[Frontiers in Bioscience E3, 11-21, January 1, 2011]

Postprandial transcriptome associated with virgin olive oil intake in rat liver

Roberto Martinez-Beamonte1, 3, Maria A. Navarro1, Natalia Guillen1, 3, Sergio Acin1, Carmen Arnal2, 3, Mario A Guzman1, Jesus Osada1, 3

1Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Veterinaria, Instituto Aragones de Ciencias de la Salud (Universidad de Zaragoza- Salud del Gobierno de Aragon), Spain. 2Departamento de Patologia Animal, Facultad de Veterinaria, Universidad de Zaragoza, Spain. 3CIBER de Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Spain

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
3.1. Rats
3.2. Study design
3.3. Plasma triglycerides
3.4. Histological analysis
3.5. Hepatic lipid analysis
3.6. RNA isolation, Affymetrix oligonucleotide array hybridization, and data analysis
3.7. Quantification of mRNA
3.8. Statistical analysis
4. Results
4.1. Plasma and hepatic triglycerides
4.2. Gene expression in livers of rats 4 hours after a fat meal
4.3. Gene expression in livers of rats 8 hours after a fat meal
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Liver has been proposed as a gatekeeper that regulates postprandial lipemia and a potential target for regulation by acute intake of virgin olive oil. To characterize the hepatic gene expression response to a fat gavage, male rats were fed a bolus of 5 ml of extra-virgin olive oil and the hepatic mRNA expression analyzed 4 hours later using DNA microarrays. To provide an initial screening of candidate genes, only twenty one with remarkably modified expression between both conditions (signal log2 ratio > 2.5 or < -2.5) were considered and confirmed by quantitative real time PCR. Those that presented biological significance were also analyzed 8 hours after the experimental approach. Hepatic A2m Slc13a5 and Nrep mRNA expressions were found significantly changed in both studied conditions and showed the highest significant associations with postprandial plasma triglycerides and lack of association with basal triglyceridemia. These results highlight new gene regulation in liver by postprandial triglyceridemia and will help to understand the complex human pathology providing the involvement of hepatic proteins and new strategies to cope with postprandial metabolism.

2. INTRODUCTION

The contribution of postprandial lipemia to atherosclerosis has been well established (1) and a large series of studies have found significant associations between impaired elimination of postprandial lipoproteins and cardiovascular diseases (2-4). Postprandial metabolism can be influenced by a lot of factors whose complete list is far behind to be known. Aspects such as genetic and hormonal influences, feeding schedule, composition of food with emphasis in cholesterol and dietary fat content, and saturation have been found to modulate postprandial responses (5-9). Cianflone et al have proposed intestine, adipose tissue, muscle and liver as the gatekeepers that regulate postprandial lipemia and potential targets for regulation (7). In this regard, Perona et al have observed that incubation of hepatocytes with different postprandial lipoproteins induces changes in the mRNA expression of receptors (10), confirming an important role of liver in regulation of the postprandial process susceptible to be tested using high-throughput analyses.

Rat is an animal model which most of its plasma cholesterol is transported into HDL, due to the absence of cholesterol ester transfer protein (11)-an activity found to parallel postprandial triglyceride response- (12). Despite this difference with the human model, this animal represents an interesting approach to study changes in the postprandial state in the absence of the mentioned protein. Development of functional approaches in this animal is particularly relevant due to its recent incorporation to the transgenic and knock-out technology (13, 14), which will bring a renew interest and a demanding use. In addition, rat lipoprotein metabolism has been found to be sensitive to chronic dietary fat amount and composition (15). Also, chronic administration of a diet enriched in 10% (w/w) olive oil has been observed to reduce plasma TG and modify hepatic gene expression in apoE- deficient mice (16). Previous experiments in mice showed that a bolus of 16 mL olive oil/ kg was sufficient to induce their plasma postprandial response (17), suggesting that this dose may also be sufficient in rats. To test the hypotheses that this dose may change plasma triglycerides and induce important changes in hepatic gene expression, rats were administered a virgin olive oil gavage and sacrificed four hours later. Then, gene expression was determined by micro array analysis to systematically examine the expression of a large number of hepatic genes. The 21-gene expressions were confirmed by quantitative real time PCR. The relevance of observed changes was further tested in animals eight hours after the fat intake, the confirmation of these results shows the association of them with hepatic- specific mechanisms involved in the postprandial regulation induced by an acute fat intake.

