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9th International Master class NutrigenomicsWageningen<br />Defining healthFrom basic science to industrial relevance <br />1<br />1<br />

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Nutrigenomics Masterclass2011 How to use Nutrigenomics & molecular nutrition research? From challenges to solutions<br />Michael MüllerNetherlands Nutrigenomics Centre<br />& Nutrition, Metabolism and Genomics GroupDivision of Human Nutrition, Wageningen University<br />

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Why we need Nutrigenomics<br />To understand nutrition & metabolic health<br />To comprehensively phenotype<br />To validate FFQs<br />To enable strategies to optimize personal health<br />To provide scientific evidence for health claims of “functional” foods<br />Mechanisms<br />Biomarkers<br />Nutritional Science 2.0<br />Personal Nutrition<br />Health claim support<br />3<br />

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Key questions<br />What is your scientific problem? Why do you need nutrigenomics?<br />What are the best suitable genomics tools for your nutrition research and how to apply them?<br />One key question: What is the role of nutrition in the genotype-phenotype relationship?<br />What is healthy and how to measure and quantify the health status?<br />What is the impact of nutrigenomics for nutrition? What are the applications? What is the impact of nutrigenomics for the food (& pharma) industry?<br />

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Challenge 1: Successful ageingStay healthy as long as possible<br />100 %<br />Health/ “Quality of life”<br />time<br />

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Challenge 2: What's healthy?<br />

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Challenge 3: We have a tsunami of health problems<br />

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Challenge 4:Our “paleolithic” genes + modern diets<br />Paleolithic era<br />Modern Times<br />1.200.000 Generations between feast en famine<br />2-3 Generations in energy abundance<br />% Energy<br />% Energy<br />100<br />100<br />Grain<br />Milk/-products<br />Isolated Carbohydrates<br />Isolated Fat/OilAlcohol<br />Low-fat meatChicken<br />Eggs<br />Fish<br />50<br />Meat<br />Chicken<br />Fish<br />50<br />Fruit<br />Vegetables (carrots)<br />Nuts<br />Honey<br />Fruit<br />Vegetables<br />Beans<br />0<br />0<br />

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We are different<br />

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You are what you eat<br />

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How many human genes do we have?Not so many but….<br />

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Paternal Haplogroup:R1b1b2a1a1 a subgroup of R1b1b2<br /> <br />Maternal Haplogroup:<br />X2c1 is a subgroup of X<br />.<br /> <br />Where I am coming from?<br />Locations of haplogroup R1b1b2 circa 500 years ago, before the era of intercontinental travel<br />Locations of haplogroupX circa 500 years ago, before the era of intercontinental travel<br />

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Genotype<br />A genotype is an individual's collection of genes. The term also can refer to the two alleles inherited for a particular gene. <br />The genotype is expressed when the information encoded in the genes. DNA is used to make protein and RNA molecules. <br />The expression of the genotype contributes to the individual's observable traits, called the phenotype.<br />

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Phenotype <br />A phenotype is an individual's observable traits, such as height, eye color, and blood type. <br />The genetic contribution to the phenotype is called the genotype. <br />Some traits are largely determined by the genotype, while other traits are largely determined by environmental factors (including nutrition).<br />

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Phenotype plasticity<br /> Phenotypic plasticity is the ability of an organism to change its phenotype in response to changes in the environment (e.g. nutrition).<br />

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1 Genotype => 5 nutritional phenotypes<br />155 kg<br />76 kg<br />

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Duality of biological information:Epigenetic & Genetic<br />

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Nutrigenomics Quantification of the nutritional genotype-phenotype <br />Lifestyle<br />Nutrition<br />Environment<br />

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Nutrigenomics will provide solutions ….but only if ask the right questions<br />Complex Nutrition<br />+<br />Complex Genotypes<br />+<br />Complex Lifestyles<br />+<br />Complex Omics Technologies<br />Simple solutions? <br />

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From mouse to men: From big to small signals<br />Small<br />Human muscle<br />High Fat Diet decreases mRNA for genes involved in OXPHOS in healthy young men and mice<br />Big<br />Mouse muscle<br />Diabetes 54:1926-1933, 2005 <br />

