Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes

Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes

Available online at www.sciencedirect.com ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx Extra virgin olive oil phenols down-...

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Available online at www.sciencedirect.com

ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx

Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes☆ Paola Priore, Luisa Siculella⁎, Gabriele Vincenzo Gnoni Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy

Received 18 March 2013; received in revised form 20 January 2014; accepted 28 January 2014

Abstract Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (−25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (−49%) = oleuropein (−48%) N tyrosol (−30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, −41%, = oleuropein, −38%, N tyrosol, −17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil. © 2014 Elsevier Inc. All rights reserved. Keywords: Acetyl-CoA carboxylase; AMP-activated protein kinase; Extra virgin olive oil; Lipid synthesis; Phenols; Rat-hepatocytes

1. Introduction The Mediterranean diet is associated with a low incidence of various chronic degenerative pathologies such as atherosclerotic cardiovascular diseases, neurological disorders and cancer [1–3]. Extra virgin olive oil (EVOO), the main fat source of this diet, is generally considered to be a major contributor to human health in the Mediterranean area [4,5]. New studies are extending EVOO action to the prevention of the metabolic syndrome, a cluster of risk factors that includes hypercholesterolemia, hypertriglyceridemia, high blood pressure, obesity, fatty liver and insulin resistance, all closely linked to diabetes and coronary heart disease [6,7]. The beneficial effects of EVOO have historically been attributed to its elevated oleic acid content; more recently, converging evidence indicates that the EVOO non-saponifiable fraction, rich in phenols, significantly promotes human health [8,9]. This fraction contains a ☆

number of phenols ranging from the simple compounds hydroxytyrosol (2,(3,4-dihydroxyphenyl)-ethanol, HTyr, Fig. 1A) and tyrosol (p-hydroxy-phenyl ethanol, Tyr, Fig. 1B), to several products of conjugation of these phenols with elenolic acid, such as oleuropein (Ole, Fig. 1C) [10]. Studies on the mechanism of action of EVOO phenols have mainly focused on their antioxidant and anti-inflammatory properties [11– 13]. However, in the last decade several lines of evidence indicate that EVOO phenolic compounds may also possess anti-atherosclerotic and anti-diabetic characteristics [5,9,14,15]. In vivo experiments have demonstrated that oral administration of HTyr [16] or Ole [17] to hypercholesterolemic rats significantly lowers the serum levels of total cholesterol, triglycerides (TG) and low density lipoprotein (LDL)-cholesterol, and increases the serum level of high density lipoprotein (HDL)-cholesterol. Diabetic rats fed a diet supplemented with EVOO showed lower serum levels of TG when compared to

Funding Statement: This work was supported by Grants from Apulia Region (Italy), POR Strategic Projects [Grant number: CIP PS_101]. ⁎ Corresponding author. Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Laboratorio di Biochimica e Biologia Molecolare, Università del Salento, Via Prov.le Lecce-Monteroni, I-73100, Lecce, Italy. Tel.: +39 0832298696; fax: +39 0832298626. E-mail address: [email protected] (L. Siculella). http://dx.doi.org/10.1016/j.jnutbio.2014.01.009 0955-2863/© 2014 Elsevier Inc. All rights reserved.

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Fig. 1. Chemical structures of EVOO phenols. Chemical structures of hydroxtyrosol (A), tyrosol (B) and oleuropein (C).

animals fed a sunflower oil-enriched diet, suggesting that EVOO provides better control of the hypertriglyceridemia accompanying diabetes [18]. Similarly, administration of Ole- and HTyr-rich extracts from olive leaves, during four weeks significantly reduced serum glucose and cholesterol levels in alloxan-induced diabetic rats [15]. Ole supplementation can attenuate liver steatosis induced by high-fat diets in mice by decreasing hepatic concentrations of cholesterol [19]. In an international study with 200 healthy male volunteers, Covas et al. [20] showed that EVOO phenols are able to increase plasma HDLcholesterol levels as well as to reduce TG level. All these studies highlight the effect of EVOO phenolic compounds on serum levels of cholesterol and TG in animals under particular nutritional and hormonal conditions. However, the EVOO hypolipidemic action has not been closely investigated. Liver is a key organ in the synthesis, the storage and the excretion of lipids through very low-density lipoprotein (VLDL); alterations in hepatic lipogenesis will have an impact on serum lipid levels. To date, there have been no reports in the literature that have assessed the direct effects of EVOO phenolic compounds on hepatic lipid metabolism under regular hormonal and nutritional conditions. In the present study we investigated this aspect showing that addition of EVOO phenols to hepatocytes from normal, untreated rats, induced short-term reduction of fatty acid, cholesterol and TG syntheses. Moreover, acetyl-CoA carboxylase (ACC), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and diacylglycerol acyltransferase (DGAT) activities as well as AMP-activated protein kinase (AMPK) were involved in tuning down lipid synthesis. 2. Methods and materials