3. MATERIAL AND METHODS

3.1. Rats

Male Wistar rats, weighing 250- 300 g (purchased from Charles River, Barcelona, Spain), were used for experiments. Rats, housed in sterile filter-top cages (3-4 per cage), were acclimatized in a room maintained at 20�C with a 12-h light-dark cycle for 10 days, allowed ad libitum access to water and standard chow diet (Pascual S.A., Barcelona, Spain), and fasted for 18 h before experiments. Animals were handled and killed, always observing criteria from the European Union for care and use of animal laboratory in research, and the protocol was approved by the Ethics Committee for Animal Research of the University of Zaragoza.

3.2. Study design

Prior to the experiment, a baseline fasting blood sample was obtained cutting a piece of tail that had been anaesthetized by subcutaneous injection with 0.5 ml of 5% lidocaine HCl (Braun Medical SA, Barcelona, Spain). Tail blood samples were collected in heparin-coated capillary tubes and centrifuged at 2000 g for 5 min. Obtained plasma was kept at 4�C for immediate triglyceride analysis. Rats were randomly allocated into 3 groups of 5 rats each. The control group did not receive any fat meal. The other two groups were fed 5 ml of extra virgin olive oil (Aceites Toledo, Spain) as a bolus and sacrificed 4 and 8 hours after the feeding, respectively. This amount represents the use of a dose of 16 mL olive oil/ kg, sufficient to induce a plasma postprandial response in mice (17). Olive oil was directly administered to stomach using a 1.1 mm diameter and 50 mm long flexible Abbocath connected to a sterile polypropylene syringe and delivered in 4 seconds. At the moment of sacrifice, rats were anesthetized with 1 ml of 8% Avertine (Aldrich Chemical Co., Madrid, Spain) in 0.1 M phosphate, pH 7.2, and blood drawn from hearts. Blood was collected in tubes containing 1 g/l sodium EDTA. Liver was removed and quickly frozen in liquid N2 until total RNA was extracted.

3.3. Plasma triglycerides

Total plasma triglyceride concentrations were enzymatically quantified in a microtiter assay using commercial kits from Sigma Chemical Co. (Madrid, Spain). Cardiolipid (Sigma) was used as quality control.

3.4. Histological analysis

A sample of liver from each rat was stored in neutral formaldehyde and embedded in paraffin wax. Sections (4 μm) were stained with hematoxylin and eosin and observed using a Nikon microscope. Hepatic fat content was evaluated by quantifying the extent of fat droplets in each liver section with Adobe Photoshop 7.0 and expressed as percentage of total liver section (18).

3.5. Hepatic lipid analysis

Tissues (10 mg) were homogenized in 1 ml of PBS. An aliquot was saved to determine protein concentration by the BioRad dye binding assay (BioRad, Madrid, Spain). One volume of homogenate was twice extracted with two volumes of chloroform: methanol (2:1). The separated organic phases of each animal were combined and evaporated under N2 stream. Extracts were dissolved in 100 �L of isopropanol to estimate cholesterol and triglyceride concentrations using commercial kits as above described.

3.6. RNA isolation, Affymetrix oligonucleotide array hybridization, and data analysis

RNA from each liver was isolated using Trigent reagent (Sigma). DNA contaminants were removed by TURBO DNAse treatment using the DNA removal kit from AMBION (Austin, TX, USA). RNA was quantified by absorbance at A260/280 (the A260/280 ratio was greater than 1.75). The integrity of the 28 S and 18 S ribosomal RNAs was verified by agarose formaldehyde gel electrophoresis followed by ethidium bromide staining and the 28S/18S ratio was greater than 2. Twenty m g aliquots of total liver RNA from each rat of each group (n= 5 for the control and n= 5 for 4h group) were pooled and purified using the RNeasy system (Quiagen, Barcelona, Spain). Eight micrograms of total liver RNA were then used for biotin labeling. Hybridization, washing, scanning and analysis with the Affymetrix GeneChip Rat Expression Array 230 version 2.0 (Affymetrix, Santa Clara, CA) were performed according to the standard Affymetrix protocols. Fluorometric data were generated by Affymetrix software, and the fluorimetric signal adjusted so that all the probe sets provided intensities within a manageable range. The data obtained in the microarray hybridizations were processed with Affymetrix® GeneChip® Command Console® 1.1 and Expression Console™ 1.1 (Affymetrix) software. Annotation was confirmed by checking the MGI Gene Ontology Browser at the Jackson's website http://www.informatics.jax.org/searches/GO.cgi?id=GO:0042416 and the Rat Genome Browser at http://rgd.mcw.edu/tools/genes/genes_view.cgi?id=631411. An initial approach of selecting genes that were up- or down-regulated in the postprandial state was carried out by first considering only those whose signal log2 ratio between control and 4 h after feeding was higher than 2.5 (up-regulated genes) or lower than -2.5 (down-regulated genes). Transcripts whose signal intensities were lower than the noise of the matrix � 3 standard deviations were not taken into account either. The complete datasets were deposited in the GEO database (accession number GSE17859).

