Oleuropein attenuates hepatic steatosis induced by high-fat diet in mice

Research Article

Oleuropein attenuates hepatic steatosis induced by high-fat dietin mice

Soyoung Park1, Youngshim Choi1, Soo-Jong Um2, Seung Kew Yoon3, Taesun Park1,⇑

1Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul 120-749, Republic ofKorea; 2Department of Bioscience and Biotechnology/Institute of Bioscience, Sejong University, Republic of Korea; 3Department of Internal

Medicine, College of Medicine, The Catholic University, Republic of Korea

Background & Aims: Oleuropein, a secoiridoid derived from signaling molecules (TLR2, TLR4, and myeloid differentiation pri-
olives and olive oil, has been known to possess antimicrobial,antioxidative, and anticancer activities. The purpose of the pres-ent study was to determine whether oleuropein has a protectiveeffect against hepatic steatosis induced by a high fat diet (HFD)and to elucidate its underlying molecular mechanisms in mice.Methods: Male C57BL/6N mice were fed a normal diet (ND), HFD,or an oleuropein-supplemented diet (OSD) for 10 weeks. Theplasma and hepatic lipid levels were determined, and the hepaticgene and protein expression levels were analysed via RT-PCR andWestern blotting, respectively.Results: The supplementation of HFD with oleuropein reversedthe HFD-induced increases in liver weight along with plasmaand hepatic lipid levels in mice. The expression of Wnt10b inhib-itor genes, such as secreted firizzed-related sequence protein 5and dickkopf homolog 2, was downregulated, whereas theb-catenin protein expression was upregulated in the liver ofOSD-fed mice compared to HFD-fed mice. Fibroblast growth factorreceptor 1 (FGFR1), phosphoextracellular-signal-regulated kinase1/2, cyclin D, and E2F transcription factor 1, along with several keytranscription factors and their target genes involved in adipogen-esis, were downregulated by oleuropein. OSD-fed mice exhibiteddecreased expression of the toll-like-receptor-(TLR)-mediated
Journal of Hepatology 20

Keywords: Oleuropein; Hepatic steatosis; High-fat diet; Lipogenesis; Inflammation.Received 9 January 2010; received in revised form 10 July 2010; accepted 15 August2010; available online 31 October 2010⇑Corresponding author. Tel.: +82 2 2123 3123; fax: +82 2 365 3118.E-mail address: [email protected] (T. Park).Abbreviations: NAFLD, non-alcoholic fatty liver disease; TG, triglyceride; FFA, freefatty acid; ALT, alanine aminotransferase; AST, aspartate aminostransferase; LXR,liver X receptor; SREBP-1c, sterol regulatory element-binding protein-1c; PPARc,peroxisome proliferator-activated receptors gamma; HFD, high fat diet; FAS, fattyacid synthase; TLR, toll-like-receptor; ND, normal diet; OSD, oleuropein-supple-mented diet; TC, total cholesterol; GAPDH, glyceraldehyde-3-phosphate dehy-drogenase; ERK, extracellular regulated MAP kinase; LPL, lipoprotein lipase; aP2,adipocyte protein 2; C/EBPa, CCAAT/enhancer binding protein alpha; Cyc-D, cy-clin D; E2F1, E2F transcription factor 1; CTSS, cathepsin S; SFRP5, secreted friz-zled-related sequence protein 5; DKK2, dickkopf homolog 2; BMPR, bonemorphogenetic protein receptor; FGFR1, fibroblast growth factor receptor 1; TL-R2, toll like receptor 2; TLR4, toll like receptor 4; MyD88, myeloid differentiationprimary response gene 88; TNFa, tumor necrosis factor a; IFNb, interferon beta;IL-1b, interleukin 1 beta; FZD receptors, frizzled receptors; LRP, low-density lip-oprotein-receptor-related protein; DSH, dishevelled; TCF/LEF, T cell factor/lym-phoid-enhancing factor; Shn2, schnurri 2; CD36, cluster of differentiation 36;a-SMA, a-smooth muscle actin; Fas, TNF receptor superfamily member 6; TRAIL,TNF-related apoptosis-inducing ligand.

