Journal of Functional Foods 31 (2017) 131–140

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Journal of Functional Foods

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Red paprika (Capsicum annuum L.) and its main carotenoid capsanthinameliorate impaired lipid metabolism in the liver and adipose tissue ofhigh-fat diet-induced obese mice

http://dx.doi.org/10.1016/j.jff.2017.01.0441756-4646/� 2017 Elsevier Ltd. All rights reserved.

Abbreviations: NAFLD, non-alcoholic fatty liver disease; WAT, white adipose tissue; ND, normal diet; HD, high-fat diet; RP, red paprika-supplemented diet; CS, capsupplemented diet; TG, triacylglycerol; TC, total cholesterol; HDL, high-density lipoprotein; NEFA, non-esterified fatty acid; LDL, low-density lipoprotein; ox-LDL,low-density lipoprotein; Apo A-1, apolipoprotein A-1; Apo B (H), apolipoprotein B (H); Lp-A, lipoprotein A; HOMA-IR, homeostatic index of insulin resistance; TNF-necrosis factor-a; PAI-1, plasminogen activator inhibitor-1; IL-6, interlukin-6; IL-1b, interlukin-1b; G6PDH, glucose-6-phosphate dehydrogenase; ME, malic enzymsuperoxide dismutase; MDA, malondialdehyde; CD36, cluster of differentiation 36; FABP-1, fatty acid-binding protein-1; PPARa, peroxisome proliferator-activated ra; LCAD, long-chain acyl-CoA dehydrogenases; MCAD, medium-chain acyl-CoA dehydrogenases; PDK-4, pyruvate dehydrogenase kinase-4; SREBP-1c, sterol reelement binding protein-1c; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthesis; SCD-1, stearoyl-CoA desaturase-1; PEPCK, phosphoenolpyruvate carboxykinaseglucose 6-phosphatase; CYP7A1, cholesterol 7-alpha-hydroxylase; PGC-1a, PPAR gamma coactivator-1a; PGC-1b, PPAR gamma coactivator-1b; Sirt-1, sirtuin-1peroxisome proliferator-activated receptor-c; C/EBPa, CAAT/enhancer-binding protein-a; aP2, adipocyte fatty acid binding protein-2; AMPK, AMP-activated proteiTBARS, thiobarbituric acid reactive substances.⇑ Corresponding author.

⇑⇑ Corresponding author.E-mail addresses: ksuna7@knou.ac.kr (S. Kim), jyan@kfri.re.kr (J. Ahn).

Ji-Sun Kim a,b, Tae-Youl Ha b, Suna Kim c,⇑⇑, Sung-Joon Lee a, Jiyun Ahn b,⇑aDepartment of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of KoreabResearch Group of Nutrition and Metabolic System, Korea Food Research Institute, Seongnam 13539, Republic of KoreacDepartment of Food and Nutrition in Human Ecology, College of Natural Science, Korea National Open University, Seoul 03078, Republic of Korea

a r t i c l e i n f o

Article history:Received 14 November 2016Received in revised form 21 December 2016Accepted 22 January 2017Available online 2 February 2017

Chemical compounds studied in this article:Capsorubin (PubChem CID: 5281229)Violaxanthin (PubChem CID: 448438)Capsanthin (PubChem CID: 5281228)Lutein (PubChem CID: 5281243)Zeaxanthin (PubChem CID: 5280899)b-Cryptoxanthin (PubChem CID: 5281235)b-Carotene (PubChem CID: 5280489)

Keywords:Red paprikaCapsanthinHigh-fat dietHepatic steatosisLipid accumulation

a b s t r a c t

Red paprika (Capsicum annuum L.) is a widely used natural food colorant and source of vital micronutri-ents. The main carotenoid in red paprika is capsanthin. In this study, the effects of red paprika and capsan-thin on impaired lipid metabolism were investigated in diet-induced obese mice. Forty male mice weredivided into 4 groups; normal diet (ND), high-fat diet (HD), HD with red paprika, and HD with capsanthin.After 8 weeks, the red paprika and capsanthin groups showed significantly reduced weight gain, and ame-liorated hypertrophy of the liver and adipose tissues. Red paprika and capsanthin treatment also improvedserum lipid profile and adipokine secretion, and ameliorated hepatic steatosis by suppressing hepatic lipo-genesis, fatty acid oxidation, and gluconeogenesis. In epididymal adipose tissue, red paprika and capsan-thin inhibited adipogenesis and decreased lipid droplet size. Taken together, red paprika and capsanthincould ameliorate the detrimental effects of diet-induced obesity by improving impaired lipid metabolism.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction hypertension and cardiovascular disease, non-alcoholic fatty liver

Obesity, caused by an imbalance between energy intake andexpenditure, is a worldwide health problem. It is known to be astrong risk factor for chronic diseases such as type 2 diabetes,

disease (NAFLD), and some forms of cancer (Kopelman, 2000).Epidemiological studies suggest that a diet rich in fat might be arisk factor for the development of obesity and insulin resistance(Bray, Paeratakul, & Popkin, 2004).