3.7. Quantification of mRNA

The difference in mRNA expression observed with the microarrays was confirmed by quantitative real-time RT-PCR (qRT-PCR) analysis of individual samples. Equal amounts of DNA-free RNA from each sample of each animal were used in qRT-PCR analyses. First-strand cDNA synthesis and the PCR reactions were performed using the SuperScript II Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, Madrid, Spain), according to the manufacturer's instructions and as previously described (19). Primers were designed by Primer Express® (Applied Biosystems, Foster City, CA) and checked by BLAST analysis (NCBI) to verify specificity and selective amplification of the target gene as well as to get amplification of the cDNA and not of genomic DNA. The sequences are shown in Table 1. Real time PCR reactions were performed in an ABI PRISM 7700 Sequence Detector (Applied Biosystems) following the standard procedure. The specificity of the PCR reaction was confirmed by observing a single dissociation curve. The relative amount of all mRNAs was calculated using the comparative 2-ΔΔCt method. Cyclophilin B (Ppib) mRNA levels were used as the invariant control.

3.8. Statistical analysis

The results are expressed as means � SD. Comparisons were made using one-way ANOVA and the Tukey-Kramer multiple comparison test (post hoc) when the distribution of the variables was normal. When the variables did not show such a distribution (according to the Shapiro-Wilk's test), or failed to show homology of variance, comparisons were made using the Mann-Whitney U test. Correlations between variables were sought using the Pearson's or Spearman's correlation tests. All calculations were performed using SPSS version 15.0 software (SPSS Inc, Chicago, IL). Significance was set at P≤ 0.05.

4. RESULTS

4.1. Plasma and hepatic triglycerides

Baseline and postprandial plasma TG concentrations of the three experimental groups are presented in Figure 1. As shown in Panel A, the basal triglyceridemia of rats showed no significant difference among the three established groups. Postprandial plasma triglycerides (Panel B) significantly increased either 4 or 8 hours after feeding compared to the control group, and the values reached at the latter time point were also significantly higher than those after 4 hours. '

Analysis of hepatic TG content, shown in panel C, indicated that this parameter increased significantly four hours after the fat meal and its levels remained significantly elevated at the eight-hour-time point. No significant change was observed between both time periods. A similar trend was observed for hepatic cholesterol (Fig 1D). Therefore, this experimental design constitutes an interesting approach to study the presence of triglyceridemia and the transitory postprandial steatotic liver as well as the potential involved mechanisms.

4.2. Gene expression in livers of rats 4 hours after a fat meal

Prior to the RNA isolation, a liver histological analysis was undertaken to discard any pathological feature in any of the rats. Once verified the previous criterion and to determine the changes in hepatic gene expression induced by the administration of a fat gavage, the expression of 31,100 transcripts represented on the Affymetrix GeneChip Rat Expression Array 230 version 2.0 was quantified in pooled liver samples of 5 fast rats as controls and 5 rats sacrificed 4 hours after having been fed them with a bolus of 5 ml extra-virgin olive oil. The livers of control group expressed 15,667 transcripts (identified as "present" by Affymetrix software), while those of the fed animals expressed 15,061. Using the Mann-Whitney ranking feature of the Affymetrix software to determine significant differences in gene expression (P<0.01), the increased expression of 1369 sequences plus the reduced expression of 1675 transcripts was identified in samples from the fed animals compared to those not fed, when no multiple test correction was applied. Based in previous works (20), a cutoff point of variation taken as signal log2 ratio higher and lower than 1.5 was introduced, showing this as the biological mRNA variation, we identified the increased expression of 170 sequences plus the reduced expression of 94 transcripts in samples from the fed animals compared to those not fed. Another source of false positives previously identified was the insensitivity of the scanner (21). For this reason, transcripts whose signal intensities were lower than the noise of the matrix � 3 standard deviations were removed as well as equally redundant expressions. When both criteria were taken into consideration, the number of genes with increased and repressed expression was reduced to 114 and 70, respectively.