mixture. The rats were housed individually in a temperature- (22 ± 1°C) and lightcontrolled (light on 08:00–20:00) environment. All rats received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, published by the National Institutes of Health (NIH Publication No. 86–23, revised 1985), as well as in accordance with Italian laws on animal experimentation (art. 4 and 5 of D.L. 116/92). 2.3. Preparation of rat-liver cells Rat-liver cells were isolated by perfusing the liver with collagenase as previously described [21]. Cells were suspended in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. Unless specified otherwise, primary hepatocytes were seeded at a density of 7×105 cells per 35 mm diameter Petri dishes; 2 h after plating, the medium was refreshed and different phenols (HTyr, Tyr and Ole dissolved in dimethyl sulfoxide, DMSO) were added to the serum-rich (10% FBS) medium for a period of 2 h. To determine the putative effects of the phenolic compounds on cell viability, a colorimetric MTT assay was performed as described in [22]. In each experiment and for each determination, control dishes incubated with DMSO were used. 2.4. Determination of fatty acid and cholesterol synthesis Lipogenic activity was determined by monitoring the incorporation of [1-14C] acetate (16 mM, 0.96 mCi/mol) into fatty acids and cholesterol essentially as reported [23]. Cells were incubated for 0.5, 1 and 2 h with the indicated phenol concentration (2.5–100 μM). To terminate the lipogenic assay, the medium was aspirated, cells were washed three times with ice-cold PBS to remove unreacted labelled substrate and the reaction was stopped with 1.5 ml of 0.5 M NaOH. The cells were scraped off with a rubber policeman and transferred to a test tube; 100 μl of cells were reserved for a protein assay [23]. The remaining cells were saponified with 4 ml of ethanol and 2 ml of double-distilled water for 90 min at 90°C. Non-saponifiable sterols and fatty acids (after acidification with 1 ml of 7 M HCl) were extracted with 3×5 ml of petroleum ether. The extracts were collected, dried under a stream of nitrogen, and counted for radioactivity.

2.1. Materials 2.5. Incorporation of radiolabelled acetate into lipid fractions HTyr (≥90% purity), Tyr (≥95% purity) and Ole (≥98% purity) were obtained from Extrasynthese (Genay Cedex, France). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, phosphate buffered saline (PBS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Gibco-Invitrogen (Paisley, UK); [1-14C]acetate was obtained from GE Healthcare (Little Chalfont, UK); [1-14C]acetyl-CoA, [3-14C]HMG-CoA and [1-14C]palmitoyl-CoA were obtained from PerkinElmer (Boston, MA, USA). Primary antibodies for ACCα, pACCα, AMPKα, pAMPKα, and α-tubulin as well as horseradish peroxidase conjugated IgGs were obtained from Cell Signaling Technologies (Boston, MA, USA). All other reagents, obtained from Sigma-Aldrich, were of analytical grade.

Newly synthesized labelled fatty acids are mainly incorporated into complex lipids, therefore, neutral lipid analysis was also carried out. Experimental conditions were the same as those for fatty acid and cholesterol synthesis assays. After 2 h of incubation with 25 μM EVOO phenols, the reaction was stopped by washing the cells three times with ice-cold PBS and treating them with 2 ml of KCl/CH3OH (1:2, v/v). Total lipids were extracted according to Giudetti et al. [23]. Neutral lipids were resolved by thin layer chromatography (TLC) on silica gel plates by using hexane:ethylether:acetic acid (70:30:10, v/v/v) as a developing system. Lipid spots, visualized by iodine vapour, were individually scraped from the plate into counting vials for radioactivity measurement.

2.2. Animals and Ethical Statement 2.6. Assay of enzymatic activities of de novo fatty acid synthesis Male Wistar rats (200–250 g) were used throughout this study. Animals had free access to tap water and were fed ad libitum with a chow diet consisting of: 18.6% crude protein, 44.2% carbohydrate, 6.2% crude fat with adequate amounts of essential fatty acids, 3.5% crude fiber, 14.7% neutral detergent fiber, 5.3% ash, and a salt and vitamin

ACC activity was determined by measuring the incorporation of radiolabelled acetyl-CoA into fatty acids in an assay coupled with the fatty acid synthase (FAS) reaction in digitonin-permeabilized hepatocytes essentially as described by Priore et al.

