Lecture2From healthy to too muchThe role of Small Intestine for metabolic flexibilityMichael MüllerNutrition, Metabolism a...
The intestine as a gatekeeperAdipokines:AdiponectinLeptinResistinANGPTL4TNFetcGI hormones:InsulinGIPGLP1PYYGhrelinANGPTL4F...
The Intestine• The small intestine in an adult human = 5 meters with a normal range of 3 -7 meters (it can measure around ...
Length of the intestineSmall intestines:• Human 6-7 m• Mouse 35 cm• Pig 15 mLarge intestines:• Human 1.5 m• Mouse 14 cm• P...
Intestine functions related to nutrition• Uptake of nutrients• Chylomicron formation• Nutrient & food bioactives metabolis...
Gut: Diseases - Disorders• Gastroenteritis is inflammation of the intestines. It is themost common disease of the intestin...
Simplified view onintestinal nutrient metabolismThiele lab 2013
The small intestine as primary organ isresponse to nutrients & food components
Gene regulation by dietary lipids in the intestine
The cellular network in intestinalhomeostasis (and inflammation)
The cellular network in intestinal(homeostasis and) inflammation.
The cellular network in intestinal(homeostasis) and inflammation
A major role for PPARain intestinal fatty acid sensingPhysiol Genomics. 2007 ;30(2):192-204
Intestinal PPAR target genes are largelyregulated by dietary PUFAS/MUFAs6h after oralgavage withOA18:1EPA20:5DHA22:6WY1464...
Longitudinal distribution of genes involved in dietary lipidmetabolism in wild-type mice on PPARα activation by WY14643Fat...
1. Esophagus2. Stomach3. Duodenum4. Jejunum5, Ileum6. Cecum7. Ascending colon8. Descending colon9. Rectum
Background• Obesity and insulin resistance are two major riskfactors underlying the metabolic syndrome. Thedevelopment of ...
Methods• Male C57BL/6J mice were fed a low-fat or ahigh-fat diet that mimicked the fatty acidcomposition of a Western-styl...
Low-fat (LF) diet High-fat (HF) dietgm% kcal% gm% kcal%Protein 19 20 24 20Carbohydrate 67 70 41 35Fat 4 10 24 45Ingredient...
Body weight and oralglucose tolerance test• (A) Body weight gain of C57BL/6Jmice during a low-fat or high-fat dietinterven...
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 res...
Secretomeanalysis
Conclusion• During dietary fat-induced development of obesity andinsulin resistance, we found substantial changes in genee...
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 nutrients...
Gut microbialdysbiosisassociatedwith disease
Factors shaping intestinal microbial compositionand effects of dysbiosis on host health
Microbiota-induced maturation of thegastrointestinal tract
Challenges for mouse studies:Several genera are significantly differentin abundance between mouse strains
Microbial impact on host physiology
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 enhancedov...
• Obesity is a highly heritable disease driven by complex interactions between geneticand environmental factors. Human gen...
Highlights of the study► Detailed analysis of diet-induced obesity in more than 100 inbred mouse strains► Identification o...
Natural Variation in Gene-by-DietInteractions
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...
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 he...
Aim of this study• To identify molecules that signal fromthe surface to the proliferative coloniccrypt to increase cell pr...
-7 0 7 14 dayschow diet n=16HF heme diet n=8HF control diet n=8Collection of:Colon-scrapings for gene expression-part of t...
Dietary heme increases fecal water cytotoxicity020406080100120controlheme*%celllysis
control heme 200xStainings with an antibody against Ki67, showing more proliferating (brown) cells on theheme diet, and de...
Effect of heme on gene expression• Around 3,700 genes differentially expressed (q<0.01 andsignal intensity > 20), determin...
Laser Capture Microdissection Technology (LCM)LCM was used to isolate colonic surface and crypt cellsLCM system and proced...