Genes, with a signal log2 ratio higher than 2.5 (for those genes up-regulated) or lower than -2.5 (for those down-regulated), were taken into account to select the most relevant differentially regulated ones. Table 2 lists the genes whose mRNAs reflected these expressions. Sixteen genes fulfilled the criterion of showing increased expression as a response to the olive oil bolus. Among these genes, 3 coded for cellular membrane proteins (Rell1, Fam134b and Coq10b), 2 were involved in immunity (Cxcl1 and A2m), 2 belonged to lipid metabolism (Ces2 and Fabp4), 6 were proteins with miscellaneous functions (e.g., 1 was involved in signal transduction (Gadd45b), another was an enzyme involved in amino acid metabolism (Sds), 1 was a metabolite transport protein (Slc13a5), 1 was an apoptotic protein (C8orf4a), 1 coded for a protein involved in circadian rhythm (Ccrn4lb), and 1 was a transcription factor (Zfp354a)). Finally, 3 EST were also found (BE113272, AI236188 and AI144796). Five genes met the criterion of showing a reduced expression as a response to the olive oil bolus (Tables 2). Of these, 2 were involved in signaling pathways (Nrep and RGD1309362), 1 coded for a hormone (Inhba), 1 was a metabolite transport protein (Slc34a2) and finally 1 coded for an ubiquitin ligase (Trim47).

To validate the results obtained with the microarray, the expressions of the 21 genes - BE113272, Gadd45b, Cxcl1, A2m, Slc13a5, Sds, Zfp354a, C8orf4a, Rell1, Ces2, AI236188, Ccrn4lb, Fam134b, Coq10b, Fabp4, AI144796, Nrep, Inhba, Slc34a2, Trim47 and RGD1309362- that were up- or down-regulated (signal log2 ratio > 2.5 or < -2.5) were individually studied by specific qRT- PCR assays. Cyclophilin B was used to normalize the results that are shown in Table 3. Among the sixteen up-regulated genes included in the validation analysis (Table 3), only eleven - BE113272, Gadd45b, A2m, Slc13a5, Sds, Zfp354a, Rell1, AI236188, Fam134b, Coq10b and AI144796- appeared significantly increased in their expressions by the administration of the olive oil bolus. Three of the five down-regulated genes selected- Nrep, Trim47 and RGD1309362- were significantly decreased in their expression after the fat gavage challenge.

Figure 2A shows the correlation between the mean values of signal log2 ratio for the above mentioned 21 genes with the microarray assay performed with pooled samples (Table 2), and the signal log2 ratio obtained by qRT- PCR of individual samples (Table 3). Good agreement between these procedures was obtained (r= 0.86, P<0.0001) and most of the samples, except three, were correctly classified, although the magnitude of the response differed between both methods. In an attempt to evaluate the accuracy of setting a cut-off point of a signal log2 ratio 2.5 in microarray analysis, the value of signal log2 ratio obtained in qRT-PCR analysis using individual samples was plotted against the probability value obtained for comparison of individual expressions for each gene (Figure 2B). A significant inverse relationship was observed where a signal log2 ratio of 2.3 was the minimum value required to obtain a P< 0.05. These results indicate that pooled samples can be successfully used to provide an initial screening of gene expression, with the attending economic and time savings although with the limitation of no information on biological variability. In addition, the high biological variation of mRNA indicates a certain threshold of change to evaluate significant changes by nutritional interventions.

4.3. Gene expression in livers of rats 8 hours after a fat meal.

To verify whether the hepatic expression changes observed after the postprandial were maintained for a longer period, a new group of rats receiving the fat meal were euthanized 8 hours after the food ingestion, and the fourteen previously selected genes were assayed. Fabp4 was also included due to its role in fatty acid transport. All these data are shown in Table 4. Three groups of genes were observed: those whose hepatic expressions were not significantly increased or decreased at this time period versus control (BE113272, Gadd45b, Sds, Zfp354a, Rell1, AI236188, Fam134b, Coq10b, AI144796, RGD1309362), those whose change persisted (Slc13a5, Nrep and Trim47), and those whose values were even more pronounced (A2m and Fabp4). As evidenced, the first category of genes was the most relevant and this suggests that the postprandial state induces a burst of gene expression at the mRNA level that is extinguished rapidly.

