Anti-obesity and anti-hepatosteatosis effects of dietary scopoletin in high-fat diet fed mice

Journal of Functional Foods 25 (2016) 433–446

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Anti-obesity and anti-hepatosteatosis effects of dietary scopoletin in high-fat diet fed mice Ju Ri Ham a, Hae-In Lee b, Ra-Yeong Choi a, Mi-Ok Sim c, Myung-Sook Choi d, Eun-Young Kwon d, Kyeong Won Yun e, Myung-Joo Kim f, Mi-Kyung Lee a,* a

Department of Food and Nutrition, Sunchon National University, Suncheon 57922, Republic of Korea Mokpo Marin Food-Industry Research Center, Mokpo 58621, Republic of Korea c National Development Institute of Korean Medicine, Jangheung 59338, Republic of Korea d Center for Food and Nutritional Genomics Research, Kyungpook National University, Daegu 41566, Republic of Korea e Department of Oriental Medicine Resource, Sunchon National University, Suncheon 57922, Republic of Korea f Department of Bakery & Barista, Suseong College, Daegu 42078, Republic of Korea b

A R T I C L E

I N F O

A B S T R A C T

Article history:

The effects of scopoletin on non-alcoholic fatty liver in obese mice were investigated. Mice

Received 1 April 2016

were fed high-fat diet (HF) with or without two doses of scopoletin (0.01 and 0.05%, w/w)

Received in revised form 21 June

for 16 weeks. Both doses of scopoletin led to similar reductions in body weight, visceral fat,

2016

serum levels of leptin, lipid, TNFα, IL-6, IFNγ and MCP-1, insulin resistance and hepatic lipid

Accepted 24 June 2016

accumulation, whereas they increased serum adiponectin and faecal lipid levels. Ingenu-

Available online

ity pathway analysis revealed that hepatic gene networks related to lipid concentrations, inflammation of organs, quantity of adipose tissue, proliferation of cell and necrosis were

Keywords:

down-regulated in the scopoletin group. The top up- or down-regulated genes were Cidea,

High-fat

Apoa4, Cyp7a1, Errfi1, Col1a1, Mmp13, Cdkn1a, Gdf15 and Saa1, which emerged as associated

Non-alcoholic liver disease

genes related to hepatic steatosis and inflammation. These results indicate that scopoletin

Obesity

may ameliorate HF-induced hepatic dysfunction via regulation of lipid metabolic and in-

Scopoletin

flammatory genes.

Transcriptome

© 2016 Published by Elsevier Ltd.

* Corresponding author. Department of Food and Nutrition, Sunchon National University, Suncheon 57922, Republic of Korea. Tel.: +82 61 750 3656, fax.: +82 61 752 3657. E-mail address: [email protected] (M.-K. Lee). Abbreviations: ALT, alanine aminotransferase; Apoa4, apolipoprotein A-IV; AST, aspartate aminotransferase; β-oxidation, fatty acid β-oxidation; Cdkn1a, cyclin-dependent kinase inhibitor 1A; Cidea, cell death-inducing DFFA-like effector A; Col1a1, collagen, type 1, alpha 1; CPT, carnitine palmitoyltransferase; Cyp7a1, cholesterol 7 alpha-hydroxylase; Errfi1, ERBB receptor feedback inhibitor 1; FAS, fatty acid synthase; FFA, free fatty acid; G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Gdf15, growth differentiation factor 15; GK, glucokinase; HDL-C, HDL-cholesterol; H&E, haematoxylin and eosin; HF, high-fat diet; HF-LS, highfat diet with 0.01% scopoletin; HF-HS, high-fat diet with 0.05% scopoletin; HOMA-IR, homeostatic index of insulin resistance; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IFNγ, interferon gamma; IPA, ingenuity pathway analysis; IPGTT, intraperitoneal glucose tolerance test; IR, insulin resistance; MCP-1, monocyte chemoattractant protein-1; Mmp13, matrix metallopeptidase 13; NAD+, nicotinamide adenine dinucleotide+; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NC, normal diet; NAFLD, nonalcoholic fatty liver disease; PAP, phosphatidate phosphohydrolase; PEPCK, phosphoenolpyruvate carboxykinase; Saa1, serum amyloid A1; TC, total cholesterol; TG, triacylglycerol; Trichrome, Masson’s trichrome; TNFα, tumour necrosis factor alpha http://dx.doi.org/10.1016/j.jff.2016.06.026 1756-4646/© 2016 Published by Elsevier Ltd.

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1.

Journal of Functional Foods 25 (2016) 433–446

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease that increases in prevalence with increasing incidence of obesity and type 2 diabetes (Vernon, Baranova, & Younossi, 2011). NAFLD is generally characterized by fat accumulation exceeding 5% of hepatic tissue, which encloses a spectrum ranging from steatosis to steatohepatitis, fibrosis and cirrhosis (Ganji, Kashyap, & Kamanna, 2015; Machado & Cortez-Pinto, 2005). Obese patients with hepatosteatosis and steatohepatitis have higher levels of inflammatory biomarkers relative to age-, sex- and obesity-matched controls (Ganji et al., 2015; Zhang, Yang, & Yu, 2015). Previous studies have indicated that systemic inflammation is strongly associated with hepatic steatosis and cardiometabolic disorders in obese individuals (Acharyya et al., 2007). Hepatic proinflammatory cytokines such as TNFα, IL-6 and IL-1β are overproduced in fatty liver (Shoelson, Lee, & Goldfine, 2006) and crucial effectors of insulin resistance (Raso et al., 2013). Although weight loss is essential to ensuring successful outcomes to the treatment of obesity-induced liver disease, numerous natural compounds have also been investigated for their treatment (Duarte et al., 2015). Scopoletin (6-methoxy-7-hydroxycoumarin) is a naturally occurring coumarin, which is found in many edible plants and fruits, such as oat (Avena sativa), elephant garlic (Allium ampeloprasum), celery (Apium graveolens), red pepper (Capsicum annuum), chili pepper (Capsicum frutescens), carrot (Daucus carota), chicory (Cichorium intybus), lemon (Citrus limon), grapefruit (Citrus paradisi) and sweet potato (Ipomoea batata) (Carpinella, Ferrayoli, & Palacios, 2005; Matsumoto, Mizutani, Sakata, & Shimizu, 2012). Scopoletin has been reported to possess antioxidant (Shaw, Chen, Hsu, Chen, & Tsai, 2003), antitumoural (Chang et al., 2012), antihypertension (Ojewole & Adesina, 1983) and anti-hyperglycaemic activity (Chang et al., 2015). Moreover, it has been shown to ameliorate synovial inflammation and destruction of cartilage and bone in adjuvant arthritis (Netzer et al., 2015). Mandukhail, Aziz, and Gilani (2010) suggested that scopoletin reduced the risk of hypercholesterolaemia, hypertriglyceridaemia and hyperglycaemia associated with high-fat diet. Gnonlonfin, Gbaguidi, Gbenou, Sanni, and Brimer (2011) reported cassava (Manihot esculenta Crantz), which is one of the most important food crops in tropical regions, contains scopoletin at between 4.1 and 11.1 mg/kg dry weight. Dietary intake of berries is known to have beneficial effects on human diseases, such as cardiovascular disease, obesity and some cancers (Firuzi, Miri, Tavakkoli, & Saso, 2011). Recently, goji berries (the fruit of Lycium barbarum) became a popular food supplement in China and southeastern Asia, and scopoletin has been reported to be one of the main phenolic components in the ethyl acetate extract of goji berries (present at 8 mg/kg on extract) (Forino, Tartaglione, Dell’Aversano, & Ciminiello, 2016). Scopoletin has been recommended as a marker constituent for the quality control of noni products (Issell, Franke, & Fielding, 2008). Noni (Morinda citrifolia Linn), which is also known as the Indian mulberry, hog apple and cheese fruit, has antitumour, hypotensive and immune enhancing effects (Nayak & Shettigar, 2010). Pandy, Narasingam, Kunasegaran, Murugan, and Mohamed (2014) reported a

scopoletin concentration in the methanol extract of noni fruit was 18.95 µg/mg. Furthermore, in a previous study, we reported that the ethyl acetate fraction of Artemisia iwayomogi exhibited hepatoprotective effects in alcohol and high-fat fed mice (Lee, Seo, Yun, Kim, & Lee, 2011) and that its main component was scopoletin (204 mg/g) (Seo, Jeong, & Yun, 2010). However, it is still unclear whether scopoletin can regulate NAFLD through metabolic and transcriptomic mechanisms. Therefore, this study was designed to elucidate the role of scopoletin in obesity-associated hepatosteatosis and inflammation via whole genome expression analysis.

