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Microbiota and Obesity Causes of Obesity

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Microbiota and Obesity Causes of Obesity Microbiota y Obesidad: Papel de los polifenoles Prof. J. Alfredo Martínez Microbiota and Obesity Causes of Obesity OBESITY Environment Genetics Lifestyle Neuroendocrine + Factors + + - - ENERGY ENERGY INTAKE EXPENDITURE GUT MICROBIO TA ? EXCESSIVE ADIPOSITY ObesityObesity andcomorbidities comorbidities OBESITY Non-communicable diseases Public health challenge Diabetes Chronic respiratory diseases Cancer GUT CVDs OBESITYMICROBIOTA Unhealthy Physical diet inactivity Socioeconomic status Environmental Genetic factors factors Neuroendocrine Perinatal Nutrition Microbiota WHO, 2015; Ng et al., 2014; Bäckhed et al., 2004 Obesity consequences ADIPOSITY Insulin Dyslipemia Inflammation Resistance Glucose Intolerance Hypertension Diabetes mellitus type II Atherosclerosis Metabolic Syndrome Obesity complications: Inflammation Type 2 Complex Hypertension Diabetes dyslipidemia TNF- alpha IL-6 Cardiovascular CRP Endothelial Disease dysfunction IL-8 IL-8 Low-grade systemic Glucose inflammation homeostasis Cancer impairment CONNECTION Changes in Intestinal Microbiota Spagnuolo et al., 2010 Obesity and gut microbiota Cecal microbiota in lean vs obese mice Ley et al., 2005(PNAS) Obesity and gut microbiota Phylogenetic diversity curves for microbiota of lean vs obese individuals Turnbaugh et al., 2009 (Nature) Obesity and gut microbiotaINTRODUCTION Santacruz et al., 2009 Correlation of different bacterial Low-grade groupsOverweight with adolescents weight loss (13- 15(kg) years) systemic n=36 inflammation 10 weeks Calorie restricted diet (10-40%) & increased physical activity (15-23 kcal/kg BW/week) CONNECTION High weight-loss group Low weight-loss group (˃4 kg) (˃2 Kg) - Increased B. fragilis & Lactobacillus No significant differences - Decreased C. coccoides -Increased Bacteroides fragilis - Decreased Clostridium coccoides Changes in Intestinal Microbiota Santacruz et al., 2009 Obesity and gut microbiota Obesity and gut microbiota WEIGHT LOSS < 2 KG INTENSIVE PHASE 2 MONTHS WEIGHT > 4 KG LOSS Obesity and gut microbiota Obesity and gut microbiota Obesity and gut microbiota Obesity and gut microbiota Diet Induced Obesity: Microbiota & gut fermentation Firmicutes/Bacteroidetes ratio Murphy et al., 2010 Erysipelotrichi class Turnbaugh et al., 2008 Bacterial species positively altered (E. cylindroides, E. ventriosum, B. wadsworthia, P. nanceiensis, EubiosisDysbiosis L. reuteri) De la Serre et al., 2010 Bacterial species negatively altered (M. jalaludini, C. fusiformis) Turnbaugh et al., 2008 Parks et al.,2013 Zhang et al., 2009 Million et al., 2012 SCFAs Yin et al., 2012 Lecomte et al., 2015 den Besten et al., 2013 Diet Induced Obesity and gut microbiotaRESULTS Firmicutes/Bacteroidetes ratio 15 t FIRMICUTES/BACTEROIDETES 10 HFS (Initial) RATIO 5 HFS (final) 0 HFS (Initial) HFS (final) Low F/B ratio GUT MICROBIOTA ? HEALTH OR DISEASE Ley et al., 2006 OBESE LEAN Turnbaugh et al., 2006 Bervoets et al, 2013 Obesity and gut microbiota Gut microbiota “new organ” within our organism FIRMICUTES (60-80%) . 