, such asperoxisome proliferator-activated receptor gamma (PPARc), sterol

H. Joo et al. / Food and Chemical Toxicology 55 (2013) 100–105 101

regulatory element-binding protein-1c (SREBP-1c), and fatty acid-binding protein (aP2) were analyzed in white adipose tissue. Addi-tionally, UCP2 mRNA level, which is related to thermogenesis wasassessed.

2. Materials and methods

2.1. Preparation of the WEG and HEG

The WEG and HEG were kindly supplied by the Korea Food Research Institute(Songnam, Gyeonggi, Korea). Fresh garlic cloves were purchased from a local garlicmarket (Seosan, Chungnam, Korea). A 550 g of fresh garlic was crushed with 450 mlof distilled water to prepare the garlic slurry. The garlic slurry was boiled in 100 �Chot water for 1 h to prepare the WEG. The supernatant was freezed-dried after cen-trifugation (5000g, 10 �C, 10 min). In order to prepare the HEG, the garlic slurry wastreated with enzymes (Viscozyme L: 191 units, Celluclast 1.5L: 1320 units, and Lip-ouozyme supra: 246 units; Novo Nordisk, Bagsvaerd, Denmark). The mixture wasadjusted to pH 5.5 and processed under 100 MPa for 24 h at 50 �C using a TFS-2L highhydrostatic pressure machine (Toyo Koatsu Co. Ltd., Hiroshima, Japan) to solubilizethe garlic cell walls and to obtain major component, such as allicin and water solublematerials. The HEG contained 1.24 mg/g of alliicin, which was higher than that of theWEG (0.86 mg/g) as determined by high performance liquid chromatography.

2.2. Animals and diets

All experimental protocols were approved by Ewha Womans University AnimalExperimentation Ethics Committee for the care and use of laboratory animals.Three-week-old male Sprague–Dawley rats, weighing 70–80 g, were purchasedfrom Daehan Experimental Animals (Eumseong, Korea). The rats were individuallyhoused in a temperature-controlled room (22 ± 2 �C) under 12 h light/dark condi-tions. Rats consumed a commercial standard diet (Rodent NIH-31 diet) and waterad libitum for 1 week to stabilize their metabolic condition. After acclimatization,the rats were divided into three groups (n = 8 each) and were maintained on a HFcontrol diet (45% of total energy as fat) based on the AIN-93 G diet (Reeves,1997) with slight modifications: high-fat diet; HF group, high-fat diet +5g/kg hotwater extract of garlic; HF + WEG group, high-fat diet +5 g/kg high hydrostatic pres-sure extract of garlic; HF + HEG group. The rats were maintained on these diets for11 weeks. Table 1 shows the experimental diet compositions. The amount of corn-starch in the HF diet was adjusted accordingly when the WEG and HEG powderwere added. Food intake and body weight of the rats were measured twice a weekduring the experimental period. At the end of the experiment, the rats were fastedovernight and anesthetized with an intramuscular injection of Zoletil/Rompun(Bayer, Leverkusen, Germany) (4:1) at a dose of 0.1 ml/80 g body weight. A centrallongitudinal incision was made in the abdominal wall, and blood samples were col-lected by cardiac puncture. Blood samples were centrifuged at 1500g for 20 min at4 �C to obtain serum, which was stored at �20 �C for biochemical analyses. Liverand WAT (epididymal and retroperitoneal) were excised, frozen immediately in li-quid nitrogen, and stored at �70 �C.

2.3. Serum biochemical measurements

Serum levels of triglycerides, total cholesterol, high density lipoprotein (HDL)-cholesterol, aspartate transaminase (AST), and alanine transaminase (ALT) weredetermined by enzymatic colorimetric methods (Salè et al., 1984) using commercial

Table 1Experimental diet compositions. (units: g/kg).

Component HF HF + WEG HF + HEG

Casein 171 171 171Sucrose 122 122 122Cornstarch 202 197 197Dyetrose 155 155 155

L-cystine 2 2 2

Cellulose 60 60 60Lard 230 230 230Mineral mixa 43 43 43Vitamin mixb 12 12 12Choline bitartrate 3 3 3WEG – 5 –HEG – – 5Total 1000 1000 1000Fat,% (calories) 45 45 45

Diets were based on the AIN-93 G diet composition and given in powdered form.a AIN-93 G mineral mixture.b AIN-93 G vitamin mixture.

kits (Asan Pharm Co., Ltd., Seoul, Korea). Low density lipoprotein (LDL)-cholesterolwas calculated using the Friedewald formula (Friedewald et al., 1972) and the ath-erogenic index (AI) was calculated by the Rosenfeld formula (Rosenfeld, 1989).