To verify whether the response of these gene expressions was linked to plasma triglycerides on each individual, an analysis of association between gene expressions (Tables 4) and the concentration of plasma triglycerides (Figure 1) was carried out. Figure 3 shows those associations found statistically significant. Interestingly, A2m and Slc13a5 mRNA expressions showed the highest positive associations with postprandial triglyceridemia (rs= 0.86, P<0.000 and rs= 0.76, P<0.001, respectively), and Nrep the lowest negative (rs= -0.907, P<0.000). No significant association was observed with initial triglyceridemia (data not shown). These results indicate that these new selected genes are associated with postprandial triglyceridemia.

To analyze to which extent the response of these three gene expressions was associated with the hepatic triglyceride and cholesterol contents, an analysis of correlation between gene expressions (Table 4) and the content of these lipids (Figure 1) was carried out and shown in Figure 4. Both lipid parameters were significantly associated with gene expression, although the value of association was stronger for hepatic cholesterol than triglycerides. These results suggest that these compounds are associated with these gene expression changes.

5. DISCUSSION

Using a fat gavage intervention in rats and postprandial microarray expression profiling and its confirmation by qRT-PCR at two independent time points, we have provided new panoply of genes whose hepatic expression is modified by an acute olive oil intake. Thereby, our work provides new resources whereby the liver may response to the handling of exogenous fat intake as well as an experimental approach to test the quality of oils in vivo.

In this work, the vast amount of information provided by microarrays has been handled by selecting only the genes with the highest expression changes and removing those with array signals within the signal noise of the matrix in agreement with our previous experience (20-22). With this criterion, only 16 genes were found to be remarkably upregulated and 5 notably downregulated. The modification of the expression of these genes was individually confirmed by qRT-PCR. Good agreement was observed between the Affymetrix chip and qRT-PCR data (Figure 2A). However, as shown in Figure 2A and Table 3, this approach might provide false positive variations regarding number of genes involved in a dietary response by the skewing effect of those highly responders. Furthermore in response to an acute intake of fat, results of Fig 2B indicate that the biological variation of mRNA when individually studied was high enough that only genes with a certain threshold of change (signal log2 ratio � 2.3) would show biological significance. Despite these limitations, the present approach provides meaningful and manageable data and is appropriate to find new candidate genes in experimental conditions.

The validation analysis carried out by qRT-PCR in individual samples (Table 3) showed that eleven genes - BE113272, Gadd45b, A2m, Slc13a5, Sds, Zfp354a, Rell1, AI236188, Fam134b, Coq10b and AI144796- were found significantly increased and three - Nrep, Trim47 and RGD1309362- were significantly decreased in their expression 4 hours after the olive oil challenge. In our experience (20-22), one single time point in RNA analysis may not be representative due to its high metabolic rate. For this reason, the variations of the previous fourteen genes and Fabp4, were further analyzed in an independent experiment where animals received the same amount of olive oil and were sacrificed eight hours after (Table 4). As indicated, 10 genes were not found increased at this time period. These results clearly indicate that RNA response following an acute olive oil intake is different at various time periods. However, five genes (A2m, Fabp4, Slc13a5, Nrep and Trim47) persisted in their significant changes at this time period as well. In an association analysis with plasma postprandial triglyceridemia (Fig 3), only four of these hepatic gene expressions were significantly associated (A2m, Fabp4, Slc13a5 and Nrep). When association analysis was carried out with hepatic cholesterol and triglycerides, only three of the previous genes (A2m, Slc13a5 and Nrep) showed significant associations (Fig 4). Using this biological procedure of selection, we have shown that expressions of these genes are highly associated with plasma triglyceridemia elicited after consuming olive oil gavage and at two time points in rats and with hepatic lipids. To our knowledge, this is the first report involving the hepatic changes of A2m, Slc13a5 and Nrep genes as a postprandial response.

