Lipid-Lowering Effects of Medium-Chain Triglyceride-Enriched

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Bioactive Constituents, Metabolites, and Functions

Lipid-lowering effects of medium-chain triglyceride-enriched coconut oil in combination with licorice extracts in experimental hyperlipidemic mice Eun-Jung Lee, Hyeongjoo Oh, Beom Goo Kang, Min-Kyung Kang, Dong Yeon Kim, Yun-Ho Kim, Jeong Yeol Lee, Joung Gun Ji, Soon Sung Lim, and Young-Hee Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04080 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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Journal of Agricultural and Food Chemistry

Lipid-lowering effects of medium-chain triglyceride-enriched coconut oil in combination with licorice extracts in experimental hyperlipidemic mice †

Eun-Jung Lee, †Hyeongjoo Oh, †Beom Goo Kang †Min-Kyung Kang, †Dong Yeon Kim, † Yun-Ho Kim, ‡Jeong Yeol Lee, ‡Joung Gun Ji, †Soon Sung Lim, †Young-Hee Kang †

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea; ‡ Korea Beauty & Health Care Co., Seoul, Korea

Eun-Jung Lee and Hyeongjoo Oh contributed to this study equally

Running Title: Lipid-lowering combined coconut oil and licorice extracts

Total words: 7791 6 Tables and 5 Figures (including 1 color figure) Author contributions E.-J. L., H. O. and Y.-H. K. designed research; E.-J. L., H. O., B.G. K., M.-K. K., D. Y. K. and Y.-H. K. (Y.H. Kim) conducted research; E.-J. L. and H. O. analyzed data; E.-J. L., H. O., S.S. L. and Y.-H. K. wrote the paper; J.Y. L. and J.G. J. prepared MCT-coconut oil; Y.-H. K. had primary responsibility for final content. All authors read and approved the final manuscript. Conflict of interests The authors declare that they have no conflict of interest.

Funding sources: This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through High Value-added Food Technology Development project, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (1160200 33 HD0 30).

Abbreviations used: ACC, acetyl CoA carboxylase; ALT, alanine aminotransferase; AMPK, AMPactivated protein kinase; AST, aspartate aminotransferase; FA, fatty acids; MCT, medium chain triglyceride; CO, coconut oil; FAS, fatty acid synthase; FATP1, fatty acid transporter 1; GU, glycyrrhiza uralensis extract; HDL, high-density lipoprotein; LCAT, lecithin–cholesterol acyltransferase; LDL, low-density lipoprotein; LXR, liver X receptor; PPAR, peroxisome proliferatoractivated receptor; PLTP, phospholipid transfer protein; SREBP, sterol regulatory element-binding protein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; VCO, virgin coconut oil; UCP1, uncoupling protein-1 To whom correspondence should be addressed: Young-Hee Kang, Ph.D Department of Food and Nutrition, Hallym University Chuncheon, Kangwon-do, 24252 Korea Phone: 82-33-248-2132 Fax: 82-33-254-1475 Email: [email protected] 1

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ABSTRACT

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Coconut oil has gained in popularity over recent years as healthy oil due to its potential

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cardiovascular benefits. Coconut oil contains medium chain triglycerides (MCT) including lauric

5

acid and capric acid that display beneficial properties in human health. Licorice (Glycyrrhiza

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uralensis) is used as a sweetener and in traditional Chinese medicine with anti-inflammatory, anti-

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microbial and antioxidant activities. This study investigated the in vivo effects of medium chain-

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triglycerides (MCT)-coconut oil (MCO) and its combination with licorice extract (LE-MCO) on serum

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lipid profile, hepatic steatosis, and local fat pad proteins in diet-induced obese mice. No liver

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toxicity was observed in 45% fat diet (HFD)-fed mice orally treated with LE, MCO and LE-MCO for

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12 weeks. Their supplementation reduced HFD-enhanced body weight, blood glucose and insulin

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in mice. Plasma levels of both PLTP and LCAT were boosted in LE-MCO-administrated mice.

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Supplementation of LE-MCO diminished plasma levels of TG and TC with concomitant reduction of

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the LDL-C level and tended to raise blood HDL-C level, compared to that of HFD alone-mice.

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Treatment of LE-MCO encumbered the hepatic induction of hepatosteatosis-related proteins of

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SREBP2, SREBP1c FAS, ACC and CD36 in HFD-fed mice. 6) Substantial suppression of this

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induction was also observed in the liver of mice treated with MCO. Oral administration of LE-MCO

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to HFD mice boosted hepatic activation of AMPK and the induction of UCP-1 and FATP1 in brown

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fat. Conversely, LE-MCO disturbed hepatic PPAR-LXR-RXR signaling in HFD-fed animals and

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reversed HFD-elevated epididymal PPARγ. Collectively, oral administration of LE-MCO may

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impede hyperlipidemia and hepatosteatosis through curtailing hepatic lipid synthesis.

22 23 24 25 26 27

Key Words: Brown fat, coconut oil, epididymal fat, licorice extracts, fatty liver, hyperlipidemia 2


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INTRODUCTION

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Hyperlipidemia is a major risk factor for cardiovascular diseases, the leading cause of

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death in the developing countries. Excessive fat in the circulation can accumulate, forming plaques

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on the walls of blood vessels and producing unstable blood flow through the vessels.1 Genetic

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predisposition, poor diet, obesity, an inactive lifestyle, and cigarette smoking can lead to

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hyperlipidemia.2 Other causes include excessive alcohol consumption, use of medications such as

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hormones or steroids, diabetes, hypothyroidism and pregnancy.2-4 Hyperlipidemia can be

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ameliorated through the appropriate use of medications and maintenance of healthy lifestyle. The

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most commonly prescribed hypercholesterolemia medicines are statins such as simvastatin,

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lovastatin, atorvastatin and rosuvastatin.5 Use of statins is the first-line therapy and one of everal

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lipid-lowering approaches.6 Occasionally, statins are not tolerated, due to the side effects of muscle

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pain.7,8 Recently, proprotein convertase subtilisin-kexin type 9 inhibitors are shown to be effective

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in the treatment of hypercholesterolemia through regulating plasma LDL-C levels.9,10 However,

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further research is needed to rule out unwanted off-target effects of its inhibition.9,11

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A growing body of preclinical, epidemiological and clinical evidence has described the

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tolerability and safety profile of bioactive natural compounds with lipid-lowering effects.12 It has

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been demonstrated that the side effects of most commonly used natural compounds are mild and

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reversible. The pharmacological mechanisms of action of nutraceuticals include inhibition of

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cholesterol synthesis, inhibition of intestinal cholesterol absorption, and stimulation of LDL-C

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excretion.12 The dietary supplementation with plant sterols and esters inhibits cholesterol

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absorption in the small intestine, an attractive approach to lowering plasma cholesterol.13 In

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addition, curcumin can be used as a safe and well-tolerated adjunct to statins to effectively control

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hyperlipidemia.14 Virgin coconut oil (VCO) has been getting the spotlight as healthy oil due to its

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potential health benefits including cardiovascular health support, weight loss and immune

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improvement.15 The potential beneficial effect of VCO on lipid-lowering capability and in vitro LDL

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oxidation may be due to its bioactive polyphenol components.16 The VCO supplementation has

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beneficial effects on lipid parameters by reducing lipogenesis and enhancing the rate of fatty acid 3



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catabolism.17 Consumption of extra VCO reduces body mass index and increased high density

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lipoprotein-cholesterol (HDL-C) level in patients with coronary artery disease.18 These observations

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indicate that VCO can be used as a nutraceutical for drug therapy against dyslipidemia.

