Nutrigenomics of the two hits: non-resolving  metabolic and pro-inflammatory stress                 Michael Müller        ...
I will not talk aboutAnti-inflammatory foods
Content The two hits:  Non-resolving metabolic & pro-inflammatory stress The liver & the two hits Obesity & NASH - Inte...
A consideration of biomarkers to be used for evaluation of inflammation in human           nutritional studies ILSI workin...
The two hitsNon-resolving metabolic & proinflammatory stress                      2003
“2 hits” in Metabolic SyndromeToo much metabolic & inflammatory stress                  2012
PPARaTHE LIVER & THE 2 HITS
More steatosis in mouse livers from PPARa -/- mice on a high fat diet         Stienstra R, Mandard S, Patsouris D, Maass C...
PPARa controls acute phase response induced by HF diets    Stienstra R, Mandard S, Patsouris D, Maass C, Kersten S, Müller...
Increased plasma levels of chemokines in                           HF-diet fed PPARa -/- mice                             ...
de Wit NJ, Afman LA, Mensink M, Müller M.     Phenotyping the effect of diet on non-            alcoholic fatty liver dise...
OBESITY & NASHINTERACTION OF WAT & LIVER
Interaction between WAT and liver tissue        essential for NASH/NAFLD in C57Bl/6 mice run-in diet                  20 w...
High fat diet-induced obesity                                                 25                                          ...
A subpopulation of mice fed HFD develops NASH
Immunohistochemical staining confirms enhanced liver    inflammation and early fibrosis in HFH mice                HFL    ...
Upregulation of inflammatory and fibrotic gene expression in HFH responder mice
Adipose dysfunction in HFH mice
Change in adipose gene expressionindicate adipose tissue dysfunction
Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 mice
Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 mice                  Multivariate analysis of associatio...
Conclusions• The data support the existence of a  tight relationship between adipose  tissue dysfunction and NASH  pathoge...
How inflammation is initiated and      developed in obesity        Annu Rev Nutr. 2012 Mar 9.
TOO MUCH SATURATED FAT& MACROPHAGES
Chylomicron          CE   /TG                           Angptl4                     LPL                                   ...
Examination of the role of Angptl4 under      conditions of lipid overload                                 Low fatAngptl4 ...
Angptl4-- mice on HFD become very ill
Systemic inflammation in Angptl4-/- mice fed HFD                                             Angptl4+/+                   ...
Inflammatory response independent of microbiota
No effect of medium chain or PUFA TGs
Massive enlargement of mesenteric lymph   nodes in Angptl4-/- mice fed HFD
Angptl4 protects against lipolysis and  subsequent foam cell formation
Angptl4 protects against lipolysis and  subsequent foam cell formation
Conclusion• A high saturated fat diet causes massive inflammation in  Angptl4-/- mice originating in mesenteric lymph node...
Nutritional regulation of immune capacity              Veldhoen M, Brucklacher-Waldert V.            Dietary influences on...
Human study:        Plasma Protein Profiling Reveals  Protein Clusters Related to BMI and Insulin  Levels in Middle-Aged O...
We are different“Personal” systemic inflammatory states            .PLoS One. 2010 Dec 23;5(12):e14422
Human nutrigenomics studyDietary fat and inflammation in adipose tissue                                                   ...
Design of the SFA vs MUFA-rich                 intervention studyT=0 wks          T=2 wks                                 ...
“Obese-linked” pro-inflammatorygene expression profile by saturated fatSFA diet   MUFA diet                       • The SF...
Fish-oil supplementation induces anti-inflammatory gene  expression profiles in human blood mononuclear cells             ...
General conclusions• (Over)nutrition and inflammation are intimately linked =>  non-resolving metabolic and pro-inflammato...
Sander KerstenRinke StienstraLydia AfmanGuido HooiveldMark BoekschotenLaetitia LichtensteinCaroline Duval& NMG groupChrist...
Nutrigenomics of the two hits: non-resolving metabolic and pro-inflammatory stress
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Nutrigenomics of the two hits: non-resolving metabolic and pro-inflammatory stress

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My 1. talk Oct3 2012 at #INCON12 Costa Rica
Nutrigenomics of the two hits: non-resolving metabolic and pro-inflammatory stress