santhin-oxidizeda, tumore; SOD,eceptor-gulatory; G6Pase,; PPARc,n kinase;

http://crossmark.crossref.org/dialog/?doi=10.1016/j.jff.2017.01.044&domain=pdfhttp://dx.doi.org/10.1016/j.jff.2017.01.044mailto:ksuna7@knou.ac.krmailto:jyan@kfri.re.krhttp://dx.doi.org/10.1016/j.jff.2017.01.044http://www.sciencedirect.com/science/journal/17564646http://www.elsevier.com/locate/jff

132 J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140

Obesity is characterized by excessive fat accumulation, and isaccompanied by dyslipidaemia. One of the major organs involvedin metabolizing fats from the diet is the liver. Dysregulation of lipidmetabolism in the liver induces abnormal accumulation of lipids,and the subsequent formation of lipid droplets, known as hep-atosteatosis. Adipose tissue, an endocrine organ that secretes anumber of adipokines known to mediate lipid metabolism, inflam-mation, and insulin sensitivity, is also critical in metabolic control(Kwon, Jung, Park, Yun, & Choi, 2015).

Paprika (Capsicum annuum L., sweet pepper) is often eaten as afresh or cooked vegetable, and is widely used as a natural foodcolouring (Jeong, Ko, Cho, Ahn, & Shim, 2006). Paprika was intro-duced to Korea in the early 1990s, and has since become a primaryexport item in Korean agriculture (Jeong, Kim, Kim, & Yun, 2008).There are many different varieties of paprika with different shapesand colours, and paprika is considered to be a good source of notonly carotenoid pigments, but also phytochemicals, such as vita-mins, minerals, and flavonoids (Kim, Ahn, Ha, Rhee, & Kim, 2011;Kim, An, Park, Lim, & Kim, 2016; Kim, Ahn, Lee et al., 2011;Matsufuji, Ishikawa, Nunomura, Chino, & Takeda, 2007). Amongthe different varieties, red paprika as a ripe fruit is preferred byconsumers, and capsanthin, one of the xanthophylls, is the majorcarotenoid present in red paprika (Oshima, Sakamoto, Ishiguro, &Terao, 1997). In paprika, capsanthin exists in a form where it isacylated with fatty acids, and as a result, it contains oxygen andis more polar than b-carotene; the different structures of capsan-thin may exert different functional effects in the human body(Kim, Lee, Rhee, & Kim, 2016). Capsanthin is effective as a free-radical scavenger (Bendich & Olson, 1989), exhibits anti-tumoractivity (Maoka, Enjo, Tokuda, & Nishino, 2004), attenuatesobesity-induced inflammation (Maeda, Saito, Nakamura, &Maoka, 2013), and raises plasma high-density lipoprotein (HDL)cholesterol levels (Aizawa & Inakuma, 2009). However, the effectsof red paprika and capsanthin on dysregulated lipid metabolism inthe liver and adipose tissue of diet-induced obese mice have notyet been fully elucidated.

In this study, we investigated whether red paprika and capsan-thin have protective effects on diet-induced obesity. We also mea-sured the expression of genes involved in adipogenesis, lipolysis, b-oxidation, and inflammation in the liver and epididymal white adi-pose tissue (WAT).

2. Material and methods

2.1. Carotenoid analysis and sample preparation

The red paprika (Capsicum annuum L. var. Special) used in thisstudy was provided by the Korea Paprika Growers Association(Daejeon, Korea).

For carotenoid analysis, capsorubin, violaxanthin, capsanthin,lutein, zeaxanthin, b-cryptoxanthin, and b-carotene were pur-chased from ChromaDex (Irvine, California, USA). HPLC-grademethanol (MeOH), acetone, and water were purchased from Bur-dick & Jackson (Kuskegon, MI, USA). All other chemicals were pur-chased from Sigma-Aldrich (St. Louis, MO, USA). Carotenoids weredetermined by high-performance liquid chromatography (HPLC)coupled to a UV/VIS detector (Jasco, Tokyo, Japan) and XTerraRPC18 column (250 � 4.6 mm, 5 lm; Waters, Milford, USA) (Kim,Ahn, Lee et al., 2011). Briefly, powdered red paprika was extractedusing acetone until the colour had completely disappeared, andthen the acetone extracts were saponified using 30% KOH in MEOHfor 18 h. Carotenoids were determined by high-performance liquidchromatography coupled to a UV/VIS detector and XTerra RP C18column. The carotenoids were detected at 450 nm. Mobile phasesconsisted of 15% water/MeOH (v/v) (A) and 50% acetone/MeOH(v/v) (B) as gradient system for 47 min at a flow rate of 1.5 mL/min.

For preparation of the experimental diet, after washing andremoving the seeds and placenta, the whole fruit was freeze-dried and ground into a powder. Capsanthin (purity: colour value100), the main carotenoid of red paprika, was purchased fromLKT Laboratories, Inc. (St. Paul, MN, USA).