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[24]. This method avoids a number of interferences associated with the classical bicarbonate fixation assay of ACC activity [25]. After incubation with EVOO phenols (2 h, 25 μM), the culture medium was removed and primary rat-hepatocytes were permeabilized using 400 μl of an isotonic assay mixture containing digitonin (4 μg/ml), 63 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES, (pH 7.5), 1.5 mM MgCl2, 0.6 mM KH2PO4, 0.6 mM MgSO4, 0.5 mM citrate, 2.5 mM EGTA, 1.25 mM CaCl2, 22.5 mM NaHCO3, 70.5 mM NaCl, 2.0 mM ATP, 0.5 mM NADPH, 0.44 mM dithioerythritol, 0.9% (w/v) bovine serum albumin, 0.062 mM [1-14C]acetyl-CoA (4 Ci/mol), 0.062 mM butyryl–CoA and 3.0 mU FAS. The reaction mixture was prepared within 15 min of use by mixing the digitonin, previously dissolved in an EGTA stock solution by heating in a boiling-water bath, with the other components of the assay mixture. Cell permeabilization with digitonin offers several advantages in assaying soluble enzymes: (i) it allows in situ intracellular enzyme activities to be assayed rapidly and directly in a nearly natural environment, (ii) it reduces the necessity for the time-consuming preparation of sub-cellular fractions for enzyme assay, and (iii) it avoids any possible post-homogenizing modifications. Simultaneous cell permeabilization and enzyme activity assays have been used for measuring the activity of a range of regulatory enzymes of intermediary metabolism in different cell types [25,26]. FAS activity was assayed by measuring the incorporation of [1-14C]acetyl-CoA into fatty acids essentially as described above for ACC activity, except that 0.2 mM malonylCoA was included and ATP, butyryl-CoA and FAS were omitted in the digitonincontaining assay mixture [24]. The assay was incubated at 37°C for 10 min. Both lipogenic assays were stopped by the addition of 100 μl of 10 M NaOH. Thereafter, the cells were scraped off the dishes with a rubber policeman and transferred to a test tube. The assay plates were washed twice with 450 μl of 0.5 M NaOH and the wash solutions were collected into the same tube. The samples were saponified by adding 5 ml of CH3OH and boiling for 45–60 min in capped tubes. After cooling and acidification with 200 μl of 12 M HCl, fatty acids were extracted three times with 4 ml of petroleum ether each time. The combined petroleum ether extracts were evaporated to dryness. Residua were dissolved in scintillation fluid and counted for radioactivity. The activities of ACC and FAS are expressed as nanomoles of [1-14C]acetyl-CoA incorporated into fatty acids per minute per milligram of protein.

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onto a nitrocellulose membrane [26]. To detect ACCα, AMPKα and their respective phosphorylated forms pACCα and pAMPKα, the blots were incubated with specific primary antibodies for 1.5 h at room temperature and then for 1 h with appropriate horseradish peroxidase-conjugated IgG (dilution 1:5000). Signals were detected by enhanced chemiluminescence using the Amersham ECL plus kit (GE Healthcare, Milan, Italy). α-Tubulin detection was used for signal normalization [26].

2.10. Statistical analysis Data are the means ± S.D. for the indicated number of experiments. The results were computed with Excel (Microsoft 7). One-way analysis of variance (ANOVA) was used to determine significant differences among groups in Figs. 2, 3, 5, 6 and 7. Twoway ANOVA was adopted to assess any differences among the treatments and the times