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Challenges in nutrigenomics research: Small signals<br />24 H fasting<br />20 W high-fat<br />6 H WY<br />KO mice vs C.<br />

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Nutrigenomics: The two strategies<br />SignaturesProfilesBiomarkers<br />Target GenesMechanismsPathways<br />Molecular Nutrition& Genomics<br />NutritionalSystems Biology<br /><ul><li>Identification of dietary signals

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Identification of dietary sensors

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Identification of target genes

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Reconstruction of signaling pathways

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Measurement of stress signatures

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Identification of early biomarkers

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Nutritional plasma proteome </li></ul> and metabolome<br />+<br />++++<br />Complexity<br />L. Afman & M. Müller J Am Diet Assoc. 2006;106:569-576.<br />

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Nutrigenomics – molecular nutrition and genomics<br />5 -10000 (?)metabolites<br />90000 (?)proteins<br />100000 (?) transcripts<br />20000 genes<br />Müller & Kersten<br />NRG 2003<br />

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24<br />A scientific question: What is the role of “free fatty acids”?<br />During fasting<br />During long endurance activity<br />In obese people <br />“Hunger” signal or<br />“Need for glucose” signal ?<br />time (hours)<br />

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Fats<br />FFA<br />Remnant<br />LPL<br />VLDL<br />Chylomicrons<br />We are what we eat<br />

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Transcription-factor pathways mediating nutrient-gene interactions<br />Müller & Kersten<br />NRG 2003<br />

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Orphans?<br />Endocrine receptorsSteroid hormonesHigh affinity<br />Endocrine receptorsLipidsLow affinity<br />Chawla, Science 2001 <br />Nuclear hormone receptors<br />

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Intestine<br />LXR Decreased cholesterol absorption<br />FXR<br /> Increased bile salt recirculation<br />PPARα<br /> Improved lipid handling<br />Regulation of Cholesterol and Lipid Handling in Metabolic Organ Systems by Nuclear Receptors<br />

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Nuclear receptors – how does it work?<br />

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Understanding NutritionHow nutrients regulate our genes: via sensing molecular switches<br />Improved organcapacity by PUFAs<br />Am J ClinNutr. 2009; 90:415-24Am J ClinNutr. 2009;90:1656-64Mol CellBiology2009;29:6257-67<br />Am J ClinNutr. 2010;91:208-17BMC Genomics2009<br />Physiol. Genomics2009Circulation 2010Diabetes 2010<br />Cell Metabolism 2010<br />Am J Clin Nutr. 2007;86(5):1515-23<br />PLOS ONE 2008;3(2):e1681 BMC Genomics 2008; 9:231BMC Genomics 2008; 9:262J Biol Chem. 2008;283:22620-7Arterioscler Thromb Vasc Biol. 2009;29:969-74.<br />Plos One 2009;4(8):e6796HEPATOLOGY 2010;51:511-522<br />J Clin Invest. 2004;114:94-103<br />J Biol Chem. 2006;28:934-44 <br />Endocrinology. 2006;147:1508-16<br />Physiol Genomics. 2007;30:192-204Endocrinology. 2007;148:2753-63 <br />BMC Genomics 2007; 8:267 Arterioscler Thromb Vasc Biol. 2007;27:2420-7 <br />

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Is PPARa the hepatic fatty acid sensor? <br />YES!<br />

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Disturbance of hepatic lipid handling leads to steatosis<br />Mitochondrial fatty acid oxidation/<br />Fatty acid binding/activation<br />ketone body synthesis<br />Lipid transport<br />Lipogenesis<br />Peroxisomal/microsomal<br />fatty acid oxidation<br />Steatosis<br />Lipases<br />