2.2. Animals and diets

Forty male C57BL/6J mice aged 4 weeks were purchased fromORIENT, Inc. (Seongnam, Korea). The animals were housed individ-ually in plastic cages in a room with controlled temperature(23 ± 1 �C), and maintained on a reverse 12 h light/dark cycle. After1 week of acclimation, the mice were randomly divided into fourgroups, and fed experimental diets for 8 weeks: normal diet (ND,5% fat, n = 10), high-fat diet (HD, 15% fat and 1% cholesterol,n = 10), HD containing 10% (w/w) powdered red paprika (RP,n = 10) and HD containing 0.025% (w/w) capsanthin (CS, n = 10).The animals were given food and distilled water ad libitum duringthe experimental period. The compositions of the experimentaldiets (Supplementary Table 1) were based on the AIN-76 semi-synthetic diet (American Institute of Nutrition, 1977, 1980). Atthe end of the experimental period, the mice were sacrificed fol-lowing a 12 h fast, and blood was collected from the orbital venouscongestion. After cervical dislocation, the liver, epididymal andperirenal WAT were immediately removed and weighed. All ofthe samples were stored at �70 �C until analysed. All of the ani-mals received care according to institutional guidelines, and allof the experiments were approved by the KFRI Institutional AnimalCare and Use committee.

2.3. Histological examination

Isolated livers and epididymal WAT were fixed in a buffer solu-tion of 5% formalin, and embedded in paraffin. Sections with athickness of 4 lm were prepared and stained with hematoxylin-eosin. The stained areas were viewed using a light microscope witha magnifying power of �200. The lipid droplet size in the epididy-mal WAT was measured using Image J (National Institute of Health,Maryland, USA).

2.4. Biochemical analysis of serum and liver tissue

The concentration of triacylglycerol (TG), total cholesterol (TC),high-density lipoprotein (HDL) cholesterol, non-esterified fattyacid (NEFA) and glucose in the serumwas determined using a com-mercial kit (SHINYANG Chemical Co., Ltd., Seoul, Korea). The serumlevel of low-density lipoprotein (LDL) cholesterol was calculatedusing the Friedewald formula (Friedewald, Levy, & Fredrickson,1972). Serum levels of oxidized-LDL (ox-LDL) and insulin weredetermined using commercial mouse ELISA kits (USCN Life ScienceInc., Wuhan, China, and Shibayagi Co., Ltd., Gunma, Japan, respec-tively). Serum levels of apolipoprotein A-1 (Apo A-1), apolipopro-tein B (H) (Apo B (H)), and lipoprotein A (Lp-A) were determinedusing a commercial kit (Nittobo Medical Co., Ltd., Kyoto, Japan).The homeostatic index of insulin resistance (HOMA-IR) was calcu-lated using the equation: [fasting insulin concentration (mU/L) �fasting glucose concentration (mg/dL) � 0.05551]/22.5 (Wallace,Levy, & Matthews, 2004). Leptin, adiponectin, and tumor necrosisfactor-a (TNF-a) were measured using a mouse ELISA kit (R&D Sys-tems, Minneapolis, MN, USA). The plasminogen activator inhibitor-1 (PAI-1) level was quantified using an ELISA kit (InnovativeResearch, Inc., MI, USA). The concentrations of interlukin-6 (IL-6)and interlukin-1b (IL-1b) were determined using ELISA kits (Invit-rogen Co. Ltd., Camarillo, CA, USA).

The hepatic lipids were extracted as described previously(Folch, Lees, and Sloan-Stanley, 1957). The TG and cholesterol

Capsanthin

Capsorubin

Violaxanthin

Lutein

Zeaxanthin

β-Carotene

β-Cryptoxanthin

(A)

(B)

Fig. 1. Chemical structures and chromatogram of carotenoids identified in the Special cultivar of red paprika. (A) Chemical structures of carotenoids. (B) Chromatogram of redpaprika extracts. peak 1, capsorubin; peak 2, violaxanthin; peak 3, capsanthin; peak 4, lutein; peak 5, zeaxanthin; peak 6, b-cryptoxanthin; peak 7, b-carotene.

J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140 133

134 J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140

concentrations in the liver were analysed using the same enzy-matic kit as used for the serum analysis.

The hepatic cytosolic and microsomal fractions were preparedusing 0.25 M sucrose/0.5 M EDTA buffer (pH 7.4) and 1.15% KClsolution, respectively. The activities of glucose-6-phosphate dehy-drogenase (G6PDH), malic enzyme (ME) (Baginski, Foa, & Zak,1974; Ochoa, 1969), catalase (Aebi, 1974), and superoxide dismu-tase (SOD) (Marklund & Marklund, 1974) were measured in thecytosolic fraction. Protein concentration was determined by theLowry method (Lowry, Rosebrough, Farr, & Randall, 1951). Theconcentration of malondialdehyde (MDA), a product of lipid perox-idation, in the microsomes was determined by reacting with thio-barbituric acid, and measuring the absorbance at 532 nm (Ohkawa,Ohishi, & Yagi, 1979).