LCM separates surface and crypt gene expressionHmox1051015202530354045LCMtotal surface cryptfoldchangeKi6702468101214LCMto...
Conclusions from LCM• Heme-related genes and stress-related genesupregulated specifically at the surface epithelium(Hmox1,...
Downregulated mitogenic surface-to-crypt signals arealso decreased at protein levelGene level Protein levelOther signals w...
Gene level Protein levelUpregulated mitogenic surface-to-crypt signalsare not translated into protein• Amphiregulin (Areg)...
Protein Translation is controlled by 4E-BP1• Protein translation is inhibited by 4E-BP1, which is surface specifically upr...
Conclusions• Signaling from the injured surface epithelium occurs via downregulation of feedbackinhibitors of proliferatio...
Background:• Colon densely populated by bacteria• Cytotox is caused by a covalently modified heme metaboliteAim:• To deter...
Heme changed the composition of microbiota0%20%40%60%80%100%Control HemeRelativecontributionTM7FirmicutesCyanobacteriaActi...
• Selective susceptibility of Gram-positives for heme fecal water• Allowed expansion of Gram-negative bacteria → ↑ LPS exp...
Study 3.Role of microbiota in heme-induced hyperproliferationBackground:• Heme diet changes microbiotaAim:• Investigate wh...
Design of antibiotics (Abx) experimentBroad spectrum antibioticsAmpicillin:1 g/LNeomycin:1 g/LMetronidazole: 0.5 g/LCollec...
Results:• heme induced cytotoxicity and ROS• Abx changes bile acid compositionfrom unconjugated to conjugated BA• Heme inc...
No induction of oncogenes 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:• Microbio...
Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flex...
Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flex...
Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flex...
Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flex...
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Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flexibility"

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"The role of Small Intestine for metabolic flexibility"

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Nanjing 2 2013 Lecture "Nutrigenomics part 2" From healthy to too much: The role of the Small Intestine for metabolic flexibility"

  1. 1. Lecture2From healthy to too muchThe role of Small Intestine for metabolic flexibilityMichael MüllerNutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University
  2. 2. The intestine as a gatekeeperAdipokines:AdiponectinLeptinResistinANGPTL4TNFetcGI hormones:InsulinGIPGLP1PYYGhrelinANGPTL4FGF15/19Food intakeLPLLPLLPLSFAGlucoseFructoseFGF21ANGPTL4Satiety
  3. 3. 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
  4. 4. 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
  5. 5. 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)
  6. 6. 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.
  7. 7. Simplified view onintestinal nutrient metabolismThiele lab 2013
  8. 8. The small intestine as primary organ isresponse to nutrients & food components
  9. 9. Gene regulation by dietary lipids in the intestine
  10. 10. The cellular network in intestinalhomeostasis (and inflammation)
  11. 11. The cellular network in intestinal(homeostasis and) inflammation.