1011- 1012 cells/mL in colon BACTEROIDETES (20-40%) ≥ 150 times more genes than the human genome . Regulates key “host” functions - Immunity . Classified from phylum to species levels - Nutrient production/availability - Energy harvesting Bäckhed et al., 2005 Obesity, microbiota and microbiome MicrobiotaSUPERORGANISM-Microbiome 90 % Microbial cells Bacterial cells Bacterial genome density ≥ 100-fold genes Gut Bacteria 14 microbiota 100 trillion (10 ) Archaea Fungi Gut microbiota composition Micro-eukaryotes Protozoa Virus GUT MICROBIOTA 10 % Human cells HMP Consortium, 2012; Qin et al., 2010; Ley et al., 2006; Bäckhed et al., 2005 Microbiota in body Habitats GutPrimary microbiota clustering composition of the microbiota in healthy by adults body area. Firmicutes Bacteroidetes Proteobacteria Actinobacteria Stomach Verrucomicrobia 102-103 cfu/mL Duodenum, jejunum <105cfu/mL Ileum 103-107 cfu/mL Large intestine 109-1012 cfu/mL HMP Consortium, 2012; Sekirov et al., 2010 Gut Microbiota and the host Metabolic functions • Production of vitamins • Antimicrobial secretion • Fermentation of non-digestible polysaccharides • Production of SCFAs • Energy source and energy harvest Gut microbiota exerts important functions for health • Epithelial cell growth and differentiation regulation • Intestinal villi and crypts development • TJPs and mucus layer properties regulation • Colonization resistance Structural & protective functions • Innate and adaptive immunity activation • Inflammatory cytokine regulation • Immune system development • B- and T-cell development Immune functions Villanueva-Millan et al., 2015; Prakash et al., 2011 Gut microbiota (metagenomics) analyses Cultured Uncultured CULTURE- INDEPENDENT MOLECULAR PHYLOGENETIC APPROACHES Microbial community Which microbes are there ? What are these microbes doing ? • 16 S rDNA based pyrosequencing • Metabolomic analysis • RT- PCR Gut Microbiota and metagenomics Gel electrophoresis and band Amplification of 16S rDNA purification Dietary intervention DNA extraction (hypervariable region, V4-V6) (amplicons of ~ 560 bp) QUIamp DNA Stool Mini Kit RT-PCR (Qiagen) Taxonomic identification of bacterial species by 454 Roche GS Statistical analysis FXL pyrosequenging Data interpretation Pool each PCR amplicon Nutrition and gut microbiota Benef Microbes, (2015); 6 (1): 97-111 doi: 10.3920/BM2013.0097 16S rDNA- based pyrosequensing Beneficial Microbes (2014) NutritionNutrition and the the gut microbiota microbiotaMATERIAL AND METHODS Tabla 1. Composition of the diets: control diet (2014, Harlan Teklad Global 14% Protein Rodent Maintenance Diet), High- fat sucrose diet (TD. 06415, Teklad Research purified diet). Control diet High-fat sucrose diet (2014) (TD. 06415) Energy (Kcal/g) 2.9 4.6 Control diet n= 5 HFS diet (Harlan, 2014) (TD. 06415) Protein (Energy %) 20 19 Carbohydrate (Energy %) 67 36 In adaptation period HFS diet Starch* (g/kg) 480 85 intervention Sucrose** (g/kg) Traces 200 Fat (Energy %) 13 45 6 weeks Saturated (%) 18 36 Baseline faeces Final faeces Monounsaturated (%) 20 47 collection collection Polyunsaturated (%) 62 17 Fiber content*** (g/kg) 221 58 * unrefined starch from wheat and corn in 2014; refined cornstarch in TD.06415 ** no added sucrose in 2014; trace amounts of simple sugars from Gut microbiota analysis by 16S rDNA grain ingredients based