2.

Materials and methods

2.1.

Animals

Four-week-old male C57BL/6J mice were purchased from Jackson Laboratory Center (Bar Harbor, ME, USA) and individually housed under a controlled temperature (22 ± 2 °C) and a 12 h lightdark cycle. After a one week adaptation period, mice were randomly divided into four groups of nine and fed a normal diet (NC), high-fat diet (HF, 20% fat and 1% cholesterol) or HF with 0.01 or 0.05% scopoletin diet (HF-LS or HF-HS; TCI Co., Ltd, Tokyo, Japan) for 16 weeks. The compositions of the experimental diets were based on the AIN-76 semisynthetic diet (American institute of nutrition, 1977). Body weight and food intake were measured once a week and daily, respectively, during the feeding period. At the end of the experimental period, mice were fasted for 12 h and then anaesthetized with ether. Blood samples were subsequently taken from the inferior vena cava for serum biomarker analysis, after which the liver and adipose tissues were removed, rinsed with a physiological saline solution and stored immediately at −70 °C until analysis. The present study was approved by the Sunchon National University Institutional Animal Care and Use Committee (SCNU IACUC-2013-11).

2.2. Serum adipokines, cytokines, chemokine and liver damage markers Serum adiponectin and leptin levels were determined using a quantitative sandwich enzyme immunoassay kit (R&D System, Minneapolis, MN, USA). Tumour necrosis factor alpha (TNFα), interleukin-6 (IL-6), interferon gamma (IFNγ), and monocyte chemoattractant protein-1 (MCP-1) levels were determined using a multi-detection kit (BioRad, Hercules, CA, USA) and Luminex 200 Labmap system (Luminex, Austin, TX, USA). The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using an automated chemistry analyser (Fuji-Dri-Chem 3500; Fujifilm, Tokyo, Japan).

2.3.

Insulin resistance biomarkers

At week 8, 12 and 16, the 6 h fasting serum glucose concentrations were measured using a glucometer (GlucoDr supersensor, Allmedicus, Anyang, Korea). Serum insulin level

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was determined using commercially available quantitative sandwich enzyme immunoassay kits (CrystalChem, Downers Grove, IL, USA). An intraperitoneal glucose tolerance test (IPGTT) was performed at 16 weeks. After 6 hours of fasting, mice were injected intraperitoneally with glucose (1g/kg body weight), and blood glucose levels were then determined using samples collected from the tail vein at 0, 30, 60, and 120 min and presented as the area under curve (AUC). The homeostatic index of insulin resistance (HOMA-IR) was calculated according to the homeostasis of assessment as follows Seo et al. (2008): HOMAIR = fasting glucose (mmol/L) × fasting insulin (µIU/mL) / 22.5.

2.4.

Serum, hepatic and faecal lipid contents

Serum triacylglycerol (TG), total cholesterol (TC), HDL-cholesterol (HDL-C) (Asan Diagnostics, Seoul, Korea) and free fatty acid (FFA) (Shinyang Diagnostics, Seoul, Korea) concentrations were determined using commercial kits. The hepatic and faecal lipids were extracted as previously described (Folch, Lees, & Sloane-Stanley, 1957), after which the cholesterol, TG and FFA contents were analysed using the same enzymatic kit that was used for serum analysis.

2.5.

Morphology of liver and adipose tissues

The liver and epididymal adipose tissues were removed and fixed in a buffer solution containing 10% formalin, after which the fixed tissues were paraffin-embedded, and 3–5 µm sections were prepared and stained with haematoxylin and eosin (H & E) or Masson’s Trichrome (Trichrome). To further confirm the hepatic lipid droplet accumulation, frozen sections were stained with Oil Red O solution and the stained area was viewed using an optical microscope at 200 × magnification.

2.6.

Hepatic enzyme activities

Fatty acid synthase (FAS) activity was determined from a spectrophotometric assay according to the method described by Nepokroeff, Lakshmanan, and Porter (1975), using a spectrophotometric assay that measured one unit of FAS activity representing the oxidation of 1 nmol of NADPH per minute at 30 °C. The phosphatidate phosphohydrolase (PAP) activity was determined using the method developed by Walton and Possmayer (1984). Fatty acid β-oxidation (β-oxidation) activity was measured spectrophotometrically by monitoring the reduction of NAD+ to NADH in the presence of palmitoyl-CoA using the method described by Lazarow (1981) with slight modification. Carnitine palmitoyltransferase (CPT) was assayed spectrophotometrically based on the release of CoA-SH from palmitoyl-CoA using the general thiol reagent 5,5′-dithiobis (2nitrobenzoate) as described by Bieber, Abraham, and Helmrath (1972), with slight modifications. The glucokinase (GK) activity was determined using a spectrophotometric continuous assay as described by Davidson and Arion (1987) and Newgard and McGarry (1995) with slight modifications, in which the formation of glucose-6-phosphate (G6P) was coupled to its oxidation by G6P dehydrogenase and NAD+ at 37 °C. The glucose6-phosphatase (G6Pase) activity was determined as previously described (Jang, Kim, Choi, Kwon, & Lee, 2010). Phosphoenolpyruvate carboxykinase (PEPCK) activity was monitored in the

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direction of oxaloacetate synthesis using the spectrophotometric assay developed by Bentle and Lardy (1976) with slight modification.

2.7.

Microarray and IPA network analysis

Total RNA was extracted from the liver tissue using TRIZOL reagent (Invitrogen Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. DNase digestion was used to remove any DNA contamination and RNA was re-precipitated in ethanol to ensure that there was no phenol contamination. For quality control, RNA purity and integrity were evaluated using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). RNA was stored at −70 °C prior to further analysis by microarray and RT-qPCR. Three pooled RNA sample sets were constructed with 9 individual samples from NC, HF and HF-LS groups as previously described (Do, Kwon, Kim, Kim, & Choi, 2010). Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, TX, USA) to yield biotinylated cRNA. A total of 750 ng biotinylated cRNA per sample was hybridized to an Illumina Mouse WG-6 v2 Expression BeadChip (Illumina, Inc., San Diego, CA, USA) for 16–18 h at 58 °C, according to the manufacturer’s instructions. Hybridized arrays were washed and stained with Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the standard protocol in the bead array manual. The quality of hybridization and overall chip performance were determined by visual inspection of both internal quality controls and the raw scanned data in the Illumina BeadStudio software. Probe signal intensities significantly higher than background intensities were determined (detection p-value < 0.05). Probe signal intensities were quantile normalized and log transformed. Microarray analysis was performed using Illumina GenomeStudio v2011.1 (Gene Expression Module v1.9.0). Statistical significance of the expression data was determined using LPE test. False discovery rate (FDR) was controlled by adjusting p-value using Benjamini–Hochberg algorithm. All data analysis and visualization of differentially expressed genes was conducted using R 2.15.3 (www.r-project.org). A p < 0.05 and Log2 ratio > 0.5 were used as the cut-off values. Ingenuity pathway analysis (Ingenuity® Systems, www .ingenuity.com, IPA version: 8.8 (2010), Content version: 3204 (2010)) is a web-based application that applies a systemic biology approach to solve various biological problems. The knowledge base of IPA has been curated from journal articles, textbooks, and other data sources. This software has many applications; however, only functional analysis of genes and their networks were conducted in this study. The p-value defines the significance of gene function in a network as well as the gene to gene relationship, and a p < 0.05 was taken to indicate a significance and non-random association. The right-tailed Fisher’s Exact Test was used to calculate the p-value.