  12. 12. The cellular network in intestinal(homeostasis) and inflammation
  13. 13. A major role for PPARain intestinal fatty acid sensingPhysiol Genomics. 2007 ;30(2):192-204
  14. 14. Intestinal PPAR target genes are largelyregulated by dietary PUFAS/MUFAs6h after oralgavage withOA18:1EPA20:5DHA22:6WY14643Genes 508 874 894 1218Apolipoproteins & relatedTAG (re)-synthesisPnliprp2 ,Pnliprp1, Mgll,Lipe,Lipa,Pla2g6,Pnpla2,Pnpla8, Daglb,Ces1,Ces3Chylomicron assembly& secretionKetone body synthesisIntestinal lipases &phospholipasesFat/Cd36,Slc27a2,Slc27a4,Acsl1,Acsl3,Acsl5,Fabp2,Fabp1,Scarb2,Scarb1Gpat1, Mogat2,Dgat1,Dgat2,Gpat3,Agpat3Mttp, Stx5a,Vti1a,Bet1,Sar1aApob,Apoa1 ,Apoa2,Angptl4,Apoa4,Apoc2,Vldlr,Apoc3,Apoe,Apol3,ApoolAcaa2,Acad10,Acad8,Acad9,Acadl,Acadm,Acads,Acadsb,Acadvl,Acot10,Acot2,Acot9,Aldh9a1,Cpt1a,Cpt2,Crat,Dci,Decr1,Hadha,Hadhb,Hibch,Slc22a5,Slc25a20,Aldh3a2,Cyp4a10,Abcd3,Acaa1a,Acaa1b,Acot3,Acot4,Acot5,Acot8,Acox1,Acox2,Crot,Decr2,Ech1,Ehhadh,Hsd17b4,Peci,Pecr,PparaAcat1, Hmgcl,Hmgcs2Mitochondrial,microsomal, peroxisomalfatty acid oxidationFatty acidtransport &bindingPPARα
  15. 15. Longitudinal distribution of genes involved in dietary lipidmetabolism in wild-type mice on PPARα activation by WY14643Fatty acid transport and binding1 2 3 4 5 6 7 8 9 100369121 2 3 4 5 6 7 8 9 100369121 2 3 4 5 6 7 8 9 1002461 2 3 4 5 6 7 8 9 1002461 2 3 4 5 6 7 8 9 10012341 2 3 4 5 6 7 8 9 100481216ACSL1MicroarrayFABP2Rel.expressionACSL3SLC27A2ACSL5SLC27A4Rel.expressionRel.expressionRel.expressionRel.expressionRel.expressionWTControl WTWY14643B** ** **** ******** ***** * ******* * *** ** ** ** ******* ****TAG (re-) synthesis1 2 3 4 5 6 7 8 9 100.00.51.01.52.02.53.01 2 3 4 5 6 7 8 9 100.00.51.01.52.02.53.01 2 3 4 5 6 7 8 9 100.00.51.01.52.02.53.0MicroarrayMicroarrayMOGAT2Rel.expressionDGAT1DGAT2Rel.expressionRel.expressionCWTControl WTWY14643******Chylomicron formation1 2 3 4 5 6 7 8 9 10012341 2 3 4 5 6 7 8 9 1004812161 2 3 4 5 6 7 8 9 1001234MicroarrayMTTPRel.expressionBET1FABP1Rel.expressionRel.expressionDWTControl WTWY14643*** * ****** *** *** **** ** ** *1 2 3 4 5 6 7 8 9 10012345Intestinal lipases and phospholipases1 2 3 4 5 6 7 8 9 1001231 2 3 4 5 6 7 8 9 1001231 2 3 4 5 6 7 8 9 1001231 2 3 4 5 6 7 8 9 100369121 2 3 4 5 6 7 8 9 1001234Microarray PNPLA2MGLLRel.expressionPLA2G6LIPAPNPLA8PLCB1Rel.expressionRel.expressionRel.expressionRel.expressionRel.expressionWTControl WTWY14643A**** ** * *** ** **** *** ** *** * **** ***** ** **** * *********
  16. 16. 1. Esophagus2. Stomach3. Duodenum4. Jejunum5, Ileum6. Cecum7. Ascending colon8. Descending colon9. Rectum
  17. 17. 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.
  18. 18. 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.