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PPARa controls lipid metabolism<br />Mitochondrial fatty acid oxidation/<br />Fatty acid binding/activation<br />ketone body synthesis<br />Acsl1<br />Acsl3<br />Acsl4<br />Lipid transport<br />Acsl5<br />Acsm3<br />Hahd2<br />Acaa2<br />Acss2<br />Hadha<br />Adad8<br />Fabp1<br />Acot2<br />Hadhb<br />Acad9<br />Fabp2<br />Acot9<br />Hadhsc<br />Acad10<br />Cpt1a<br />Fabp3<br />Abca1<br />Hibch<br />Acads<br />Slc27a1<br />Cpt1b<br />Fabp4<br />Abcb4<br />Npc1<br />Hmgcl<br />Acadm<br />Slc27a2<br />Fabp5<br />Cpt2<br />Vldlr<br />Abcb11<br />Hmgcs2<br />Acadl<br />Crat<br />Slc27a4<br />Abcg5<br />Lrp4<br />Slc25a20<br />Cd36<br />Acadvl<br />Dci<br />Abcg8<br />Slc22a5<br />Acat1<br />Decr1<br />Lipogenesis<br />Miscellaneous<br />Acaca<br />Elovl5<br />Acacb<br />Peroxisomal/microsomal<br />Elovl6<br />Agpat2<br />Elovl7<br />Acot1<br />fatty acid oxidation<br />Agpat3<br />Acot7<br />Gpam<br />Agpat5<br />Hsd17b12<br />Acot10<br />Agpat6<br />Mod1<br />Acot12<br />Fads1<br />Adfp<br />Mogat1<br />Scd2<br />Fads2<br />Mttp<br />Adipor2<br />Slc25a10<br />Acaa1a<br />Fasn<br />Bdh<br />Acox1<br />Scd1<br />Srebf1<br />Cyp4a10<br />Acaa1b<br />Dgat1<br />Crot1<br />G0s2<br />Cyp4a12<br />Acot3<br />Ech1<br />Lepr<br />Cyp4a14<br />Acot4<br />Ehhadh<br />Lpin2<br />Aldh3a<br />2<br />Acot5<br />Lrp4<br />Decr2<br />Acot8<br />Hsd17b4<br />Mlycd<br />Abcd2<br />Peci<br />Scrab2<br />Glycerol metabolism<br />Abcd3<br />Pctp<br />Ppar<br />a<br />Pdk4<br />Gpd1<br />Pltp<br />Pnlpa2<br />Gpd2<br />Ppargc1a<br />Mgll<br />Gyk<br />Ucp2<br />Lipe<br />Aqp3<br />Ucp3<br />Aqp7<br />Lipases<br />Lipl<br />Lipg<br />Angptl4<br />

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DIETome database forevidence-basednutrition<br />Evidence-basedNutrition<br />Genes regulated by fatty acidsGenes regulated by high fat<br />Genes also regulated by inflammation<br />Query<br />DIET<br />GenomeEpigenomeTranscriptomeProteomeMetabolome<br />“DIETome”database<br />Query<br />Nutrigenomics<br />Potential BiomarkersOrgan-specific secreted proteins<br />

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Adipocytes at the crossroads of energy homeostasis<br />

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Nutrition<br />Pharma<br /> Insulin ± oral agents<br />Oral combination<br />Oral monotherapy<br />Diet & exercise<br />Nutritional prevention of complex diseases needs new biomarkers<br />Complex Disease<br />100<br />Different & similar targets<br />80<br />60<br />DISEASE STATE (%) <br />40<br />20<br />0<br />TIME (months/years)<br />HomeostasisHealth<br />L. Afman & M. Müller J Am Diet Assoc. 2006;106:569-576.<br />

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Metabolic defects leading to the development of hepatic steatosis<br />

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Metabolism & Inflammation<br />

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Liver, FAT & NASH/NAFLD<br /><ul><li>Nonalcoholic Fatty Liver Diseases (NAFLD):Liver component of Metabolic Syndrome

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Different stages in NAFLD progression:

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Molecular events involved in NASH pathogenesis:

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Role of PPARa (Endocrinology 2008 & Hepatology 2010)

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Role Kupffer cells (Hepatology 2010)

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Role of macrophages in lipid metabolism (JBC 2008; Cell Metabolism 2010)</li></ul>hepatic steatosis steatohepatitis (NASH) & fibrosis cirrhosis<br />

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Interaction between WAT and liver tissue essential for NASH/NAFLD in C57Bl/6 mice<br />Objective: <br />Nonalcoholic fatty liver disease (NAFLD) is strongly linked to obesity and diabetes, suggesting an important role of adipose tissue in the pathogenesis of NAFLD. <br />Here we aimed to investigate the interaction between adipose tissue and liver in NAFLD, and identify potential early plasma markers that predict NASH. <br />

