Î±-Mangostin Regulates Hepatic Steatosis and Obesity through SirT1

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#-Mangostin Regulates Hepatic Steatosis and Obesity through SirT1AMPK and PPAR# Pathway in High Fat Diet induced Obese Mice Young Hee Choi, Jin Kyung Bae, Hee-Sung Chae, Young-Mi Kim, Yim Sreymom, Ling Han, Ha Young Jang, and Young-Won Chin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01637 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 21, 2015

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α-Mangostin Regulates Hepatic Steatosis and Obesity through

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SirT1-AMPK and PPARγγ Pathway in High Fat Diet induced

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Obese Mice

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Young Hee Choi,† Jin Kyung Bae,† Hee-Sung Chae,† Young-Mi Kim,† Yim Sreymom, † Ling

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Han,† Ha Young Jang,§ Young-Won Chin†,*

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†

College of Pharmacy and BK21Plus R-Find Team, Dongguk University-Seoul, 32 Dongguk-

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lo, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, South Korea

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§

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ro, Dong-gu, Daegu, 701-310, South Korea

Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, 80 Dongnae-

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Running title: α-Mangostin Regulates Hepatic Steatosis and Obesity

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ABSTRACT: Previous studies have shown that α-mangostin (α-MG) suppresses

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intracellular fat accumulation and stimulation of lipolysis in in vitro systems. Together with

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the relatively high distribution of α-MG in liver and fat, these observations made it possible

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to propose a plausible hypothesis that an α-MG supplement may regulate hepatic steatosis

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and obesity. An α-MG supplement (50 mg/kg) reduced the body weight gain (13.8%) and

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epidymal and retroperitoneal fat mass accumulation (15.0% and 11.3%, respectively), as well

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as the biochemical serum profiles such as cholesterol [TC (26.9%), LDL-C (39.1%), and

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HDL-C (15.3%)], glucose (30.2%), triglycerides (29.7%), and fatty acid (30.3%) levels in

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high-fat fed mice compared with the high-fat diet-treated group, indicating that α-MG may

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regulate lipid metabolism. In addition, an α-MG supplement upregulated the hepatic AMPK,

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SirT1, and PPARγ levels compared with the high-fat diet states, suggesting that α-MG

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regulates hepatic steatosis and obesity through the SirT1-AMPK and PPARγ pathways in

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high-fat diet-induced obese mice.

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Keywords: α-mangostin, hepatic steatosis, obesity, SirT1, AMPK

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INTRODUCTION

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The obesity-induced metabolic disorders have been known mainly to be caused by an

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imbalance of dietary intake such as excess fat and lack of exercise, which are related to

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hyperglycemia, hyperlipidemia, hepatic steatosis and cardiovascular diseases, and hence,

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have become a global health threat.1,2 The general characteristics of obesity include an

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increase in adipose tissue masses from increased fat cell numbers (hyperplasia) and size

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(hypertrophy).3 In addition, lipid accumulation and changes in lipid forms (e.g. triglycerides,

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fatty acids, and various cholesterols) in adipose tissue, blood, and other tissues are associated

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with the development of obesity.4-6 Thus, along with adipose tissue, the modulation of

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various targets in referential tissues may be able to regulate body weight and metabolic

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disorders (e.g. hepatic steatosis and diabetic mellitus). It seemed to be valuable to investigate

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the changes in fat mass in adipose tissue, the circulating lipid fuels in the blood, and signaling

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pathway in the liver, a representative organ related to fatty acid oxidation.7-9

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A high-fat diet is associated with impairment of the hepatic sirtuin 1 (SirT1)-AMP-

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activated protein kinase (AMPK) axis, a central signaling system controlling the lipid

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metabolism pathways.10 In particular, the activation of the SirT1-AMPK pathway in

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metabolic tissues, including liver, has been found to increase rates of fatty acid oxidation and

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to repress lipogenesis, largely by modulating peroxisome proliferator-activated receptor γ

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(PPARγ) and retinoid X receptor (RXR) activities.11

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Mangosteen, Garcinia mangostana L., is a tropical fruit that has been used for hundreds