2.8.

Quantitative real-time PCR (RT-qPCR) analysis

Total RNA (1 µg) was reverse-transcribed into cDNA using a QuantiTect reverse transcription kit (Qiagen, Hilden, Germany). The mRNA expressions were then quantified by real-time

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quantitative PCR using a SYBR green PCR kit (Qiagen, Hilden, Germany) and a CFX96TM real-time system (BioRad, Hercules, CA, USA) with the primers shown in supplementary data. Cycle thresholds were determined based on the SYBR green emission intensity during the exponential phase. Ct values were normalized to Gapdh, which was stably expressed in NC, HF and HF-LS mice. Relative gene expression was calculated using 2−ΔΔCt method (Livak & Schmittgen, 2001).

2.9.

Statistical analysis

All data are presented as the mean ± S.E. Data were evaluated by one-way analysis of variance (ANOVA) using SPSS (Chicago, IL, USA) and differences between means were determined by Duncan’s multiple-range test. Values were considered statistically significant at p < 0.05.

3.

Results

3.1. Scopoletin reduced body weight, visceral fat weight and liver damage marker After 13 weeks, body weight was significantly higher in the HF group than the NC group; however, it was significantly lower in the HF-LS group than the HF group (Fig. 1A) at the end of experiment. Both doses of scopoletin significantly reduced body weight gain, adipocyte size and white adipose tissue weight (epididymal and perirenal), which were increased by the HF diet (Fig. 1A-C). Serum AST and ALT levels in the HF group were significantly elevated compared with the NC group; however, these levels were decreased in the HF-LS (AST 25% and ALT 45%) and HF-HS (AST 13% and ALT 34%) groups (Fig. 2A & B).

Fig. 1 – Effect of scopoletin supplementation on body weight, body weight gain (A), adipocyte size (B), and visceral fat weight (C) in high-fat diet-induced obese mice. Values are expressed as the mean ± S.E. abcdNot sharing a common letter are significantly different between groups (p < 0.05).

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Fig. 2 – Effect of scopoletin supplementation on serum AST (A) and ALT (B) activities and hepatic histological analysis (C) in high-fat diet-induced obese mice. abcNot sharing a common letter are significantly different between groups (p < 0.05). Haematoxylin and Eosin (H & E), Oil Red O and Masson’s trichrome stained. Yellow arrows indicated lipid droplets. Green arrows indicated fibrosis (200 × magnification).

3.2. Scopoletin lowered obesity-induced inflammation and hyperlipidaemia HF diet feeding caused increased serum cytokines (TNFα, IL-6 and IFNγ), chemokine (MCP-1) and adipokine (leptin) levels relative to the NC group. Both doses of scopoletin led to similar reductions in these levels, whereas they increased the serum adiponectin level compared with the HF group (Table 1). At 16 weeks, HF diet induced hyperlipidaemia (TG, TC and FFA) (Fig. 3A). However, serum TG and TC levels were significantly lower in both scopoletin groups than the HF group at

16 wk and 12 wk, respectively. The FFA level was also significantly reduced in both scopoletin groups than the HF group. Although scopoletin did not change the HDL-C level, it significantly elevated the HTR that was decreased by HF (Fig. 3A).

3.3. Scopoletin attenuates hepatic lipid accumulation by faecal lipid excretion and lipid metabolic enzyme activities H & E and Oil Red O stains revealed that HF increased fat droplets; however, scopoletin improved this fat accumulation. Trichrome stain also revealed that deposition of collagen fibres

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Table 1 – Effect of scopoletin supplementation on serum adipokines, cytokines and chemokine levels and hepatic enzyme activities in high-fat diet-induced obese mice*.

Serum Leptin (ng/mL) Adiponectin (µg/mL) TNFα (pg/mL) IL-6 (pg/mL) IFNγ (pg/mL) MCP-1 (pg/mL) Hepatic lipid metabolic enzymes FAS1 PAP2 β-oxidation1 CPT1 Hepatic glucose metabolic enzymes GK1 G6Pase1 GK/G6Pase PEPCK1

NC

HF

HF-LS

HF-HS

13.70 ± 1.69a 6.04 ± 0.34b 62.96 ± 3.80a 1.75 ± 0.12a 8.11 ± 0.89a 172.56 ± 14.23a

25.41 ± 1.43b 4.74 ± 0.10a 103.56 ± 9.91b 3.08 ± 0.46b 14.55 ± 1.25b 276.36 ± 16.30b

16.44 ± 3.35a 5.60 ± 0.33b 68.27 ± 6.94a 1.72 ± 0.25a 9.06 ± 1.25a 166.59 ± 24.90a

18.89 ± 1.65a 5.64 ± 0.20b 67.99 ± 5.73a 1.55 ± 0.33a 8.32 ± 1.98a 157.83 ± 25.35a

13.70 ± 1.69a 6.04 ± 0.34b 62.96 ± 3.80a 1.75 ± 0.12a

25.41 ± 1.43b 4.74 ± 0.10a 103.56 ± 9.91b 3.08 ± 0.46b

16.44 ± 3.35a 5.60 ± 0.33b 68.27 ± 6.94a 1.72 ± 0.25a

18.89 ± 1.65a 5.64 ± 0.20b 67.99 ± 5.73a 1.55 ± 0.33a

13.83 ± 1.15a 56.19 ± 6.27a 0.24 ± 0.04ab 51.34 ± 6.05

14.04 ± 0.69a 96.61 ± 5.62b 0.16 ± 0.01a 38.82 ± 3.99

33.19 ± 4.67b 60.75 ± 3.25a 0.54 ± 0.09c 45.47 ± 3.66

28.63 ± 3.36b 69.21 ± 4.15a 1.40 ± 0.07bc 44.23 ± 3.18

* Values are mean ± S.E. abc In the same row not sharing a common superscript letter are significantly different between groups (p < 0.05). 1 nmol/min/mg protein. 2 µmol/min/mg protein.

around the congested central vein was increased in the HF group, indicating that HF diet induced fibrosis. However, scopoletin decreased fibrosis relative to the HF group (Fig. 2C). Hepatic TG, FFA and cholesterol levels were significantly increased in the HF group by 81, 27 and 94%, respectively, when compared with the NC group. Faecal TG and cholesterol levels also increased in response to the HF diet. However, the two doses of scopoletin led to similar reductions in hepatic lipid levels and elevated faecal excretion (Fig. 3B & C). FAS activity did not change significantly between the NC group and HF group; whereas, PAP activity was significantly higher in the HF group than the NC group. Both doses of scopoletin led to dose-independent decreases in lipid biosynthetic enzymes, FAS and PAP activities compared with the HF group. The activities of the fatty acid oxidation enzymes, β-oxidation and CPT increased in response to both doses of scopoletin (Table 1).

