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

https://doi.org/10.1016/j.jhep.2010.08.019Get rights and content

Background & Aims

Oleuropein, a secoiridoid derived from olives and olive oil, has been known to possess antimicrobial, antioxidative, and anticancer activities. The purpose of the present study was to determine whether oleuropein has a protective effect 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. The plasma and hepatic lipid levels were determined, and the hepatic gene and protein expression levels were analysed via RT-PCR and Western blotting, respectively.

Results

The supplementation of HFD with oleuropein reversed the HFD-induced increases in liver weight along with plasma and hepatic lipid levels in mice. The expression of Wnt10b inhibitor genes, such as secreted firizzed-related sequence protein 5 and dickkopf homolog 2, was downregulated, whereas the β-catenin protein expression was upregulated in the liver of OSD-fed mice compared to HFD-fed mice. Fibroblast growth factor receptor 1 (FGFR1), phosphoextracellular-signal-regulated kinase 1/2, cyclin D, and E2F transcription factor 1, along with several key transcription factors and their target genes involved in adipogenesis, were downregulated by oleuropein. OSD-fed mice exhibited decreased expression of the toll-like-receptor-(TLR)-mediated signaling molecules (TLR2, TLR4, and myeloid differentiation primary-response gene 88) and proinflammatory cytokines, in their livers, as compared to HFD mice.

Conclusions

These results suggest that the protective effects of oleuropein against HFD-induced hepatic steatosis in mice appear to be associated with the Wnt10b- and FGFR1-mediated signaling cascades involved in hepatic lipogenesis, along with the TLR2- and TLR4-mediated signaling implicated in hepatic steatosis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) can be considered as a spectrum of liver pathologies, with, on the one hand, simple steatosis with absence of necrosis or signs of inflammation and, on the other hand, severe signs of inflammation with fibrosis or cirrhosis [1]. An excessive and inappropriate dietary-fat intake, combined with peripheral insulin resistance, continued triglyceride (TG) hydrolysis via lipoprotein lipase, and other genetic alterations in the key lipid metabolic pathways, results in increased blood free-fatty-acid (FFA) concentration [2], leading to increased TG concentration in the liver. The in vivo activation of liver X receptor (LXR), sterol-regulatory-element-binding protein 1c (SREBP1c), and peroxisome-proliferator-activated receptors gamma (PPARγ) affects the lipid accumulation in the liver induced by a high-fat diet (HFD) [3], [4]. LXR has been shown to activate SREBP1c [5], which stimulates the key lipogenic genes, including those encoding the acetyl-CoA carboxylase and the fatty-acid synthase [6], [7]. In animal models with fatty liver, PPARγ is transcriptionally upregulated and consequently activates the lipogenic target genes, thus exacerbating hepatic steatosis [8].

Oleuropein is a nontoxic secoiridoid derived from olives and olive oil, which influences their sensory organoleptic properties and 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 that oleuropein has a high antioxidant activity comparable to a hydrosoluble analog of tocopherol [11], and inhibits the proliferation and migration of various cancer cell lines, such as leukemia, melanoma, colon, breast, and kidney cancer cells, in a dose-responsive manner [12]. More recently, it has been reported that oleuroepin significantly decreased the body weight, body fat accumulation, and plasma TG concentrations in rats with diet-induced obesity. Moreover, uncoupling-protein-1 contents of the interscapular brown adipose tissue and rates of urinary noradrenaline and adrenaline excretions were significantly decreased in rats fed oleuropein as opposed to HFD-fed control animals [13]. Although a number of studies have been carried out to investigate the biochemical roles of oleuropein, the protective activity of oleuropein against NAFLD has never been reported. Therefore, the purpose of the present study was to determine whether oleuropein has a protective effect against hepatic steatosis induced by a HFD in mice. The regulatory effect of oleuropein, on the expression of several key transcription factors and their target genes involved in adipocyte differentiation, and toll-like-receptor-(TLR)-mediated signaling molecules involved in metabolically triggered inflammation, were also investigated in a mouse model of HFD-induced hepatic steatosis.

Section snippets

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 a room 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 libitum for 1 week prior to their division into three weight-matched groups (n = 8 per group): the normal diet (ND), HFD, and oleuropein-supplemented diet (OSD) groups. ND was a purified diet based on the AIN-76 rodent diet

Body and liver weights

After 10-week feeding, HFD-fed mice showed significantly higher final body weight and cumulative body weight gain compared to ND-fed mice. Oleuropein supplemented to HFD significantly reduced 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 food intake (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),

Discussion

Macroscopic and microscopic results demonstrated that, in HFD-fed mice, lipid accumulation in the liver was observed as early as 8 weeks up to 16 weeks, establishing a novel mouse model of diet-induced hepatic steatosis [15], [16]. In a previous study, the plasma TC, TG, and FFA concentrations of rats fed a 0.2%-(18.4 mg/kg body weight/day)-oleuropein-supplemented diet for 28 days were found to be significantly lower than those of HFD-fed rats [13]. Furthermore, the 6-week treatment of

Conflict of interest

The authors declared that they do not have anything to disclose regarding funding from industry or conflict of interest with respect to this manuscript.

Acknowledgements

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

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