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Several studies consistently demonstrate that consumption of coconut oil (CO) elevates

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LDL-C and thus may cause adverse cardiovascular health despite healthy oil with relatively high

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concentration of medium-chain triglycerides (MCT).19,20 Accordingly, its consumption should be

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considered as a saturated fat not exceeding the USDA's daily recommendation. Evidence

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suggests that replacing CO with unsaturated plant oils may influence blood lipid profiles in a

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manner consistent with a reduction in risk factors for cardiovascular disease.20 The herb licorice

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(the roots and underground stems of Glycyrrhiza species) has been used in food and medicinal

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remedies for the treatment of various diseases of lung and liver.21 Major bioactive components

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present in licorice roots possess antioxidant, antitumor, anti-diabetic and anti-inflammatory

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properties.21-24 Our previous studies revealed that the licorice extracts and isoliquiritigenin inhibited

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glomerulosclerosis-mimetic mesangial matrix expansion triggered by chronic hyperglycemia due to

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their anti-proliferative and anti-fibrotic activities.25,26 Given the emerging health-promoting roles of

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CO and the diverse bioactivity of licorice, the present investigation was conducted to exploit

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therapeutic potential to improve blood lipid profiles and to impede hepatic steatosis in high-fat diet

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(HFD)-fed mice receiving MCT-CO (MCO) and MCO containing licorice extract (LE-MCO). This

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study examined body weight loss, blood lipid profiles, local fat pads and hepatosteatosis-

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associated gene proteins in diet-induced obese mice. Oral supplementation of LE-MCO may

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display lipid-lowering effects to a greater extent than MCO-alone of LE-alone, and could be used

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as a therapeutic approach treating dyslipidemia and hepatosteatosis.

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MATERIALS AND METHODS

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Materials Hematoxylin-eosin (H&E), carboxymethylcellulose, and oil red O were purchased form

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Sigma-Aldrich chemical (St. Louis, MO, USA) as well all reagents, unless specifically stated

85

elsewhere. Antibodies of CD36, sterol regulatory element-binding proteins 1c (SREBP1c),

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peroxisome proliferator-activated receptor α (PPARα) and uncoupling protein 1 (UCP1) were

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provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies of PPARγ, fatty acid

88

synthase (FAS), phospho-acetyl CoA carboxylase (ACC) and 5'-adenosine monophosphate-

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activated protein kinase α (AMPKα) were purchased from Cell Signaling Technology (Beverly, CA,

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USA). Rabbit antibodies of sterol regulatory element-binding protein 2 (SREBP2) and liver X

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receptor α (LXRα) were obtained from Abcam Biochemicals (Cambridge, UK). Rabbit antibody of

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fatty acid transport protein 1 (FATP1) was supplied by Biorbyt (Canmbridge, UK). Horseradish

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peroxidase (HRP)-conjugated goat anti-rabbit IgG was supplied from Jackson ImmunoResearch

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Laboratory (West Grove, PA, USA).

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Preparation of licorice extracts Two-year-old dried licorice (Glycyrrhiza uralensis) used in this study was purchased from

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Halim Farm (Young-Ju, Korea). The voucher specimen (RIC-HU1204) was deposited at the Center

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for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University. The

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specimens were authenticated by Emeritus Prof. H.J. Chi, Seoul National University, Korea. Dried

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powdered roots of Glycyrrhiza uralensis (40 kg) were extracted in a 10-fold volume of water by

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shaking at 121°C for 7 h. The total filtrate was concentrated at 50°C to dryness in vacuo, and

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lyophilized in a freeze-drier to render the licorice extract. Medium chain triglyceride (MCT)-coconut

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oil (Cerin® LCO-SL5 MILD COCO) was purchased from Chemrez Technologies, Inc. (Quezon,

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Philippines). MCT-coconut oil (9.84 μl) and licorice extract (1.125 mg) were mixed by adding 0.5%

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carboxymethylcellulose (6.3 ml/kg) as an excipient.

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Animal experiments C57BL/6J mice (5 weeks old) were obtained from DBL (Eumsung, Korea). Mice were kept

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on a 12-h light/12-h dark cycle at 23 ± 1°C with 50 ± 10% relative humidity under specific

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pathogen-free conditions, fed a standard pellet laboratory chow diet (Cargill Agri Purina, Biopia,

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Korea), and were provided with water ad libitum at the animal facility of Hallym University. Mice

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were acclimatized for 1 week before commencing the feeding experiments, at which period they

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were divided into five subgroups (n = 9-10 for each subgroup). Subsequently, their diets were

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changed to either low-fat diet (LFD; 10% dietary fat) for controls or HFD (45% dietary fat)

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purchased from Research Diets (New Brunswick, NJ, USA, Table 1). The HFD-fed mice were

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further divided into four subgroups. Each group of HFD mice were administrated for 12 weeks with

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vehicle (HFD-alone) and 37.5 mg/kg licorice extract (LE), 337.6 μl/kg MCO, or 337.6 μl/kg MCO

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containing 37.5 mg/kg LE (LE-MCO) daily via gavage. All experiments were approved by the

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Committee on Animal Experimentation of Hallym University and performed in compliance with the

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University’s Guidelines for the Care and Use of Laboratory Animals (Hallym2017-55). No mice died

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and no apparent signs of exhaustion were observed during the experimental period. Visceral fat,

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posterior abdominal fat, epididymal fat, subcutaneous fat and brown fat were weighed on sacrifice

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and frozen at -80°C.

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The animal body weight (BW) was measured at the beginning of the experiment and at 1-

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week intervals for 12 weeks. The BW of HFD group became greater from the 2nd week of the fat

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diet feeding, as compared with that of LFD-fed mice (Figure 1B). From the 7th week of the feeding

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the BWs of LE, MCO and LE-MCO groups were significantly less than that of HFD-alone-fed mice

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(Figure 1B). There was no significant difference among the BW gains of LE, MCO and LE-MCO

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groups after feeding for 12 weeks (Table 2). The food intake was measured every week for 12

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weeks. At the 12th week mice were sacrificed to collect blood samples, fats and tissue specimens.

