Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flexibility"
Lecture2From healthy to too muchThe role of Small Intestine for metabolic flexibilityMichael MüllerNutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University
The intestine as a gatekeeperAdipokines:AdiponectinLeptinResistinANGPTL4TNFetcGI hormones:InsulinGIPGLP1PYYGhrelinANGPTL4FGF15/19Food intakeLPLLPLLPLSFAGlucoseFructoseFGF21ANGPTL4Satiety
The Intestine• The small intestine in an adult human = 5 meters with a normal range of 3 -7 meters (it can measure around 50% longer at autopsy because of loss ofsmooth muscle tone after death).• It is approximately 2.5-3 cm in diameter. Although the small intestine ismuch longer than the large intestine (typically around 3 times longer), it getsits name from its comparatively smaller diameter.• Although as a simple tube the length and diameter of the small intestinewould have a surface area of only about 0.5m2, the surface complexity ofthe inner lining of the small intestine increase its surface area by a factor of500 to approximately 200m2, or roughly the size of a tennis court.• The small intestine is divided into three structural parts:• Duodenum 26 cm in length• Jejunum 2.5 m• Ileum 3.5 m
Length of the intestineSmall intestines:• Human 6-7 m• Mouse 35 cm• Pig 15 mLarge intestines:• Human 1.5 m• Mouse 14 cm• Pig 5 m
Intestine functions related to nutrition• Uptake of nutrients• Chylomicron formation• Nutrient & food bioactives metabolism• Barrier function– Mucosal immunity– Host-microbe interaction• Gland: Satiety signaling (incretines,enterokines)
Gut: Diseases - Disorders• Gastroenteritis is inflammation of the intestines. It is themost common disease of the intestines.• Colitis is an inflammation of the large intestine.• Celiac disease is a common form of malabsorption,affecting up to 1% of people of northern Europeandescent. Allergy to gluten proteins, found in wheat,barley and rye, causes villous atrophy in the smallintestine. Life-long dietary avoidance of these foodstuffsin a gluten-free diet is the only treatment.• Crohns disease and ulcerative colitis are examples ofinflammatory bowel disease (IBD). While Crohns canaffect the entire gastrointestinal tract, ulcerative colitis islimited to the large intestine.
Background• Obesity and insulin resistance are two major riskfactors underlying the metabolic syndrome. Thedevelopment of these metabolic disorders isfrequently studied, but mainly in liver, skeletalmuscle, and adipose tissue.• To gain more insight in the role of the smallintestine in development of obesity and insulinresistance, dietary fat-induced differential geneexpression was determined along the longitudinalaxis of small intestines of C57BL/6J mice.
Methods• Male C57BL/6J mice were fed a low-fat or ahigh-fat diet that mimicked the fatty acidcomposition of a Western-style human diet.• After 2, 4 and 8 weeks of diet intervention smallintestines were isolated and divided in threeequal parts.• Differential gene expression was determined inmucosal scrapings using Mouse genome 4302.0 arrays.
Body weight and oralglucose tolerance test• (A) Body weight gain of C57BL/6Jmice during a low-fat or high-fat dietintervention of 8 weeks.• (B) An oral glucose tolerance testwas performed after 7 weeks of dietintervention. After an oral gavage of100 mg glucose, blood glucoselevels were monitored for 150minutes.• The changes in blood glucose levels(upper figure) and the area under hecurve were calculated (lower figure).In (A) and (B), data are means ± SE.* p < 0.05.• LF = low-fat diet, HF = high-fat diet
Dietary fat-induced differential geneexpression along the longitudinal axis of thesmall intestine
Heat map diagrams of differentiallyexpressed genes on a high-fat diet
Heat map diagrams of differentiallyexpressed genes on a high-fat diet
Heat map diagrams of differentiallyexpressed genes on a high-fat diet
Relative mRNA expression of nuclear receptorsalong the longitudinal axis of the small intestineunder basal conditions
Results• The high-fat diet significantly increased body weight and decreasedoral glucose tolerance, indicating insulin resistance.• Microarray analysis showed that dietary fat had the mostpronounced effect on differential gene expression in the middle partof the small intestine.• By overrepresentation analysis we found that the most modulatedbiological processes on a high-fat diet were related to lipidmetabolism, cell cycle and inflammation.• Our results further indicated that the nuclear receptors Ppars, Lxrsand Fxr play an important regulatory role in the response of thesmall intestine to the high-fat diet.• A secretome analysis revealed differential gene expression ofsecreted proteins, such as Il18, Fgf15, Mif, Igfbp3 and Angptl4.• Finally, we linked the fat-induced molecular changes in the smallintestine to development of obesity and insulin resistance.