3.4. Scopoletin improved insulin resistance by lowering serum glucose and insulin levels and glucose metabolic enzyme activities Serum glucose and insulin concentrations were markedly elevated from 12 wk to the end of the experimental period in the HF group; however, they were significantly lowered in both scopoletin groups (Fig. 4A & B). HOMA-IR was significantly elevated in the HF group compared with the NC group. Low and high doses of scopoletin reduced HOMA-IR by 62.1 and 57.3%, respectively (Fig. 4C). IPGTT revealed that HF diet significantly impaired glucose tolerance, but scopoletin improved its value to one similar to the NC group (Fig. 4D). Both doses of scopoletin increased GK activity relative to the HF group. G6Pase activity was higher in the HF group than the NC group; however, both doses of scopoletin suppressed

the activity. Therefore, the GK/G6Pase ratio was increased by scopoletin supplementation. PEPCK activity did not differ among groups (Table 1).

3.5.

Scopoletin changes hepatic gene expression profiling

This study showed that scopoletin dose-independently (0.01 and 0.05% in diet) ameliorates non-alcoholic fatty liver, inflammation and insulin resistance in HF diet-fed mice. Therefore, we analysed global gene expression using the microarray to elucidate the molecular mechanism of scopoletin. When the HF group was compared with NC, 284 genes were up (172)- or down (112)-regulated. Of the 172 up-regulated genes in HF, scopoletin up-regulated 2 genes (Cyp7a1 and Gvin1) and down-regulated 19 genes (Gpnmb, Cidea, Gdf15, Pdk4, Cyp2b9, Ly6d, Mmp13, Anxa2, Saa1, Sdcbp2, S100a11, Col1a1, Uap111, Gsta2, Cd63, E030040G24Rik, Snhg18, LOC673589 and Gpc1), while among the 112 down-regulated genes, four (Moxd1, Prei4, Hsd3b5 and C8b) were up-regulated and one (Mmd2) was down-regulated by scopoletin. Conversely, 15 and 8 of the genes that did not differ between the NC and HF group were up- and downregulated by scopoletin, respectively (Table 2). The three significant gene networks identified by scopoletin supplementation are depicted Fig. 5. The biofunctions of these networks were related to (1) lipid concentration, organ inflammation and adipose tissue quantity (Fig. 5A), (2) rheumatic disease (Fig. 5B) and (3) proliferation of cells and necrosis (Fig. 5C), which were down-regulated in the scopoletin group. The top up- or down-regulated genes were Cidea, Apoa4, Cyp7a1, Errfi1, Col1a1, Mmp13, Cdkn1a, and Saa1. The expression of major genes was confirmed by RTqPCR analysis of individual RNA samples (Fig. 6). All genes showed expression patterns consistent with the microarray results.

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Fig. 3 – Effect of scopoletin supplementation on serum (A), hepatic (B) and faecal(C) lipid levels in high-fat diet-induced obese mice. Values are expressed as the mean ± S.E. abcNot sharing a common letter are significantly different between groups (p < 0.05).

4.

Discussions

Chronic HF intake leads to insulin resistance in humans and rodents, which can easily develop to NAFLD (Wang et al., 2014; Woo et al., 2010). We also confirmed feeding mice a HF diet for 16 weeks resulted in increased serum glucose and insulin levels, as well as hepatic lipid accumulation. However, two doses of scopoletin (0.01 and 0.05% in diet) effectively reduced fatty liver, inflammation markers and insulin resistance by modulating hepatic lipid and glucose metabolic enzyme activities and

increasing faecal lipid excretion in HF diet-induced obese mice. Both doses of scopoletin significantly inhibited hepatic lipogenic enzyme (FAS and PAP) activities, while they increased the activities of fatty acid oxidation enzyme (β-oxidation and CPT) simultaneously with excretion of faecal lipid compared with the HF group. These effects resulted in suppression of body weight gain, visceral obesity and hepatic steatosis in HF dietinduced obese mice. Leptin and adiponectin are two major adipokines that are secreted from adipose tissue and can influence glucose and lipid metabolism (Im et al., 2015; Sim, Ham, Lee, Seo, & Lee,

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Fig. 4 – Effect of scopoletin supplementation on serum glucose (A), insulin (B), HOMA-IR (C) and IPGTT (D) in high-fat dietinduced obese mice. Values are expressed as the mean ± S.E. abNot sharing a common letter are significantly different between groups (p < 0.05).

2014). Leptin has been shown to be positively correlated with adiposity and body weight in humans and rodents (Yoon et al., 2015), and its profibrogenic role has been characterized in liver disease (Ikejima et al., 2002). On the other hand, adiponectin led to insulin sensitivity and fatty acid oxidation (Im et al., 2015; Marra & Bertolani, 2009). Hernandez Vera, Vilahur, FerrerLorente, Pena, and Badimon (2012) showed that adiponectin improved hyperglycaemia in obese mice by decreasing the hepatic TG content. Adiponectin has hepatoprotective and antifibrogenic effects during liver injury (Marra & Bertolani, 2009). The current study demonstrated that both doses of scopoletin significantly decreased leptin concentration and increased adiponectin concentration in the serum when compared with the HF group. In addition, two doses of scopoletin were found to have potential G6Pase inhibitory activity, but did not influence PEPCK in HF-fed mice. G6Pase is a major gluconeogenic enzyme that catalyses dephosphorylation of glucose-6-phosphate to glucose in the liver (Cho et al., 2013). Taken together, these results indicate that scopoletin may attenuate HF-induced insulin resistance via inhibition of hepatic glucose production. Thus, scopoletin dose-independently (0.01 and 0.05% in diet) ameliorates non-alcoholic fatty liver and insulin resistance in HF diet-fed mice. Scopoletin is relatively nontoxic and humans appear to be able to tolerate high doses without significant side effects. Lethal doses 50% (LD50) of scopoletin have been determined to be 3800 mg/kg in rats (oral) and 350 mg/kg in mice (intravenous) (Mishkinsky, Goldschmied, Joseph, Ahronson, & Sulman, 1974; Nieschulz & Schmersahl, 1968). The low dose of scopoletin used in our study was equivalent to 10.96 mg/kg/day in mice based on average food intake.

For the average 70 kg human, this dose translates to 62 mg/ day using an allometric scaling factor of 0.081 (Reagan-Shaw, Nihal, & Ahmad, 2008). Next, the molecular mechanisms associated with scopoletin (0.01%)-mediated changes in gene expression during the development of NAFLD were investigated by microarray analysis of diet-induced obese mice. Scopoletin up- or down-regulated 49 genes in the liver relative to the HF group that fell into three gene networks. The top biofunction of the differentially expressed genes was associated with lipid concentration, organ inflammation and quantity of adipose tissue. Overall, the following associated genes were identified by network analysis: Cidea, ApoA4, Cyp7a1, Errfi1, Mmp13, Col1a1, Gdf15, Cdkn1a and Saa1. Gene expression profiling showed that scopoletin modified genes including Cidea and Apoa4 were involved in triacylglycerol synthesis and storage. Cidea was one of the most downregulated genes involved in lipid accumulation by scopoletin. Cidea is a critical regulator of TG storage and the development of large lipid-droplets in the liver (Puri et al., 2008; VerHague, Cheng, Weinberg, & Shelness, 2013). Zhou et al. (2012) reported that Cidea expression was markedly increased in human hepatic steatosis and genetically modified animal models. Here, we demonstrated a significant relationship between hepatic Cidea gene level and hepatic lipids (TG, r = 0.680, p < 0.01; cholesterol, r = 0.634, p < 0.01), serum lipids (TG, r = 0.481, p < 0.05; TC, r = 0.452, p < 0.01; FFA, r = 0.431, p < 0.05) and body weight (r = 0.393, p < 0.05). This finding is consistent with the results observed for in high-fat diet or ob/ob mice (Gummesson et al., 2007; Matsusue et al., 2008; Zhou et al., 2012). Our previous study

