Ginger Essential Oil Ameliorates Hepatic Injury and Lipid

Feb 21, 2016 - The objective of this study was to investigate the hepatoprotective efficacy and mechanism of action of ginger essential oil (GEO) agai...
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Ginger essential oil ameliorates hepatic injury and lipid accumulation in high fat diet-induced nonalcoholic fatty liver disease Yi-Syuan Lai, Wan-Ching Lee, Yu-En Lin, Chi-Tang Ho, Kuan-Hung Lu, Shih-Hang Lin, Suraphan Panyod, Yung-Lin Chu, and Lee-Yan Sheen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06159 • Publication Date (Web): 21 Feb 2016 Downloaded from http://pubs.acs.org on February 21, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Ginger essential oil ameliorates hepatic injury and lipid accumulation in high fat

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diet-induced nonalcoholic fatty liver disease

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Yi-Syuan Lai,§, ⊥ Wan-Ching Lee,§ Yu-En Lin,§ Chi-Tang Ho,§,& Kuan-Hung Lu§,

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Shih-Hang Lin,§ Suraphan Panyod,§ Yung-Lin Chu,# and Lee-Yan Sheen*,§,∇,○

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§

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Education and Research,

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University, Taipei 10617, Taiwan

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Institute of Food Science and Technology,





National Center for Food Safety



Center for Food and Biomolecules, National Taiwan

Department of Hospitality Management, Yu Da University of Science and

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Technology, Miaoli 36143, Taiwan

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&

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08901, USA

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University of Science and Technology, Pingtung 91201, Taiwan

Department of Food Science, Rutgers University, New Brunswick, New Jersey

International Master’s Degree Program in Food Science, National Pingtung

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* Corresponding author: Prof. Lee-Yan Sheen, Institute of Food Science and

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Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei

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10617,

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[email protected]

Taiwan.

Tel:

886-2-33664129;

Fax:

886-2-2362-0849;

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ABSTRACT

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The objective of this study was to investigate the hepatoprotective efficacy and

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mechanism of action of ginger essential oil (GEO) against the development of

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nonalcoholic fatty liver disease (NAFLD). Mice were maintained on either a control

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diet or high-fat diet (HFD) supplemented with GEO (12.5, 62.5, and 125 mg/kg) or

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citral (2.5 and 25 mg/kg) for 12 weeks. We demonstrated that GEO and its major

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component (citral) lowered HFD-induced obesity in a dose-dependent manner,

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accompanied by antihyperlipidemic effects by reducing serum free fatty acid,

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triglyceride, and total cholesterol levels. Moreover, liver histological results showed

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that administration of 62.5 and 125 mg/kg GEO and 25 mg/kg citral significantly

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reduced hepatic lipid accumulation. Further assessment by western blotting and

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investigation of the lipid metabolism revealed that hepatic protein expression of sterol

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regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC),

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fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase

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(HMGCR), and cytochrome P450 2E1 (CYP2E1) were down-regulated by GEO and

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citral, indicating that GEO and citral suppressed HFD-stimulated lipid biosynthesis

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and oxidative stress. Furthermore, GEO and citral effectively enhanced the

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antioxidant capacities and reduced inflammatory response in mouse liver, which

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exerted protective effects against steatohepatitis. Collectively, GEO and citral

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exhibited potent hepatoprotective effects against NAFLD induced by HFD in obese

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mice. Thus, GEO might be an effective dietary supplement to ameliorate

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NAFLD-related metabolic diseases, and citral could play a vital role in its

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management.

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Keywords: ginger essential oil, citral, non-alcoholic fatty liver disease, lipogenesis,

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obesity

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INTRODUCTION

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Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver

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disease and its prevalence is rapidly increasing due to strong association with obesity,

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dyslipidemia, type 2 diabetes mellitus, and hypertension.1 Epidemiologic surveys

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indicate that the prevalence of NAFLD is approximately 6–35% in the general

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population, but is 30–63% in obese adults.2 Moreover, in 25% of this high-risk 2

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population, NAFLD may result in nonalcoholic steatohepatitis (NASH), and 42% of

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this group had advanced fibrosis. In severe cases, liver failure or cancer may develop.

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Thus, the prevalence of NAFLD is much higher in patients with metabolic diseases

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caused by obesity than in non-obese populations.3

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Several factors are involved in the pathologic metabolic mechanisms that

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ultimately lead to the accumulation of liver fat and NAFLD progression. However, the

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mechanisms involved in the initiation and progression of NAFLD are yet to be fully

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clarified. Consolidated data indicate that lipid metabolism and oxidative stress may

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play a critical role in the development of obesity-triggered NAFLD. Research has

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shown that high calorie diets high in saturated fats can result in storage of excess fat

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in adipose tissue, causing adipocyte hypertrophy and adipose tissue expansion leading

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to obesity. During metabolic processing, free fatty acids (FFA) released into the liver

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stimulate the expression of the key factor controlling cholesterol synthesis,

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3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), as well as the critical fatty

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acid synthesis factors, sterol regulatory element-binding protein-1c (SREBP1-c),

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acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS). Together, these

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enzymes promote synthesis of liver lipids, such as cholesterol and triglycerides (TG).