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of years around the world as a traditional herbal medicine, containing biologically active

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compounds including xanthones, terpenes, anthocyanins, tannins, phenols, and multiple

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vitamins.12 Recently, mangosteen has gained popularity as a functional food and botanical

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dietary supplement due to its purported health benefits. α-mangostin (α-MG), a major 3


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xanthone derivative, possesses a variety of pharmacological efficacies such as antioxidant,

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anti-inflammatory, and anti-diabetic activities.12-15 Moreover, AMPK activation by α-MG in

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glioblastoma cells has been reported to be beneficial to autophagy.16 Interestingly, the

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suppression of intracellular fat accumulation and the stimulation of lipolysis by α-MG in

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3T3-L1 cells,17 as well as the high distribution of α-MG in liver and fat in vivo,18 provide a

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substantial hypothesis that α-MG may be valuable in the prevention or treatment of obesity-

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related diseases such as hepatic steatosis. To date, the role of α-MG in a high-fat diet-induced

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obesity model has not yet been established, thus, here, we focus on the regulatory effects of

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α-MG on hepatic steatosis and obesity in high-fat diet fed mice, associated with the SirT1-

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AMPK and PPARγ pathways in an in vivo system, along with the regulation of body weight,

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fat mass, and lipid metabolism.

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Materials and Methods

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Chemicals. α-MG was purified (> 98.0% of purity) at the College of Pharmacy, Dongguk

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University (Seoul, South Korea) according to the previously reported protocol.14

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Polyethylene glycol 400 (PEG 400) was from Showa Chemical Company (Tokyo, Japan).

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Antibodies against β-actin, SirT1, p-AMPK, AMPK, PPARγ, RXRα were purchased from

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Cell Signaling Technology, Inc. (Danvers, MA).

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Animals. The protocols for the animal studies were approved by the Institute of

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Laboratory Animal Resources of Dongguk University, Seoul, South Korea (# 2012-0673;

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July 25, 2012). Male C57BL/6 mice were obtained from Charles River Orient (Seoul, South

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Korea) and acclimated for 1 week before starting the study. Upon arrival, animals were

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randomized and housed at three per cage under strictly controlled environmental conditions

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(20–25°C and 48–52% relative humidity). A 12-hour light/dark cycle was used at an intensity

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of 150 to 300 Lux. C57BL/6 mice were started on either a normal diet (ND, 10% of

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kilocalories as fat; Product N12450B), or a high-fat diet (HD, 60% of kilocalories as fat;

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Product D12592, Research Diets, New Brunswick, NJ, USA) for 80 days. After 5 weeks (at

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day 35) of ND or HD, the mice were distributed into six treatment groups. α-MG (10 or 50

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mg/kg/day) dissolved in PEG 400 and distilled water (6:4, v/v) was orally administered to

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mice with ND (NMG10 and NMG50 groups, respectively; n = 8 for each) or HD (HMG10

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and HMG50 groups, respectively; n = 8 for each) every day during the last six weeks of diet

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feeding. ND and HD groups (n = 8 for each) were orally administered a vehicle instead of α-

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MG.

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Measurement of liver, fats, and kidney weights, and triglyceride (TG) levels in the

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liver. At the end of the experimental period, the liver, fats, and kidney were extracted and the

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weights were measured. In case of fats, the weights of total, epididymal, and retroperitoneal

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fats were measured separately. For the measurement of hepatic TG, 0.3 g of liver was

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homogenized in 0.1 M Tris-acetate buffer (pH 7.4), and six volumes of chloroform:methanol

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(2:1) were added. After vigorous stirring, the mixtures were incubated on ice for 1 hour and

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centrifuged at 800 x g for 3 minutes. The resulting lower phase was aspirated. The hepatic

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TG content was determined using an Enzychrom TG assay kit.