Conclusion• During dietary fat-induced development of obesity andinsulin resistance, we found substantial changes in geneexpression in the small intestine, indicating modulationsof biological processes, especially related to lipidmetabolism.• Moreover, we found differential expression of potentialsignaling molecules that can provoke systemic effects inperipheral organs by influencing their metabolichomeostasis. Many of these fat-modulated genes couldbe linked to obesity and/or insulin resistance.• Together, our data provided various leads for a causalrole of the small intestine in the etiology of obesity and/orinsulin resistance.
Immune mechanisms that limit bacteria–epithelial cell interactions
Microbialcommunitycomposition atdifferent bodylocations in ahealthy human
A summary of the functional changes seen in various regions of thegut for different microbiome colonizations
Assembly and stability of the gut microbiota, andenvironmental factors affecting the gut microbiome duringlife
Major metabolites involved in host-microbe communication,originating from synthesis from microbial conversion of nutrientsand host metabolites in the gut lumen
Saturated fat affects obesity & liver TGsCorrelation between body weight gain and epididymal fat pads
HF-PO diet reduced microbial diversity and increasedthe Firmicutes-to-Bacteroidetes ratio
Lipid metabolism-related gene expression in the distalsmall intestine after 8 weeks of diet intervention
Conclusions• Saturated fat stimulates obesity and hepatic steatosisand affects gut microbiota composition by an enhancedoverflow of dietary fat to the distal intestine.• Unsaturated fat is more effectively taken up by the smallintestine, likely by more efficiently activating nutrientsensing systems (PPARs) and thereby contributing tothe prevention the development of early pathology (e.g.NASH).
• Obesity is a highly heritable disease driven by complex interactions between geneticand environmental factors. Human genome-wide association studies (GWAS) haveidentified a number of loci contributing to obesity; however, a major limitation of thesestudies is the inability to assess environmental interactions common to obesity. Usinga systems genetics approach, we measured obesity traits, global gene expression,and gut microbiota composition in response to a high-fat/high-sucrose (HF/HS) diet ofmore than 100 inbred strains of mice. Here we show that HF/HS feeding promotesrobust, strain-specific changes in obesity that are not accounted for by food intakeand provide evidence for a genetically determined set point for obesity. GWASanalysis identified 11 genome-wide significant loci associated with obesity traits,several of which overlap with loci identified in human studies. We also show strongrelationships between genotype and gut microbiota plasticity during HF/HS feedingand identify gut microbial phylotypes associated with obesity.
Highlights of the study► Detailed analysis of diet-induced obesity in more than 100 inbred mouse strains► Identification of 11 genetic loci associated with obesity and dietaryresponsiveness► Significant overlap between mouse loci with human GWAS loci for obesity► Strain-specific shifts in gut microbiota composition in response to dietaryintervention
Robust Shifts in Gut MicrobiotaComposition after HF/HS Feeding
Plasticity of Gut Microbiota Is StrainSpecific
Conclusions• The intestine plays a crucial role as barrier & gatekeeper• Responsible for the efficient uptake of nutrients & foodbioactives• Symbiosis with large sets of microbiota that have asignificant impact on the health status of the host(human)• Interaction between host (phenotype/genotype),microbiota, foods/diet, environmental factors• Likely plays a key role in development of many recentlyemerging diseases. Link to immunity & inflammation• Careful with animal models (strong genotype effects)!
The effects of heme on the colonicmucosa and the microbiota in miceINCON, 1-4 Oct 2012, San Jose
Heme as a nutritional stressorNutritional stressor: heme• Heme is the color pigment of red meat• Known that heme and/or heme-metabolite(s):- are luminal irritants in the colon- catalyse the production of reactive oxygen species (ROS)- are cytotoxic for epithelial cells and induces necrosis- increase proliferation of epithelial cellshyperplasia of the epitheliumde Vogel et al., 2008, Carcinogenesis 29:398
Aim of this study• To identify molecules that signal fromthe surface to the proliferative coloniccrypt to increase cell proliferationupon stress induced by hemeExfoliation ornecrotic celldeathCelldivisionApoptosis ordifferentiationExfoliation ornecrotic celldeathCelldivisionApoptosis ordifferentiation
-7 0 7 14 dayschow diet n=16HF heme diet n=8HF control diet n=8Collection of:Colon-scrapings for gene expression-part of the tissue for IHC and LCMColonic contentsFecesStudy 1.Effects of heme on the colonic mucosaHigh-risk western purified dietmale C57Bl6J mice (8 per group)high fat 40%*heme 0.5 μmol/g
Dietary heme increases fecal water cytotoxicity020406080100120controlheme*%celllysis
control heme 200xStainings with an antibody against Ki67, showing more proliferating (brown) cells on theheme diet, and deeper crypts.200xDietary heme increases cell proliferation
Effect of heme on gene expression• Around 3,700 genes differentially expressed (q<0.01 andsignal intensity > 20), determined with microarray• Processes in which these genes were involved:• Can we determine more precisely where these changes take place, in surfacecells or in crypt cells?