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Table 2 – Genes up- or down-regulated in the liver by scopoletin among high-fat responsive genes. Among the 172 up-regulated genes by HF, 21 genes were changed in comparison of scopoletin vs HF. Gene symbol

Gene title

Log2

P-value

Cyp7a1 Givin1 Gpnmb Cidea Gdf15 Pdk4 Cyp2b9 Ly6d Mmp13 Anxa2 Saa1 Sdcbp2 S100a11 Col1a1 Uap1l1 Gsta2 Cd63 E030040G24Rik Snhg18 LOC673589 Gpc1

Cholesterol 7 alpha-hydroxylase Unknown gene Glycoprotein (transmembrane) nmb Cell death-inducing DFFA-like effector A Growth differentiation factor 15 Pyruvate dehydrogenase kinase, isoenzyme 4 Cytochrome P450, family 2, subfamily b, polypepti de 9 Lymphocyte antigen 6 complex, locus D Matrix metallopeptidase 13 Annexin A2 Serum amyloid A 1 Syndecan binding protein (syntenin) 2 S100 calcium binding protein A11 Collagen, type I, alpha 1 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 Glutathione S-transferase, alpha 2 CD63 antigen Unknown gene Small nucleolar RNA host gene 18 Unknown gene Glypican 1

0.91 0.97 −1.61 −1.47 −1.45 −1.36 −1.22 −1.06 −1.05 −1.00 −0.99 −0.87 −0.83 −0.75 −0.71 −0.65 −0.65 −0.55 −0.55 −0.52 −0.52

1.16 × 10−4 9.26 × 10−3 8.46 × 10−10 2.12 × 10−2 1.02 × 10−7 1.67 × 10−6 5.73 × 10−5 2.55 × 10−2 6.04 × 10−3 9.82 × 10−3 2.98 × 10−5 2.60 × 10−2 1.68 × 10−2 4.45 × 10−2 4.32 × 10−2 2.73 × 10−2 3.22 × 10−2 5.33 × 10−3 7.73 × 10−3 2.06 × 10−2 3.31 × 10−2

Among the 112 down-regulated genes by HF, 5 genes were changed in comparison of scopoletin vs HF. Gene symbol

Gene title

Log2

P-value

Moxd1 Prei4 Hsd3b5 C8b Mmd2

Monooxygenase, DBH-like 1 Glycerophosphocholine phosphodiesterase GDE1 homolog Hydroxy-delta-5-steroid dehydrogenase, 3 beta – and steroid delta-isomerase 5 Complement component 8, beta polypeptide Monocyte to macrophage differentiation-associated 2

0.97 0.93 0.86 0.69 −0.82

9.36 × 10−3 1.66 × 10−2 1.66 × 10−2 1.22 × 10−2 2.28 × 10−2

Among the unchanged genes by HF, 23 genes were changed in comparison of scopoletin vs HF. Gene symbol

Gene title

Log2

P-value

Usp2 Irgb10 Per1 Gbp1 Selenbp2 LOC667597 Gadd45g Upp2 Igtp Tgtp Errfi1 Cxcl13 Mettl20 Nmrk1 Apol9b Apoa4 Fgf21 Xlr4a Tubb2b Cib3 Tceal8 Cdkn1a Serpina7

Ubiquitin specific peptidase 2 Unknown gene Period circadian clock 1 Guanylate binding protein 2b Selenium binding protein 2 Unknown gene Growth arrest and DNA-damage-inducible 45 gamma Uridine phosphorylase 2 Interferon gamma induced GTPase T cell specific GTPase 1 ERBB receptor feedback inhibitor 1 Chemokine (C-X-C motif) ligand 13 Methyltransferase like 20 Nicotinamide riboside kinase 1 Apolipoprotein L 9b Apolipoprotein A-IV Fibroblast growth factor 21 X-linked lymphocyte-regulated 4A Tubulin, beta 2B class IIB Calcium and integrin binding family member 3 Transcription elongation factor A (SII)-like 8 Cyclin-dependent kinase inhibitor 1A Serine (or cysteine) peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7

1.09 1.00 1.00 0.99 0.98 0.98 0.97 0.95 0.95 0.94 0.87 0.86 0.66 0.60 0.59 −1.61 −1.30 −1.21 −1.20 −1.01 −0.82 −0.77 −0.74

1.14 × 10−3 5.26 × 10−3 5.34 × 10−3 6.77 × 10−3 5.51 × 10−3 8.10 × 10−3 8.10 × 10−3 1.03 × 10−2 3.61 × 10−3 1.52 × 10−2 3.46 × 10−9 4.99 × 10−3 4.42 × 10−2 3.23 × 10−2 4.43 × 10−2 3.39 × 10−2 7.38 × 10−6 2.56 × 10−5 7.09 × 10−5 4.21 × 10−3 4.58 × 10−2 2.42 × 10−2 9.20 × 10−3

Differentially expressed genes based on HF vs. NC and scopoletin vs HF comparison according to p < 0.05, log2 ratio > 0.5.

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Fig. 5 – The hepatic gene network 1 (A), network 2 (B) and network 3 (C) in response to scopoletin supplementation in highfat diet-induced obese mice. Intensity of node colour indicates magnitude of up-regulation (red) or down-regulation (green). Solid arrow – induction and/or activation; dashed arrow – suppression and/or inhibition. Network number assigned in the order of the significant score. Biofunction is the most significant function for each network.

Journal of Functional Foods 25 (2016) 433–446

443

Fig. 6 – Comparison of microarray and quantitative RT-qPCR on gene expression levels by scopoletin supplementation. Fold changes in mRNA expression based on NC group. Microarray and RT-qPCR data shown as means ± S.E. Significant differences between HF versus NC and HF-LS versus HF are indicated; *p < 0.05, **p < 0.01, ***p < 0.001 and #p < 0.05, ## p < 0.01, ###p < 0.001, respectively.

also showed that hepatic Cidea gene expression decreased dramatically in response to scopoletin supplementation in an alcoholic fatty liver animal model (Lee & Lee, 2015). Therefore, Cidea down-regulation of scopoletin likely contributes to the decrease of hepatic lipid droplets in both alcoholic and nonalcoholic steatosis. Here, we also found that scopoletin downregulated Apoa4 expression relative to the HF group. Apoa4 is a lipid binding protein expressed in the mammalian intestine and rodent liver (VerHague et al., 2013; Xu, Park, So, Hur, & Lee, 2014). Recently, VerHague et al. (2013) demonstrated that hepatic steatosis induces hepatic Apoa4 expression in mice, which in turn promotes TG secretion to reduce hepatic TG burden. Xu, Park, So, Hur, and Lee (2014) also reported that hepatic Apoa4 greatly up-regulated hepatic steatosis and steatohepatitis and was potentially related to TG homeostasis. On the other hand, the increased expression of Cyp7a1 and Errfi1 (Mig6) may have contributed to the decreased hepatic cholesterol level in NAFLD mice. Cholesterol metabolism may play a role in the development of NAFLD (Renaud, Cui, Lu, & Klaassen, 2014). Dietary cholesterol exacerbates hepatic ste-

atosis and inflammation in mice (Subramanian et al., 2011), while Cyp7a1 catalyses the rate-limiting step in the biosynthesis of bile acid from cholesterol in the liver (Russell & Setchell, 1992). In the present study, Cyp7a1 gene expression was up-regulated by scopoletin, suggesting that scopoletin promotes bile acid production from excessive cholesterol in HF diet fed mice. Here, we also found that faecal cholesterol and TG levels were greater in mice supplemented with scopoletin than the HF group. Additionally, bile acids in the liver inhibit both TG synthesis and gluconeogenesis (Watanabe et al., 2004). Ku et al. (2012) recently reported that the Errfi1 (Mig6) gene plays an important role in cholesterol homeostasis and bile acid synthesis. They showed that liver-specific ablation of Mig6 developed hepatomegaly and fatty liver from decreased daily excretion of faecal bile acid. In the present study, there was no significant difference between the NC group and HF group; nevertheless, scopoletin significantly up-regulated Errfi1 gene expression relative to the HF group. The expression of several inflammation markers, including Mmp13, Col1a1, Saa1, Gdf15 and Cdkn1a, were significantly