  19. 19. Low-fat (LF) diet High-fat (HF) dietgm% kcal% gm% kcal%Protein 19 20 24 20Carbohydrate 67 70 41 35Fat 4 10 24 45Ingredients gm kcal gm kcalCasein, lactic 200 800 200 800L-Cystine 3 12 3 12Corn Starch 427.2 1709 72.8 291Maltodextrin 100 400 100 400Sucrose 172.8 691 172.8 691Cellulose, BW200 50 0 50 0Soybean Oil 25 225 25 225Palm oil 20 180 177.5 1598Mineral Mix S10026 10 0 10 0DiCalcium Phosphate 13 0 13 0Calcium Carbonate 5.5 0 5.5 0Potassium Citrate, 1 H2O 16.5 0 16.5 0Vitamin Mix V10001 10 40 10 40Choline Bitartrate 2 0 2 0Total 1055 4057 858.15 4057Diets used in this study
  20. 20. 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
  21. 21. Dietary fat-induced differential geneexpression along the longitudinal axis of thesmall intestine
  22. 22. Heat map diagrams of differentiallyexpressed genes on a high-fat diet
  23. 23. Heat map diagrams of differentiallyexpressed genes on a high-fat diet
  24. 24. Heat map diagrams of differentiallyexpressed genes on a high-fat diet
  25. 25. Relative mRNA expression of nuclear receptorsalong the longitudinal axis of the small intestineunder basal conditions
  26. 26. 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.
  27. 27. Secretomeanalysis
  28. 28. 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.
  29. 29. Immune mechanisms that limit bacteria–epithelial cell interactions
  30. 30. Microbialcommunitycomposition atdifferent bodylocations in ahealthy human
  31. 31. A summary of the functional changes seen in various regions of thegut for different microbiome colonizations
  32. 32. Assembly and stability of the gut microbiota, andenvironmental factors affecting the gut microbiome duringlife
  33. 33. Major metabolites involved in host-microbe communication,originating from synthesis from microbial conversion of nutrientsand host metabolites in the gut lumen
  34. 34. Gut microbialdysbiosisassociatedwith disease
  35. 35. Factors shaping intestinal microbial compositionand effects of dysbiosis on host health
  36. 36. Microbiota-induced maturation of thegastrointestinal tract
  37. 37. Challenges for mouse studies:Several genera are significantly differentin abundance between mouse strains
  38. 38. Microbial impact on host physiology
  39. 39. Saturated fat affects obesity & liver TGsCorrelation between body weight gain and epididymal fat pads
  40. 40. HF-PO diet reduced microbial diversity and increasedthe Firmicutes-to-Bacteroidetes ratio
  41. 41. Lipid metabolism-related gene expression in the distalsmall intestine after 8 weeks of diet intervention
  42. 42. 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).
  43. 43. • 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.
  44. 44. 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
  45. 45. Natural Variation in Gene-by-DietInteractions
  46. 46. Robust Shifts in Gut MicrobiotaComposition after HF/HS Feeding
  47. 47. Plasticity of Gut Microbiota Is StrainSpecific
  48. 48. 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)!
  49. 49. The effects of heme on the colonicmucosa and the microbiota in miceINCON, 1-4 Oct 2012, San Jose
  50. 50. 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
  51. 51. 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
  52. 52. -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
  53. 53. Dietary heme increases fecal water cytotoxicity020406080100120controlheme*%celllysis
  54. 54. control heme 200xStainings with an antibody against Ki67, showing more proliferating (brown) cells on theheme diet, and deeper crypts.200xDietary heme increases cell proliferation
  55. 55. 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?
  56. 56. Laser Capture Microdissection Technology (LCM)LCM was used to isolate colonic surface and crypt cellsLCM system and procedure
  57. 57. 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**
  58. 58. 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
  59. 59. Downregulated mitogenic surface-to-crypt signals arealso decreased at protein levelGene level Protein levelOther signals with a similar pattern: Bmp2, Wif1, Ihh
  60. 60. 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
  61. 61. Protein Translation is controlled by 4E-BP1• Protein translation is inhibited by 4E-BP1, which is surface specifically upregulated byheme.
  62. 62. 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)
  63. 63. 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
  64. 64. 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.
  65. 65. • 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
  66. 66. 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.
  67. 67. 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
  68. 68. 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
  69. 69. No induction of oncogenes on heme + Abx
  70. 70. • Several cytotoxicity sensors not induced by heme + AbxHeme induced cytotox not seen by mucosa on Abx
  71. 71. • 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

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