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Blood chemistry. At the end of the experimental period, plasma samples were collected

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and total protein, albumin, aspartate aminotransferase (AST), alanine aminotransaminase

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(ALT), creatinine, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-

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density lipoprotein cholesterol (LDL-C), TG, free fatty acid (FFA), glucose, blood urea

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nitrogen (BUN), and creatinine levels were analyzed (analyzed by Green Cross Reference

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Lab., Seoul, South Korea). 5


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Hematoxylin and eosin staining in the liver and various sites of fat. The left lateral

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lobe of the liver, and epididymal and retroperitoneal fat were excised,19 fixed in 10%

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formalin, and embedded in paraffin. 4µm-thick sections of liver, and epididymal and

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retroperitoneal fat were obtained and stained with hematoxylin and eosin (H&E) for the

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histological examination of adipocytes, as previously described.19

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Immunoblotting analysis. Protein expression of hepatic SirT1, AMPK, p-AMPK, PPARγ,

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and RXRα were assessed by Western blotting analysis according to the manufacturer’s

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instructions. After the determination of protein concentration, 20 µg protein was mixed 1:1

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with 2× sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromophenol blue, and

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1.25 M Tris-buffer, pH 6.8), loaded onto an SDS-PAGE gel (8% or 15%) and run at 150 V

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for 90 minutes. The tissue proteins were transferred to an ImmunoBlot polyvinylidene

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diﬂuoride membrane using a Bio-Rad semidry transfer system. Following blocking,

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membranes were incubated with primary antibody at dilutions of 1:500–1:1000 at 4°C

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overnight, washed 3 times in PBS, and further incubated with a HRP-conjugated secondary

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anti-IgG antibody at dilutions of 1:2000–1:20,000 for 1 hour. After washing 3 times, the

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immunoreactive proteins were visualized by ECL chemiluminescence detection (Amersham

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Biosciences, Piscataway, NJ). Equal loading of proteins was verified by β-actin

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immunoblotting.

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Statistical analysis. A P value < 0.05 was deemed to be statistically significant using

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Duncan’s multiple range test of the Social Package of Statistical Sciences (SPSS) posteriori

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analysis of variance (ANOVA). All data are expressed as the mean ± standard errors.

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Results Effects of α-MG on body weight gain and food intake. Effects of α-MG on body weight 6



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changes in C57BL/6 mice fed a normal diet (ND) or a high-fat diet (HD) are shown in Figure

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1. The body weight gains in the high-fat diet groups were significantly greater than those in

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the normal diet groups from day 14 to the end of this experiment (e.g., the body weight gain

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compared with the initial body weight: 12.3% vs 28.8% at day 14, and 39.6% vs 130% at day

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80, respectively, for the ND and HD groups). The body weight loss appeared 26 days after

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starting the treatment with 50 mg/kg α-MG (at day 61 after the start of the HD) in the

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HMG50 group compared with the HD group. However, the body weight loss did not appear

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in the HMG10 group compared with the HD group. The mean value of body weight in the

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NMG50 group seemed to be greater than in the ND or NMG10 group, however there was no

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significant difference. The food intakes were comparable among all groups of mice (data not

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shown).

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Effects of α-MG on hepatic steatosis. In an effort to assess the effect of α-MG on hepatic

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steatosis, the measurements of liver weight and hepatic TG level, and histopathological

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analysis of the liver were conducted. In the HMG50 group, the high-fat diet induced an

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increase in liver weight and hepatic TG level in the HD group (by 44.1% and 262% increase

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for liver weight and hepatic TG, respectively, vs the ND group), and the increase in liver

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weight and hepatic TG level were restored (by 12.2% and 44.1% decrease, respectively, vs

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the HD group) (Figures 2A and 2B). As shown in Figure 2C, the histopathological analysis

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of the liver revealed that HD caused the accumulation of fat droplets in the liver,

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accompanying the hepatic steatosis. Arrestingly, the hepatic fat accumulation decreased in the

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HMG50 group according to liver histology. The conditions of liver weight, hepatic TG level,

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and hepatic steatosis in the HMG10 group were still abnormal and similar to the HD group.

