Oleuropein reduces free fatty acid-induced lipogenesis via lowered extracellular signal-regulated kinase activation in hepatocytes

doi:10.1016/j.nutres.2012.06.017

Nutrition Research

Volume 32, Issue 10, October 2012, Pages 778-786

https://doi.org/10.1016/j.nutres.2012.06.017 Get rights and content

Abstract

Oleuropein, a bitter glucoside found in green olive leaves, and its metabolite hydroxytyrosol display powerful antioxidant activity both in vivo and in vitro. In this study, we hypothesized that the antioxidant activity of oleuropein could attenuate hepatic steatosis. To test this hypothesis, we established steatotic hepatocytes using HepG2 and FL83B cells treated with free fatty acids (FFAs) (oleate:palmitate, 2:1). To confirm hepatic steatosis, the intracellular lipid levels were quantitatively measured by Nile Red staining, and the sizes and distributions of lipid droplets were visualized by transmission electron microscopy. The expression of PAT family proteins as well as of adipose differentiation-related protein and tail interacting protein (TIP47) was evaluated by reverse transcriptase polymerase chain reaction and immunoblotting. To examine the cellular and molecular events associated with oleuropein, annexin V/propidium iodide staining and immunoblotting were performed. Oleuropein decreased the number and size of lipid droplets in FFA-treated cells and reduced intracellular triglyceride accumulation. However, it did not affect the expression of lipid droplets-associated PAT family proteins, including adipose differentiation-related protein and TIP47. In addition, oleuropein reduced FFA-induced extracellular signal-regulated kinase activation but had no effect on c-Jun N-terminal kinase or AKT activation. Given its protective effects against FFA-induced hepatocellular steatosis, oleuropein may be a lipid-lowering agent.

Introduction

Non-alcoholic fatty liver disease (NAFLD), one of the most common causes of chronic liver disease in the world [1], [2], results from excessive fat accumulation, not alcohol abuse, in the liver. It is associated with nonalcoholic steatohepatitis (NASH), which is characterized by steatosis, various degrees of inflammation, and fibrosis/cirrhosis. The pathogenesis and progression of NASH can be explained by the “two-hit hypothesis” [3], [4]. According to this theory, the first hit involves the accumulation of fat in hepatocytes owing to insulin resistance. The progression of hepatic steatosis to NASH is then induced by hepatic injury attributable to oxidative stress, reactive oxygen species, lipid peroxidation, and cytokines (the second hit). These factors result in hepatocellular injury and subsequent progression toward hepatic fibrosis.

Olive oil, an important component of the Mediterranean diet, is known to be effective in the prevention of cancer, cardiovascular disease and chronic degenerative disease [5], [6], [7]. Oleuropein, the most abundant compound in olive leaves, can produce other bioactive substances, including oleanolic acid and hydroxytyrosol, by hydrolysis. Corona et al [8] first demonstrated that although oleuropein is not absorbed in the small intestine but it is rapidly degraded by the colonic microflora to yield hydroxytyrosol, which may then be absorbed. However, how oleuropein reaches the liver and whether it is effective in vivo are unclear. Recently, Poudyal et al [9] reported that an olive leaf extract reduced the weight of the liver in a high-fat-fed rat model by reducing portal collagen deposition and fat deposition in the liver.

Previous research has demonstrated the preventive effect of oleuropein against steatohepatitis induced by a high-fat diet in rodents [10]. However, the mechanism underlying its hepatic lipid-lowering effect remains unclear. Based on previous results, we hypothesized that oleuropein prevents hepatic steatosis induced by free fatty acids (FFA). To test our hypothesis, we established FFA-induced steatotic cells using the human hepatoma-derived cell line HepG2 and normal mouse hepatocyte FL83B cells. HepG2 cells were selected because the cell line has been successfully used to establish a stable in vitro steatosis model in previous studies. However, the epigenetic and genetic alterations in this cell line confer many properties that are distinct from those of normal hepatocytes, including differences in metabolism regulation [11], [12]. Accordingly, we employed normal mouse hepatocytes to further test our hypothesis in this study. Based on our hypothesis, this investigation determined the anti-steatotic effects, oleuropein on steatotic hepatocytes at the cellular and molecular level.

Section snippets

Cell culture and FFA treatment

HepG2 and FL83B cells in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, and 0.25 μg/mL streptomycin were maintained at 37°C in a humidified incubator with 5% CO₂. Fatty acid-free bovine serum albumin (BSA, 1%; Sigma-Aldrich, St. Louis, MO, USA) was used as a vehicle for FFA treatment. After reaching 70% confluence, cultured cells were serum-starved and exposed to0.5 mmol/L FFAs (oleic acid:palmitic acid, 2:1)

FFA treatment induces cellular steatosis

Recently, we reported that oleuropein displayed a protective effect against high-fat diet–induced hepatic steatosis in a mouse model [8]. In the present study, we examined whether oleuropein could prevent FFA-induced steatosis in an in vitro model.

To determine the optimal concentration of FFA to induce steatosis, HepG2 or FL83B cells were cultured with FFAs (oleate:palmitate, 2:1) at a concentration of 0.5, 1, 1.5, or 2 mmol/L for 24 hours. Treatment with 0.5 or 1 mmol/L FFAs (oleate:palmitate,

Discussion

NAFLD, one of the most common hepatic disorders in developed countries, encompasses a spectrum of liver diseases, ranging from simple steatosis to steatosis combined with various degrees of necroinflammation and fibrosis. Patients with NAFLD display increased lipolysis and increased delivery of FFAs to the liver. Higher concentrations of FFAs are associated with more severe liver disease[18]. Generally, animal models have been used to study NAFLD, but these models are difficult to prepare, and

Acknowledgment

This study was supported by a grant from the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A090282) and the National Research Foundation of Korea grant funded by the Korean government (MEST) (No. 2011-0014620).

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Oleuropein reduces free fatty acid-induced lipogenesis via lowered extracellular signal-regulated kinase activation in hepatocytes

Abstract

Introduction

Section snippets

Cell culture and FFA treatment

FFA treatment induces cellular steatosis

Discussion

Acknowledgment

Am J Gastroenterol

Gastroenterology

Eur J Cancer

Lancet Oncol

J Nutr

J Hepatol

Chem Biol Interact

J Immunol Methods

J Lipid Res

Cell

Gastroenterology

Biochem Biophys Res Commun

Phytochemistry

J Biol Chem

Curr Opin Cell Biol

Curr Opin Cell Biol

Curr Opin Cell Biol