2.7. DGAT activity assay DGAT activity was determined by measuring the incorporation of [1-14C]palmitoylCoA into TG using endogenous diglycerides (DG) as the second substrate [27]. Hepatocytes, pre-incubated for 2 h with 25 μM HTyr, Tyr or Ole, were incubated with 150 μl of preheated (37°C) assay medium, supplemented with digitonin. The assay was terminated after 4 min by adding 4 ml CHCl3:CH3OH (1:1, v/v). The final assay mixture contained the following components at the indicated concentrations: 50 mM 4morpholineethanesulfonic acid (pH 6.5), 5 mM EDTA, 1 mM dithioerythritol, 65 mM NaCl, 0.1 mM [1-14C]palmitoyl-CoA (0.2 μCi/assay), 0.25 mg bovine serum albumin, and 64 μg digitonin/mg cell protein. TG were isolated from a lipid extract [23] by TLC on Silica G using petroleum ether:diethylether:acetic acid (80:20:2, v/v/v) as the developing solvent. The silica, containing the TG fraction, was scraped off the plate and the radioactivity was counted. 2.8. HMGCR activity assay The HMGCR activity assay was performed essentially as described in [28]. Hepatocytes, pre-incubated for 2 h with 25 μM HTyr, Tyr or Ole, were incubated with 150 μl preheated (37°C) assay medium, supplemented with digitonin. Primary rat-hepatocytes were permeabilized with digitonin to quantify [3-14C]HMG-CoA (0.33 mM, 2 dpm/pmol) conversion to radiolabelled mevalonolactone. The final assay mixture contained the following components at the indicated concentrations: 16.7 mM imidazole (pH 7.2), 30 mM EDTA, 0.3 mM EGTA, 50 mM KF, 2.2 mM dithiothreitol, 0.33 mM [3-14C]HMG-CoA (2 dpm/pmol), 64 μg digitonin/mg cell protein, 33.5 mM glucose 6-phosphate, 3.4 mM NADP+, and 157.5 mU glucose 6-phosphate dehydrogenase for NADPH + H+ generation. The assays were performed for 8 min at 37°C and the reactions were stopped by the addition of 20 μl of 7 M HCl. Mevalonate formed during the reaction was lactonized for at least 30 min at 37°C. As an internal standard [3H] mevalonolactone was added to all samples [25,26]. The radioactive product was isolated by TLC using toluene:acetone (1:1, v/v) as the mobile phase. Silica spots were recovered and subjected to scintillation counting. 2.9. Western blot analysis Primary rat-hepatocytes were seeded at a density of 1×106 cells per 100 mm diameter Petri dish. 2 h after plating, the medium was changed and incubations were carried out in serum-rich (10% FBS) medium for 2 h. The medium from each Petri dish was discarded and the cells were washed twice with 4 ml of ice-cold PBS. Cells were scraped with a rubber policeman into 0.5 ml of lysis buffer containing 50 mM HEPES, 250 mM mannitol, 10 mM citrate, 4 mM MgCl2, 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 0.5 μM phenylmethanesulfonylfluoride, PMSF, 0.05% Tween 20, 0.5% βmercaptoethanol and protease inhibitors. The extracts were heat-denaturated for 5 min and samples containing 30 μg of total protein were loaded on 7% sodium dodecyl sulfate polyacrylamide gels. Following electrophoresis, the proteins were transferred

Fig. 2. Dose-dependent effect of hydroxytyrosol, tyrosol and oleuropein on cholesterol synthesis in rat-hepatocytes. After an initial 2 h plating period, primary rathepatocytes, growing in serum-rich medium, were incubated for 1 h with labelled acetate and different concentrations (0–100 μM) of hydroxtyrosol (A), tyrosol (B) or oleuropein (C). The data, expressed as nmol [1-14C]acetate incorporated into cholesterol/h/mg protein, are means±S.D. of six independent experiments. In each experiment determinations were carried out in triplicate. Values with superscripts are significantly different at P b.05.

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2 h) compared to the controls (0 μM, 0.5–2 h); thus, no toxic effect could be ascribed to any of the treatments (data not shown). Acetate in the cell is transformed into acetyl-CoA, which represents a common precursor for both fatty acid and cholesterol synthesis. Hence, both these metabolic pathways were assayed by using labelled acetate as a precursor. Bar graphs in Fig. 2 and 3 (2.5–100 μM EVOO phenols) show a significant reduction of [1- 14C]acetate incorporation into total cholesterol and fatty acids, respectively. This inhibition, measured after 1 h of EVOO phenol incubation, is dose dependent and already evident at a concentration of 25 μM. Fig. 4 shows the time-courses of labelled acetate incorporation into cholesterol (Panel A) and fatty acids (Panel B) both in the absence (control) and presence of 25 μM HTyr, Tyr or Ole. Cholesterol synthesis is significantly inhibited by all of the tested compounds already after 1 h of EVOO phenol treatment (Fig. 4A). In particular, at 2 h of incubation, all three phenols inhibited cholesterogenesis by about 30% compared to controls with no significant differences in cholesterol levels observed among HTyr-, Tyr- or Ole-treated hepatocytes. The phenol treatments also inhibited fatty acid synthesis. At 2 h of incubation, HTyr and Ole caused an ~50% reduction of labelled acetate incorporation into fatty acids while Tyr inhibited lipogenesis by about 30% (Fig. 4B). All further experiments were performed on cells treated with 25 μM phenol and incubated for 2 h in order to obtain significant inhibition of lipid and fatty acid synthesis while at the same time using the lowest effective phenol concentration. 3.2. Effect of EVOO phenolic compounds on neutral lipid synthesis Since newly synthesized fatty acids are mainly incorporated into complex lipids, the effect of EVOO phenol addition to primary cultured rat-hepatocytes on [1-14C]acetate incorporation into neutral lipids was tested. TLC analysis of neutral lipids revealed that TG, DG and free cholesterol are the main fractions that incorporated labelled acetate (Fig. 5). After cell treatment with each of the three EVOO phenol compounds, neutral lipid synthesis was mainly reduced in TG and cholesterol fractions (Fig. 5). All phenolic compounds significantly lowered TG synthesis compared to control, with HTyr and Ole being similarly potent (−35%) and Tyr having an intermediate effect (−20%). No significant changes were detected in label incorporation into phospholipids following any of the treatments (data not shown). 3.3. EVOO phenol modulation of lipogenic enzyme activities