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According to Figures 2D and 2E for the measurement of liver functions, the serum ALT and

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AST levels were significantly higher in the HD group (by 144% and 128% increase, 7


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respectively, vs the ND group), but were recovered to control levels in the HMG10 (by 45.7%

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and 42.3% decrease, respectively, vs the HD group) and HMG50 (by 59.8% and 53.6%

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decrease, respectively, vs HD group) groups. The albumin levels were comparable among the

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three groups of mice fed HD (data not shown). With the ND, in both the NMG10 and

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NMG50 groups, all conditions of the liver maintained a normal state. Collectively, these

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results demonstrate that 50 mg/kg α-MG treatment attenuated hepatic steatosis in high-fat

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diet fed mice.

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Effects of α-MG on fat accumulation. To investigate whether α-MG regulated the fat

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accumulation in adipose tissues, the weight and size of epididymal and retroperitoneal fats

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were examined. The weights of total, epididymal, and retroperitoneal fats were increased in

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the HD group (by 614%, 549%, and 804% increase, respectively, vs the ND group), which

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were significantly reduced (by 13.8%, 15.0%, and 11.3% decrease, respectively, vs HD

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group) in the HMG50 group (Figures 3A-3C). As shown in Figures 3D and 3E, the sizes of

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epididymal and retroperitoneal fats in the HD group were significantly increased, by 64.9%

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and 104.6%, respectively, compared with those of the ND group. In the HMG50 group, the

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sizes of adipose tissue were slightly reduced, by 24.2% and 44.5 % in both epididymal and

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retroperitoneal fats, respectively, as compared with the HD group (Figure 3F). However, 10

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mg/kg α-MG treatment did not significantly affect the fat weights and sizes from the HD

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group. In normal diet groups (ND, NMG10 and NMG50), there were no changes in fat

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weights or sizes regardless of treatment with α-MG.

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Effects of α-MG on lipid metabolism. To determine whether α-MG improved lipid

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metabolism, the serum glucose, FFA, TG, TC, HDL-C, and LDL-C levels were measured

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(Figure 4). All parameters in the HD group were higher (by 76.6%, 45.7%, 37.1%, 111%,

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24.8%, and 172% increase, respectively) than in the ND group, and all of them were 8



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decreased in the HMG50 group (by 30.2%, 30.3%, 29.7%, 26.9%, 15.3%, and 39.1%

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decrease, respectively, vs the HD group). In the HMG10 group, FFA, TC, and LDL-C levels

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were reduced (by 28.5%, 23.3%, and 18.4% decrease, respectively, vs the HD group). In

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particular, in the HMG50 group, the reduction in the LDL-C level was greater than that of the

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HDL-C level (by 39.1% and 15.3% decrease for LDL-C and HDL-C levels, respectively, vs

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the HD group). The ratio of HDL-C/LDL-C in the HMG50 group was significantly increased

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(by 38.5%) compared with the HD group. These results suggest that α-MG improved the

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serum lipid profile, which may result from the decrease in lipogenesis.

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Effects of α-MG on hepatic fatty acid metabolism through the SirT1-AMPK pathway.

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To investigate the involvement of α-MG in hepatic fatty acid metabolism, Western blotting

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analysis was performed as shown in Figure 5. Firstly, the protein levels of hepatic PPARγ

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and AMPK were increased in the HD group (vs the ND group), however those of hepatic

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SirT1 and phosphorylated AMPK were much the same in the ND and HD groups. When ND-

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fed mice were supplied with 10 or 50 mg/kg α-MG, the protein levels of hepatic PPARγ and

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AMPK were remarkably amplified, however, α-MG seemed to negligibly affect the level of

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SirT1 and phosphorylated AMPK proteins. In HD-fed mice, an α-MG supplement

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upregulated the hepatic PPARγ protein level in HD-fed mice, suggesting that α-MG acted as

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a hepatic PPARγ agonist. Interestingly, the protein levels of hepatic SirT1 and

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phosphorylated AMPK showed a different phenomenon with the α-MG supplement between

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ND- and HD-fed mice. In the HMG10 and HMG50 groups, protein levels of both hepatic

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SirT1 and phosphorylated AMPK were outstandingly increased compared with the HD group,

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suggesting that the hepatic SirT1 and AMPK axis was implicated in changes in fat

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accumulation in the liver, in parallel with the induction of hepatic PPARγ by α-MG. Based on

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these results, α-MG regulated the hepatic fatty acid metabolism through the SirT1 and AMPK 9


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pathways. Subsequently, the effects of α-MG treatment on the levels of lipogenic genes

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involved in fatty acid oxidation were determined in the liver. The protein expression of RXRα

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was stimulated in the HMG10 and HMG50 groups compared with the HD group, suggesting

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that the activated PPARγ and RXRα may form a heterodimer and bind to responsive DNA

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elements more effectively, thereby stimulating the transcription of genes related to lipid

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metabolism in the liver.