2.5. Quantitative RT-PCR

Total RNA was extracted from the liver and epididymal WATusing a NucleoSpin RNAII kit (Macherey-Nagel, Düren, Germany).The tissue was homogenized using a FastPrep�-24 Sample Prepara-tion System (MP Biomedicals, Irvine, CA, USA), and the total RNAwas isolated according to the manufacture’s protocol. The RNAwas then diluted with RNase-free water. The RNA quantity andquality were determined using an ND-1000 spectrophotometer(NanoDrop Technologies, Wilmington, DE, USA). After cDNA syn-thesis (Maxime RT PreMix Kit with Random Primer; iNtRONBiotechnology, Seongnam, Korea), real-time PCR amplificationwas conducted using a Light Cycler� 480 real-time PCR system(Roche, Mannheim, Germany). SYBR� Green real-time PCR MasterMix (TOYOBO Co. Ltd., Osaka, Japan) was used to quantify thedesired PCR products in real-time. The real-time PCR reaction mix-ture consisted of cDNA, master mix, and forward and reverse pri-mers (Supplementary Table 2). Three repeats were performed,and the mRNA level of each sample was normalized to the b-actin mRNA level.

Table 1Carotenoid content in Special cultivar of red paprika.a

2.6. Western blotting

The liver and epididymal WAT were lysed with ice-cold RIPAbuffer (Ahn, Cho, Kim, Kwon, & Ha, 2008). The protein content ofthe samples was determined by the Bradford method, using a pro-tein assay kit (Bio-Rad, Richmond, CA, USA). Equal amounts of pro-tein (30 lg) were separated using SDS-PAGE, and then transferredto a PVDF membrane. The membrane was then blocked with Tris-buffered saline containing 5% skim milk and 0.1% Tween 20(Amresco Inc., Solon, OH, USA), and probed with the following anti-bodies: anti-phospho-AMPK, anti-AMPK (Cell Signaling Technol-ogy, Inc., Beverly, MA, USA), and anti-b-actin (Sigma-Aldrich, St.Louis, MO, USA). The membranes were then washed, and incubatedwith a horseradish peroxidase-conjugated secondary antibody for1 h at room temperature. Immunodetection was carried out usingECL detection reagent (Amersham Biosciences, Uppsala, Sweden).All of the figures showing results of quantitative analysis (ImageJ, National Institutes of Health) include data from at least threeindependent experiments.

Carotenoids Contents

Capsorubin 2.54 ± 0.30Violaxanthin 0.23 ± 0.03Capsanthin 50.49 ± 0.46Lutein 3.58 ± 0.46Zeaxanthin 0.21 ± 0.06b-Cryptoxanthin 0.62 ± 0.15b-Carotene 4.08 ± 0.35Total carotenoids, mg/100 g dry weight 61.75 ± 0.47

a Each value represents the mean ± standard deviation (SD) of three independentreplications.

2.7. Statistical analysis

The results are expressed as the mean ± standard error of themean (SEM). Statistical analyses were performed using SAS version8.0 for Windows (SAS Inst., Cary, NC, USA). One-way analysis ofvariance (ANOVA) and Duncan’s multiple-range test were used tocompare the quantitative data from the different groups. P < 0.05was considered statistically significant.

3. Results

3.1. Quantification of carotenoids

Carotenoid profile and contents were analysed using HPLC, aspreviously described (Kim, Ahn, Lee et al., 2011). We identifiedseven carotenoid peaks in the order of capsorubin, violaxanthin,capsanthin, lutein, zeaxanthin, b-cryptoxanthin, and b-carotene.The structures of each carotenoid and carotenoid compositions ofred paprika are shown in Fig. 1. The carotenoid contents of redpaprika are shown in Table 1. The total carotenoid content of redpaprika was 61.75 ± 0.47 mg/100 g dry weight (dw), and capsan-thin (50.49 ± 0.46 mg/100 g dw) was the main carotenoid in redpaprika.

3.2. Effects of RP and CS on body weight, food intake, and organ weight

The changes in the body weights of the mice during the 8-weekexperimental period are shown in Fig. 2A. The initial average bodyweight was 17.52 ± 0.21 g. After 8 weeks, the body weights of theND and HD groups increased to 22.78 ± 0.28 g and 26.35 ± 0.58 g,respectively. The RP and CS groups showed significantly lessweight gain. RP was especially effective in inhibiting HF-inducedweight gain, despite the increased food intake (Fig. 2B and C) andinhibited hypertrophy of the liver and WAT (Fig. 2D). The CS groupexhibited decreased weights of the liver, similar to the ND group.These data indicate that diet-induced obesity was inhibited inthe RP and CS groups.

After 8 weeks, HD-induced hypertrophy and lipid accumulationin the liver and epididymal WAT were ameliorated in the RP and CSgroups. H&E staining confirmed that storage of hepatic fat and epi-didymal WAT in lipid droplets obviously decreased in the RP andCS groups (Fig. 2E). The RP and CS groups exhibited significantlydecreased epididymal adipocyte size, compared to that reportedfor the HD group (Fig. 2F).