Fig. 3. Dose-dependent effect of hydroxytyrosol, tyrosol and oleuropein on fatty acid synthesis in primary rat-hepatocytes. Primary rat-hepatocytes were incubated for 1 h with labelled acetate and different concentrations (0–100 μM) of hydroxtyrosol (A), tyrosol (B) or oleuropein (C). The data, expressed as nmol [1-14C]acetate incorporated into fatty acids/h/mg protein, are means ± S.D. of six independent experiments. In each experiment determinations were carried out in triplicate. Values with superscripts are significantly different at P b .05.

in Fig. 4. When significant values were found (Pb.05), post-hoc comparisons of means were made using Tukey-Kramer test. All statistical analyses were performed with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA).

3. Results 3.1. Effect of EVOO phenols on cholesterol and fatty acid synthesis Firstly, MTT test, morphological observation, protein assay and trypan blue exclusion showed that hepatocyte viability was not affected by the phenolic treatments (HTyr, Tyr or Ole, 2.5-100 μM, 0.5-

In order to determine the underlying mechanism of action of HTyr, Tyr or Ole on lipid biosynthetic pathways, experiments were carried out to assay the activities of the following enzymes: (i) ACC and FAS for de novo fatty acid synthesis, (ii) DGAT for TG synthesis and (iii) HMGCR for cholesterol synthesis. All enzymatic activities were measured by in situ assays using digitonin-permeabilized rat-hepatocytes. The specific activity of ACC was markedly reduced (~40%) in cells incubated for 2 h with 25 μM HTyr or Ole compared to untreated cells (Fig. 6A). Incubation of the hepatocytes with 25 μM Tyr resulted in a modest but significant reduction in ACC activity (Pb.05 vs control, Fig. 6A). The ACC product, malonyl-CoA, is the substrate for the multienzyme complex FAS in the cytosolic process of long-chain fatty acid synthesis. FAS activity showed no significant change after any EVOO phenol treatment (Fig. 6B). DGAT activity, which catalyses long-chain fatty acid addition to DG for TG formation, was measured by monitoring the rate of [1-14C]palmitoyl-CoA incorporation into TG. DGAT activity was reduced (~30%) in HTyr- and Ole-treated cells compared to untreated cells (Fig. 6C). Similarly to what was observed in the ACC assay, Tyr induced a smaller but statistically significant reduction of DGAT enzymatic activity (Fig. 6C). HMGCR activity was affected to a similar extent by each one of the phenolic compounds

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Fig. 4. Time-dependent effect of hydroxytyrosol, tyrosol and oleuropein on cholesterol and fatty acid synthesis in primary rat-hepatocytes. Primary rat-hepatocytes were incubated for 0.5, 1 or 2 h with 25 μM hydroxytyrosol, tyrosol or oleuropein and labelled acetate, the incorporation of which into cholesterol (A) and fatty acids (B) was followed. The data, expressed as nmol [1-14C] acetate incorporated/h/mg protein, are means ± S.D. of six independent experiments. In each experiment determinations were carried out in triplicate. Values not sharing the same superscript are significantly different (P b .05, two-way ANOVA followed by Tukey’s test). Ctr, control, untreated cells; HTyr, hydroxytyrosol; Tyr, tyrosol; Ole, oleuropein.

tested, and treated cells showed a ~20% reduction in activity compared to untreated cells (Fig. 6D). Note, all these findings are in accordance with the results in Figs. 2–5 which show the phenolinduced reduction in total synthesis of cholesterol, fatty acids and neutral lipids.