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DISCUSSION

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The present study provides the first in vivo evidence that α-MG attenuated hepatic steatosis

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through hepatic SirT1-AMPK and PPARγ pathways. As found in the experimental data, the

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hepatic fat accumulation was significantly attenuated, and the increase in hepatic TG content

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was decreased by α-MG compared with the HD group. All of these results were consistent

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with the changes in metabolic parameters.

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A metabolic syndrome is defined as the combination of various symptoms of abdominal

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obesity, dyslipidemia, impaired glucose metabolism, hypertension, inflammatory responses,18

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and hepatic steatosis, the latter of which is referred to as a representative hepatic component

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of metabolic syndrome.2,20 Typically, the high-fat diet is emerging as the most common cause

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for the overproduction and accumulation of lipids (mostly TG) within hepatocytes, which

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evolves into a line of diagnostic symptoms in hepatic steatosis with obesity patients.19-222

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Hepatic steatosis leads to the elevated influx of fatty acids, hepatic fat accumulation, and

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impaired export of TG and other cytotoxic factors, including oxidative stress, lipid

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peroxidation, and inflammatory cytokines19 after excess intake of nutrients. Thus, the hepatic

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steatosis model in obese mice induced by a high-fat diet was utilized in this study.

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The gross inspection of the HMG50 group indicate a clear recovery in body weight, fat 10



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mass, hepatic steatosis, and serum biochemical profiles relevant to lipid metabolism. The

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average values of food intake for 80 days were comparable among all groups, indicating that

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α-MG did not control appetite, but had a beneficial influence on fatty acid oxidation or

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cholesterol homeostasis.20,23 In particular, the upregulated serum glucose, FFA, TC, LDL-C,

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and TG were gradually reduced by α-MG in the HMG10 and HMG50 groups, and notably a

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significantly increased HDL-C and HDL/LDL-C ratio were observed. Moreover, the

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decreased TG content may result from decreased lipid synthesis. Considering that the high

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HDL-C/LDL-C ratio showed a positive correlation with a lower risk of cardiovascular

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disease, 50 mg/kg α-MG treatment showed potential efficacy to ameliorate metabolic

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diseases.24,25 In the HMG50 group, hepatic steatosis and abnormal liver function seemed to

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be recovered by 50 mg/kg α-MG supplementation, but 10 mg/kg α-MG seemed to reduce

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hepatic steatosis negligibly. The elevated ALT and AST levels in the HD groups were restored

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in the HMG10 and HMG50 groups, indicating that a high-fat diet resulted in abnormal liver

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function, and α-MG attenuated hepatic injury. With supplementation of 10 and 50 mg/kg α-

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MG with the ND, the normal liver state was maintained, suggesting that α-MG itself did not

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cause liver injury. In addition, hepatic steatosis was recovered in the HMG50 group with

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respect to morphological aspects. Furthermore, no renal injuries were observed in any group

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of mice (supplementary information 1). Taken together with body weight changes, hepatic

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steatosis, fat accumulation, and/or lipid metabolism, α-MG seemed to regulate hepatic

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steatosis and obesity. In particular, the body weight and liver weight changes seemed to be

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dose-dependent, because 50 mg/kg α-MG treatment significantly decreased body and liver

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weights, but there was no significant decrease at 10 mg/kg α-MG treatment compared to

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those in the HD group. 50 mg/kg of α-MG administered to mice is equivalent to

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approximately 320 mg/kg in the human adult, according to the interspecies dose conversions 11


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of NOAELs and previous reports.26-28

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The Western blotting results clearly indicate that the hepatic SirT1 and phosphorylated

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AMPK levels were activated by α-MG in the HMG10 and HMG50 groups. Moreover, the

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protein expression of hepatic PPARγ and RXRα was stimulated by α-MG in the HMG10 and

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HMG50 groups.