mary-response gene 88) and proinflammatory cytokines, in theirlivers, as compared to HFD mice.Conclusions: These results suggest that the protective effects ofoleuropein against HFD-induced hepatic steatosis in mice appearto be associated with the Wnt10b- and FGFR1-mediated signalingcascades involved in hepatic lipogenesis, along with the TLR2-and TLR4-mediated signaling implicated in hepatic steatosis.� 2010 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Non-alcoholic fatty liver disease (NAFLD) can be considered as aspectrum of liver pathologies, with, on the one hand, simple ste-atosis with absence of necrosis or signs of inflammation and, onthe other hand, severe signs of inflammation with fibrosis or cir-rhosis [1]. An excessive and inappropriate dietary-fat intake,combined with peripheral insulin resistance, continued tri-glyceride (TG) hydrolysis via lipoprotein lipase, and other geneticalterations in the key lipid metabolic pathways, results inincreased blood free-fatty-acid (FFA) concentration [2], leadingto increased TG concentration in the liver. The in vivo activationof liver X receptor (LXR), sterol-regulatory-element-binding pro-tein 1c (SREBP1c), and peroxisome-proliferator-activated recep-tors gamma (PPARc) affects the lipid accumulation in the liverinduced by a high-fat diet (HFD) [3,4]. LXR has been shown toactivate SREBP1c [5], which stimulates the key lipogenic genes,including those encoding the acetyl-CoA carboxylase and thefatty-acid synthase [6,7]. In animal models with fatty liver, PPARcis transcriptionally upregulated and consequently activates thelipogenic target genes, thus exacerbating hepatic steatosis [8].

Oleuropein is a nontoxic secoiridoid derived from olives andolive oil, which influences their sensory organoleptic propertiesand is responsible for their typically bitter and pungent aroma[9]. This secoiridoid compound has a variety of biochemical roles,including antimicrobial [10], antioxidative [11], and anticancer[12] activities. Several in vitro studies have demonstrated thatoleuropein has a high antioxidant activity comparable to ahydrosoluble analog of tocopherol [11], and inhibits theproliferation and migration of various cancer cell lines, such asleukemia, melanoma, colon, breast, and kidney cancer cells, in a

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JOURNAL OF HEPATOLOGY
dose-responsive manner [12]. More recently, it has been reportedthat oleuroepin significantly decreased the body weight, body fataccumulation, and plasma TG concentrations in rats withdiet-induced obesity. Moreover, uncoupling-protein-1 contentsof the interscapular brown adipose tissue and rates of urinarynoradrenaline and adrenaline excretions were significantlydecreased in rats fed oleuropein as opposed to HFD-fed controlanimals [13]. Although a number of studies have been carriedout to investigate the biochemical roles of oleuropein, the protec-tive activity of oleuropein against NAFLD has never beenreported. Therefore, the purpose of the present study was todetermine whether oleuropein has a protective effect againsthepatic steatosis induced by a HFD in mice. The regulatory effectof oleuropein, on the expression of several key transcriptionfactors and their target genes involved in adipocyte differentia-tion, and toll-like-receptor-(TLR)-mediated signaling moleculesinvolved in metabolically triggered inflammation, were alsoinvestigated in a mouse model of HFD-induced hepatic steatosis.
Materials and methods

Animal care and experimental protocol

Male C57BL/6N mice (five-week-old) were purchased from Orient Bio(Gyeonggi-do, South Korea) and were housed in standard cages placed in aroom at 21 ± 2.0 �C temperature, 50 ± 5% relative humidity, and a 12 h-light/12 h-dark cycle. All mice consumed a commercial diet and tap water ad libitumfor 1 week prior to their division into three weight-matched groups (n = 8 pergroup): the normal diet (ND), HFD, and oleuropein-supplemented diet (OSD)groups. ND was a purified diet based on the AIN-76 rodent diet composition.HFD was identical to ND, except that 200 g fat/kg (170 g lard plus 30 g cornoil) and 1% cholesterol were added to it. Therefore, the HFD was calorically den-ser than the ND (4616 kcal/kg vs. 3929 kcal/kg). OSD was identical to HFD andcontained 0.03% (w/w) oleuropein (with approximately 80% purity; Extrasyn-these Genay, France). The experimental diets were given ad libitum for10 weeks, in the form of pellets.

The mice were weighed every 7 days and their food intake was recordeddaily during the feeding period. At the end of the experimental period, the ani-mals were anesthetized with ether, following 12-h fasting. Blood was drawnfrom the abdominal aorta into an EDTA-coated tube and the plasma wasobtained by centrifuging the blood at 2000g for 15 min at 4 �C. The entire liverwas dissected out, weighed, and snap frozen in liquid nitrogen prior to its stor-age at �70 �C.