3.3. Effects of RP and CS on serum lipid profile, glucose, insulin, andadipokines

The serum biochemical values associated with lipid metabolismare shown in Table 2. The RP and CS groups showed significantlydecreased TG (32% and 25%, respectively) and TC (41% and 26%,respectively) levels compared to the values reported for the HDgroup. However, there was no significant difference in the serumHDL-cholesterol level among these groups. The serum levels ofLDL, oxLDL-cholesterol, Apo A-1, Apo B (H), Lp-A, and NEFA signif-icantly decreased in the RP and CS groups, compared to those inthe HD group. The elevated glucose and insulin levels in the HDgroup decreased in the RP and CS groups. HOMA-IR analysis indi-cated that HD-induced insulin resistance was ameliorated in the

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Fig. 2. Effects of RP and CS on body weight, food intake, and organ weight. (A) Change of body weight during the experimental period. (B) Food intake. (C) Weight gain. (D)Organ weight. (E) Representative images of the liver gross appearance (top row), along with histological sections after staining with hematoxylin and eosin viewed at amagnification �200 in the liver (middle row) and epididymal WAT (bottom row). (F) Measurement of lipid droplet size in epididymal WAT. Values (means ± SEM; n = 10) notsharing a common letter are significantly different (P < 0.05). ND, normal diet; HD, high fat diet; RP, red paprika-supplemented HD; CS, capsanthin-supplemented HD.

J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140 135

RP and CS groups. RP and CS also reversed dysregulation of serumadipokines, such as leptin, adiponectin, PAI-1, TNF-a, IL-6, andIL-1b. These data indicate that RP and CS improved HD-induceddyslipidaemia, and reversed the changes in serum adipokine levels.

3.4. Effects of RP and CS on hepatic lipid profile and enzyme levels

The hepatic lipid profile and enzyme levels are shown in Table 3.After 8 weeks, the hepatic total lipid, TG, and cholesterol content

136 J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140

markedly decreased in the RP and CS groups. The activities of thehepatic enzymes involved in FA and TG synthesis (G6PDH andME) significantly decreased in the RP and CS groups, comparedwith the values reported for the HD group. In the case of hepaticantioxidant enzymes, RP and CS significantly suppressed SODactivity, with no difference in catalase activity. The TBARS levelincreased in the HD group, and recovered in the RP and CS groups.These data suggest that RP and CS reduced HD-induced hep-atosteatosis by regulating lipogenesis and anti-oxidant activity.

3.5. Effects of RP and CS on hepatic mRNA and protein expression

We examined the expression of a number of genes involved inhepatic lipid metabolism (Fig. 3). The RP and CS groups exhibitedsignificantly decreased expression of mRNAs associated with lipiduptake and transport (CD36 and FABP-1), and reduced the expres-sion of genes involved in fatty acid oxidation (LCAD, MCAD, and

Table 3Effects of RP and CS on the lipid profile, activities of lipogenic and antioxidant enzymes, a

ND

Lipid profileTotal lipid, mg/g liver 72.63 ± 3.34d

Triacylglycerol, mg/g liver 32.98 ± 1.07d

Cholesterol, mg/g liver 11.21 ± 0.42d

Lipogenic enzyme activityG6PDH2, lmole/min/mg hepatic protein 1.99 ± 0.12b

ME3, lmole/min/mg hepatic protein 0.62 ± 0.04b

Antioxidant enzyme activity and lipid peroxidation levelCatalase, nmole/min.mg protein 32.44 ± 0.61NS7

SOD4, unit/min.mg protein 36.78 ± 1.12a

TBARS5, MDA6 nmole/g tissue 79.92 ± 4.74b

1 Values (means ± SEM; n = 10) in a row not sharing a common superscript are signifi2 G6PDH, glucose-6-phosphate dehydrogenase.3 ME, malic enzyme.4 SOD, superoxide dismutase.5 TBARS, thiobarbituric acid reactive substances.6 MDA, malondialdehyde.7 NS, not significant.

Table 2Effects of RP and CS on serum lipid profile, glucose, insulin, and adipokines.1

ND HD

Lipid profile, glucose and insulinTriacylglycerol, mg/dL 66.18 ± 2.54b 85Total cholesterol, mg/dL 90.03 ± 2.74c 16HDL-cholesterol, mg/dL 50.43 ± 2.46NS7 45LDL-cholesterol, mg/dL2 34.22 ± 1.05c 10oxLDL-cholesterol, ng/mL 57.18 ± 1.04b 68Apolipoprotein A1, mg/dL 5.08 ± 0.42a 2.2Apolipoprotein B(H), mg/dL 0.65 ± 0.21b 2.2Lipoprotein A, mg/dL 21.46 ± 1.84b 51NEFA3, mEq /L 1.51 ± 0.05c 1.8Glucose, mg/dL 163.22 ± 10.73b 22Insulin, lU/mL 7.91 ± 0.63c 16HOMA-IR 4 2.62 ± 0.43c 6.1