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The food and energy intakes in all the LE, MCO and LE-MCO groups significantly increased, as

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compared with those of LFD control group (Table 3). In contrast, these intakes were significantly

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reduced, when compared with those of HFD-alone group. The organ weights of kidney, heart,

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pancreas, spleen and liver were measured on sacrifice. The organ weights of kidney, pancreas, 6


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spleen and liver highly increased in the HFD-alone group, while in LE, MCO and LE-MCO groups

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these organ weights were lower than those of HFD-alone group (Table 4). Especially, the liver

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weights in LE, MCO and LE-MCO groups were much lower than that of LFD group.

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Measurements of serum lipids and biochemical parameters

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The blood contents of total cholesterol (TC) and triglycerides (TG) were determined by

142

using commercial kits (Asan Pharmaceutical, Anseong, Korea). Optical density was measured

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using a Microplate reader at λ = 550 nm and λ = 500 nm (Bio-Rad Laboratories, Hercules, CA,

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USA). High density lipoprotein cholesterol (HDL-C) in serum was obtained from Wako Pure

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Chemical Industries, Ltd (Osaka, Japan) and measured using a Microplate reader at λ = 600 nm

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(Bio-Rad Laboratories). Low density lipoprotein cholesterol (LDL-C) concentration and very low-

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density lipoprotein cholesterol (VLDL-C) in serum were calculated using the following equation;

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LDL-C = TC-(HDL-C+VLDL-C) and VLDL-C = TG/5.

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Aspartate transaminase (AST) and alanine transaminase (ALT) in serum, and blood

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glucose levels after an overnight fast were determined using a Kornelab 20XT (Thermo Fisher

151

Scientific Inc., Waltham, MA, USA).

152 153

Sandwich enzyme linked immunosorbent assays (ELISA)

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Plasma insulin was determined by using an ELISA kit (FUJIFILM Wako Shibayagi Co.,

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Osaka, Japan), according to the manufacture's instruction. Plasma level of lecithin-cholesterol

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acyltransferase (LCAT) was measured using an ELISA kit (Cloud-Clone Co., TX, USA), and

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plasma level of phospholipid transfer protein (PLTP) was examined by using an ELISA kit

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(MyBioSource, CA, USA), according to the respective manufacturer’s instructions.

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H&E staining Mouse epididymal fat tissues were fixed in 4% paraformaldehyde for 24 h, and then

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gradually dehydrated in a graded series of ethanol solution for 18 h, followed by paraffin

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embedding. Paraffin-embedded tissues were horizontally cut into 5 μm in thickness with a razor 7



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blade. The tissue sections were deparaffinized and hydrated with xylene and graded ethanol. The

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H&E staining was applied, and the stained tissue sections were observed using an optical

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Axioimager microscope system (Zeiss, Oberkochen, Germany), and the relative adipocyte size

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was measured using by ImageJ program for each section.

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Oil red O staining Mouse liver specimens were fixed in 4% paraformaldehyde for 24 h, and then dehydrated

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in 30% sucrose/phosphate buffered saline for overnight, followed by embedding liver tissues by

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optimal cutting temperature (OCT). For histological analysis, OCT-embedded tissues were

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horizontally cut into 10 μm in thickness. The tissue sections were hydrated with distilled water,

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placed in absolute propylene glycol (PG) for 5 min and stained in oil red O/PG solution for 1 h.

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After washing with tap-water, the hepatocyte nuclei were counter-stained with hematoxylin. The

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stained tissue sections were observed using an optical Axioimager microscope system and images

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were obtained for each section. The staining intensity was obtained for each section using Auto-

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measure AxioVision program.

179 180 181

Western blot analysis Western blot analysis was carried out using macrophage lysates and tissue extracts

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prepared from mouse organ tissue extracts. Organ tissue extracts were prepared in 1 M Tris–HCl

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(pH 6.8) lysis buffer containing 1 M β-glycerophosphate, 1% β-mercaptoethanol, 0.5 M NaF, 0.1 M

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Na3VO4 and protease inhibitor cocktail. Cell lysates and tissue extracts containing equal amounts

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of total proteins were electrophoresed on 6-10% SDS-PAGE and transferred onto a nitrocellulose

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membrane. Non-specific binding was blocked by soaking the membrane in a TBS-T buffer [50 mM

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Tris–HCl (pH 7.5), 150 mM NaCl and 0.1% Tween 20] supplemented 3% bovine serum albumin

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BSA for 3 h. The membrane was incubated with a specific antibody of target proteins. The

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membrane was then incubated with a secondary antibody of goat anti-rabbit IgG, goat anti-mouse

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IgG or donkey anti-goat IgG conjugated to HRP. Each protein level was determined by using

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Supersignal West Pico Chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, 8


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USA) and Konica X-ray film (Konica Co., Tokyo, Japan). Incubation with β-actin antibody was

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conducted for the comparative control. For the measurements of the relative intensity of bands of

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interest, densitometric analyses of the blots were performed by Molecular Imaging Software

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Version 4.0.4 (Kodak, Connecticut, USA).

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RT-PCR analysis Total RNA was isolated from liver tissues using a commercially available Trizol reagent kit

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(invitrogen, Carlsbad, CA, USA). The RNA (5 μg) was reversibly transcribed with 200 units of

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reverse transcriptase (Promega Corporation, Madison, WI) and 0.5 mg/ml oligo-(dT)15 primer

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(Bioneer, Daejeon, Korea). RT-PCR analysis was also performed for semi-quantifying the levels of

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mRNA transcript of retinoid X receptor α (RXRα). The PCR condition for RXRα [5’-CAA TGG CGT

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CCT CAA GGT TC-3’ (forward), 5’-ACTCCACCTCGTTCTCATTC-3’ (reverse, 326 bp) was 94°C (3

204

min) and 30 cycles at 94°C (30 s), 55°C (45 s), and 72°C (45 s). The housekeeping gene GAPDH

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[5’-AAC TTT GGC ATT GTG GAA GGG-3’ (forward), 5’-GAC ACA TTG GGG GTA GGA ACA C-3’

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(reverse, 224 bp)] was used for an internal normalization for the co-amplification with respective

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gene. The PCR condition for CD36 [5’-GCT TGC AAC TGT CAG CAC AT-3’ (forward), 5’-GCC TTG

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CTG TAG CCA AGA AC-3’ (reverse, 134 bp) was 95°C (10 min) and 30 cycles at 95°C (15 s), 56°C

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(15 s), and 72°C (15 s).

