Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling

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Abstract

Background

Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis.

Methods

We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a 1H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways.

Results

Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation.

Conclusions

Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation.

General significance

NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.

Highlights

► NMR-based metabolomics of culture medium discriminated resveratrol treatment. ► Resveratrol reduced glycolysis and aminoacid utilisation for energy purpose. ► Mitochondrial succinate utilisation was increased by resveratrol treatment. ► 13C NMR spectroscopy shows an increase in mitochondrial oleate beta oxidation.

Introduction

Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenol found in grapes, grape products and other plant products that has been the subject of intense scientific interest in recent years on account of its multiple protective and therapeutic effects [1], [2], [3], [4], [5]. Reported health benefits include cardioprotection, antiaging effects, defence against metabolic and neurodegenerative diseases, cancer prevention and therapy [6], [7]. Some important cellular processes, such as cell cycle regulation and apoptosis are deeply influenced by this compound and its preventive and/or therapeutic properties against cancer have often been ascribed to antiproliferative and pro-apoptotic effects [8].

More recent research on the molecular mechanisms by which resveratrol might exert many of its biological effects has emphasised the importance of its interaction with sirtuins. In particular, resveratrol has been shown to increase Sirt1 activity and to enhance Sirt1-dependent metabolic processes both in vivo and in vitro [9], [10], [11].

Resveratrol can affect different metabolic pathways depending on the organ or cell type, on the cell state, and on the duration and dosage of treatment. At the same time, it has been shown that resveratrol has numerous intracellular targets and affects the expression and activity of different transcription factors/cofactors, thus regulating metabolic homeostasis and resulting in multiple pleiotropic effects. Among the numerous metabolic effects exhibited by resveratrol, the topic of “calorie restriction mimicry” has aroused considerable interest and several studies on cell cultures have been performed forcing the system toward either a fed or a fasted condition by increasing or decreasing nutritional supplies in the culture medium [10], [12], [13], [14].

These considerations prompted us to study the metabolic modifications induced by resveratrol treatment in an in vitro system, using a metabolomic approach that allows the simultaneous screening of multiple metabolic pathways. Considering that the liver is one of the main target organs of resveratrol action in vivo, we analysed the effects of resveratrol on HepG2 cells, a human hepatoblastoma line that, although actively proliferating, exhibits many features specific to human differentiated hepatocytes, and is also widely used in resveratrol metabolic studies [15], [16], [17].

We first verified in our experimental conditions whether cellular responses to the treatment were similar to those reported in the literature, particularly in regard to antiproliferative and apoptotic effects [17], [18]. Next, we studied the metabolic profile in relation to resveratrol treatment. Changes in the exo-metabolome were evaluated using 1H-NMR spectroscopy on medium and Multivariate Data Analysis (MVDA). The utilisation of substrates and the concurrent release of metabolites into the extracellular space reflect the metabolic pathways that operate during a given set of physiological activities and in response to metabolic perturbations, either chemical or physical, leading to the characterisation of a specific metabolic profile (or fingerprint). Previously, this approach allowed us to discriminate two different metabolic profiles in HepG2 cells in relation to different phases of cell growth [19].

Section snippets

Cell culture and viability

Human HepG2 cells were purchased from the American Type Culture Collection (ATCC) and were used within twenty passages. Cells, suspended in RPMI 1640 medium (Sigma) supplemented with 10% foetal calf serum, 2 mM l-glutamine, 1% sodium pyruvate, 100 μg/mL streptomycin and 100 U/mL penicillin, were seeded in culture plates at 8 × 104 cells/cm2 at 37 °C in a humidified incubator in the presence of 5% CO2. Resveratrol (Sigma), 20–200 μM in DMSO, was added to the cells and cultures were incubated for 24 or

Effects of resveratrol treatment on cell cycle, apoptosis and Sirt1 expression

We first investigated in our experimental system the effects of resveratrol on proliferation, apoptosis and Sirt1 expression and localisation, in order to choose the best conditions for the metabolomic analysis. Cells were plated and cultured to high density and then treated with 20–200 μM resveratrol for 24 or 48 h. The decrease of cell growth was dose- and time-dependent, with an apparent GI50 of 80 μM and 40 μM after 24 and 48 h of culture, respectively. Treatment with resveratrol resulted in a

Discussion

Resveratrol has been shown to influence several metabolic pathways in both in vivo and in vitro experimental systems, creating great interest in applications that could be useful for human health. We focused on energy metabolism, since this molecule exerts an effect that mimics calorie restriction [1], [12], [24].

Many different molecular mechanisms of action have been shown to influence cell metabolism with a long list of involved up or down regulated target genes and activated or suppressed

Acknowledgment

We gratefully acknowledge the help of Dr. Giorgio Capuani for statistical multivariate analysis. The research was supported by a grant from the Italian Ministry of Economy and Finance to the CNR for the Project “FaReBio di Qualita”.

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