The animals used in this study were treated in accordance with the Guide forthe Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources,Commission on Life Sciences, National Research Council, 1996), as approved byYonsei University’s Institutional Animal Care and Use Committee.

Biochemical analysis

The plasma concentrations of total cholesterol (TC), TG, and FFA were determinedenzymatically using commercial kits (Bio-Clinical System, Gyeonggi-do, SouthKorea). Hepatic lipids were extracted as described, using the method developedby Folch et al. [14], and dried lipid residues were dissolved in 2 ml ethanol. Theconcentrations of cholesterol, TG, and FFA in the hepatic-lipid extracts were mea-sured using the same enzymatic kits used for the plasma analysis. Alanine amino-transferase (ALT) and aspartate aminostransferase (AST) levels were determinedwith an automatic analyzer (Express Plus, Chiron Diagnostics, East Walpole,MA, USA).

Histological examination

The liver tissue specimens were fixed in 10% buffered formalin and embedded inparaffin, cut at thicknesses of 5 lm, and later stained with hematoxylin and eosinfor the histological examination of fat droplets. Steatosis and inflammation werenumerically scored following semi-quantitative pathological standards.


RNA extraction and semi quantitative RT-PCR

The total RNA was isolated from the liver tissue of each mouse, using Trizol (Invit-rogen, CA, USA), and reverse-transcribed using the Superscript II kit (Invitrogen,CA, USA), according to the manufacturer’s recommendations. The forward (F)and reverse (R) primers for mice genes are shown in Table 1. The PCR was pro-grammed as follows: 10 min at 94 �C, 30–33 cycles of 94 �C for 30 s, 55 �C for30 s; 72 �C for 1 min, and 10-min incubation at 72 �C. Four microliters of eachPCR reaction was mixed with 1-ll sixfold-concentrated loading buffer and wasloaded onto 2% agarose gel containing ethidium bromide. The measured mRNAlevels were normalized to the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) mRNA levels.

Western blot analysis

Liver tissues of each mouse were pooled and homogenized at 4 �C in an extractionbuffer containing 100 mM Tris–HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mMsodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100,1 mM phenylmethanesulohonylfluoride, 2 lg/ml aprotinin, 1 lg/ml pepstatin A,and 1 lg/ml leupeptin. The tissue homogenates were centrifuged (1300g,20 min, 4 �C) and the resulting supernatants (whole-tissue extracts) were usedfor the Western blot analysis. The total protein concentrations of the whole-tissueextracts were determined by Bradford assay (Bio-Rad, CA, USA). Protein sampleswere separated with 8% SDS–PAGE, transferred onto a nitrocellulose membrane(Amersham, Buckinghamshire, UK) and hybridized with primary antibodies(diluted 1:1000), overnight at 4 �C. The membrane was then incubated with therelative secondary antibody, and immunoreactive signals were detected usingthe chemiluminescent detection system (Amersham, Buckinghamshire, UK) andquantified using the Quantity One analysis software (Bio-Rad, CA, USA). Antibod-ies to the following proteins were purchased from the indicated sources: extracel-lular regulated MAP kinase (ERK), phospho-ERK (Thr202/Tyr204), and b-cateninfrom Cell-signaling Technology (Beverly, MA, USA); b-actin from Santa CruzBiotechnology (Santa Cruz, CA, USA).

Statistical analysis

Results were expressed as the mean ± SD of eight animals. The RT-PCR data werepresented as the mean ± SD of the triplicate analysis of the RNA samples pooledfrom eight mice per group. Comparisons among groups were made using one-way ANOVA. The differences between mean values in the three groups weretested through the Duncan’s multiple-range test and were considered significantwhen the p value was less than 0.05.