LDL� cholesterol ¼ total cholesterol�HDL� cholesterol� ðTriglycerides=5Þ

AI ¼ ðtotal cholesterol�HDL� cholesterolÞ=HDL� cholesterol

2.4. Hepatic and fecal lipids analysis

Hepatic and fecal lipids were extracted using the method described by Bligh andDyer (1959). Triglycerides and total cholesterol were determined by enzymatic col-orimetric methods using commercial kits, as described above.

2.5. Histological analysis

Epididymal WAT was dissected from rats, and fixed in 10% formalin for 24 h.The fixed samples were washed and embedded in paraffin, and 5 lm thick sectionswere mounted on slides. The histology sections were stained with hematoxylin-eo-sin. The tissue sections were observed under a microscope (Olympus, Tokyo, Japan)at 200� magnification.

2.6. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was isolated from epididymal WAT using Trizol reagent (Invitrogen,Carlsbad, CA, USA). The corresponding cDNA was synthesized from 4 lg of totalRNA using M-MLV reverse transcriptase. After cDNA synthesis, real-time qPCR wasperformed using universal SYBR Green PCR Master Mix on a fluorometric thermal cy-cler (Rotor-Gene 2000, Corbett Research, Mortlake, Australia). Primers were designedusing an online program (Primer3. cgi v 0.6) (Rozen and Skaletsky, 2000). The se-quences of the sense and antisense primers used for amplification were as follows:aP2; 50-TCACCCCAGATGACAGGAAA-30 and 50-CATGACACATTCCACCACCA-30

(NM_053365), b-actin; 50-GGCACCACACTTTCTACAAT-30 and 50-AGGTCTCAAACAT-GATCTGG-30 (NM_031144), PPARc; 50-TGTGGGGATAAAGCATCAGC-30 and 50-CAAGGCACTTCTGAAACCGA-3 0 (NM_001145366), SREBP-1c; 5 0-AGGAGGCCATCTTGTTGCTT-30 and 50-GTTTTGACCCTTAGGGCAGC-30 (AF286470), UCP2; 50-ACTGTCGAAGCCTACAAGAC-30 and 50-CACCAGCTCAGTACAGTTGA-30 (NM_019354).The DDCt method was used for relative quantification (Livak and Schmittgent,2001). The DDCt value for each sample was determined by calculating the differencebetween the Ct value of the target gene and the Ct value of the b-actin reference gene.The normalized level of target gene expression in each sample was calculated usingthe formula 2�DDCt. Values were expressed as fold-change over the control.

2.7. Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). Differencesamong the three groups were assessed by one-way analysis of variance (ANOVA)and Tukey’s multiple range test using SAS software version 9.2 (SAS Inc., Cary,NC, USA). Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Body weight, energy intake, and fat accumulation

Initial body weights at the beginning of the experiment werenot different among the three groups. At 11 weeks, the final bodyweight was significantly lower in the HEG supplemented groupby 8.9% compared with that in the HF control group (Fig. 1 and Ta-ble 2). The final body weight of the WEG supplemented grouptended to be lower than that in the HF group, but the differencewas not statistically different. However, energy intake and energyefficiency ratio did not differ among the groups (Table 2). The rel-ative weight of WAT was lower by 16% in the HEG supplementedgroup compared to that in the HF control group (p < 0.05), whereasthe WEG supplemented group was not different (Table 2).

3.2. Serum lipid profiles

The HEG supplemented group had significantly lower serumconcentrations of triglycerides (24.3%), total cholesterol (16.3%),and LDL-cholesterol (43.3%) compared to those in the HF group(Table 3). The AI in the HEG supplemented group were alsodecreased by 32.7% compared to that in the HF group (p < 0.05).

Fig. 1. Effect of WEG and HEG on body weight in rats fed a high-fat diet. Data aremean ± SEM (n = 8). �significantly different from control (p < 0.05).

Table 3Effects of WEG and HEG on serum lipid profiles.