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Data analysis The results are presented as mean ± SEM for each treatment group. Statistical analyses

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were performed using Statistical Analysis Systems statistical software package (SAS Institute Inc.,

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Cary, NC, USA). Significance was determined by one-way ANOVA, followed by Duncan range test

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for multiple comparisons. Differences were considered significant at P < 0.05.

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RESULTS

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Improvement of serum biomarkers by LE-MCO in HFD-fed mice

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After the 12 week-fat feeding, this study evaluated whether oral supplementation of LE,

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MCO or LE-MCO caused hepatotoxicity. The levels of serum ALT and AST are considered to be

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highly sensitive biomarkers of hepatotoxicity. The blood levels of ALT and AST were enhanced in

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HFD, where a greater increase in ALT than AST was observed (Figure 1C). When HFD-fed mice

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were treated with LE, MCO and LE-MCO, the levels of ALT and AST in serum were reduced

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(Figure 1C). Especially, the ALT level in LE-MCO group was incomparable to that of LFD-fed mice

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(Figure 1C). Serum levels of PLTP and LCAT, both involved in controlling plasma HDL levels, were

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measured in fat groups of LE, MCO and LE-MCO. In HFD-fed mice the levels of PLTP and LCAT

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markedly declined (Figure 1D). However, LE-MCO-treated mice highly enhanced the serum levels

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of both PLTP and LCAT. In addition, the LCAT level was boosted in MCO group (Figure 1D). On

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the other hand, the blood levels of glucose and insulin were elevated in HFD-alone group (Figure

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1E and 1F). In LE-, MCO- and LE-MCO-treated mice hypoglycemic effects were observed along

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with reduction of insulin levels. The serum level of insulin in LE-MCO animal group further

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decreased, as compared with that of MCO-treated animals (Figure 1F). Collectively, the treatment

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of LE-MCO reduced the homeostatic model assessment-insulin resistance (HOMA-IR) value

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increased by HFD, indicating that this dietary intervention improved insulin resistance (Figure 1G).

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Effect of LE-MCO on blood lipid contents of HFD-fed mice Table 5 shows the changes in the blood levels of TG, TC, VLDL-C, LDL-C and HDL-C of

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HFD-fed mice treated with LE, MCO, or LE-MCO for 12 weeks. The blood TG levels highly

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elevated by HFD were significantly reduced in mice receiving LE, MCO and LE-MCO (Table 5).

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The TG content of LE-MCO-treated mice was restored to that of LFD control group. The levels of

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blood TC and LDL-C were significantly enhanced in HFD-fed mice (Table 5). The levels of TC and

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LDL-C were not influenced by MCO, whereas the presence of LE-MCO curtailed these levels.

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Interestingly, the LDL-C level in LE-MCO-treated mice was even less than that of LFD control 10


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(Table 5). In addition, treating LE, MCO, and LE-MCO to animals diminished the blood VLDL-C

246

levels of HFD-fed mice. When LE-MCO was treated to mice, the VLDL-C level was restored to that

247

of LFD-fed mice. Thus, LE-MCO may be a potent lipid-lowering agent under a condition of

248

hypertriglyceridemia or hypercholesterolemia. On the other hand, in mice fed HFD for 12 weeks

249

blood HDL-C level was significantly elevated, as compared with that of LFD-fed mice (Table 5).

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The HDL-C level was not influenced by treating LE or MCO, but LE-MCO tended to further elevate

251

blood HDL-C level (Table 5).

252 253 254

Inhibitory effects of LE-MCO on white fat weights and hepatic steatosis The weights of visceral, posterior abdominal, subcutaneous and epididymal white fat pads

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were highly elevated in HFD groups (Table 6). When the obese animals were treated with LE,

256

MCO or LE-MCO, all the white fat weights declined. Especially, these fat weights in LE-MCO

257

groups plummeted to those of LFD-fed animals (Table 6). The overall weight of white fat pads in

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the LE-MCO group plunged by ≈50%, as compared with that of HFD-fed mice. Histological

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observation from epididymal fat pads revealed that HFD induced fat hypertrophy, evidenced by

260

H&E staining (Figure 2A). However, the epididymal adipocyte sizes of LE, MCO and LE-MCO

261

groups were significantly reduced, as compared with that of the HFD-alone group.

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To investigate hepatic fat accumulation, the liver tissue specimens were stained with oil

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red O. Lipid droplets were markedly observed in HFD-fed mice (Figure 2B). In contrast, the fat

264

accumulation was dampened in all the LE, MCO and LE-MCO groups. This was consistent with

265

reduction of the liver weights in LE, MCO and LE-MCO groups (Table 4).

266 267 268

Inhibition of hepatic lipid synthesis by LE-MCO SREBP2 is required in the regulation of genes required for cholesterol synthesis, while

269

SREBP1c is a key regulator for hepatic lipid accumulation through activation of enzymes involved

270

in the fatty acid biosynthesis.27,28 LE-MCO reduced blood levels of TG and TC in diet-induced

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hypertriglyceridemic and hypercholesterolemic obese animals (Table 5). This study revealed that

272

MCO and LE-MCO attenuated the hepatic induction of SREBP2 and SREBP1c in HFD-fed mice 11



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(Figure 3A). Especially, their hepatic expression levels of LE-MCO-treated mice were similar to

274

those of LFD-fed mice (Figure 3A). This was consistent with the substantial reduction of cholesterol

275

synthesis and hepatic lipid accumulation (Figure 2B).

276

SREBP1c are crucial in the regulation of FAS and ACC required for hepatic TG

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synthesis.28 HFD augmented hepatic induction of FAS and phospho-ACC in mice by ≈2-fold, which

278

was inhibited by treating LE, MCO, or LE-MCO (Figure 3B). Additionally, this study found that HFD

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markedly induced hepatic expression of the fatty acid translocase CD36 in mice (Figure 3B).

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However, the expression of hepatic CD36 was significantly attenuated by supplementing LE, MCO

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or LE-MCO to HFD-fed mice at the transcriptional level (Figure 3C). When HFD-fed mice received

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LE-MCO via gavage, the induction FAS and CD36, and ACC activation were restored to that of

283

LFD group. Accordingly, the marked blockade of hepatic FAS, ACC and CD36 by LE-MCO may

284

contribute to the inhibition of the development of fatty liver (Figure 2B).

285 286 287

Effects of LE-MCO on AMPK and PPAR signaling in HFD-fed mice AMPK plays a key role in controlling metabolic pathways in response to energy demand.29

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HFD reduced hepatic activation of AMPK in mice, while LE-, MCO or LE-MCO-treated mice

289

promoted the AMPK activation (Figure 4A). The hepatic activation of AMPK in LE-MCO-treated

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animals was much greater than that of LE- or MCO-treated group. Accordingly, LE-MCO may block

291

steatosis through liver-specific activation of AMPK in HFD-fed mice.