Results

Body and liver weights

After 10-week feeding, HFD-fed mice showed significantly higherfinal body weight and cumulative body weight gain compared toND-fed mice. Oleuropein supplemented to HFD significantlyreduced final body weight (ND, 42 ± 2.4 g vs. HFD, 30 ± 3.2 g vs.OSD, 28 ± 2.4 g) and body weight gain (ND, 8 ± 0.7 g vs. HFD,22 ± 1.9 g vs. OSD, 10 ± 2.2 g) in mice (Fig. 1A and B). Daily foodintake (2.7–2.9 g/day) did not differ among experimental groups.Since the HFD is calorically denser than the ND (4616 vs.3929 kcal/kg), the cumulative energy intake for 10 weeks was13% greater in HFD mice than in ND mice (872 vs. 770 kcal).The absolute and relative weights of the liver were significantlygreater in HFD-fed mice than in ND-fed mice. The addition ofoleuropein to HFD resulted in a significant reduction in the abso-lute weight of the liver compared to HFD-fed mice (ND,1.0 ± 0.07 g vs. HFD, 2.3 ± 0.26 g vs. OSD, 1.3 ± 0.22 g) (Fig. 1Cand D). Livers of HFD-fed mice were lighter in color than thoseof ND- and OSD-fed mice (Fig. 1E). The histopathological analysesshowed increased lipid deposition in livers of HFD-fed mice asopposed to those of ND-fed mice. Livers of OSD-fed mice showed

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Table 1. Primer sequences used for RT-PCR.

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Fig. 1. Effects of OSD on the body, liver weight, and liver histology of the experimental mice. (A) Changes in body weight, (B) body weight gain, (C) liver weight, and (D)relative weight of the liver. (E) Representative photographs, and (F) photomicrographs of the liver from the ND-, HFD-, and OSD-fed mice (magnification 100�). (G) Steatosisscore. (H) Inflammation score. The values are means ± SEM of eight mice. The values with different superscripts are significantly different at p <0.05.


a marked decrease in lipid accumulation compared to HFD-fedmice (Fig. 1F). HFD-induced hepatic steatosis was significantlyreduced in OSD-fed mice, as shown by the decrease in the steato-


sis score (ND, 1.0 ± 0.50 vs. HFD, 5.3 ± 0.20 vs. OSD, 1.77 ± 1.15)(Fig. 1G). Although the inflammation score followed the samepattern shown in the steatosis score of liver tissues, the

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Fig. 2. Effect of long-term oleuropein treatment on the plasma and liver biomarkers of the mice fed HFD for 10 weeks. (A) Plasma total cholesterol, (B) plasmatriglyceride, (C) plasma free fatty acid, (D) liver cholesterol, (E) liver triglyceride, and (F) liver free fatty acid. The values are means ± SD of eight mice. The values withdifferent superscripts are significantly different at p <0.05.

Research Article

differences among groups were not statistically significant (ND,1.0 ± 0.5 vs. HFD, 2.0 ± 0.58 vs. OSD, 0.9 ± 0.58) (Fig. 1H).

Plasma and hepatic-lipid levels

HFD-fed mice showed significantly higher levels of plasma TC,TG, and FFA and of hepatic cholesterol, TG, and FFA than didthe ND-fed mice. Oleuropein-supplemented HFD significantlyattenuated this HFD-induced elevation in plasma concentrationsof TC, TG, and FFA. HFD-induced elevations in plasma activities ofALT and AST were significantly reversed by supplementing theHFD with oleuropein (Fig. 2A). The hepatic accumulation of cho-lesterol, TG, and FFA induced by the HFD was also significantly

988 Journal of Hepatology 201

alleviated by feeding the mice oleuropein (Fig. 2B). These resultsindicate that the dietary supplementation of oleuropein in HFDhad marked effects on the improvement of both the blood andhepatic lipid levels, in the experimental mice.

Expression of hepatic genes related to lipogenesis and fibrosis

HFD-fed mice were characterized by drastic increases in thehepatic mRNA expression of LXR, PPARc2, lipoprotein lipase(LPL), adipocyte protein 2 (aP2), CCAAT/enhancer-binding-protein alpha (C/EBPa), cyclin D (Cyc-D), E2F transcriptionfactor 1 (E2F1), cathepsin S (CTSS), secreted frizzled-relatedsequence protein 5 (SFRP5), dickkopf homolog 2 (DKK2), bone

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Fig. 3. Effect of dietary oleuropein supplementation on the expression of genes involved in lipogenesis and liver fibrosis in the liver of HFD-fed mice. The mRNAexpression level was measured via RT-PCR. (A) LXR, PPARc2, LPL, aP2, C/EBPa, Cyc-D, and E2F1. (B) CTSS, SFRP5, DKK2, BMPR, and FGFR1. (C) a-SMA and collagen mRNAexpression levels in the liver. The RNA samples used for RT-PCR were pooled from eight mice per group. A representative image of triplicate experiments is shown in the leftpanel. The right panel shows the intensity of the bands that were densitometrically measured and normalized to the mRNA expression levels of GAPDH. The values withdifferent superscripts are significantly different at p <0.05.