Laser Capture Microdissection Technology (LCM)LCM was used to isolate colonic surface and crypt cellsLCM system and procedure
LCM separates surface and crypt gene expressionHmox1051015202530354045LCMtotal surface cryptfoldchangeKi6702468101214LCMtotal surface cryptcontrolhemefoldchangeheme control hemecontrolHeme oxygenase is highly induced in thesurfaceKi67 is highly induced in the crypt**
Conclusions from LCM• Heme-related genes and stress-related genesupregulated specifically at the surface epithelium(Hmox1, Creb3l3)• Cell proliferation upregulated in the crypt (Ki67 andcyclins)• Apoptosis is downregulated (Birc5, Xiap, Casp3)• Heme exerts its effect on the surface, and does notdirectly act on the proliferating cells in the crypt• Therefore signaling molecules from surface to crypt haveto start the proliferation
Downregulated mitogenic surface-to-crypt signals arealso decreased at protein levelGene level Protein levelOther signals with a similar pattern: Bmp2, Wif1, Ihh
Gene level Protein levelUpregulated mitogenic surface-to-crypt signalsare not translated into protein• Amphiregulin (Areg) and Epiregulin (Ereg):epithelial growth factors which play a role in human carcinogenesis
Protein Translation is controlled by 4E-BP1• Protein translation is inhibited by 4E-BP1, which is surface specifically upregulated byheme.
Conclusions• Signaling from the injured surface epithelium occurs via downregulation of feedbackinhibitors of proliferation, e.g. Wif1, Ihh, Bmp2 and IL-15• Upregulated molecules are not translated into proteins, because of the surface specificupregulation of the translation inhibitor 4E-BP1.• If validated in humans, Wif1, Ihh, Bmp2 and IL-15 may be used as early biomarkers of diet-modulated colon cancer risk.IJssennagger et al., Gut (2012)
Background:• Colon densely populated by bacteria• Cytotox is caused by a covalently modified heme metaboliteAim:• To determine the changes in microbiota upon heme consumption• Do microbiota play a role in the heme induced surface to crypt signaling?Results:• Heme induced hyperproliferationStudy 2.Effects of heme on the microbiota
Heme changed the composition of microbiota0%20%40%60%80%100%Control HemeRelativecontributionTM7FirmicutesCyanobacteriaActinobacteriaVerrucomicrobiaFusobacteriaFibrobacteresDeferribacteresProteobacteriaBacteroidetesVerrucomicrobia, proteobacteria and bacteroideteswere more abundant on the heme dietRatio Control HemeGram-negative to Gram-positive bacteria0.72 ± 0.09 2.16 ± 0.27 *Bacteroidetes to Firmicutes0.63 ± 0.09 1.87 ± 0.30*Heme increased the ratio of gram-negative to gram-positive bacteria.
• Selective susceptibility of Gram-positives for heme fecal water• Allowed expansion of Gram-negative bacteria → ↑ LPS exposure• No functional change in the sensing of the bacteria by the mucosa, as changes ininflammation pathways and Toll- like receptor signaling were not detected.Conclusion:• Changes in microbiota does not cause the observed hyperproliferation and hyperplasia viainflammation pathwaysHeme alters microbiota and mucosa, but withoutfunctional changes in host-microbe cross-talk
Study 3.Role of microbiota in heme-induced hyperproliferationBackground:• Heme diet changes microbiotaAim:• Investigate whether there is a causal role for microbiota in the heme induced cytotoxicity andhyperproliferation.
Design of antibiotics (Abx) experimentBroad spectrum antibioticsAmpicillin:1 g/LNeomycin:1 g/LMetronidazole: 0.5 g/LCollection of colon contents and mucosa-7 0 7 14 dayschow diet n=36 HF heme dietHF control dietN=9N=9N=9N=9Control + AbxControlHemeHeme + AbxHigh-risk western purified dietmale C57Bl6J mice (9 per group)high fat 40%*heme 0.5 μmol/g
Results:• heme induced cytotoxicity and ROS• Abx changes bile acid compositionfrom unconjugated to conjugated BA• Heme increases proliferation,but not when Abx given simultaneouslyNo hyperproliferation on heme + Abx
• Several cytotoxicity sensors not induced by heme + AbxHeme induced cytotox not seen by mucosa on Abx
• Still luminal cytotoxicity but not sensed by the mucosa.• Thus an increased mucus barrier with Abx.Conclusion:• Microbiota facilitate the heme induced hyperproliferation by breaking the mucus barrierPossible Mechanism:• Several mucin degrading bacteria (e.g. Akkermansia Muciniphila) decreased by Abx, andthis decrease might contribute to the increased mucus barrier.Microbiota facilitate heme induced hyperproliferation