HF HF + WEG HF + HEG

Serum lipids (mmol/L)Triglyceride 1.11 ± 0.08a 0.91 ± 0.06ab 0.84 ± 0.06b

Total cholesterol 2.76 ± 0.10a 2.83 ± 0.13a 2.31 ± 0.1b

HDL cholesterol 1.52 ± 0.10 1.51 ± 0.06 1.43 ± 0.08LDL cholesterol 0.90 ± 0.08a 0.76 ± 0.13ab 0.51 ± 0.10b

Atherogenic index (AI) 1.04 ± 0.11a 0.91 ± 0.08ab 0.70 ± 0.09b

AST (IU/L) 56.86 ± 2.71 56.55 ± 2.07 53.68 ± 2.73ALT (IU/L) 10.35 ± 0.35 10.27 ± 0.53 9.49 ± 0.35

Values are mean ± SEM (n = 8). abThe means without a common superscript letterare significantly different at the p < 0.05 level by Tukey’s multiple range test. HF,high-fat diet group; HF + WEG, high-fat diet +5 g/kg hot water extract of garlic;HF + HEG, high-fat diet +5 g/kg high hydrostatic pressure extract of garlic.

102 H. Joo et al. / Food and Chemical Toxicology 55 (2013) 100–105

However, the levels of triglycerides, total cholesterol, LDL-cholesterol, and the AI in the WEG supplemented group were notsignificantly different compared to those in the HF group (Table 3).

3.3. Hepatic and fecal lipid profiles

The results of hepatic and fecal lipids analyses are shown in Ta-ble 4. The concentrations of hepatic lipids (38.9%) and total choles-terol (33.6%) in the HEG supplemented group were lower thanthose in the HF group (p < 0.05). Additionally, the level of fecal tri-glycerides in the HEG supplemented group were increased by53.4% compared to that in the HF group (p < 0.05). The WEG sup-plemented group tended to have lower levels of hepatic lipidsand hepatic total cholesterol and a higher level of fecal triglycer-ides than those in the HF group, but the differences were not sta-tistically significant.

3.4. Liver weight and serum AST and ALT activities

The WEG and HEG did not cause a rise in serum AST and ALTactivities when compared to those in the HF group (Table 3). Inaddition, liver weight was unaffected by the WEG and HEG treat-ment (Table 2). These data indicate that the doses of WEG andHEG were well tolerated by the rats.

3.5. Histological analysis

The histological analysis of epididymal WAT is shown in Fig. 2.Both WEG and HEG supplementation decreased adipocyte size

Table 2Effects of WEG and HEG on physiological variables.

Variables HF

Initial body weight (g) 78.38 ± 1Final body weight (g) 534.42 ± 1Body weight gain (g/11 week) 450.37 ± 1Food intake (g/day) 21.03 ± 0Energy intake (kcal/day) 97.68 ± 1Energy efficiency ratio (EER) 0.058 ± 0Relative liver weight(g/100 g body weight) 2.43 ± 0Relative adipose tissue weight (g/100 g body weight) 7.22 ± 0Epididymal 3.69 ± 0Retroperitoneal 3.56 ± 0

Energy efficiency ratio (EER) = body weight gain (g/day)/energy intake (kcal/day).Values are mean ± SEM (n = 8). abThe means without a common superscript letter are sigdiet group; HF + WEG, high-fat diet + 5 g/kg hot water extract of garlic; HF + HEG, high-

compared to that in the HF group. In particular, the HEG showeda better inhibitory effect than that of the WEG (Fig. 2).

3.6. mRNA levels for adipogenesis-related genes and the UCP2 gene

We next analyzed whether WEG or HEG supplementation af-fected the mRNA expression of genes involved in adipogenesis.The mRNA levels of adipogenic genes such as PPARc, SREBP-1c,and aP2 were significantly decreased in both the WEG and HEGsupplemented groups compared with those in the HF group(Fig. 3). UCP2 mRNA level was determined to examine the effectof the extracts on thermogenesis. As a result, UCP2 mRNA levelwere significantly increased in the HEG supplemented group com-pared with that in the HF group (Fig. 4).

4. Discussion

Traditional plant material extraction methods are based on theproper choice of solvents, heat, and/or pressure to increase the sol-ubility of bioactive compounds. HHP extraction is carried out atroom temperature, so heat damage and the loss of volatile com-pounds can be avoided (Iberl et al., 1990). The bioactive organosulfurcompounds in garlic are easily affected by cooking and processingmethods; thus, a suitable processing technique is very important.Therefore, the aim of this study was to investigate the effects of aWEG and HEG on the obesity, particularly whether they affect bodyweight, serum lipid profiles, and adipogenic gene expression.