morphogenetic protein receptor (BMPR), and fibroblast growthfactor receptor 1 (FGFR1) as compared to ND-fed mice. Oleurop-ein supplementation significantly reversed the HFD-inducedupregulation of the expression of LXR, PPARc2, LPL, aP2, Cyc-D,E2F1, CTSS, SFRP5, DKK2, and FGFR1 genes in the mice liver. Nosignificant difference in the hepatic BMPR mRNA level wasobserved between HFD- and OSD-fed mice (Fig. 3A and B).


HFD-fed mice were characterized by drastic increases inthe hepatic mRNA expression of a-smooth muscle actin(a-SMA) and collagen, both being markers of liver fibrosis.Oleuropein significantly reversed the HFD-induced upregula-tion of the expression of a-SMA (HFD, 1.9 vs. OSD, 1.1)and collagen (HFD, 2.1 vs. OSD, 1.3) genes in the liver ofmice (Fig. 3C).

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Fig. 4. Effect of dietary oleuropein supplementation on the (A) b-catenin and(B) phospho-ERK protein levels measured by Western blot in the liver of HFD-fed mice. The protein samples used for Western blot analysis were pooled fromeight mice per group. A representative image of the triplicate experiments isshown in the upper panel. The lower panel shows the intensity of the bandsdensitometrically measured and normalized to the protein expression levels ofthe b-actin and total ERK. The values with different superscripts are significantlydifferent at p <0.05.

Research Article

Phospho-ERK and b-catenin protein expressions in the liver

The Western blot analyses of the hepatic tissues of the experi-mental mice revealed that the b-catenin protein expression wassignificantly decreased, whereas the phospho-ERK protein levelwas significantly increased in livers of HFD-fed mice as comparedto ND-fed mice. Oleuropein supplementation significantlyreversed these HFD-induced changes in the expressions of b-cate-nin (HFD, 0.3 vs. OSD, 0.8) and phospho-ERK (HFD, 3.3 vs. OSD, 1)(Fig. 4).

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Fig. 5. Effect of dietary oleuropein supplementation on the expression of genes invoinflammatory cytokines and (B) Fas and TRAIL in the liver of HFD-fed mice. The mRNwere pooled from eight mice per group. A representative image of the triplicate experimmeasured and normalized to the mRNA expression levels of GAPDH. The values with di


Expression of TLR-mediated signaling molecules in the liver

The mRNA levels for the following genes were significantlyhigher in the liver of HFD-fed mice compared to the liver ofND-fed mice: toll-like receptor 2 (TLR2), toll-like receptor 4(TLR4), myeloid differentiation primary-response gene 88(MyD88), tumor necrosis factor a (TNFa), interferon beta (IFNb),and interleukin 1 beta (IL-1b). By contrast, oleuropein supple-mentation significantly decreased the mRNA levels for TLR2,TLR4, MyD88, TNFa, IFNb, and IL-1b in the liver of the experimen-tal mice (Fig. 5A).

In contrast to what is observed in ND-fed mice, HFD-fed micehad shown an increase in the hepatic mRNA expression of theTNF receptor superfamily member 6 (Fas) and the TNF-relatedapoptosis inducing ligand (TRAIL), a death receptor and ligand,respectively. Oleuropein significantly reversed the HFD-inducedupregulation of the expression of Fas (HFD, 2.1 vs. OSD, 1.2)and TRAIL (HFD, 2.2 vs. OSD, 1.1) in the mice liver (Fig. 5B).

Discussion

Macroscopic and microscopic results demonstrated that, in HFD-fed mice, lipid accumulation in the liver was observed as early as8 weeks up to 16 weeks, establishing a novel mouse model ofdiet-induced hepatic steatosis [15,16]. In a previous study, theplasma TC, TG, and FFA concentrations of rats fed a 0.2%-(18.4 mg/kg body weight/day)-oleuropein-supplemented dietfor 28 days were found to be significantly lower than those ofHFD-fed rats [13]. Furthermore, the 6-week treatment of hyper-cholesterolemic rabbits with 10 and 20 mg/kg body weight/daydoses of oleuropein reduced their plasma TC and TG concentra-tions in a dose-dependent manner [11]. Based on these publishedreports, the mice in the present study were fed a 0.03%-oleuropein-supplemented diet, which is estimated to be about30 mg oleuropein/kg body weight of the mouse (assuming thata 3 g/day feed intake of a mouse weighs 30 g). In acute toxicity

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lved in the (A) TLRs-mediated pro-inflammatory signaling cascades and pro-A expression level was measured via RT-PCR. The RNA samples used for RT-PCR

ents is shown. Each histogram shows the intensity of the bands densitometricallyfferent superscripts are significantly different at p <0.05.