pyrosequensing *** calculated neutral detergent fiber in 2014 & cellulose in TD.06415 & Short- chain fatty acid analysis by GC- MS metabolomic analysis • In collaboration with University of the Basque Country Diet Induced Obesity and the gut microbiotaRESULTS Phenotypical- related measurements of male Wistar rats (n=5) fed a HFS diet for 6 weeks. (a) Weekly body weight changes of Wistar rats fed a HFS diet (n=5). (b) Weight-related parameters and plasma values after 6 weeks of HFS dietary intervention. All results are expressed as the mean± standard error of the mean. HFS, high-fat sucrose diet; WAT, white adipose tissue. Etxeberria U et al., Benef Microbes, 2015; 6 (1): 97-111 2015 Diet Induced Obesity and gut microbiotaRESULTS Observations (axis F1 y F2: 47.67 %) 4 HFS1-f Samples at baseline HFS3-f 3 HFS3-b 2 Samples after the HFS1-b intervention 1 0 HFS2-b -1 HFS2-f HFS4-b HFS5-f -2 F2 (15.85%) -3 HFS4-f -4 -5 HFS5-b -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 F1 (31.82 %) Principal component analysis (PCA) generated from 16S rDNA pyrosequencing bacterial taxa from control diet-fed lean rats and HFS diet-fed rats. PCA revealed grouping of samples as a result of the HFS dietary intervention. HFS, high- fat sucrose. XLSTAT software http://www.xlstat.com Etxeberria U et al., Benef Microbes, 2015; 6 (1): 97-111 2015 Diet Induced Obesity and gut microbiotaRESULTS Samples at baseline Variables (axis F1 y F2: 47.67 %) Samples after the 1 intervention Peptostreptococcaceae 0.75 Bdellovibrionaceae Acholeplasmataceae Eubacteriaceae Xanthomonadaceae Anaeroplasmataceae 0.5 Rikenellaceae Peptococcaceae Verrucomicrobiaceae Aerococcaceaeunclassified Flavobacteriaceae Ruminococcaceae Veillonellaceae unclassified Ruminococcaceae 0.25 PaenibacillaceaeOscillospiraceae Desulfovibrionaceae Lachnospiraceae ThermoanaerobacteraleEnterococcaceae s Family III incertae sedis Bacillaceae Acidaminococcaceae Thermoactinomycetacea unclassified Caldicoprobacteraceae Bifidobacteriaceae Prevotellaceae 0 e Clostridiaceae unclassified Planococcaceae Porphyromonadaceae F2 (15.85 %) (15.85F2 Erysipelotrichiciae Lachnospiraceae Deferribacteraceae Clostridiales incertaeGraciibacteraceae -0.25 Clostridiaceae sedis Lactobacillaceae Erysipelotrichaceae Clostridiales Family XIII Cytophagaceae incertae sedis -0.5 Catabacteriaceae Hyphomicrobiaceae Clostridiales Family XII Incertae Sedis Spiroplasmataceae Haloplasmataceae -0.75 Enterobacteriaceae Sutterellaceae Streptococcaceae -1 -1 -0.75 -0.5 -0.25 F1 (31.820 %) 0.25 0.5 0.75 1 Principal component analysis (PCA) generated from 16S rDNA pyrosequencing bacterial taxa from control diet-fed lean rats and HFS diet-fed rats. Differentially identified bacterial families from faeces samples of control diet-fed lean and HFS diet- fed obese rats. HFS-b, standard diet-fed lean rats (baseline samples); HFS-f, high-fat sucrose diet-fed obese rats (final samples). Etxeberria U et al., Benef Microbes, 2015; 6 (1): 97-111 2015 Diet Induced Obesity and the gut microbiotaRESULTS PHYLUM LEVEL a Predominant phyla b Non- Predominant phyla 1.2 90.0 * 1.0 80.0 70.0 0.8 HFS baseline 60.0 0.6 HFS final 50.0 HFS baseline * HFS final 40.0 0.4 30.0 Relative Abundance(%) 20.0 0.2 Relative
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