The dose of garlic used in our study was tolerated by the rats, asdemonstrated by the fact that the serum levels of AST and ALTwere unaffected by garlic supplementation. Body weights in theHEG supplemented group decreased significantly from 5 weekafter beginning the HEG diet and also the mass of adipose tissuein the HEG group decreased compared to those in the HF control

HF + WEG HF + HEG

.49 78.38 ± 1.83 78.14 ± 1.846.02a 498.43 ± 6.78ab 486.61 ± 6.31b

4.49a 424.86 ± 7.35ab 411.25 ± 6.34b

.43 20.28 ± 0.36 20.88 ± 0.29

.98 93.77 ± 1.66 96.59 ± 1.35

.001 0.058 ± 0.001 0.056 ± 0.001

.05 2.37 ± 0.04 2.43 ± 0.04

.33a 6.51 ± 0.26ab 6.06 ± 0.34b

.12a 3.41 ± 0.11ab 3.18 ± 0.12b

.22 3.22 ± 0.15 3.00 ± 0.16

nificantly different at the p < 0.05 level by Tukey’s multiple range test. HF, high-fatfat diet + 5 g/kg high hydrostatic pressure extract of garlic.

Table 4Effects of WEG and HEG on hepatic and fecal lipid profiles.

HF HF + WEG HF + HEG

Hepatic lipidsTotal lipid (mg/liver) 304.87 ± 34.44a 211.03 ± 26.73ab 186.35 ± 28.26b

Triglyceride (lmol/gliver)

15.06 ± 0.75 12.81 ± 0.81 12.33 ± 0.83

Total cholesterol(lmol/g liver)

4.34 ± 0.41a 3.27 ± 0.42ab 2.88 ± 0.34b

Fecal lipidsTotal lipid (mg/day) 37.03 ± 6.53 44.12 ± 3.99 43.68 ± 5.34Triglyceride (lmol/

day)1.89 ± 0.26a 2.19 ± 0.22ab 2.87 ± 0.24b

Total cholesterol(lmol/day)

10.99 ± 0.82 12.06 ± 0.85 12.61 ± 0.92

Values are mean ± SEM (n = 8). abThe means without a common superscript letterare significantly different at the p < 0.05 level by Tukey’s multiple range test. HF,high-fat diet group; HF + WEG, high-fat diet +5 g/kg hot water extract of garlic;HF + HEG, high-fat diet +5 g/kg high hydrostatic pressure extract of garlic.

Fig. 3. Effects of WEG and HEG on adipogenic genes expression. Values aremean ± SEM (n = 8). abThe Means without a common superscript letter aresignificantly different at the p < 0.05 level by Tukey’s multiple range test. Levelsof mRNA were analyzed by real-time PCR assays and were normalized to b-actin.(A) PPARc, (B) SREBP-1c, (C) aP2.

H. Joo et al. / Food and Chemical Toxicology 55 (2013) 100–105 103

group. Energy intake and energy efficiency ratio did not differamong the groups. These results suggest that the reduction in bodyweight was not associated with an anorectic effect of the garlic ex-tracts. The WEG and HEG supplemented groups showed a decreasein the size of adipocyte lipid droplets. WAT stores excess energy astriglycerides under conditions of energy excess, and expansion ofadipocyte size and number are key characteristics of obesity (Flier,1995). Thus, a reduction in body weight, which could be attributedpartially to a decrease in the mass of the fat pad and size of adipo-cytes in the garlic-supplemented groups, reflected antiobesity ef-fect of the garlic extracts. The final body weight and adiposetissue mass of the WEG supplemented group tended to be lowerthan that in the HF group, but the difference was not statisticallydifferent. These results suggested that HEG more efficiently re-duced body weight gain than WEG.

Organosulfur compounds in garlic, such as allicin and its deriv-atives, contribute to the beneficial effects of garlic (Jisawa et al.,2008). Allicin is very unstable and disappears or is transformedto another series of compounds, such as sulfides, ajoene, vinyldi-thinins, and other types of organosulfur compounds (Jisawaet al., 2008; Amagase, 2006). Allicin effectively reduces bodyweight gain in fructose-fed rats (Elkayam et al., 2003), and ajoenedecreases fat accumulation in 3T3-L1 adipocytes (Yang et al.,2006), while 1,2-vinyldithiin inhibits differentiation of human pre-adipocytes (Keophiphath et al., 2009). In the present study, theamount of allicin in the HEG was higher relative to that in theWEG by 30%. Thus, it can be assumed that allicin might be one ofreasons to explain the greater efficacy of the HEG on suppressedfat accumulation in adipose tissue resulting in reduced bodyweight gain compared to that of the WEG.