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Fig. 6. The possible molecular mechanisms of dietary oleuropein in attenuating hepatic steatosis induced by HFD. Dietary oleuropein reverses the HFD-inducedchanges in SFRP5, DKK2, and b-catenin expression, which are involved in Wnt10b-mediated signaling, and in the FGFR1 gene and phospho-ERK protein expressions, alongwith the subsequent changes in the Cyc-D and E2F1 expressions, which are implicated in the FGF1-mediated lipogenesis cascades, in the liver. The downstream adipogenic-transcription factors (LXR and PPARc2) and their target genes (LPL and aP2) were suppressed by oleuropein in the livers of HFD-fed mice, which may have contributed to thelower hepatic-lipid accumulation. There was no significant difference in the BMPR or C/EBPa gene expression in the liver between HFD- and OSD-fed mice.


studies for oleuropein, no lethality or adverse effects wereobserved in the experimental mice even when the compoundwas administered at doses as high as 1000 mg/kg body weight.Thus, LD50 could not be determined [17].

There are many pathways that influence lipogenesis, such asWnt10b-[18], BMP- [19], and FGF1- [20] mediated signaling.The Wnt/b-catenin signaling pathway plays an important rolein liver physiology and its aberrant activation is observed inhepatocellular cancers [21]. In addition, a research conductedover the last decade has established the Wnt/b-catenin signalingpathway as an important regulator of lipogenesis. Wnt10bbinding to frizzled (FZD) receptors and low-density lipoprotein-receptor-related protein 5 (LRP5) or LRP6 coreceptors leads todishevelled (DSH) phosphorylation [22]. This, in turn, resultsin the hypophosphorylation of b-catenin, which accumulates inthe cytoplasm and translocates to the nucleus where it binds tothe T-cell-factor/lymphoid-enhancing factor (TCF/LEF) transcrip-tion factors and inhibits lipogenesis by suppressing C/EBPa andPPARc [22]. Wnt10b signaling is known to be modulated byextracellular antagonists, such as SFRP5 and DKK2 [23,24]. SFRP5directly binds and sequesters Wnt10b, whereas DKK2 inhibitsWnt10b signaling by binding, as a high-affinity antagonist, to


LRP5/LRP6 coreceptors. HFD induces the epigenetic activation ofSFRP5 expression in white adipose tissues, which increases thesusceptibility to obesity [25]. In the present study, HFD appearedto exert inhibitory effects on Wnt10b signaling, whereby theexpression of Wnt10b inhibitors, such as SFRP5 and DKK2, wasupregulated, and b-catenin protein expression was downregu-lated, resulting in increased lipogenesis (Fig. 6).

Upon BMP stimulation, Schnurri 2 (Shn2) is required for theefficient transcription of PPARc, possibly as a scaffold protein toform a ternary complex with SMAD1/4 and C/EBPa [20]. FGF1promotes both the proliferation and differentiation of humanpreadipocytes and FGFR1 therein, resulting in increased ERK1/2phosphorylation and PPARc, following FGF-1 stimulation [22].Another current study in the literature states that the Cyc-D/pRB/E2F1 pathway lies downstream the mitogenically activatedRas/Raf/MEK/ERK cascade [26]. It has been found that HFD-fedmice had higher hepatic mRNA levels of BMPR, FGFR1, Cyc-D,E2F1, and phosphor-ERK protein than did ND-fed mice. The hepa-tic gene expression of adipogenic transcription factors, such as C/EBPa, PPARc2, LXR, and CTSS, and their target genes, such as LPLand aP2, were also significantly increased by HFD (Fig. 6). CTSS,a potent cysteine protease that has the ability to degrade several