292

PPARα highly expressed in the liver and skeletal muscle regulates pathways germane to

293

fatty acid oxidation.30 The current study examined whether the oral administration of LE-MCO

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blocked PPARα-LXRα signaling in HFD-fed animals. The hepatic expression of PPARα and PPARγ

295

was minimal in LFD-fed mice (Figure 4B). The HFD-fed mice induced hepatic PPARα and PPARγ,

296

which was reversed by treating MCO and LE-MCO. The reduction of hepatic PPARα and PPARγ

297

was much greater in LE-MCO-treated animals, as compared with that of LE or MCO group.

298

Hepatic sterol biosynthesis regulated by SREBP2 is coupled to LXR activation, which promotes

299

fatty acid synthesis by SREBP-1c.27 Hepatic LXRα was highly induced in HFD-fed mice, but such

300

induction was attenuated in obese mice exposed to LE, MCO and LE-MCO (Figure 4B). 12


Page 13 of 42


301

Additionally, the enhanced transcription of hepatic RXRα, the heterodimer partner of LXRα, was

302

attenuated in LE-MCO-treated mice (Figure 4C). Thus, the diminution of hepatic SREBP2 and

303

LXRα-RXRα may entail the inhibition of sterol synthesis in the liver by LE-MCO.

304 305

Modulation of white fat-PPARγ and brown fat-UCP1 and FATP1 by LE-MCO in HFD-fed mice

306

PPARγ is preferentially expressed in adipose tissues and influences fatty acid synthesis in

307

adipose tissues.30 This study examined whether the oral administration of LE-MCO deterred the

308

PPARγ induction in the epididymal adipose tissues of HFD-fed animals. The HFD-fed mice

309

elevated the epididymal induction of PPARγ (Figure 5A). In contrast, treating LE-MCO to HFD

310

group encumbered such elevation (Figure 4B).

311

The weight of brown fat pads was enhanced in HFD-fed mice, which was reduced by

312

treating LE-MCO but not by LE (Table 6). UCP1 present in the mitochondria of brown adipocytes is

313

capable of altering energy expenditure and fuel metabolism,31 and FATP1 is an insulin-sensitive

314

fatty acid transporter involved in diet-induced obesity.32 This study found that HFD induced UCP1

315

and FATP1 in brown fat tissues of mice (Figure 5B). Oral administration of LE, MCO or LE-MCO to

316

HFD-fed mice further enhanced the induction of UCP1 and FATP1 in brown fat. The induction of

317

UCP1 and FATP1 was much further elevated in mice receiving LE-MCO, as compared with that of

318

MCO group (Figure 5B).

319

13



320

Page 14 of 42

DISCUSSION

321 322

Nine major findings were observed from this study. 1) Plasma levels of ALT and AST were

323

reduced in HFD-fed mice treated with LE, MCO and LE-MCO for 12 weeks. 2) Glucose tolerance

324

was reduced in HFD-fed mice supplementing MCO or LE-MCO. 3) LE-MCO deterred the plasma

325

insulin level in HFD-fed mice and boosted plasma levels of PLTP and LCAT. 4) Plasma levels of

326

both TG and TC declined in LE- or LE-MCO-administrated mice with concomitant reduction of LDL-

327

C and VLDL-C, as compared with those of HFD alone-mice. 5) Supplementation of LE-MCO

328

tended to further raise blood HDL-C level in HFD-fed mice. 6) The hepatic induction of SREBP2

329

and SREBP1c was encumbered in fatty liver-bearing mice receiving MCO or LE-MCO. 7) The FAS

330

and CD36 induction and ACC activation were suppressed in the liver of HFD-fed mice treated with

331

LE, MCO, or LE-MCO. 8) Oral administration of LE, MCO or LE-MCO disturbed hepatic AMPKα

332

activation and PPAR-LXR/RXR signaling in animals with fatty liver, and LE-MCO inhibited HFD-

333

induced epididymal hypertropy and PPARγ induction. 9) The expression of UCP-1 and FATP1 was

334

enhanced in brown adipose tissues exposed to HFD, which was further enhanced by the

335

administration of LE-MCO to HFD-fed mice. Therefore, the interventions with LE, MCO and LE-

336

MCO may dampen diet-induce hyperlipidemia and hepatosteatosis, possibly through improving

337

insulin resistance and diminishing hepatic lipid synthesis. Among these interventions, the LE-MCO

338

supplementation had greatest lipid-lowering effects. However, it cannot be ruled out the possibility

339

that the reduced food intakes in LE-MCO-fed mice may be at least partly attributed to the

340

improvement of hyperlipidemia and hepatosteatosis.

341

Various kinds of statins have been commonly prescribed as lipid-lowering drugs for

342

dyslipidemia and hypercholesterolemia as being the first-line therapy.5,6 However, occasionally

343

statins are not tolerated due to detrimental side effects such as muscle pain due to its

344

discontinuation.7,8 Recently, it has been clinically accepted that proprotein convertase subtilisin-

345

kexin type 9 inhibitors exhibit cholesterol-lowering efficacies through regulating plasma LDL-C

346

levels as a potential pharmacologic target.9-11 Although unwanted off-target effects of PCSK9

347

inhibition are mild and reversible in the most part of cases, unfavorable side effects need to be 14


Page 15 of 42


348

clearly defined for pharmacological managements. A growing body of preclinical, epidemiological

349

and clinical evidence has described that several bioactive natural compounds display lipid-lowering

350

effects with the tolerability and safety profile.12 Recent literature supports the role of nutraceuticals

351

as LDL-C-lowering therapy in statin-intolerant patients, with evidence from clinical practices.33

352

Such nutraceuticals are phytosterols, polyphenols, plant fibers and policosanol. Curcuminoids and

353

berberine may be effective in lowering LDL‐C by increasing the expression of LDL receptor.34-36

354

The dietary supplementation of bioactive natural compounds with lipid-lowering effects is currently

355

encouraged for cardiovascular disease prevention. Pharmacological action mechanisms of

356

nutraceuticals include inhibition of cholesterol synthesis and intestinal cholesterol absorption, and

357

stimulation of LDL-C excretion.12

358

VCO is a food supplement enriched with MCT and polyphenolic antioxidants.37 VCO

359

polyphenols altering cellular antioxidant status inhibit LDL oxidation and exhibit lipid-lowering

360

capability.16,37 Virgin CO polyphenols attenuate cadmium-induced dyslipidemia and cardiovascular

361

risk ratios in rats by improving antioxidant defense systems.38 In addition, VCO polyphenols

362

attenuate the inflammatory response in oxidized LDL-activated peripheral monocytes.39 The

363

current study investigated the inhibition of lipidemia and hepatic lipid accumulation by CO enriched

364

with MCT. Unfortunately, this study did not examine the contents of polyphenols present in MCO

365

employed in this study. Polyphenols in MCO could exert inhibitory effects on TG accumulation in

366

the liver. However, MCT in CO may result in weight loss through increased energy expenditure and

367

lipid oxidation in HFD animals. It has been reported that replacement of long-chain triglycerides

368

with MCT in the diet could potentially induce modest reductions in body weight and composition

369

without adversely affecting lipid profiles.40,41 Consistently, oral treatment of MCO evoked weight

370

loss in HFD-induced obese mice and reduced plasma and hepatic TG contents without influence

371

on plasma total cholesterol level. The reduction of hepatic TG accumulation by MCO appeared to

372

entail the inhibition of SREBP1c-mediated lipogenesis involving FAS induction and ACC activation

373

in the liver.