Garlic supplementation decreases plasma levels of triglycerides,total cholesterol, and LDL-cholesterol in humans and animals (Yehand Liu, 2001). Moreover, fecal levels of triglycerides and totalcholesterol increase in high temperature/high pressure-processed

Fig. 2. Histological analysis of epididymal white adipose

garlic-treated rats (Sohn et al., 2012). The present results showedthat the HEG effectively reduced serum levels of triglycerides, totalcholesterol, and LDL-cholesterol. Additionally, hepatic total lipidsand total cholesterol concentrations were decreased, whereas fecaltriglycerides were increased in the HEG supplemented group.However, those levels were not different between the HF controlgroup and the WEG supplemented group. These data implied thatincrease of triglyceride excretion through the lower intestinal tri-glyceride absorption might resulted in reduction of body weightgain and serum lipids levels in the HEG group. Moreover, higher fe-cal triglyceride excretion in the HEG group might explain thegreater efficacy of HEG on body weight reduction compared to thatof WEG.

The mRNA levels of genes related to adipogenesis wereanalyzed in WAT to elucidate the anti-obesity action of the garlic

tissue; hematoxylin and eosin, scale bars = 50 lm.

Fig. 4. Effect of WEG and HEG on expression of UCP2 gene. Values are mean ± SEM(n = 8). abThe Means without a common superscript letter are significantly differentat the p < 0.05 level by Tukey’s multiple range test. Level of mRNA was analyzed byreal-time PCR assays and was normalized to b-actin.

104 H. Joo et al. / Food and Chemical Toxicology 55 (2013) 100–105

extracts. Adipogenesis is the process by which preadipocytes be-come adipocytes and is induced through expression of various adi-pogenic transcription factors (Ntambi and Kim, 2000; Rosen et al.,2000). PPARc and SREBP-1c are the major transcription factors thatdirectly influence fatty acid synthesis and adipogenesis. A targetgene of PPARc, aP2, is a carrier protein for fatty acids expressedin adipocytes (Duplus and Forest, 2002). Garlic supplementationreduces body weight by downregulating PPARc, SREBP-1c, andaP2 expression in mice (Lee et al., 2011b). In our data, the mRNAlevels of the adipogenic genes such as PPARc, SREBP-1c, and aP2also decreased significantly in both the WEG and HEG supple-mented groups. These results suggest that the WEG and HEG reg-ulated fat accumulation by downregulating the expression ofgenes associated with adipogenesis and adipocyte differentiation.

UCP2 is mitochondrial protein which provides a pathway forproton re-entry to the mitochondrial matrix, thereby dissipatesthe energy as heat (Horvath et al., 2003). UCP2 is associated withobesity and energy metabolism in rodents and humans, which playkey role in energy expenditure (Yonezawa et al., 2009). Hence,UCP2 increases thermogenesis and remains important target forthe treatment of obesity (Brand and Esteves, 2005). Dietary garliceffectively increases body temperature and UCP2 mRNA levels in li-ver, WAT, BAT, and muscle (Lee et al., 2011b). It has been suggestedthat garlic supplementation increases thermogenesis, which in-creases energy expenditure (Lee et al., 2011b). In the present study,HEG supplementation resulted in a decrease in body weight gainwithout any difference in energy intake, suggesting that HEG hada physiological effect on energy expenditure. The HEG also signifi-cantly increased UCP2 mRNA level in epididymal WAT, whereasWEG did not. This result suggests that suppression of weight gainin the HEG supplemented group might have been mediated, at leastpartially, by upregulation of the UCP2 gene, which increases energyexpenditure. Additionally, the antiobesity effect of HEG might bemore effective than WEG through this higher energy expenditurewith upregulation of the expression of UCP2.

5. Conclusion

In conclusion, HEG supplementation resulted in reduced bodyweight with decreased mass of adipose tissue. The final bodyweight of the WEG supplemented group tended to be lower thanthat in the HF group, but the difference was not statistically differ-ent. Serum levels of triglycerides, total cholesterol, and LDL-cholesterol also decreased in the HEG group, whereas WEG didnot. The level of fecal triglycerides in the HEG supplemented groupincreased compared to those in the HF group, whereas WEG didnot. The mRNA levels of adipogenic genes decreased significantlyin both the WEG and HEG supplemented groups, whereas the

UCP2 mRNA level clearly increased in the HEG supplementedgroup compared to that in the HF group. These results demon-strated that HEG more efficiently reduced body weight gain thanthat of WEG, which was at least partially mediated by increase ofthe fecal triglyceride and downregulation of the expressionof genes involved in adipogenesis together with upregulation ofUCP2 gene expression in rats fed a high-fat diet.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by the Food High Pressure Technol-ogy Development Project, the Korea Food Research Institute(202007-03-03-WT011), and the National Research Foundation ofKorea (NRF-2010-0023066).

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