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lower in scopoletin supplemented groups than the HF group. Hepatic low grade inflammation in obesity resulted in progressive disease and fibrosis (de Meijer, Sverdlov, Le, Popov, & Puder, 2012). Indeed, our results showed that Mmp13 and Col1a1 gene levels were dramatically increased in the HF group relative to the NC group. Increases in Mmp, an extracellular matrix gene, are a common phenomenon in liver fibrosis (San-Miguel et al., 2010). The Col1a1 gene is also up-regulated in subjects with NAFLD, and its deposition results in progressive hepatic fibrosis (Catalan et al., 2009; Gawrieh et al., 2010). The current study demonstrated that scopoletin may effectively prevent progressive liver fibrosis in HF diet-induced obese mice by reduction of extracellular matrix deposition. Gdf15, a member of the TGF-β superfamily, plays an important role in regulation of the inflammatory response, growth and cell differentiation. Inflammatory cytokines such as TNFα and IL-6 trigger production of Gdf15 (Bootcov et al., 1997; Hsiao et al., 2000). Bonaterra et al. (2012) suggested that Gdf15 deficiency reduced atherosclerosis progression and lipid accumulation in hypercholesterolaemic mice. The results of the present study showed that scopoletin also down-regulated Gdf15 gene levels and serum cytokine levels (TNFα and IL-6) compared with the HF group. Another inflammatory response gene, Saa1, was upregulated by HF diet. Yang et al. (2006) suggested that Saa acts locally to induce cytokine production and fat metabolism, as well as systemically in the liver, muscle, immune system and vasculature, thereby impacting insulin resistance and atherosclerosis. In this study, we showed that scopoletin downregulated Saa1 in the liver of HF diet-induced hepatic steatosis. Although Cdkn1a gene expression in the HF group did not change significantly, scopoletin down-regulated its expression compared with the HF group. Cdkn1a (p21) is known to be a p53 target gene and its function is related to liver regeneration (Inoue et al., 2005). Some research showed that Cdkn1a overexpression accelerated adipocyte hypertrophy, hepatic lipid accumulation, insulin resistance and NAFLD (Inoue et al., 2005; Takasaki et al., 2012). In the present study, Cdkn1a gene expression was inhibited by scopoletin, suggesting that scopoletin may promote liver regeneration. In conclusion, this study demonstrated that low dose of scopoletin (0.01%, w/w) attenuated NAFLD and prevented hepatic fibrosis development in diet-induced obese mice. Supplementation of scopoletin in the HF-induced model of NAFLD resulted in lower serum and hepatic lipid contents, amelioration of insulin resistance and inflammation, which may explain hepatic transcriptional analysis and gene expression. Accordingly, these findings suggest scopoletin could be safely used as a functional food resource for NAFLD.

Conflict of interest The authors have no financial conflicts of interest to disclose.

Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2012R1A1A2041931).

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2016.06.026.

REFERENCES

Acharyya, S., Villalta, S. A., Bakkar, N., Bupha-Intr, T., Janssen, P. M., Carathers, M., & Guttridge, D. C. (2007). Interplay of IKK/ NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. The Journal of Clinical Investigation, 117(4), 889–901. American Institute of Nutrition (1977). Report of the American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies. The Journal of Nutrition, 107, 1340–1348. Bentle, L. A., & Lardy, H. A. (1976). Interaction of anions and divalent metal ions with phosphoenolpyruvate carboxykinase. The Journal of Biological Chemistry, 251(10), 2916– 2921. Bieber, L., Abraham, T., & Helmrath, T. (1972). A rapid spectrophotometric assay for carnitine palmitoyltransferase. Analytical Biochemistry, 50(2), 509–518. Bonaterra, G. A., Zugel, S., Thogersen, J., Walter, S. A., Haberkorn, U., Strelau, J., & Kinscherf, R. (2012). Growth differentiation factor-15 deficiency inhibits atherosclerosis progression by regulating interleukin-6-dependent inflammatory response to vascular injury. Journal of the American Heart Association, 1(6), e002550. Bootcov, M. R., Bauskin, A. R., Valenzuela, S. M., Moore, A. G., Bansal, M., He, X. Y., Zhang, H. P., Donnellan, M., Mahler, S., Pryor, K., Walsh, B. J., Nicholson, R. C., Fairlie, W. D., Por, S. B., Robbins, J. M., & Breit, S. N. (1997). MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proceedings of the National Academy of Sciences of the United States of America, 94(21), 11514–11519. Carpinella, M. C., Ferrayoli, C. G., & Palacios, S. M. (2005). Antifungal synergistic effect of scopoletin, a hydroxycoumarin isolated from Melia azedarach L. fruits. Journal of Agricultural and Food Chemistry, 53(8), 2922–2927. Catalan, V., Gomez-Ambrosi, J., Rodriguez, A., Ramirez, B., Silva, C., Rotellar, F., Gill, M. J., Cienfuegos, J. A., Salvador, J., & Frühbeck, G. (2009). Increased adipose tissue expression of lipocalin-2 in obesity is related to inflammation and matrix metalloproteinase-2 and metalloproteinase-9 activities in humans. Journal of Molecular Medicine: Official Organ of the Gesellschaft Deutscher Naturforscher und Ärzte , 87(8), 803–813. Chang, T., Deng, J., Chang, Y., Lee, C., Jung-Chun, L., Lee, M., Peng, W. H., Huang, S. S., & Huang, G. (2012). Ameliorative effects of scopoletin from Crossostephium chinensis against inflammation pain and its mechanisms in mice. Evidencebased Complementary and Alternative Medicine, 2012, 595603. Chang, W., Wu, S., Xu, K., Liao, B., Wu, J., & Cheng, A. (2015). Scopoletin protects against methylglyoxal-induced hyperglycemia and insulin resistance mediated by suppression of advanced glycation endproducts (AGEs) generation and anti-glycation. Molecules: A Journal of Synthetic Chemistry and Natural Product Chemistry, 20(2), 2786–2801. Cho, Y., Chung, J. H., Do, H. J., Jeon, H. J., Jin, T., & Shin, M. (2013). Effects of fisetin supplementation on hepatic lipogenesis and glucose metabolism in Sprague-Dawley rats fed on a high fat diet. Food Chemistry, 139(1), 720–727. de Meijer, V. E., Sverdlov, D. Y., Le, H. D., Popov, Y., & Puder, M. (2012). Tissue-specific differences in inflammatory infiltrate and matrix metalloproteinase expression in adipose tissue