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Experimental Design<br />tissue collection<br />run-in diet<br />20 weeks diet intervention<br /><ul><li>plasma collection</li></ul>multiple proteinassays<br /><ul><li>liver

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stratification on body weight</li></ul>frozen sections: histological feat.<br />lipid content<br />RNA extraction:Affx microarrays<br />10 LFD<br />0<br />2<br />4<br />8<br />12<br />16<br />20 weeks<br />20 LFD<br />-3<br />quality control & data analysis pipeline<br />10 HFD<br />Mouse genome <br />430 2.0<br />10% low <br />fat diet <br />(palm oil)<br />45% high fat diet (palm oil)<br /><ul><li>ep. white adipose tissue</li></ul>paraffin sections: histological feat.<br />RNA extraction: real-time PCR<br />

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High fat diet-induced obesity<br />0<br />2<br />4<br />8<br />12<br />16<br />20<br />HFL<br />LFL<br />HFH<br />LFH<br />25<br />20<br />*<br />*<br />15<br />**<br />BW gain (g)<br />*<br />10<br />*<br />*<br />*<br />*<br />5<br />0<br />weeks under diet intervention<br />Liver TG content<br />Hepatomegaly<br />ALT plasma activity<br />200<br />10<br />100<br />***<br />***<br />**<br />160<br />8<br />80<br />**<br />120<br />6<br />60<br />*<br />Ratio LW/BW (%)<br />mg TG/g liver<br />ALT activity (UI)<br />80<br />4<br />40<br />*<br />*<br />40<br />2<br />20<br />0<br />0<br />0<br />LFL<br />LFH<br />HFL<br />HFH<br />

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Adipose dysfunction in HFH mice<br />Leptin<br />

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A subpopulation of mice fed HFD develops NASH<br />

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Immunohistochemicalstaining confirms enhanced inflammation and early fibrosis in HFH mice<br />Macrophage CD68<br />Collagen<br />Stellate cell GFAP<br />

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Results I<br />Mice exhibited pronounced heterogeneity in liver histological scoring, leading to classification into 4 subgroups: <br />LF-low (LFL) responders displaying normal liver morphology, <br />LF-high (LFH) responders showing benign hepatic steatosis, <br />HF-low (HFL) responders displaying pre-NASH with macrovesicular lipid droplets, <br />HF-high (HFH) responders exhibiting overt NASH characterized by ballooning of hepatocytes, presence of Mallory bodies, and activated inflammatory cells. <br />

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Upregulation of inflammatory and fibrotic gene expression in HFH responder mice<br />

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Adipose dysfunction in HFH mice<br />

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Change in adipose gene expression indicate adipose tissue dysfunction<br />

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Plasma proteins as early predictive biomarker for NASH in C57Bl/6 mice<br />

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Conclusions<br />Our data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis.<br />It points to several novel potential predictive biomarkers for NASH.<br />Diabetes. 2010;59:3181-91.<br />

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Nutrigenomics enables us<br /><ul><li>To understand how nutrition precisely works (evidence-based nutrition);

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To quantify the nutritional needs for optimized fitness at different life stages (“personalized” nutrition);

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To improve early diagnostics of nutrition related disorders (“challenge tests”);

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To support the development of “smart healthy food patterns” for modern mankind (healthy and tasty, sustainable, affordable)

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To enable the transition of nutritional science to nutritional science 2.0.</li></li></ul><li>How to stay healthy and keep our organs fit and vital?<br />By “challenging” our organs with a diverse and healthy food pattern to improve their capability, capacity, and performance.<br />

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2 Meals a day, work as long as possible & embrace challenge<br />Walter Breuning (1896 - 2011)<br />

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Thanks<br />Lydia Afman<br />Mark Bouwens<br />Susan van Dijk<br />DiederikEsser<br />Sergio Lopez<br />Lisette de Groot<br />Marianne Geleijnse<br />Ondine van de Rest<br />MariekeBos<br />Edith Feskens<br />RikHeijligenberg<br />Dianne Hoelen<br />Jeanne de Vries<br />Geert Heidema<br />

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Visit the Kröller-Müller Museum!<br />56<br />