Plasma postprandial levels of alpha-2 macroglobulin one hour after the meal intake in humans were not found significantly elevated (23), which would be in agreement with time dependence observed in our results (Table 4). An alpha-2 macroglobulin increase might modulate the levels of chylomicron remnants due to the fact that both compete for the low density receptor related protein (LRP) in their removal (24). Alpha-2 macroglobulin, as a major endoprotease inhibitor in mammalian blood, controls metalloproteinases and disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS-7 and ADAMTS-12) (25). The A2m- protein complex binds to the cell surface clearance receptor LRP and triggers rapid internalization of the complex by receptor- mediated endocytosis. Due to the fact that endoproteases are implicated in many diseases, including those of the cardiovascular system, arthritis and cancer (26), the observed increase after the postprandial fat meal may provide a new insight into the pathophysiology of degradative diseases.

Slc13a5 or Na+- coupled citrate transporter (NaCT) is a member of the vertebrate SLC13 gene family (27), mainly expressed in the liver and highly similar among mammalians (28). The expression of NaCT in human (HepG2 and Huh-7), rat liver primary hepatocytes and cell lines, showed robust activity for Na+- coupled citrate uptake in the sinusoidal membrane of hepatocytes (29). In a postprandial experiment using stable isotopes of triolein, Metges et al (30) observed different metabolites from these fatty acids pointing out to beta-oxidation, citric acid cycle and the phosphoenolpyruvate carboxykinase reactions in peripheral tissues. Citrate is present in blood at 135 �M (28) and the stimulation of the hepatic expression of NaCT in this postprandial design raises interesting questions regarding the utilization of circulating citrate for the generation of metabolic energy or for the synthesis of fatty acids and cholesterol in liver.

Nrep (also known as chromosome 5 open reading frame 13, C5orf13, D0H4S114, NM31, P311 or PTZ17) is an 8-kDa, polypeptide highly expressed in late-stage embryonic brain and adult cerebellum, hippocampus and olfactory bulb (31). In growing pigs fed a diet- restricted (20% less protein and 7% less energy) and showing an increase in intramuscular lipids, there was a concomitant increase of P311 (32). P311 expression also increased intracellular cholesterol and triglyceride levels, and intracellular lipid droplets in fibroblasts. Therefore, P311 has been proposed as an alternative pathway of lipid-droplet accumulation particularly induced by retinoic acid (33). Likewise, the opposite was observed in skeletal muscle atrophy in different experimental models such as fasted mice, cachetic, diabetic and uremic rats (34). In this sense, the changes of expressions of the hepatic P311/Nrep mRNA levels associated with hepatic cholesterol and triglycerides indicate that some of the processes observed in other tissues could be also involved in lipid handling in hepatic cells.

As a whole, our results provide new evidences to advance in the knowledge of hepatic management of postprandial administration of virgin olive oil. Through microarray analysis, hepatic gene expressions not previously associated with this phenomenon have been found differentially expressed and significantly associated. In this respect, our findings in rats will help to understand the complex human pathology providing hepatic proteins involved in postprandial regulation and new markers to evaluate the quality of olive oils in vivo.

6. ACKNOWLEDGMENTS

We thank Drs. C. Junquera and L. Osaba of Progenika Biopharma for performing the microarray analyses and Dr. Martinez for critical reading of the manuscript. We also thank Jesus Cazo, Jesus Navarro, Carmen Navarro and Clara Tapia of the Unidad Mixta de Investigacion for their invaluable help in maintaining the animals and Rosario Puyo for her technical assistance. This research was funded by grants CIBER Fisiopatologia de la Obesidad y Nutricion as an initiative of ISCIII, CICYT-FEDER (SAF2007-60173), Gobierno de Aragon (PI025/08) and Redes DGA (B-69). M.A.N., S.A. and MAG were recipients of Miguel Servet contracts and PROMEP fellowships. No competing financial interests exist.

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Abbreviations: A2m, alpha2- macroglobulin; LRP, low density receptor related protein; NaCT, Slc13a5 or Na+- coupled citrate transporter; OO olive oil; PCR, polymerase chain reaction; RT, reverse transcriptase; qRT, quantitative real time; TG, triglycerides; TRL, triglyceride rich lipoproteins

Key Words: Postprandial triglyceridemia, olive oil, hepatic gene expression, DNA microarrays

Send correspondence to: Jesus Osada, Department of Biochemistry and Molecular Biology, Veterinary School, University of Zaragoza, Miguel Servet, 177, E-50013 Zaragoza, Spain, Tel: 34-976-761-644, Fax: 34-976-761-612, E-mail:Josada@unizar.es