Journal of Functional Foods 25 (2016) 433–446

and liver of mice with diet-induced obesity. Hepatology Research, 42(6), 601–610. Davidson, A. L., & Arion, W. J. (1987). Factors underlying significant underestimations of glucokinase activity in crude liver extracts: Physiological implications of higher cellular activity. Archives of Biochemistry and Biophysics, 253(1), 156–167. Do, G., Kwon, E., Kim, E., Kim, H., & Choi, M. (2010). Hepatic transcription response to high-fat treatment in mice: Microarray comparison of individual vs. pooled RNA samples. Biotechnology Journal, 5(9), 970–973. Duarte, N., Coelho, I. C., Patarrão, R. S., Almeida, J. I., PenhaGonçalves, C., Macedo, M. P., & Macedo, M. P. (2015). How inflammation impinges on NAFLD: A role for Kupffer cells. BioMed Research International, 2015, 984578. Firuzi, O., Miri, R., Tavakkoli, M., & Saso, L. (2011). Antioxidant therapy: Current status and future prospects. Current Medicinal Chemistry, 18(25), 3871–3888. Folch, J., Lees, M., & Sloane-Stanley, G. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226(1), 497–509. Forino, M., Tartaglione, L., Dell’Aversano, C., & Ciminiello, P. (2016). NMR-based identification of the phenolic profile of fruits of Lycium barbarum (goji berries). isolation and structural determination of a novel N-feruloyl tyramine dimer as the most abundant antioxidant polyphenol of goji berries. Food Chemistry, 194, 1254–1259. Ganji, S. H., Kashyap, M. L., & Kamanna, V. S. (2015). Niacin inhibits fat accumulation, oxidative stress, and inflammatory cytokine IL-8 in cultured hepatocytes: Impact on nonalcoholic fatty liver disease. Metabolism: Clinical and Experimental, 64(9), 982–990. Gawrieh, S., Baye, T. M., Carless, M., Wallace, J., Komorowski, R., Kleiner, D. E., Andris, D., Makladi, B., Cole, R., Charlton, M., Curran, J., Dyer, T. D., Charlesworth, J., Wilke, R., Blangero, J., Kissebah, A. H., & Olivier, M. (2010). Hepatic gene networks in morbidly obese patients with nonalcoholic fatty liver disease. Obesity Surgery, 20(12), 1698–1709. Gnonlonfin, B. G., Gbaguidi, F., Gbenou, J. D., Sanni, A., & Brimer, L. (2011). Changes in scopoletin concentration in cassava chips from four varieties during storage. Journal of the Science of Food and Agriculture, 91(13), 2344–2347. Gummesson, A., Jernås, M., Svensson, P., Larsson, I., Glad, C. A., Schéle, E., Gripeteg, L., Sjöholm, K., Lystig, T. C., Sjöström, L., Carlsson, B., Fagerberg, B., & Carlsson, L. M. (2007). Relations of adipose tissue CIDEA gene expression to basal metabolic rate, energy restriction, and obesity: Population-based and dietary intervention studies. The Journal of Clinical Endocrinology & Metabolism, 92(12), 4759–4765. Hernandez Vera, R., Vilahur, G., Ferrer-Lorente, R., Pena, E., & Badimon, L. (2012). Platelets derived from the bone marrow of diabetic animals show dysregulated endoplasmic reticulum stress proteins that contribute to increased thrombosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(9), 2141– 2148. Hsiao, E. C., Koniaris, L. G., Zimmers-Koniaris, T., Sebald, S. M., Huynh, T. V., & Lee, S. J. (2000). Characterization of growthdifferentiation factor 15, a transforming growth factor beta superfamily member induced following liver injury. Molecular and Cellular Biology, 20(10), 3742–3751. Ikejima, K., Takei, Y., Honda, H., Hirose, M., Yoshikawa, M., Zhang, Y., Lang, T., Fukuda, T., Yamashina, S., Kitamura, T., & Sato, N. (2002). Leptin receptor–mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat. Gastroenterology, 122(5), 1399–1410. Im, J., Ki, H., Xin, M., Kwon, S., Kim, Y. H., Kim, D., Hong, S., Jin, J., & Lee, Y. (2015). Anti-obesity effect of Triticum aestivum sprout extract in high-fat-diet-induced obese mice. Bioscience, Biotechnology, and Biochemistry, 1–8, [ahead-of-print].

445

Inoue, N., Shimano, H., Nakakuki, M., Matsuzaka, T., Nakagawa, Y., Yamamoto, T., Sato, R., Takahashi, A., Sone, H., Yahagi, N., Suzuki, H., Toyoshima, H., & Yamada, N. (2005). Lipid synthetic transcription factor SREBP-1a activates p21WAF1/CIP1, a universal cyclin-dependent kinase inhibitor. Molecular and Cellular Biology, 25(20), 8938–8947. Issell, B. F., Franke, A., & Fielding, R. M. (2008). Pharmacokinetic study of Noni fruit extract. Journal of Dietary Supplements, 5(4), 373–382. Jang, S., Kim, M., Choi, M., Kwon, E., & Lee, M. (2010). Inhibitory effects of ursolic acid on hepatic polyol pathway and glucose production in streptozotocin-induced diabetic mice. Metabolism: Clinical and Experimental, 59(4), 512– 519. Ku, B. J., Kim, T. H., Lee, J. H., Buras, E. D., White, L. D., Stevens, R. D., Ilkayeva, O. R., Bain, J. R., Newgard, C. B., DeMayo, F. J., & Jeong, J. W. (2012). Mig-6 plays a critical role in the regulation of cholesterol homeostasis and bile acid synthesis. PLoS ONE, 7(8), e42915. Lazarow, P. B. (1981). Assay of peroxisomal beta-oxidation of fatty acids. Methods in Enzymology, 72, 315–319. Lee, H., & Lee, M. (2015). Coordinated regulation of scopoletin at adipose tissue–liver axis improved alcohol-induced lipid dysmetabolism and inflammation in rats. Toxicology Letters, 237(3), 210–218. Lee, H., Seo, K., Yun, K. W., Kim, M., & Lee, M. (2011). Comparative study of the hepatoprotective efficacy of Artemisia iwayomogi and Artemisia capillaris on ethanol-administered mice. Journal of Food Science, 76(9), T207–T211. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods : A Companion to Methods in Enzymology, 25(4), 402–408. Machado, M., & Cortez-Pinto, H. (2005). Non-alcoholic fatty liver disease and insulin resistance. European Journal of Gastroenterology & Hepatology, 17(8), 823–826. Mandukhail, S. U., Aziz, N., & Gilani, A. H. (2010). Studies on antidyslipidemic effects of Morinda citrifolia (Noni) fruit, leaves and root extracts. Lipids in Health and Disease, 9, 88. Marra, F., & Bertolani, C. (2009). Adipokines in liver diseases. Hepatology (Baltimore, Md.), 50(3), 957–969. Matsumoto, S., Mizutani, M., Sakata, K., & Shimizu, B. (2012). Molecular cloning and functional analysis of the orthohydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato, Ipomoea batatas (L.) lam. Phytochemistry, 74, 49– 57. Matsusue, K., Kusakabe, T., Noguchi, T., Takiguchi, S., Suzuki, T., Yamano, S., & Gonzalez, F. J. (2008). Hepatic steatosis in leptindeficient mice is promoted by the PPARγ target gene Fsp27. Cell Metabolism, 7(4), 302–311. Mishkinsky, J. S., Goldschmied, A., Joseph, B., Ahronson, Z., & Sulman, F. G. (1974). Hypoglycaemic effect of Trigonella foenum graecum and Lupinus termis (leguminosae) seeds and their major alkaloids in alloxan-diabetic and normal rats. Archives Internationales de Pharmacodynamie et de Therapie, 210(1), 27–37. Nayak, S., & Shettigar, R. (2010). Morinda citrifolia: A review. Journal of Pharmacy Research, 3(8), 1872–1874. Nepokroeff, C. M., Lakshmanan, M. R., & Porter, J. W. (1975). Fatty-acid synthase from rat liver. Methods in Enzymology, 35, 37–44. Netzer, N., Gatterer, H., Faulhaber, M., Burtscher, M., Pramsohler, S., & Pesta, D. (2015). Hypoxia, oxidative stress and fat. Biomolecules, 5(2), 1143–1150. Newgard, C. B., & McGarry, J. D. (1995). Metabolic coupling factors in pancreatic β-cell signal transduction. Annual Review of Biochemistry, 64(1), 689–719.