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AMPK, a downstream component of a protein kinase cascade, acts as an intracellular

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energy sensor to maintain energy balance. This pivotal role of AMPK makes it an ideal

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candidate for regulating whole-body energy metabolism such as glucose homeostasis and

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lipid metabolism, and additionally AMPK may play a role in protecting the body from

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metabolic diseases with obesity.29,30 Along with AMPK phosphorylation, SirT1, a nuclear

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protein, plays a key role in controlling the transcription factors related to energy

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metabolism.31 The specific SirT1 activator enhances oxidative metabolism in liver and

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skeletal muscle,29 which may regulate whole-body glucose metabolism at both systemically

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and locally.31,32 Activation of SirT1 mimics several metabolic aspects of calorie restriction

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that enhance selective nutrient utilization and mitochondrial oxidative function.32 The

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AMPK/SirT1 signalling pathway is, furthermore, the mechanism by which hormones

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enhance mitochondrial metabolism.32,33 Regulation of AMPK and SirT1 are potential

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therapeutic targets for attenuation of fatty acid oxidation in liver.33 The functions of these

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enzymes are as like a fuel gauge: it is activated under conditions of high-energy depletion.33

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Phosphorylation of AMPK leads to the down-regulation of lipid biosynthesis by

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phosphorylating of acetyl-CoA carboxylase, which decreases the amount of malonyl-CoA,

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and in turn, suppresses carnitine palmitoyl-CoA transferase, resulting in an increase of fatty

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acid oxidation.34 In addition, PPARγ forms heterodimers with the RXR that regulate the lipid

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metabolism.35,36 SirT1 and PPARγ might coordinate the regulation of adipogenesis.37-39 We 12



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have found that α-MG suppressed lipid accumulation in the mouse model. Although α-MG

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has also been found to be effective in fatty liver by PPARγ activation, the interplay between

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the AMPK signalling pathway and regulation of lipid disorders remains unclear.

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The administration of α-MG to HF-induced obese mice reduced body-weight gain,

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adipose mass, serum triglyceride, total cholesterol, and low-density lipoprotein concentration.

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Our results suggest that α-MG exerts anti-obesity effects in HF-induced obese mice by

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activating hepatic AMPK, SirT1, and PPARγ expression. The anti-obesity effect of α-MG in

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HF-induced mice may support the potential of α-MG to reduce the risks of metabolic

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diseases.

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AUTHOR INFORMATION

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Corresponding author: Young-Won Chin, Tel: 82-31-961-5218 (fax: 82-31-961-5218); e-

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mail address: [email protected]

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There is no conflict of interest for this manuscript.

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ACKNOWLEDGMENTS

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We acknowledge the persons involved in sample production, sampling, and data collection:

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Hee Chul Ahn and colleagues from the NMR and animal facilities.

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Funding

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This study was supported by the grant of National Research Foundation of Korea (NRF)

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funded by the government of Korea (NRF-2012R1A1A2004424, Y. H. Choi), the GRRC

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program of Gyeonggi province [GRRC DONGGUK2014–B03, Development of functional

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food to alleviating metabolic syndromes and circulatory disorders] and the National Research

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Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-

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2015R1A2A2A01006736).

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Legends for figures

Figure 1. Effects of α-MG on body weight gain in ND, NMG10, NMG50, HD, HMG10, HMG50 groups. *Significantly different from ND group; #Significantly different from HD group; †Significantly different between MG10 and MG50 groups.

Figure 2. Effects of α-MG on liver from ND, NMG10, NMG50, HD, HMG10, HMG50 groups. The weights of liver and hepatic TG contents were measured (A and B). Also the histology of liver was compared after H&E staining. Vein (blue arrow), Bile duct (red arrow) (C). The liver functions were determined by ALT and AST in serum (D and E). *The means not sharing a common superscript differ, P

Î±-Mangostin Regulates Hepatic Steatosis and Obesity through SirT1

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