3.4. Activation of AMPK by EVOO phenols Since short-term alterations in lipogenic enzyme activities could depend on modifications of their phosphorylation state [29], we investigated whether an AMPK/ACC signalling mechanism might be

Fig. 5. Neutral lipid synthesis in rat-hepatocytes treated with EVOO phenols. Primary rat-hepatocytes were incubated for 2 h with 25 μM of hydroxytyrosol, tyrosol or oleuropein. During the last hour of incubation labelled acetate was added and its incorporation into neutral lipids was assessed using thin layer chromatography. The data, expressed as cpm/mg protein, are means± S.D. of five independent experiments. In each experiment determinations were carried out in triplicate. For each lipid class superscripts indicate values that are significantly different at P b .05. FFA, free fatty acids; DG, diglycerides; TG, triglycerides; CHOL, cholesterol; CHOL EST, cholesterol esters. Ctr, control, untreated cells; HTyr, hydroxytyrosol; Tyr, tyrosol; Ole, oleuropein.

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Fig. 6. Effect of EVOO phenols on ACC, FAS, DGAT and HMGCR activities. After 2 h incubation with 25 μM of hydroxytyrosol, tyrosol or oleuropein, the selected enzyme activities were assayed in digitonin-permeabilized hepatocytes. The values presented, expressed as a percentage of the control, are means±SD of five independent experiments. Control specific activity: ACC, 0.22±0.05 nmol [1-14C]acetyl-CoA inc/min/mg protein; FAS, 0.71 ± 0.08 nmol [1-14C]acetyl-CoA inc/min/mg protein; DGAT, 0.11 ± 0.03 nmol [1-14C]palmitoyl-CoA inc/ min/mg protein; HMGCR, 43.0 ± 5.8 pmol [3-14C]HMG-CoA inc/min/mg protein. Superscripts indicate values that are significantly different at P b .05. Ctr, control, untreated cells; HTyr, hydroxytyrosol; Tyr, tyrosol; Ole, oleuropein.

involved in the observed reduction of ACC activity. This was carried out by assessing the relative levels of phosphorylated ACC and AMPK to total ACC and AMPK in phenol-treated and control cells by Western blot analysis. Hepatoytes treated with EVOO phenolic compounds (2 h, 25 μM) showed significant differences in the ratio of phosphorylated/total ACC (Fig. 7A) and phosphorylated/total AMPK (Fig. 7B) compared to control cells. HTyr and Ole were

markedly effective in stimulating the phosphorylation of ACC (+ 40%) and AMPK (+60%) in treated rat-liver cells; whereas Tyr was less effective, inducing however, a significant increase in the levels of the phosphorylated forms of ACC (+19%) and AMPK (+25%). These results are in accordance with the findings that all three phenols significantly inhibited ACC activity, albeit HTyr and Ole more so than Tyr (Fig. 7A).

Fig. 7. Effect of hydroxytyrosol, tyrosol and oleuropein on ACC and AMPK phosphorylation. After 2 h incubation with 25 μM of hydroxytyrosol, tyrosol or oleuropein, hepatocytes were lysed and total protein was isolated. ACC, AMPK and their phosphorylated forms (pACC and pAMPK, respectively) were assessed by Western blotting and quantified by densitometry. pACC/ACC/α-tubulin protein ratio (A) as well as pAMPK/AMPK/α-tubulin protein ratio (B) were calculated. The ratios shown, expressed as a percentage of the control, are means±S.D. of five independent experiments and are presented in the two associated bar-charts. Superscripts indicate values that are significantly different at P b .05. Ctr: control, untreated cells; HTyr: hydroxytyrosol; Tyr: tyrosol; Ole: oleuropein.

