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1Designed by Júlia Muñoz Carreras

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Molecular mechanisms involved in the protective effect of the Mediterranean diet and olive oil consumption in humans Valentini Konstantinidou, PhD Thesis director: Dr. Maria Isabel Covas Planells Cardiovascular Risk and Nutrition Group, Institut Municipal d´Investigació Mèdica (IMIM-Hospital del Mar) PhD in Biomedicine, Universitat Pompeu Fabra, Department of Experimental and Health Sciences, 2 Barcelona, Spain

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Presentation Outline Introduction in Nutrigenomics Objective-Hypothesis Methods and Results Conclusions 3

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Introduction – Nutrigenomic era 4

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Introduction – NutrigenomicsPrinciples 5 Müller and Kersten, Nature Reviews 2003

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Introduction – Nutritional Genomics Nutrigenomic studies have focused on investigating the molecular mechanisms of action of several foods and nutrients, particularly lipids, on cardiovascular risk factors and other complex traits. Intervention studies, in which subjects receive a controlled dietary intake, provide the best approach for conducting cause- effect relationships between gene expression and diet. Limitations: - Small number of participants - Brief intervention duration - Lack of replication Ordovas et al. Mol Nutr Food Res 2007 6

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Introduction – Nutrigenomic studies generalGoals  To understand the molecular mechanisms of genes-diet interaction.  To prevent diet-related diseases.  To develop evidence-based nutrition.  To contribute to public health.State of the art  Mechanisms by which dietary patterns, foods or food components elicit their beneficial/harmful effects on human health are partially unknown.  One of the mechanisms could be the modulation of disease-related genes by dietary patterns, foods or food components.  At present, the knowledge concerning the role of diet in modulating atherosclerosis-related genes is limited. 7

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Introduction – Nutrigenomic era 8

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Introduction – Nutrigenomic studies IIIn peripheral blood mononuclear cells (PBMNCs)• Higher increase in TNFα expression after butter-rich breakfast vs olive oil.• Higher increase in IL6 mRNA response after butter-rich breakfast vs walnut. (Jimenez-Gomez et al. Atherosclerosis 2009)• Postprandial activation of NFκ B after butter- and walnut-rich meals vs olive oil. (Bellido et al. Am J Clin Nutr 2004)In human adipocytes• Anti-inflammatory gene expression profile after MUFA-diet vs SFA-diet. (van Dijk et al. Am J Clin Nutr 2009)• Higher TNFα expression after conjugated linoleic acid (CLA) vs olive oil.• Lower expression in GLUT4 (glucose transporter 4), LEPTIN, LPL (lipoproteinlipase) after CLA vs olive oil. (Raff et al. J Nutr 2009) 9

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Hypothesis The consumption of a TMD, VOO and its PC, can modify the human in vivo gene expression. The gene expression changes will be towards a protective mode for cardiovascular disease development. 10

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Methods - Task 1: VOO intervention study-pilot (n=11)Design : Linear Study Wash out period 1-4 days: habitual diet 5-7 days: diet with very controlling excess of low phenolic content, antioxidants, sunflower sunflower oil for raw oil for raw and cooking and cooking purposes purposes • Intervention daySamples collection 0h 1h 6h 50ml of VOO ingestion • 3 weeks (Khymenets et al. OMICS 2009) 11

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Results - Task 1: VOO intervention study Characterization of Human Gene Expression Mononuclear Transcriptome Changes after Olive Oil Ingestion: an Response after Sustained Virgin Exploratory Approach. Olive oil Consumption in humans Konstantinidou, V. et al. (2009) Khymenets, O. et al. (2009) Folia Biologica (Praha) 55, 85-91 OMICS 13, 7-19 7 insulin sensitivity-related genes were modulated by VOO ingestion Task 1.2 Postprandial time course of changes in the expression of those genes after VOO ingestion (qRT-PCR) 12

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Methods Characterization of Human Gene Mononuclear Transcriptome Response Expression Changes after Olive Oil after Sustained Virgin Olive oil Ingestion: an Exploratory Approach. Consumption in humans. Konstantinidou et al. Khymenets et al. OMICS 2009 Folia Biologica (Praha) 2009 Time Course of Changes in the Expression of Insulin-Sensitivity Related Genes after an Acute Load of Virgin Olive Oil. Konstantinidou, V. et al. OMICS 2009 Literature Selection of 47 review atherosclerosis-related genes Task 2: TMD intervention 13

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Methods - Task 2: TMD intervention studyStudy Flow diagram Invited to be screened (n=99) Ineligible (n=9) Did not meet inclusion criteria (n=7) Declined to participate (n=2) Randomly assigned (n=90) Declined to follow up (n=1) Control Group (n=29) TMD+WOO (n=30) TMD + VOO (n=30) (n=20 in gene expression) (n=16 in gene expression) (n=20 in gene expression) 14

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Results - Task 2: TMD intervention study, Three-group analysesGene expression changes after TMD+VOO intervention 15 p<0.05 for linear trend in all cases * p<0.05 vs. control

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Results - Task 2: TMD intervention studyVolunteer’s dietary compliance • Volunteers compliance was good as it is reflected in urinary T and HT levels † † p=0.007 for quadratic trend Hydroxytyrosol (ng/ml)Tyrosol (ng/ml) * Control group TMD+WOO TMD+VOO Control group TMD+WOO TMD+VOO* p<0.05 vs control; † p<0.05 vs TMD+WOO 16

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ConclusionsStrengths To work in real life conditions in all interventions To use both the whole dietary pattern and the single component approach To work against our hypothesis by using a Spanish dietary pattern in the control groupLimitations- A lack of control group for the VOO intervention- The inability to assess potential interactions- As expected, we observed modest changes in gene expression- Unknown effects over longer intervention periods 18

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Conslucions IFNγ ADAM17 OGT IL7R ALOX5AP ARHGAP15 LIAS ADRB2 CD36 PPARBP POLK Inflammation Oxidative stress Insulin resistance The gene expression changes were observed in a protective mode for counteracting these situations. 19

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Discussion These results provide, for the first time, evidence on: An in vivo human nutrigenomic effects of the TMD, in healthyvolunteers. An in vivo human nutrigenomic effect of olive oil phenoliccompounds down-regulating atherosclerosis-related genes. 20

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Thank you for your attention 21