AdipokinesLeptin, ng/mL 4.68 ± 0.38c 9.7Adiponectin, ng/mL 10.01 ± 0.21a 8.7PAI-15, pg/mL 22.97 ± 0.28c 31TNF-a6, ng/mL 0.90 ± 0.08b 1.8Interlukin-6, ng/mL 0.27 ± 0.01b 0.3Interlukin-1b, ng/mL 1.49 ± 0.04b 1.6

1 Values (means ± SEM; n = 10) in a row not sharing a common superscript are signifi2 LDL-cholesterol was calculated as total cholesterol - [(HDL cholesterol - triglyceride3 NEFA, non-esterified free fatty acids.4 HOMA-IR, homeostatic index of insulin resistance, was calculated as [fasting insulin5 PAI-1, plasminogen activator inhibitor-1.6 TNF-a, tumor necrosis factor-a.7 NS, not significant.

PDK-4), lipogenesis (SREBP-1c, ACC, FAS, and SCD-1), glucosemetabolism (PEPCK4 and G6Pase), and bile acid metabolism(CYP7A1), compared with the values reported for the HD group.In contrast, the expression of PPARa, PGC-1b, and Sirt-1 mRNA sig-nificantly increased in the RP and CS groups, compared to thatreported for the HD group. Western blot analysis revealed thatphosphorylation of AMPK significantly increased in the RP and CSgroups, compared to that reported for the HD group (Fig. 3F). Thesedata confirmed that RP and CS reversed dysregulation of lipidmetabolism by regulating genes involved in lipid metabolism.

3.6. Effects of RP and CS on adipocyte size and mRNA expression

We examined the effects of RP and CS on the genes involved inlipid metabolism in the epididymal WAT. The expression ofadipogenesis-associated genes, such as PPARc, C/EBPa, SREBP-1c,FAS, and aP2, significantly decreased in the RP and CS groups,

nd lipid peroxidation level in the liver1

HD RP CS

204.71 ± 8.65a 108.18 ± 6.08c 128.39 ± 4.92b

75.39 ± 3.36a 38.62 ± 2.32c 49.95 ± 2.92b

17.22 ± 0.83a 13.09 ± 0.39c 13.41 ± 0.40b

5.88 ± 0.35a 1.75 ± 0.63b 1.09 ± 0.12b

1.06 ± 0.07a 0.42 ± 0.02c 0.39 ± 0.02c

34.59 ± 0.43 34.36 ± 1.50 34.37 ± 1.2328.22 ± 0.51b 38.55 ± 1.95a 34.28 ± 1.46a

111.40 ± 3.07a 78.23 ± 3.80b 78.36 ± 1.85b

cantly different (P < 0.05).

RP CS

.36 ± 5.22a 57.78 ± 5.51b 64.43 ± 1.61b

1.97 ± 6.54a 94.99 ± 4.22c 119.56 ± 5.39b

.02 ± 4.25 47.14 ± 2.15 48.36 ± 2.624.47 ± 2.85a 43.94 ± 2.53b 50.94 ± 3.11b

.44 ± 1.97a 60.09 ± 0.78b 59.85 ± 2.20b

1 ± 0.28b 4.33 ± 1.19a 4.86 ± 0.36a

5 ± 0.58a 0.77 ± 0.27b 0.88 ± 0.13b

.96 ± 3.93a 26.74 ± 3.15b 26.82 ± 2.77b

8 ± 0.04a 1.63 ± 0.03b 1.64 ± 0.04b

1.88 ± 11.88a 174.95 ± 3.73b 177.44 ± 5.11b

.11 ± 0.40a 13.70 ± 0.52b 14.5 ± 0.41b

3 ± 0.50a 3.90 ± 0.29b 4.77 ± 0.43b

7 ± 0.93a 5.77 ± 1.02bc 7.86 ± 1.10b

8 ± 0.23b 10.03 ± 0.23a 9.54 ± 0.29a

.57 ± 1.60a 26.20 ± 0.49bc 29.71 ± 1.50ab

8 ± 0.14a 0.77 ± 0.04b 0.93 ± 0.04b

1 ± 0.01a 0.27 ± 0.01b 0.30 ± 0.01ab

9 ± 0.06a 1.46 ± 0.01b 1.44 ± 0.03b

cantly different (P < 0.05).)/5].

� fasting glucose concentration � 0.05551]/22.5.

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Fig. 3. Effects of RP and CS on hepatic gene expression. The mRNA expression of genes associated with (A) lipid uptake and transport, (B) fatty acid oxidation, (C) lipogenesis,(D) glucose metabolism, and (E) bile acid and mitochondrial metabolism were measured by quantitative real-time PCR relative to b-actin (n = 10). (F) The expression of totaland phosphorylated AMPK was measured relative to b-actin by western blotting. Total protein samples (30 lg) were prepared as described in the Materials and Methodssection. Values (means ± SEM; n = 4) not sharing a common letter are significantly different (P < 0.05). ND, normal diet; HD, high fat diet; RP, red paprika-supplemented HD;CS, capsanthin-supplemented HD.