446

Journal of Functional Foods 25 (2016) 433–446

Nieschulz, O., & Schmersahl, P. (1968). On choleretic agents from Artemisia abrotanum L. Arzneimittel-Forschung, 18(10), 1330– 1336. Ojewole, J., & Adesina, S. (1983). Mechanism of the hypotensive effect of scopoletin isolated from the fruit of Tetrapleura tetraptera. Planta Medica, 49(9), 46–50. Pandy, V., Narasingam, M., Kunasegaran, T., Murugan, D. D., & Mohamed, Z. (2014). Effect of Noni (Morinda citrifolia Linn.) fruit and its bioactive principles scopoletin and rutin on rat vas deferens contractility: An ex vivo study. TheScientificWorldJournal, 2014, 909586. Puri, V., Ranjit, S., Konda, S., Nicoloro, S. M., Straubhaar, J., Chawla, A., Chouinard, M., Lin, C., Burkart, A., Corvera, S., Perugini, R. A., & Czech, M. P. (2008). Cidea is associated with lipid droplets and insulin sensitivity in humans. Proceedings of the National Academy of Sciences of the United States of America, 105(22), 7833–7838. Raso, G. M., Simeoli, R., Russo, R., Iacono, A., Santoro, A., Paciello, O., Ferrante, M. C., Conani, R. B., Calignano, A., & Meli, R. (2013). Effects of sodium butyrate and its synthetic amide derivative on liver inflammation and glucose tolerance in an animal model of steatosis induced by high fat diet. PLoS ONE, 8(7), e68626. Reagan-Shaw, S., Nihal, M., & Ahmad, N. (2008). Dose translation from animal to human studies revisited. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 22(3), 659–661. Renaud, H. J., Cui, J. Y., Lu, H., & Klaassen, C. D. (2014). Effect of diet on expression of genes involved in lipid metabolism, oxidative stress, and inflammation in mouse liver-insights into mechanisms of hepatic steatosis. Russell, D. W., & Setchell, K. D. (1992). Bile acid biosynthesis. Biochemistry, 31(20), 4737–4749. San-Miguel, B., Crespo, I., Kretzmann, N. A., Mauriz, J. L., Marroni, N., Tunon, M. J., & Gonzalez-Gallego, J. (2010). Glutamine prevents fibrosis development in rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid. The Journal of Nutrition, 140(6), 1065–1071. Seo, K., Choi, M., Jung, U. J., Kim, H., Yeo, J., Jeon, S., & Lee, M. (2008). Effect of curcumin supplementation on blood glucose, plasma insulin, and glucose homeostasis related enzyme activities in diabetic db/db mice. Molecular Nutrition & Food Research, 52(9), 995–1004. Seo, K., Jeong, H., & Yun, K. (2010). Antimicrobial activity and chemical components of two plants, Artemisia capillaris and Artemisia iwayomogi, used as Korean herbal Injin. Journal of Ecology and Environment, 33(2), 141–147. Shaw, C., Chen, C., Hsu, C., Chen, C., & Tsai, Y. (2003). Antioxidant properties of scopoletin isolated from Sinomonium acutum. Phytotherapy Research, 17(7), 823–825. Shoelson, S. E., Lee, J., & Goldfine, A. B. (2006). Inflammation and insulin resistance. The Journal of Clinical Investigation, 116(7), 1793–1801. Sim, M., Ham, J. R., Lee, H., Seo, K., & Lee, M. (2014). Long-term supplementation of umbelliferone and 4-methylumbelliferone alleviates high-fat diet induced hypertriglyceridemia and hyperglycemia in mice. ChemicoBiological Interactions, 216, 9–16. Subramanian, S., Goodspeed, L., Wang, S., Kim, J., Zeng, L., Ioannou, G. N., Yeh, M. M., Kowdley, K. V., O’Brien, K. D.,

Pennathur, S., & Chait, A. (2011). Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice. Journal of Lipid Research, 52(9), 1626– 1635. Takasaki, M., Honma, T., Yanaka, M., Sato, K., Shinohara, N., Ito, J., Tanaka, Y., Tsuduki, T., & Ikeda, I. (2012). Continuous intake of a high-fat diet beyond one generation promotes lipid accumulation in liver and white adipose tissue of female mice. The Journal of Nutritional Biochemistry, 23(6), 640– 645. VerHague, M. A., Cheng, D., Weinberg, R. B., & Shelness, G. S. (2013). Apolipoprotein A-IV expression in mouse liver enhances triglyceride secretion and reduces hepatic lipid content by promoting very low density lipoprotein particle expansion. Arteriosclerosis, Thrombosis, and Vascular Biology, 33(11), 2501–2508. Vernon, G., Baranova, A., & Younossi, Z. (2011). Systematic review: The epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Alimentary Pharmacology & Therapeutics, 34(3), 274–285. Walton, P. A., & Possmayer, F. (1984). The role of Mg2+-dependent phosphatidate phosphohydrolase in pulmonary glycerolipid biosynthesis. Biochimica et Biophysica Acta, 796(3), 364–372. Wang, X., Ren, Q., Wu, T., Guo, Y., Liang, Y., & Liu, S. (2014). Ezetimibe prevents the development of non-alcoholic fatty liver disease induced by high-fat diet in C57BL/6J mice. Molecular Medicine Reports, 10(6), 2917–2923. Watanabe, M., Houten, S. M., Wang, L., Moschetta, A., Mangelsdorf, D. J., Heyman, R. A., Moore, D. D., & Auwerx, J. (2004). Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c. The Journal of Clinical Investigation, 113(10), 1408–1418. Woo, M., Jeon, S., Kim, H., Lee, M., Shin, S., Shin, Y. C., Park, Y. B., & Choi, M. (2010). Fucoxanthin supplementation improves plasma and hepatic lipid metabolism and blood glucose concentration in high-fat fed C57BL/6N mice. ChemicoBiological Interactions, 186(3), 316–322. Xu, X., Park, J. G., So, J. S., Hur, K. Y., & Lee, A. H. (2014). Transcriptional regulation of apolipoprotein A-IV by the transcription factor CREBH. Journal of Lipid Research, 55(5), 850– 859. Yang, R., Lee, M., Hu, H., Pollin, T. I., Ryan, A. S., Nicklas, B. J., Snitker, S., Horenstein, R. B., Hull, K., Goldberg, N. H., Goldberg, A. P., Shuldiner, A. R., Fried, S. K., & Gong, D. W. (2006). Acute-phase serum amyloid A: An inflammatory adipokine and potential link between obesity and its metabolic complications. PLoS Medicine, 3(6), e287. Yoon, Y., Chung, M. Y., Hwang, J., Han, M. S., Goo, T., & Yun, E. (2015). Allomyrina dichotoma (Arthropoda: Insecta) larvae confer resistance to obesity in mice fed a high-fat diet. Nutrients, 7(3), 1978–1991. Zhang, L., Yang, B., & Yu, B. (2015). Paeoniflorin protects against nonalcoholic fatty liver disease induced by a high-fat diet in mice. Biological and Pharmaceutical Bulletin, 38(7), 1005– 1011. Zhou, L., Xu, L., Ye, J., Li, D., Wang, W., Li, X., Wu, L., Wang, H., Guan, F., & Li, P. (2012). Cidea promotes hepatic steatosis by sensing dietary fatty acids. Hepatology (Baltimore, Md.), 56(1), 95–107.