374

Major bioactive components present in licorice roots are triterpene glycosides of

375

glycyrrhizin, flavanones, chalcones, saponins, and isoflavonoids with antioxidant, antitumor, anti15



Page 16 of 42

376

diabetic and anti-inflammatory properties.21-24 Glycyrrhiza chalcones show antiobesity and lipid

377

lowering effects as a source of pancreatic lipase inhibitors.42 Additionally, glycyrrhizic acid coupled

378

with glycyrrhetinic acid and 18-β-glycyrrhetic acid is a therapeutic drug for liver diseases with

379

diverse actions including inhibition of hepatic apoptosis and necrosis, anti-inflammatory and

380

immune regulation, antiviral effects, and antitumor effects.43 This study found that LE-fortified MCO

381

can be used as a lipid-lowering nutraceutical against dyslipidemia and hepatosteatosis. The

382

present study suggested the AMPKα- or PPARα-regulation of hepatic FA and plasma lipoprotein

383

metabolism during fat-diet transition as underlying mechanistic actions of LE-MCO. The activation

384

of AMPKα and inhibition PPARα in hepatocytes leading to a reduction of lipid formation contributes

385

to the beneficial action of LE-MCO in atherosclerotic lipid profiles. Hepatic sterol biosynthesis

386

regulated by SREBP2 is coupled to LXR/RXR activation, which promotes SREBP1c expression

387

and FA synthesis.27 The treatment of LE-MCO reduced hepatic fat, and lowered plasma levels of

388

TG and TC along with VLDL-C/LDL-C levels, which may be associated with its inhibition of

389

SREBP2 expression, activation of LXR-RXR and SREBP1c expression. In addition, inhibition of

390

PPARγ in epididymal adipose tissues could be responsible for TG formation.

391

The role of SREBP pathway is expanded to induction of inflammation,44 which suggests a

392

novel metabolic strategy to control hepatic steatosis of LE-MCO. Inhibition of hepatocyte-specific

393

CD36 directly contributes to reduction in liver lipids that correlates with an improvement in the

394

inflammatory markers of ALT and AST.45 Therefore, one can assume that the anti-inflammatory

395

activity of LE-MCO may disturb hepatic SREBP and CD36, leading to inhibition of the rate of FA

396

uptake of hepatocytes under elevated free FA from HFD. On the other hand, this study revealed

397

that LE-MCO increased HDL-C in the hyperlipidemic blood with enhanced levels of PLTP and

398

LCAT that play important roles in controlling plasma HDL levels. A clinical study shows that

399

nutritional consumption of extra VCO reduces body mass index and increased HDL-C level in

400

patients with coronary artery disease.18 Also, daily consumption of VCO in young healthy adults

401

significantly increased HDL-C without major safety issues of taking VCO daily for 8 weeks.46

402

Collectively, these observations indicate that virgin coconut oil can be used as a nutraceutical for

403

drug therapy against dyslipidemia. Furthermore, this study found that that the induction of heat16


Page 17 of 42


404

producing UCP1 and insulin-sensitive FATP1 was positively stimulated in brown fat pads exposed

405

to LE-MCO. In this study a strong protection against diet-induced obesity and insulin resistance

406

was observed in LE-MCO-treated animals, suggesting UCP1 and FATP1 as novel antiobesity and

407

antidibetic targets.47

408

In summary, this study investigated the comparative competence of LE-MCO in diet-

409

induced obesity, hyperlipidemia and hepatosteatosis in mice. The treatment of MCO inhibited HFD-

410

induced obesity and hypertriglyceridemia, and disturbed lipid formation in the liver. Furthermore,

411

LE-MCO inhibited hypercholesterolemia with the concomitant reduction of LDL-C and elevation of

412

HDL-C. Collectively, the capability of LE-MCO to improve dyslipidemia and hepatosteatosis may be

413

promising in hampering liver and cardiovascular diseases via direct and indirect mechanisms. The

414

mechanistic actions of LE-MCO for dyslipidemia and hepatosteatosis included the AMPKα- or

415

PPARα-regulation of SREBP-mediated hepatic FA formation and plasma lipoprotein metabolism.

416

Despite statistically significant results, the detailed knowledge of and strict caution to

417

pharmacological applications for LE-MCT are needed for the managements of a healthy body

418

weight and composition and for specific health risks of patients, because the available evidence is

419

not of the highest quality. A pair feeding study should be performed to eliminate the possibility that

420

the reduction in food and energy intakes may influence the incidence of hyperlipidemia and

421

hepatosteatosis.

422

17



423

Page 18 of 42

REFERENCES

424 425

1

van Hinsbergh, V. W.; Eringa, E. C.; Daemen, M. J. Neovascularization of the atherosclerotic

426

plaque: interplay between atherosclerotic lesion, adventitia-derived microvessels and

427

perivascular fat. Curr. Opin. Lipidol. 2015, 26, 405-411.

428

2

Manfredini, F.; D'Addato, S.; Laghi, L.; Malagoni, A.M.; Mandini, S.; Boari, B.; Borghi, C.;

429

Manfredini, R. Influence of lifestyle measures on hypertriglyceridaemia. Curr. Drug Targets.

430

2009, 10, 344-355.

431

3

432 433

metabolic biomarkers of cardiovascular disease. Nat. Rev. Endocrinol. 2014, 10, 659-672. 4

434 435

Jensen, M. K.; Bertoia, M. L.; Cahill, L. E.; Agarwal, I.; Rimm, E. B.; Mukamal, K. J. Novel

Katsiki, N.; Mantzoros, C.; Mikhailidis, D. P. Adiponectin, lipids and atherosclerosis. Curr. Opin. Lipidol. 2017, 28, 347-354.

