Mid Term Review QLK1-CT-2000-00618

Mid Term Review QLK1-CT-2000-00618

NUGENOB: The study, the database, and perspectives for integration

Focus

• The interactions between macronutrient composition of the diet, and specific genetic variants in human obesity.

• Combining clinical/physiological response to a high-fat test meal and a long-term hypo-energetic low-fat or medium-fat diet with knowledge of genetic make up and expression levels of individual genes.

Aims - I

• Identification and characterisation of novel nutrient-sensitive candidate genes for obesity.

• Assessment of the effects of the variants and combinations of variants in nutrient-sensitive genes on the response to a high-fat test meal

Aims - II• Identification of predictors of the changes in body weight during a 10-week

hypo energetic low-fat or medium-fat dietary intervention programme:

– Anthropometrical characteristics,– Obesity-related life style factors, – Physiological functions observed at the test meal challenge, – Variants or combinations of variants of the nutrient-sensitive genes, – Gene expression in adipose tissue, – Gene-phenotype or gene-environment interactions based on combinations of these

predictors.

To identify predictors of differantial response to the two diets

• Assessment of the combined effects of– variants of nutrient-sensitive genes – short- and long-term alterations in dietary fat content on the differential expression of selected genes in adipose tissue.

Clinical investigation day

Habitual and run-in diet,life style factors

Hypoenergetic 10-week low- or high-fat diet

WP4

WP3

WP5

WP6 Sampling and processing of DNA

Genotypic screeningWP9

Adipose tissue cDNA analysis

WP11

Adipose tissue mRNA analysis

WP10

WP7 Identification of candidate genes

Analysis of candidate genes

WP8

Source populationsWP1

Recruitment ofstudy population

WP2

Databank and statistical analysis

WP12

Contribution to evidence-based

dietary guidelines

Nutrient-gene interaction in human obesity

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Data:

Study population 771 obese – 119 lean subjects

• Inclusion criteria for obese subjects – BMI >= 30 – Intention to include white Europeans – Age 20-50 years– Pre-menopausal women

• Exclusion criteria for obese subjects – Medical conditions and medication– Pregnant women– Alcohol or drug abuse – Weight change of > 3 kg within 3 months.




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dietary guidelines


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Data:

Data I - Habitual diet, life style factors

• 3-day, weighed food record

• Questionnaire addressing: – social variables,– physical activity, – drinking and smoking habits, – weight and dieting history, – health and illness, – family history of obesity and obesity related diseases– SF-36




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WP11


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dietary guidelines


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Data:

Clinical Investigation day

EE EE EE EE EE

Appetite-scoresBlood samples

Test meal

7.30 8.30 9.00 9.30 10.00 10.30 11.00 11.30 12.00 12.30 Time (h)

-30 ” 0 ” 0 30 30 60 60 90 90 120 120 150 150 180

Fat biopsy

EEEE




10 weeks

AnthropometricsBody composition

Fasting blood AnthropometricsBody composition

Data II – Clinical Investigation Day

Timepoints (fasting since, times for hood measures, vas, biopsies, etc)

Weight, height, waist, hip, FM, FFM, fat%, impedance 5,50,100,200 khz

Fatbiposy in fasting and 3-h post prandial (time, amount, reason for not taking it)

Estimated BMR (WHO 1985: use Weight and Height), basis for energy in test meal (50%)

Amount of cream and butter in test meal

VAS for hunger, fullness, satiety, w ish to eat, and thirst at baseline, + 1,2,3 h after test meal

Vo2 and Vco2 at baseline at measurements ending 30, 60, 90, 120, 150, and 180 after test meal

RQ and energy expenditure calcuated from Vo2 and Vco2 (baseline, 30,60,90,120,150,180)

Calculated substrate utilisation for fat and carbohydrates (according to Frayn)

Remarks during test day

Well bening

Subject getting sick

Blood: insulin, glucose, free fatty acids, total glycerol, free glycerol, triglycerides

total cholesterol, HDL cholesterol, LDL cholesterol, Cortisol, VLDL, Leptin, IGF

Hypo-energetic 10 week low or high fat diet

Clinicalinvestigation day



WP4

WP3

WP5




WP11


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WP8



WP2


WP12

Contribution toevidence-based

dietaryguidelines

Nutrient-gene interaction in human obes ity

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Samples:

Data:

Results of WP 5Weight loss by diet

Weight loss intervention

• 10 weeks

• - 600 kcal per day

• Either low fat or medium fat diet

Data III – Dietary intervention

Weight for week 0 (either CID weight or new fasting weight enetered) corresponding body composition

Weight at week 1-10 (week 0 and week 10 are fasting weights)

Variables indicating whether weight from week 1-9 were self reported

Compliance assessed by subject and dietitian at week 1-10 ( categori - 5 categories)

Notes at week 0-10Dropout: time, reason from CRF, reasons summarised into overall categories Food recording: week 2,5,7,10

Identification ofcandidate genes and gene variants




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Data:

Publiched results • GAD2 on Chromosome 10p12 Is a Candidate Gene for

Human Obesity. Boutin P et al. PLoS Biol. 2003 Dec;1(3):E68. Epub 2003 Nov 03

• Association studies between microsatellite markers within the gene encoding human 11 beta-hydroxysteroid dehydrogenase type 1 and body mass index, waist to hip ratio, and glucocorticoid metabolism. Draper Net al. Journal of Clinical Endocrinology and Metabolism, Vol. 87 (11) pp. 4984-4990 (2002)

• Mutational analysis of the UCP2 core promoter and relationships of variants with obesity. Dalgaard LT et al. Obes Res 2003;11:1420-1427

• A genome-wide scan for childhood obesity-associated traits in French families shows significant linkage on chromosome 6q22.31-q23.2. Meyre et al.Diabetes. 2004 Mar;53(3):803-811.

• The FOXC2 -512C>T variant is associated with hypertriglyceridemia and elevated serum C-peptide in glucose tolerant subjects Yanagisawa et al. Diabetologia, 2003 Nov;46(11):1576-80

Genotypic screening




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dietaryguidelines


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Samp

les:

Data:

Data IV – Genotyping43 SNPs in 27 genes

Adipocyte secretory peptides Regulation of adiposcyte differantiationMitocondrial proteins Nuclear receptors and transcription factors Hormones and receptors involved in appetite regulationInsulin signalling and glucose regulationLipoprotein metabolism




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dietaryguidelines

Nutrient-gene interaction in human obes ity

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Samples:

Data:

Quantitative gene expression

Data V – Quantitative gene expression

Gene Expression data (before and after weight loss, fasting biopsies)

Genes related to:

Signal transduction

Adipocyte secretion

Glucose metabolism

Lipid metabolism

Mitochondria/ Energy metabolism

Transcription (co-)factors

Reference mRNAs

Quantitative gene expression analyses for 40 candidate genes in 2 x 25 subjects (25 from each diet group)




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Data:

Gene expression profiling

Data VI – Gene expression profiling

• Affymetrix chip analyses on 13+10 women from a single center

• Custom made metabolic chip in 25+25 selected from all centres

• Data stored in local centres

Databank and statisticalAnalyses




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Data:


The NUGENOB database – formant

• The database has a simple flat format with 1 row per subject and one column per variable

• SPSS, STATA, SAS...... (Not Excel)

The NUGENOB database – Content

• Habitual diet (not ’raw’)• Questionnaire• Anthropometrics• Appetite• Blood parameters• Vo2 and Vco2

• Weight during weight loss

• Diet during weight loss• Genotypes• Quantitative gene

expression• Gene expression profiling

Raw data Derived measures

• Percentage and avarage• Summary scores • BMI, W/H

• AUC, AUCi• RQ, EE, macronutrien oxidation• Weight loss from week 0 to 10• Percentage and avarage• BMI, W/H

• Ratios

The NUGENOB database – Selection of sub-populations

• Subjects with high versus low increase in fat oxidation following the high-fat test meal

• Subjects completing the weight loss intervention, and following the diet prescriptions

• Subjects being successful or un-successful in loosing weight when following the dietary intervention

• Subjects with no versus high improvement in insulin sensitivity following weight loss

Samples in the NUGENOB biobank

• Serum and plasma samples taken in the fasting state, and every hour during a 3-hour period following a high-fat test meal. In obese subjects fasting serum and plasma samples were obtained following the 10-week intervention.

• Purified DNA in 96-well master plates (two sets) from all but 10 of the 771

obese and 119 lean subjects. • Buffy coat samples from all but ~120 of the 771 obese and 119 lean subjects

• Extracted mRNA from abdominal subcutaneous adipose tissue biopsies obtained in fasting before and after the 10-week dietary intervention in ~ 700 obese subjects. mRNA from biopsies obtained 3 hours after the test meal in a small group of obese subjects and mRNA from biopsies obtained in a small group of lean subjects.

SOPs of NUGENOB• Anthropometrical measurements • Questionnaire (Translated)• Blood sampling (obtaining, packing, storing, shipment)• Dietary Intervention• Needle subcutaneous abdominal adipose tissue biopsy• Preparation of the high fat liquid test meal.• Validating, calibrating and conducting indirect calorimetry (Hood).• Clinical investigation day, including blood sampling, energy and substrate

metabolism in fasting and flowing a high fat test meal.• Randomisation.• Data entry and data transfer into the freeware programme EpiData• Delivery of data for analyses

• Database on genes for which there is a known nutrient-gene interaction and a putative

role in obesity

• Info on genes for which gene variatns determine response to diet

• Info on genes for which the expression is changed in responds to calorie restriction, overfeeding or changes in diet composition (animal and human).

– Data from quantitative gene expression – Data from micro array studies.

• Animal studies on obesity, DIO or protection from DIO in KO, TG and mutation models

• Human gene variants affecting gene expression (with or without in vitro functional effects)

• Gene-gene interaction for nutrient sensitive genes

Database on nutrient-sensitive genes

For further information see:

www.nugenob.org

http://www.nugenob.org/

Mid Term Review QLK1-CT-2000-00618

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