Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling
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”.
References (49)
- et al.
Cardioprotective actions of grape polyphenols
Nutr. Res.
(2008) - et al.
Mechanism of human SIRT1 activation by resveratrol
J. Biol. Chem.
(2005) - et al.
SIRT 1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase
J. Biol. Chem.
(2008) - et al.
Resveratrol inhibits the expression of SREB1 in cell model of steatosis via Sirt1-FOXO1 signaling pathway
Biochem. Biophys. Res. Commun.
(2009) - et al.
Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells
Biochem. Biophys. Res. Commun.
(2009) - et al.
In vitro antiproliferative effect of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on human hepatocellular carcinoma cell line, SK-HEP-1 cells
Cancer Lett.
(2001) - et al.
Resveratrol-induced apoptosis is mediated by p53-dependent pathway in HepG2 cells
Life Sci.
(2002) - et al.
Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses
Biochimie
(2008) - et al.
Structure-based profiling of metabolites and isotopomers by NMR
Prog. Nuclear Magn. Res. Spect.
(2008) - et al.
Metabolic profiling by 13C NMR spectroscopy: [1,2-13C2] glucose reveals a heterogeneous metabolism in human leukemia T cells
Biochimie
(2006)
Resveratrol delays age-related deteriration and mimics transcriptional aspects of dietary restriction without extending life span
Cell Metab.
Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation
Biochim. Biophys. Acta
Resveratrol inhibits glucose metabolism in human ovarian cancer cells
Gynecol. Oncol.
Inhibition of phospahatidylinositol 3-kinase-mediated glucose metabolism coincides with resveratrol-induced cell cycle arrest in human diffuse large B-cell lymphomas
Biochem. Pharmacol.
Sensing of energy and nutrients by AMP-activated protein kinase
Am. J. Clin. Nutr.
Metabolic adaptations through the PGC-1α and SIRT1 pathways
FEBS Lett.
SIRT1 deacetylates and inhibits SREBP-1C activity in regulation of hepatic lipid metabolism
J. Biol. Chem.
Sirtuins regulate key aspects of lipid metabolism
Biochim. Biophys. Acta
Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling
Trends Biochem. Sci.
Substrates and regulation mechanisms for the human mitochondrial sirtuins Sirt3 and Sirt5
J. Mol. Biol.
SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells
Cell
Biochemical effects of SIRT1 activators
Biochim. Biophys. Acta
Therapeutic potential of resveratrol: the in vivo evidence
Nat. Rev. Drug Discov.
Resveratrol as an antioxidant and pro-oxidant agent: mechanism and clinical implications
Biochem. Soc. Trans.
Cited by (52)
Breaking the circulus vitiosus of neuroinflammation: Resveratrol attenuates the human glial cell response to cytokines
2023, Biomedicine and PharmacotherapyEnhanced oral permeability of Trans-Resveratrol using nanocochleates for boosting anticancer efficacy; in-vitro and ex-vivo appraisal
2021, European Journal of Pharmaceutics and BiopharmaceuticsLinking metabolic profiling, resveratrol, the gut microbiota, and antioxidant potential
2020, Toxicology: Oxidative Stress and Dietary AntioxidantsThe effects of jolkinolide B on HepG2 cells as revealed by <sup>1</sup>H-NMR-based metabolic profiling
2019, European Journal of PharmacologyCitation Excerpt :The separation that was obtained from the OPLS-DA scores plots between the control and JB-treated samples is already evident in the PCA scores plot. The parameters from the HD and NC groups of the cell extracts and cell culture media were R2Y = 98.9%, Q2Y = 0.927 for the HD group and R2X = 98.3%, Q2Y = 0.673 for the NC group in the OPLS-DA model, which presented the explained variance and high predictive capability, respectively (Liu et al., 2014; Massimi et al., 2012; Westerhuis et al., 2010). The color-coded coefficient plots demonstrate metabolite changes in detail after JB treatment.
In vitro Metabolomic Approaches to Investigating the Potential Biological Effects of Phenolic Compounds: An Update
2017, Genomics, Proteomics and BioinformaticsResveratrol Alleviates the Early Challenges of Implant-Based Drug Delivery in a Human Glial Cell Model
2024, International Journal of Molecular Sciences