5

Chauvin, B.; Drouot, S.; Barrail-Tran, A.; Taburet, A. M. Drug-drug interactions between HMG-

436

CoA reductase inhibitors (statins) and antiviral protease inhibitors. Clin. Pharmacokinet. 2013,

437

52, 815-831.

438

6

Cicero, A. F. G.; Bove, M.; Borghi, C. Pharmacokinetics, pharmacodynamics and clinical

439

efficacy of non-statin treatments for hypercholesterolemia. Expert. Opin. Drug Metab. Toxicol.

440

2018, 14, 9-15.

441

7

Rosenson, R. S.; Baker, S.; Banach, M.; Borow, K. M.; Braun, L. T.; Bruckert, E.; Brunham, L.

442

R.; Catapano, A. L.; Elam, M. B.; Mancini, G. B. J.; Moriarty, P. M.; Morris, P. B.; Muntner, P.;

443

Ray, K. K.; Stroes, E. S.; Taylor, B. A.; Taylor, V. H.; Watts, G. F.; Thompson, P. D. Optimizing

444

cholesterol treatment in patients with muscle complaints. J. Am. Coll. Cardiol. 2017, 70, 1290-

445

1301.

446

8

447 448

Apostolopoulou, M.; Corsini, A.; Roden, M. The role of mitochondria in statin-induced myopathy. Eur. J. Clin. Invest. 2015, 45, 745-754.

9

Okere, A. N.; Serra, C. Evaluation of the potential role of alirocumab in the management of

449

hypercholesterolemia in patients with high-risk cardiovascular disease. Pharmacotherapy 2015,

450

35, 771-779. 18


Page 19 of 42

451 452 453 454 455 456 457


10 Pokrywka, G. S. PCSK9 inhibitors: a non-statin cholesterol-lowering treatment option. Postgrad. Med. 2018, 130, 287-298. 11 Pirillo, A.; Catapano, A.L. Proprotein Convertase Subtilisin Kexin 9 Inhibitors. Cardiol. Clin. 2018, 36, 241-256. 12 Cicero, A. F. G.; Colletti, A. An update on the safety of nutraceuticals and effects on lipid parameters. Expert. Opin. Drug Saf. 2018, 17,303-313. 13 Carden, T. J.; Hang, J.; Dussault, P. H.; Carr, T. P. Dietary Plant sterol esters must be

458

hydrolyzed to reduce intestinal cholesterol absorption in hamsters. J. Nutr. 2015, 145, 1402-

459

1407.

460

14 Panahi, Y.; Ahmadi, Y.; Teymouri, M.; Johnston, T. P.; Sahebkar, A. Curcumin as a potential

461

candidate for treating hyperlipidemia: A review of cellular and metabolic mechanisms. J. Cell.

462

Physiol. 2018, 233, 141-152.

463 464 465 466 467

15 Babu, A. S.; Veluswamy, S. K.; Arena, R.; Guazzi, M.; Lavie, C. J. Virgin coconut oil and its potential cardioprotective effects. Postgrad. Med. 2014, 126, 76-83. 16 Nevin, K. G.; Rajamohan, T. Beneficial effects of virgin coconut oil on lipid parameters and in vitro LDL oxidation. Clin. Biochem. 2004, 37, 830-835. 17 Arunima, S.; Rajamohan, T. Influence of virgin coconut oil-enriched diet on the transcriptional

468

regulation of fatty acid synthesis and oxidation in rats - a comparative study. Br. J. Nutr. 2014,

469

111, 1782-1790.

470

18 Cardoso, D. A.; Moreira, A. S.; de Oliveira, G. M.; Raggio Luiz, R.; Rosa, G. A coconut extra

471

virgin oil-rich diet increases HDL cholesterol and decreases waist circumference and body

472

mass in coronary artery disease patients. Nutr. Hosp. 2015, 32, 2144-2152.

473 474 475 476 477 478

19 Sankararaman, S.; Sferra, T. J. Are We Going Nuts on Coconut Oil? Curr. Nutr. Rep. 2018, doi: 10.1007/s13668-018-0230-5. 20 Eyres, L.; Eyres, M. F.; Chisholm, A.; Brown, R. C. Coconut oil consumption and cardiovascular risk factors in humans. Nutr. Rev. 2016, 74, 267-280. 21 Yang, R.; Yuan, B. C.; Ma, Y. S.; Zhou, S.; Liu, Y. The anti-inflammatory activity of licorice, a widely used Chinese herb. Pharm. Biol. 2017, 55, 5-18. 19



479

Page 20 of 42

22 Li, G.; Nikolic, D.; Van Breemen, R. B. Identification and chemical standardization of licorice

480

raw materials and dietary supplements using UHPLC-MS/MS. J. Agric. Food Chem. 2016, 64,

481

8062-8070.

482

23 Chin, Y. W.; Jung, H. A.; Liu, Y.; Su, B. N.; Castoro, J. A.; Keller, W. J.; Pereira, M. A.; Kinghorn,

483

A. D. Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra). J. Agric.

484

Food Chem. 2007, 55, 4691-4697.

485 486 487

24 Chen, X.; Liu, Z.; Meng, R.; Shi, C.; Guo, N. Antioxidative and anticancer properties of

licochalcone A from licorice. J. Ethnopharmacol. 2017, 198, 331-337. 25 Li, J.; Lee, Y. S.; Choi, J. S.; Sung, H. Y.; Kim, J. K.; Lim, S. S.; Kang, Y. H. Roasted licorice

488

extracts dampen high glucose-induced mesangial hyperplasia and matrix deposition through

489

blocking Akt activation and TGF-beta signaling. Phytomedicine 2010, 17, 800-810.

490

26 Li, J.; Kang, S. W.; Kim, J. L.; Sung, H. Y.; Kwun, I. S.; Kang, Y. H. Isoliquiritigenin entails

491

blockade of TGF-beta1-SMAD signaling for retarding high glucose-induced mesangial matrix

492

accumulation. J. Agric. Food Chem. 2010, 58, 3205-3212.

493

27 Rong, S.; Cortés, V. A.; Rashid, S.; Anderson, N. N.; McDonald, J. G.; Liang, G.; Moon, Y. A.;

494

Hammer, R. E.; Horton, J. D. Expression of SREBP-1c requires SREBP-2-mediated

495

generation of a sterol ligand for LXR in livers of mice. Elife 2017, 6, e25015.