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Meanwhile, fatty acid metabolism and beta-oxidation decrease due to inhibition of the

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regulatory factors peroxisome proliferator–activated receptor-α (PPARα) and

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carnitine palmitoyltransferase I (CPT-1). This results in abnormal liver lipid

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metabolism and leads to the development of hepatic steatosis, further hepatic lipid

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accumulation, and NAFLD.4 In addition, fatty acid activation by excess hepatic FFA,

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upregulates the expression of cytochrome P450 isoform 2E1 (CYP2E1) and increases

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the production of reactive oxygen species (ROS). Subsequently, acute oxidative stress

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and increased liver inflammation results in hepatocyte damage, necrosis, and

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progression of fibrosis.5 Given the high prevalence of NAFLD and the lack of

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satisfactory treatments, many studies aim to develop diet replacement therapies for

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preventing and treating the disease.6

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Ginger (Zingiber officinale Roscoe), the rhizomes of ginger, is a popular spice

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and herbal medicine for the improvement of obesity,7 cancer,8 diabetes,9,

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atherosclerosis,11 and fructose-induced fatty liver.12 Recently, studies have

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demonstrated that ginger essential oil (GEO), the volatile oil of fresh ginger, which is

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produced by steam distillation, has the potential of reducing experimental colitis 3

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symptoms,13 inhibiting gastric ulcers in the stomach,14 and protecting hepatocytes

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against acute ethanol-induced fatty liver.15 The chemical composition of GEO has

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been characterized, of which the main components are citral,α-zingiberene, camphene,

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α-farnesene, and β-sesquiphellandrene.16 Also, citral has been proven to be the major

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component and has antiadipogenic effects against energy-intense diet-induced

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obesity.17

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However, limited studies have been performed on the effect of GEO and its

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bioactive compound on the molecular biological mechanism of NAFLD induced by

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HFD.18 Therefore, the purpose of this study was to assess the efficacy of GEO or

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citral on HFD-induced NAFLD in C57BL/6J mice and to identify the possible

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mechanisms involved in preventing the progression of NAFLD. The present study can

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provide experimental evidence of GEO and citral as functional food ingredients.

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MATERIALS AND METHODS

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GEO Extraction and Mainly Ingredient Analysis

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Samples of ginger (Zingiber officinale Roscoe) were purchased from the

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Mingjian Township Farmers' Association of Nantou County (Nantou, Taiwan). The

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collected ginger was thoroughly washed under running water to remove all dirt.

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Whole rhizome of ginger was cut into small pieces (5 mm of thickness) and stirred

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with 3 volumes of distilled water. The mixture was subjected to steam distillation for

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2–3 h, and the volatile extracts were then dehydrated to obtain GEO. The resulting oil

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was stored at a temperature of -20°C for further analysis. The yield of the obtained oil

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from fresh ginger was 0.12% (w/w), calculated according to the weight of ginger used

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in the extraction procedure. GEO analyses were performed using a gas chromatograph

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(GC, Thermo Scientific Focus GC) equipped with an AI 3000 II autosampler, a

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Stabilwax (Crossbond Carbowax-PEG, Restek) capillary column (60 m length, 0.32

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mm internal diameter, 1.0 µm-thick film), and a flame ionization detector. Nitrogen

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was used as the carrier gas at a flow rate of 30 mL/min with a split ratio of 80:1. The

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analysis conditions were as follows: initial column temperature 50°C; an increase of

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5°C/min to 200°C held for 10 min; injector temperature 200°C and detector

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temperature 200°C; and an injection volume of 1 µL (diluted 1:100 in acetone).

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Calibration curves were constructed with reference standards and used to determine

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the concentration of citral in GEO. Citral (purity 95%) was purchased from

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Sigma-Aldrich Corp. (St. Louis, MO, USA).

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Animals and Experimental Design

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Six-week-old male C57BL/6J mice were obtained from the Animal Center of the

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College of Medicine, National Taiwan University (Taipei, Taiwan). The animals were

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maintained in accordance with the National Taiwan University guidelines for the care

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and use of laboratory animals. The animals were housed in an alternate light/dark

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cycle (12 h) room with a temperature of 23 ± 2°C and a relative humidity of 50–70%.

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All mice were fed experimental diets ad libitum with free access to drinking water at

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all times. After two weeks of adaptive feeding, the mice were randomly assigned to

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groups of 8 animals each and fed different experimental diets as follows: mice in the

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control group were fed a normal chow diet with 13.5% kcal fat content (Laboratory

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Rodent Diet 5001, Lab Diet/PMI Nutrition International, Purina Mills LLC, Gray

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Summit, MO), while mice in the negative control group and treatment groups were

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fed an HFD with 60% kcal fat content (D12492, Research Diets, Inc., New Brunswick,

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NJ) for 12 weeks to induce a steatosis condition. Mice in the treatment groups were

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orally administered either GEO (12.5, 62.5, or 125 mg/kg) or citral (2.5 or 25 mg/kg).

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Citral was comparable amount with the current GEO sample (19.8% of GEO). The

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control and HFD groups of mice were subjected to gavage with the same volume of

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olive oil. Food intake and body weight were measured weekly for the duration of the

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experiment. At the study endpoint, mice were fasted overnight and sacrificed by

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carbon dioxide asphyxiation. Blood samples was collected by cardiac puncture and

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separated by centrifugation at 1000 × g for 10 min at 4°C to collect serum samples.

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The liver, subcutaneous adipose, epididymal adipose, and perirenal adipose were

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harvested and weighed.

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Serum Biochemical Analysis The

levels

of

serum

biochemical

parameters,

including

aspartate

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aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and

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TG were measured colorimetrically and assayed by an automatic biochemistry

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analyzer (SPOTCHEM EZ SP-4430, ARKRAY, Inc., Kyoto, Japan) according to the

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manufacturer’s instructions. Serum levels of insulin and FFA were measured using 5

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commercial kits obtained from Mercodia AB and BioVision, respectively.

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Histological Staining

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To examine the morphology, the largest lobe of the liver tissue was fixed with

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the 10% neutral-buffered formalin solution and then embedded in paraffin serially

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sectioned at 5 µm stained with hematoxylin and eosin (H&E). Histological scoring, as

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an indicator of the degree of fatty liver, was performed by an experienced pathologist.

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Hepatic steatosis was graded as follows: grade 1 = minimal (