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Research Article
extracellular matrix proteins [27], was recently identified as anovel biomarker of adiposity [28]. The CTSS inhibitor reducedlipid accumulation and the gene expression of adipocyte markers,including PPARc, aP2, cluster of differentiation 36 (CD36), andSREBP1c, in differentiated human preadipocytes [28]. Takentogether, these findings suggest that HFD may induce hepatic ste-atosis through multiple signaling pathways involving Wnt10b-,BMP-, and FGF1-mediated lipogenesis signaling.
In the present study, we observed that oleuropein treatmentsignificantly attenuated the HFD-induced elevation in the hepaticconcentration of cholesterol, TG, and FFA. The hepatic expressionof adipogenic nuclear-transcription factors (e.g., PPARc2 andLXR), their target genes (LPL and aP2), and CTSS was significantlydecreased by OSD, as opposed to HFD. The gene expression ofWnt10b inhibitors (e.g., SFRP5 and DKK2) was downregulated,whereas the b-catenin protein expression was upregulated, inthe liver of OSD-fed mice as compared to HFD-fed mice. Besides,oleuropein-supplemented HFD resulted in decreased mRNA lev-els of FGF1-mediated signaling molecules (e.g., FGFR1, CycD, andE2F1) and ERK phosphorylation along with the unaltered BMPRmRNA level in livers of experimental mice. These results indicatethat the protective effects of oleuropein against hepatic lipogen-esis can be mediated, at least in part, by both Wnt10b- and FGF1signaling.

Although no inflammation was detected in liver histology,feeding mice HFD for 10 weeks provoked increased expressionof TLR2 and TLR4 and their target genes such as TNFa, IFNb, andIL-1b, in their liver. This effect is considered as an early eventassociating hepatic lipogenesis and inflammatory stress.

TLR2 and TLR4 belong to the innate immune response and theligand-induced activation of TLR2 or TLR4 leads to the commondownstream activation of TNF-receptor-associated factor 6 viathe adapter molecule MyD88 [29]. This cascade of events culmi-nates in the activation of the nuclear factor kappa B and theinduction of proinflammatory cytokines [29]. A previous studydemonstrated that TLR2 and TLR4 bind lipid-based structuresvia their long-chain saturated FFA moieties [30]. The hepaticexpression of TLR4 was significantly upregulated in patients withNAFLD, as compared to controls [31].

It was shown, herein, that HFD elevated the mRNA levels ofTLR2, TLR4, and MyD88, leading to the expression of proinflamma-tory cytokines such as TNFa, IL-1b, and IFNb, in the liver of theexperimental mice. Oleuropein is known to inhibit the biosynthe-sis of proinflammatory cytokines such as TNFa and interleukin-6(IL-6) in human monocytes and neutrophils treated with Pseudo-monas aeruginosa [32]. In the present study, oleuropein-supple-mented HFD decreased the hepatic mRNA expression ofproinflammatory cytokines, including TNFa, IL-1b, and IFNb, andtheir upstream signaling molecules, such as TLR2, TLR4, andMyD88. Based on these results, we speculated that in mice feda HFD, oleuropein can attenuate hepatic steatosis, probablythrough the suppression of the TLR2- and TLR4-mediated proin-flammatory signaling cascades. The liver is comprised of hetero-geneous cell types including hepatocytes, stellate cells,endothelial cells, and immune cells and it is probable that diet-induced changes in the TLRs and downstream molecule expres-sions may reflect a regulation of both liver cell type populationand mRNA level, within a specific cell type.

In conclusion, the results of this study demonstrate, for thefirst time, that the addition of oleuropein to HFD decreases bodyweight gain and liver weight and improves the lipid profiles in


both the plasma and the liver of mice. The evidence obtained inthe present study suggest that the protective effects of oleuropeinagainst hepatic steatosis appear to be mediated, at least in part,by the Wnt10b- and FGFR1-mediated lipogenesis signaling cas-cades along with the TLR2- and TLR4-mediated proinflammatorysignaling pathways. This is the first in vivo experimental studythat encourages the future use of a novel natural agent, oleurop-ein, for the prevention and treatment of hepatic steatosis.

Conflict of interest

The authors declared that they do not have anything to discloseregarding funding from industry or conflict of interest withrespect to this manuscript.

Acknowledgements

This work was supported by a grant of the Korea Health 21 R&DProject, Ministry of Health & Welfare (#090282), Republic ofKorea and by the SRC program of the Korea Science andEngineering Foundation (KOSEF) grant funded by the Koreagovernment (#2009-0063409).

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Oleuropein attenuates hepatic steatosis induced by high-fat diet in mice

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