496 497 498 499 500 501 502 503 504

28 Horton, J. D.; Goldstein, J. L.; Brown, M. S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest. 2002, 109, 1125-1131. 29 Carling, D.; Thornton, C.; Woods, A.; Sanders, M. J. AMP-activated protein kinase: new regulation, new roles? Biochem. J. 2012, 445,11-27 30 Smith, S. A. Peroxisome proliferator-activated receptors and the regulation of mammalian lipid metabolism. Biochem. Soc. Trans. 2002, 30, 1086-1090. 31 Porter, C. Quantification of UCP1 function in human brown adipose tissue. Adipocyte 2017, 6, 167-174. 32 Wu, Q.; Ortegon, A. M.; Tsang, B.; Doege, H.; Feingold, K. R.; Stahl, A. FATP1 is an insulin-

505

sensitive fatty acid transporter involved in diet-induced obesity. Mol. Cell. Biol. 2006, 26, 3455-

506

3467. 20


Page 21 of 42

507


33 Ward, N. C.; Pang, J.; Ryan, J. D. M.; Watts, G. F. Nutraceuticals in the management of

508

patients with statin-associated muscle symptoms, with a note on real-world experience. Clin.

509

Cardiol. 2018, 41, 159-165.

510 511 512

34 Barbagallo, C. M.; Cefalù, A. B.; Noto, D.; Averna, M. R. Role of nutraceuticals in hypolipidemic therapy. Front. Cardiovasc. Med. 2015, 2, 1-22. 35 Sahebkar, A.; Serban, M. C.; Gluba‐Brzózka, A.; Mikhailidis, D. P.; Cicero, A. F.; Rysz, J.;

513

Banach, M. Lipid-modifying effects of nutraceuticals: An evidence-based approach. Nutrition

514

2016, 32, 1179-1192.

515

36 Tai, M. H.; Chen, P. K.; Chen, P. Y.; Wu, M. J.; Ho, C. T.; Yen, J. H. Curcumin enhances cell‐

516

surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene

517

expression in HepG2 cells. Mol. Nutr. Food Res. 2014, 58, 2133-2145.

518 519

37 Illam, S. P.; Narayanankutty, A.; Raghavamenon, A. C. Polyphenols of virgin coconut oil prevent pro-oxidant mediated cell death. Toxicol. Mech. Methods 2017, 27, 442-450.

520

38 Famurewa, A. C.; Ejezie, F. E. Polyphenols isolated from virgin coconut oil attenuate cadmium-

521

induced dyslipidemia and oxidative stress due to their antioxidant properties and potential

522

benefits on cardiovascular risk ratios in rats. Avicenna J. Phytomed. 2018, 8, 73-84.

523

39 Jose, S. P.; Krishnakumar, I. M.; Ratheesh, M.; Asha, S.; Sandya, S.; Rajmohan, V.

524

Polyphenolic fraction of virgin coconut oil inhibits the inflammatory response in oxidized LDL

525

activated human peripheral blood mononuclear cells by modulating TLR/NF-kB signaling

526

pathways. Eur. J. Integrative Med. 2017, 10, 59-65.

527

40 Mumme, K.; Stonehouse, W. Effects of medium-chain triglycerides on weight loss and body

528

composition: a meta-analysis of randomized controlled trials. J. Acad. Nutr. Diet. 2015, 115,

529

249-263.

530

41 Bueno, N. B.; de Melo, I. V.; Florêncio, T. T.; Sawaya, A. L. Dietary medium-chain

531

triacylglycerols versus long-chain triacylglycerols for body composition in adults: systematic

532

review and meta-analysis of randomized controlled trials. J. Am. Coll. Nutr. 2015, 34, 175-183.

533 534

42 Birari, R. B.; Gupta, S.; Mohan, C. G.; Bhutani, K. K. Antiobesity and lipid lowering effects of Glycyrrhiza chalcones: experimental and computational studies. Phytomedicine 2011, 18, 79521



535 536 537

Page 22 of 42

801. 43 Li, J. Y.; Cao, H. Y.; Liu, P.; Cheng, G. H.; Sun, M. Y. Glycyrrhizic acid in the treatment of liver diseases: literature review. Biomed Res. Int. 2014, 2014, 872139.

538

44 Li, N.; Zhou, Z. S.; Shen, Y.; Xu, J.; Miao, H. H.; Xiong, Y.; Xu, F.; Li, B. L.; Luo, J.; Song, B. L.

539

Inhibition of the sterol regulatory element-binding protein pathway suppresses hepatocellular

540

carcinoma by repressing inflammation in mice. Hepatology 2017, 65, 1936-1947.

541

45 Wilson, C. G.; Tran, J. L.; Erion, D. M.; Vera, N. B.; Febbraio, M.; Weiss, E. J. Hepatocyte-

542

specific disruption of CD36 attenuates fatty liver and improves insulin sensitivity in HFD-fed

543

mice. Endocrinology 2016, 157, 570-585.

544

46 Chinwong, S.; Chinwong, D.; Mangklabruks, A. Daily consumption of virgin coconut oil

545

increases high-density lipoprotein cholesterol levels in healthy volunteers: A randomized

546

crossover trial. Evid. Based Complement. Alternat. Med. 2017, 2017, 7251562.

547 548

47 Townsend, K. L.; Tseng, Y. H. Brown fat fuel utilization and thermogenesis. Trends Endocrinol. Metab. 2014, 25, 168-177.

22


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549


FIGURE LEGENDS

550 551

Figure 1: Animal experimental design (A), effects of LE-MCO on body weight (B), liver toxicity (C),

552

plasma levels of PLTP and LCAT (D), blood glucose (E) and insulin (F), and HOMA-IR values (G).

553

C57BL/6J mice were fed either low fat diet (LFD, 10% kcal fat) or high fat diet (HFD, 45% kcal fat).

554

Mice were divided into 5 subgroups (n=8-10 for each subgroup). The first group of mice was LFD

555

control mice, and HFD mice were divided into 4 groups. Mice were fed LFD or HFD for 12 weeks.

556

Each group of HFD mice daily was administrated licorice extract (LE), medium chain triglycerides

557

(MCT)-coconut oil (MCO) or combination of LE and MCT-coconut oil (LE-MCO) via gavage for 12

558

weeks. The animal body weight (BW) was measured at the beginning of the experiment and at 1-

559

week intervals for 12 weeks. Values were expressed as mean ± SEM (n=8-10). †, compared to

560

LFD-fed mice; , compared to HFD-fed mice. Aspartate transaminase (AST) and alanine

561

transaminase (ALT) and blood glucose after an overnight fast were determined. Plasma levels of

562

insulin, phospholipid transfer protein (PLTP) and lecithin-cholesterol acyltransferase (LCAT) were

563

measured using ELISA kits. Calculation of insulin resistance (HOMA-IR), fasting plasma glucose

564

(mg/dl) x insulin (μU/ml) / 405. Bar graphs (mean ± SEM, n = 3-4 separate experiments), and

565

values in bar graphs (same achromatic colored) not sharing a same lower case alphabet letter

566

indicate significant different at P

Lipid-Lowering Effects of Medium-Chain Triglyceride-Enriched

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