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4. Discussion The low incidence of atherosclerotic disease in the Mediterranean area has led to the suggestion that high consumption of EVOO is protective against the development of this pathology [30,31]. Recent interest has focused on EVOO phenols, which are thought to have more effect on human health than once believed [1–4,8,32]. These phenolic compounds possess strong antioxidant properties [11,12] and show anti-inflammatory [13], anti-diabetic [15] and anti-atherosclerotic effects [5,9,14]. There are multiple mechanisms by which phenolic compounds might impact on the development of atherosclerosis, such as the inhibition of LDL oxidation, platelet aggregation, endothelial expression of tissue factor and adhesion molecules [5,33,34]. Several lines of evidence have shown that, besides monocyte adhesion to the endothelium, high plasma levels of cholesterol and TG play a critical role in the onset of atherosclerosis since their metabolism determines the fate of lipoproteins such as LDL and HDL [35]. Given this evidence, three of the main EVOO phenolic compounds, HTyr, Ole and Tyr, have attracted considerable attention because of their lipid-lowering effects observed in different hyperlipidemic animal models [16,19,36]. However, the biochemical mechanisms underlying the reported hypolipidemic effects, in terms of lipid metabolism in the liver, remain largely unknown as well as the relative differences in action among different phenols, thus limiting their therapeutic potential. In previous studies EVOO phenols were administered for several weeks before changes in plasma and hepatic lipid content were observed [16,19,36]. Since long-term changes are probably preceded by short-term control events, this work set out to study the short-term effects of HTyr, Tyr and Ole treatments on selected markers of lipid metabolism in hepatocytes of normal rats. We chose to focus on these short-term effects because it is known that the levels of EVOO phenolic compounds rise early after virgin olive oil ingestion, reaching a peak at around 1 h-post ingestion in plasma [37,38] and within 2 h in urine [37,39,40]. The present study represents the first report of a rapid and direct effect of EVOO phenols on lipid synthesis in normal liver cells. Here we show that in primary-cultured rat-hepatocytes EVOO phenolic compounds cause a short-term inhibition that is dose- and time-dependent of both cholesterol and fatty acid syntheses. After 2 h of a 25 μM phenol treatment, rat-liver cells showed approximately a 30% and 50% reduction in cholesterogenesis and fatty acid synthesis, respectively. Another intriguing result obtained in this study is the finding that, despite the putative antioxidant inactiveness of Tyr compared to other EVOO phenols [41,42], its effect on cholesterogenesis and lipogenesis in rat-liver cells was easily detectable and significant. TG synthesis was inhibited by both 25 μM HTyr and Ole after 2 h of incubation, whereas the effect of Tyr on label incorporation into TG was modest althought statistically significant compared to untreated cells (Pb.05 vs control). Since biological activity and chemical structure of EVOO phenolic compounds are closely correlated [43], the observed differences in their relative potencies could be attributable to the structure of elenolic acid (open or close form) and the number and position of hydroxyl groups on the aromatic ring (see Fig. 1). Indeed, current findings definitely support the notion that the ability of naturally occurring phenols to affect several kinases involved in signal transduction [44], or the cellular antioxidant machinery [43], largely depend on their chemical structure. These studies have shown that the position and number of the hydroxyl group on the 2-phenyl ring strongly influence the conformation of the phenolic molecules which, in turn, dictate their effects. Thus, even if beyond the scope of this study, it is reasonable to suggest that structure-activity relationship should be taken into account when assessing differences in the EVOO phenols-induced lipid lowering effect.