J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140 137

α

SREBP-1c

AMPK

SCD-1

FAS

ACC

P

SIRT1

PGC-1

PPAR

Fatty acid oxidation

Lipogenesis

Adipogenesis

PEPCK

G6Pase

Gluconeogenesis

Steatosis Lipid droplet

SREBP-1c

FAS CEBPaP2

PPAR

Fig. 5. Summary of the beneficial effects of red paprika and capsanthin in improving impaired lipid metabolism in the liver and white adipose tissue of high-fat diet-inducedobese mice.

(B)(A)

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Fig. 4. Effects of RP and CS on gene expression of epididymal WAT. The mRNA expression of genes associated with (A) adipogenesis, and (B) adipocyte-derived hormonalfactors were measured by quantitative real-time PCR relative to b-actin (n = 10). Values (means ± SEM; n = 4) not sharing a common letter are significantly different (P < 0.05).ND, normal diet; HD, high fat diet; RP, red paprika-supplemented HD; CS, capsanthin-supplemented HD.

138 J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140

compared to that observed for the HD group. In addition, the levelsof adipocyte-derived hormonal factors, such as leptin and resistin,were significantly decreased by RP and CS supplementation(Fig. 4A and B). These data suggest that RP and CS effectivelydecreased HD-induced WAT hypertrophy by inhibiting adipogene-sis and improving adipokine secretion.

4. Discussion

In this study, we demonstrated that red paprika, and its maincarotenoid, capsanthin, attenuated diet-induced obesity in

C57BL/6Jmice. Capsanthin, one of the xanthophylls, has eleven con-jugated double bonds ending in one or two polar ketones and effi-ciently absorbs green light to give a red-orange hue (Guzman,Hamby, Romero, Bosland, & O’Connell, 2010; Shah, Shi-Lin, Gong,& Arisha, 2014). Carotenoids such as xanthophylls are lipid-soluble molecules that follow the absorption pathway of dietaryfats (Fraser & Bramley, 2004; Sajilata, Singhal, & Kamat, 2008). Cap-santhin is a polar carotenoid and is likely to be localized at the polarsurface of lipoproteins consisting of phospholipids and apoprotein(Fraser & Bramley, 2004; Oshima et al., 1997). Red paprika and cap-santhin treatments resulted in improvements in serum lipid profile,adipokine secretion, hepatic steatosis and adipogenesis. Therefore,

J.-S. Kim et al. / Journal of Functional Foods 31 (2017) 131–140 139

our data suggest that red paprika and capsanthin have anti-obesityand hepatoprotective capabilities, and are effective in amelioratingthe effects of a high-fat diet in mice.

The total carotenoid, ascorbic acid, and tocopherol content inred paprika is higher than that in paprika of other colours. Amongthem, red paprika is known to have a high capsanthin content(Oshima et al., 1997). We determined that the capsanthin contentwas approximately 81% of total carotenoid content and capsanthinwas the main carotenoid in red paprika. In a previous report, cap-santhin content in the ‘‘Special” cultivar of red paprika was58.33 ± 3.91 mg/100 g dw (Kim, Ahn, Lee et al., 2011). Further-more, the antioxidant activity of red paprika is higher than thatof paprika of other colours, and capsanthin has higher antioxidantactivity than b-carotene does (Kim, Ahn, Ha et al., 2011; Kim, Ahn,Lee et al., 2011). These differences are related to different struc-tures having different oxygen quenching abilities, due to differ-ences in the number of conjugated double bonds, the chainstructure, and the functional groups in the structures (Hirayama,Nakamura, Hamada, & Kobayasi, 1994). In the ripe fruits of redpaprika, capsanthin is esterified with fatty acids. The amount ofesterified capsanthin increases as the fruit ripens, and in the ripefruit, esterified capsanthin accounts for 70–80% of the total capsan-thin (Minguez-Mosquera & Hornero-Mendez, 1994). Hepaticsteatosis is an early and simple form of fatty liver disease, andaccumulation of TG-rich lipid droplets within hepatocytes canoccur without hepatic inflammation or evidence of injury. In ani-mal models, hepatic steatosis appears when the hepatic TG contentexceeds 55 mg/g of liver, or cytoplasmic lipid droplets are presentin more than 5% of hepatocytes (Cohen, Horton, & Hobbs, 2011;Szczepaniak et al., 2005). A high-fat diet is a useful tool to inducemetabolic alterations and NAFLD (Kanuri & Bergheim, 2013). Inthe present study, the hepatic TG content in the HD group was75.39 ± 3.36 mg/g of liver, confirming that the experimental designresulted in obesity, including hepatic steatosis. Hepatic TG forma-tion is related to diet, de novo lipogenesis, and adipose tissue lipol-ysis (Bedogni et al., 2005; Fan, 2013; Gan, Xiang, Xie, & Yu, 2015;Masarone, Federico, Abenavoli, Loguercio, & Persico, 2014). In thisstudy, the red paprika and capsanthin treatments significantlydecreased the level of hepatic TG, and upregulated PPARa, therebydecreasing fatty acid synthesis-associated gene expression (SREBP-1c, ACC, FAS, and SCD-1), resulting in decreased lipogenesis. Thesetreatments also decreased expression of the FA uptake proteinCD36 in the liver. Furthermore, hepatic activation of AMPK throughphosphorylation promotes mitochondrial b-oxidation and sup-presses fatty acid synthesis (Foretz & Viollet, 2011; Hardie,2008). In this study, we confirmed that red paprika and capsanthintreatments induced AMPK phosphorylation in liver tissues.