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The findings that primary hepatocytes are responsive to EVOO phenols suggest that the effects of these compounds observed in vivo [16,19,36] might be directly in the liver rather than secondary to their effects in other tissues or to the antiinflammatory/antioxidant responses. To determine the underlying mechanism of action of EVOO phenols on lipid synthesis, the activities of enzymes catalysing pace-setting steps of fatty acid, TG and cholesterol syntheses were investigated by in situ assays using digitonin-permeabilized rathepatocytes. This tool offers the advantage of rapid measurement of intracellular enzyme activities in a nearly natural environment, thus circumventing the necessity of preparing cellular fractions for enzyme assays [25]. De novo lipogenesis is catalysed by two enzymatic systems working in sequence: ACC, catalysing the carboxylation of acetyl-CoA to form malonyl-CoA which represents a substrate for FAS, and donates two carbon units during each sequential round of condensation in the synthesis of palmitate. Data presented in Fig. 6 indicate that only ACC activity is significantly reduced by the addition of phenols to hepatocytes. With regard to the apparent insensitivity of FAS to HTyr, Tyr or Ole, it is worth noting that while ACC is regulated by both short- and long-term mechanisms, only the latter is involved in FAS modulation [45]. Based on these results we hypothesise that reduced ACC activity could be responsible, at least in part, for the rapid EVOO phenolinduced decrease of total fatty acid biosynthesis in rat-liver cells. Since ACC activity is known to be short-term regulated by allosteric modulators and/or by phosphorylation/dephosphorylation processes [29], we examined the effect of EVOO phenols on the relative level of ACC phosphorylation in treated cells compared to untreated controls. Other studies using both cell lines and animal models have shown that dietary phenolic compounds may have protective roles against steatosis, carcinogenesis and adipogenesis, through the activation of the AMPK signalling pathway [46–49]. AMPK works as a sensor of cellular energy status, being activated by increased cellular AMP/ATP ratio or by upstream kinases depending on tissue responsiveness or the dose and the time of incubation with its agonist [29]. In the liver, activation of AMPK (through phosphorylation of its α-subunit on Thr172) switches off fatty acid synthesis by phosphorylating and consequently inactivating ACC [29]. This mechanism seems to exert a protective role in metabolic syndrome-associated diseases and may account for the lipid-lowering effects of phenols [29,50,51]. The present work shows that within 2 h of 25 μM EVOO phenol treatment, the ratios of phosphorylated/total ACC and phosphorylated/total AMPK are altered in treated cells compared to control cells. In particular, HTyr and Ole were highly effective in stimulating the phosphorylation of AMPK, and consequently of ACC (+40%), thus inhibiting its activity (Fig. 6A). These results are similar to those obtained in vascular endothelial cells [47] and in human adipocytes [49] after HTyr treatment. It is possible that phenols activate AMPK by regulating AMP/ATP cellular ratio or targeting SIRT1 deacetylase (silent information regulator 1) pathway as already reported for resveratrol (a major phenol in red wine) in human HepG2 liver cells [50]. Whether such mechanisms have a role in the regulation of lipid metabolism induced by EVOO phenols is worthy of additional investigation. Regarding lipid synthesis, it was also found that the EVOO phenolinduced decrease of cholesterogenesis in rat-hepatocytes was, at least in part, attributable to a reduced activity of HMGCR, which is a key enzyme in this metabolic pathway. This finding adds additional support to a previous observation that Ole treatment significantly attenuated hepatic cholesterol levels produced by administering a high-fat diet to mice [19]. A potential mechanism underlying the reduced TG synthesis in treated cells was investigated by measuring the activity of DGAT. This enzyme, which catalyses long-chain fatty acid addition to DG for TG

8

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formation, is the only enzyme exclusively involved in this pathway and is considered to play a key regulatory role. A good correlation of cellular enzyme activity with the rate of TG synthesis has been reported in digitonin-permealized hepatocytes [25]. In line with the reduced TG synthesis induced by EVOO phenol treatments (see Fig. 5), DGAT activity was also significantly reduced by phenol treatment of cells. These findings indicate that, besides reduction in the activity of ACC, DGAT modulation could represent an additional control step for TG formation in EVOO phenol-treated hepatocytes. Availability of TG represents the most important source of secreted VLDLs that are produced in the liver primarily for the blood transport of newly synthesized TG to peripheral tissues. Thus, it is plausible that the observed EVOO phenol-induced reduction in hepatic lipid synthesis may decrease VLDL secretion, which may be an additional route to the observed hypolipidemic effect of HTyr, Ole and Tyr [16,17,19]. Data on plasma phenol concentrations that can be achieved in humans after consumption of olive oil are poor [40] and controversial [52]. This may be due to a number of factors: the very variable levels of phenols found in EVOOs (50–800 mg/kg) [53]; the level of dietary intake of olive oil; the method and the time chosen for plasma phenol quantification [52]. However, the effective phenol concentration used in this study is in the μM range and similar phenol concentrations have been shown to exert anti-atherogenic and antioxidant effects in several cell lines [14,47,49,52]. The 25 μM EVOO phenol concentration used in the present study could be considered a pharmacological dose, as is the case for other antioxidants [54], because it is unlikely that this concentration can be achieved in plasma after EVOO oral intake. Anyhow, it must be underlined that liver together with gut are the first sites of phenol metabolism and biotransformation after the intake. This process is very relevant to the extent that phenols in their free form are deemed undetectable in biological matrices such as body fluids [52]. Considering the above, the question of EVOO phenol bioavailability in liver remains a matter of debate. The results of this study clearly demonstrate a short-term and direct inhibitory effect of 25 μM of EVOO phenols on fatty acid, cholesterol and TG synthesis in rat-liver cells; ACC, HMGCR and DGAT, the respective key regulatory enzymes, are mainly involved in this inhibition. Therefore, a reduction in hepatic lipid synthesis may be envisaged when assessing the potential benefits of EVOO phenols. However, the physiological relevance of these in vitro findings needs to be tested in appropriate animal models and in humans.

Acknowledgments The authors thank Dr. Math J. H. Geelen for critically reading the manuscript; Dr. Alberto Basset, University of Salento (Italy) for statistical assistance; Dr. Jackie Nugent, National University of Ireland Maynooth (Ireland) for English-language editing of the manuscript.

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