Adipose tissues also play a crucial role in fatty acid mobilizationand fat storage. The mobilization and transport of free fatty acidsfrom adipose tissue to the liver may amplify hepatic lipogenesis,leading to hepatic steatosis (Browning & Horton, 2004; Byrne,2010). PPARc and CEBPa are the key transcription factors involvedin adipocyte differentiation and lipid accumulation (Shao & Lazar,1997), and regulate the expression of adipogenic genes(Schoonjans, Martin, Staels, & Auwerx, 1997). In the present study,the red paprika and capsanthin treatments significantly altered theexpression of adipogenic genes, such as PPARc, CEBPa, SREBP-1c,FAS, and aP2. In addition, the red paprika treatment significantlyattenuated impaired lipid metabolism in the liver and WAT.

It is well known that abnormal lipid metabolism in obesity is amajor cause of dyslipidaemia, insulin resistance, inflammation, andhepatic steatosis (Jung, Cho, & Choi, 2016; Kim, Choi et al., 2016).Insulin is one of the most important hormones. It inhibits glyco-neogenesis, and directly suppresses the transcription and activityof hepatic gluconeogenic enzymes, such as PEPCK and G6Pase(Barthel & Schmoll, 2003). In this study, the red paprika and cap-

santhin treatments significantly decreased the levels of glucose,insulin, and HOMA-IR, a substitute marker for insulin resistance,in the high-fat diet-fed mice. In addition, the hepatic mRNAexpression of PEPCK and G6Pase was significantly decreased bythe red paprika and capsanthin treatments. Inflammation is oneof the main causes of impaired insulin action (Shoelson, Lee, &Goldfine, 2006). The production of adipokines is related to ectopicfat accumulation, and decreased insulin sensitivity in the liver andadipose tissue. Interestingly, the red paprika and capsanthin treat-ments ameliorated the dysregulation of serum adipokines, such asleptin, adiponectin, PAI-1, TNF-a, IL-6, and IL-1b. In addition, thered paprika and capsanthin treatments significantly reduced themRNA expression of leptin and resistin in epididymal WAT. TNF-a and IL-6 play a role in insulin resistance in obesity by inhibitinginsulin receptor signalling (Jarrar et al., 2008). The expression ofleptin may be of importance in the regulation of body fat(Schoonjans et al., 1997; Zhang et al., 1994). Resistin is expressedin adipocytes, and encodes a protein proposed to link obesity andtype 2 diabetes (Schoonjans et al., 1997; Steppan et al., 2001).

5. Conclusion

In conclusion, we showed that red paprika, and its main carote-noid, capsanthin, exert protective effects against lipid accumulationin the liver and WAT via modulation of genes involved in lipogene-sis, b-oxidation, and adipogenesis, as summarized in Fig. 5. Theseresults suggest that red paprika and capsanthin may exert helpfulprotective effects against diet-induced lipid accumulation. As aresult, we suggest that dietary intake of fresh red paprika containingcapsanthin might be helpful for lowering the risk of deteriorativediseases caused by high fat and cholesterol consumption.

Acknowledgment

This research was supported by Golden Seed Project, Ministry ofAgriculture, Food and Rural Affairs (MAFRA), Ministry of Oceansand Fisheries (MOF), Rural Development Administration (RDA),Korea Forest Service (KFS) and Korea Food Research Institute(KFRI).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jff.2017.01.044.

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Red paprika (Capsicum annuum L.) and its main carotenoid capsanthin ameliorate impaired lipid metabolism in the liver and adipose tissue of high-fat diet-induced obese mice1 Introduction2 Material and methods2.1 Carotenoid analysis and sample preparation2.2 Animals and diets2.3 Histological examination2.4 Biochemical analysis of serum and liver tissue2.5 Quantitative RT-PCR2.6 Western blotting2.7 Statistical analysis

3 Results3.1 Quantification of carotenoids3.2 Effects of RP and CS on body weight, food intake, and organ weight3.3 Effects of RP and CS on serum lipid profile, glucose, insulin, and adipokines3.4 Effects of RP and CS on hepatic lipid profile and enzyme levels3.5 Effects of RP and CS on hepatic mRNA and protein expression3.6 Effects of RP and CS on adipocyte size and mRNA expression

4 Discussion5 ConclusionAcknowledgmentAppendix A Supplementary materialReferences