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  • A subpopulation of mice fed HFD develops NASH. Haematoxylin and eosin staining (D) and oil red O staining (E) of representative liver sections of the 4 subgroups
  • (Immuno)histochemical staining confirms enhanced inflammation and early fibrosis in HFH miceImmunohistochemical staining of macrophage activation in representative liver section of HFL and HFH mice using antibody against the specific macrophagemarker Cd68Collagen staining using fast green FCF/sirius red F3B. Staining of stellate cell activation using antibody against GFAP.
  • - Number of genes up- or down-regulated in the various subgroups in comparison to the LFL mice, as determined by Affymetrix GeneChip analysis. Genes with a p-value below 0.05 were considered significantly regulated. - Heat map showing changes in expression of selected genes involved in lipid metabolism, inflammation and fibrosis in liver. Changes in gene expression of selected genes as determined by real-time quantitative PCR. Mean expression in LFL mice was set at 100%. Error bars reflect standard deviation. Bars with different letters are statistically different (P<0.05 according to Student’s t-test). Number of mice per group: n=4 (LFL, HFL, HFH), n=6 (LFH).
  • Haematoxylin and eosin staining of representative adipose tissue sections. Immunohistochemical staining of macrophages using antibody against Cd68. Collagen staining using fast green FCF/sirius red F3B.
  • Adipose tissue mRNA expression of a selected group of genes was determined by quantitative real-time PCR after 21 weeks of dietary intervention. Mean expression in LFL mice was set at 100%. Error bars reflect standard deviation. * = significantly different from HFL mice according to Student’s t-test (P<0.05). Number of mice per group: n=4 (LFL, HFL, HFH), n=6 (LFH).
  • . A) Plasma concentration of haptoglobin, TIMP-1, IL-1β, leptin and insulin were determined by multiplex assay at specific time points during the 21 weeks of dietary intervention after a 6h fast. White squares: LFL, Light grey squares: LFH, dark grey squares: HFL, black squares: HFH. Error bars reflect standard deviation. * = significantly different from HFL mice according to Student’s t-test (P<0.05). Number of mice per group: n=4 (LFL, HFL, HFH), n=6 (LFH).
  • Graphs illustrating the result of multivariate analysis showing the association of protein plasma concentrations at various time points with final liver triglyceride content. Significant proteins display an inverse RSD value higher than 2 (bold line indicates the inverse RSD threshold value of 2).RSD = Relative standard deviation.
  • Nutrigenomics of the two hits: non-resolving metabolic and pro-inflammatory stress

    1. 1. Nutrigenomics of the two hits: non-resolving metabolic and pro-inflammatory stress Michael Müller Netherlands Nutrigenomics Centre & Nutrition, Metabolism and Genomics Group Division of Human Nutrition, Wageningen University @nutrigenomics
    2. 2. I will not talk aboutAnti-inflammatory foods
    3. 3. Content The two hits: Non-resolving metabolic & pro-inflammatory stress The liver & the two hits Obesity & NASH - Interaction of the white adipose tissue & the liver Too much saturated fat & macrophages Human Nutrigenomics examples Conclusions
    4. 4. A consideration of biomarkers to be used for evaluation of inflammation in human nutritional studies ILSI working group BJN 2012 submitted
    5. 5. The two hitsNon-resolving metabolic & proinflammatory stress 2003
    6. 6. “2 hits” in Metabolic SyndromeToo much metabolic & inflammatory stress 2012
    7. 7. PPARaTHE LIVER & THE 2 HITS
    8. 8. More steatosis in mouse livers from PPARa -/- mice on a high fat diet Stienstra R, Mandard S, Patsouris D, Maass C, Kersten S, Müller M PPAR{alpha} protects against obesity-induced hepatic inflammation Endocrinology. 2007 Jun;148(6):2753-63
    9. 9. PPARa controls acute phase response induced by HF diets Stienstra R, Mandard S, Patsouris D, Maass C, Kersten S, Müller M PPAR{alpha} protects against obesity-induced hepatic inflammation Endocrinology. 2007 Jun;148(6):2753-63
    10. 10. Increased plasma levels of chemokines in HF-diet fed PPARa -/- mice Wildtype PPARα-/-Chemokine Low fat diet High fat diet Low fat diet High fat dietMCP-1 (CCL2) 18 29 37 75Mip1α (CCL3) 260 260 200 160Mip-1β (CCL4) 28 12 43 69MCP-3 (CCL7) 52 54 52 70Eotaxin (CCL11) 793 780 753 879MCP-5(CCL12) 23 22 25 36Mip-1γ (CCL15) 8.6 9.8 14 14Mip-3β (CCL19) 140 110 110 160MDC (CCL22) plasma (n=5) was used for determining the concentration of multiple chemokines Pooled mouse 82 84 92 99 (Rules Based Medicine). pg/ml plasma, except for Mip-1γ (ng/ml plasma).Mip-2 (CXCL2) 12 15 14 18IP-10 (CXCL10) 28 31 46 109
    11. 11. de Wit NJ, Afman LA, Mensink M, Müller M. Phenotyping the effect of diet on non- alcoholic fatty liver disease J Hepatol 2012
    12. 12. OBESITY & NASHINTERACTION OF WAT & LIVER
    13. 13. Interaction between WAT and liver tissue essential for NASH/NAFLD in C57Bl/6 mice run-in diet 20 weeks diet intervention tissue collection • stratification • plasma collection • liver on body weight frozen sections: histological feat. multiple protein assays lipid content RNA extraction: Affx microarrays 10 LFD-3 20 LFD 0 2 4 8 12 16 20 weeks quality control & 10 HFD Mouse data analysis 10% low genome pipeline fat diet 45% high 430 2.0 (palm oil) fat diet (palm oil) • ep. white adipose tissue paraffin sections: histological feat. RNA extraction: real-time PCR
    14. 14. High fat diet-induced obesity 25 LFL HFL LFH HFH 20 BW gain (g) * * 15 ** * 10 * * * 5 * 0 0 2 4 8 12 16 20 weeks under diet intervention Liver TG content Hepatomegaly ALT plasma activity 200 10 100 Ratio LW/BW (%) *** ALT activity (UI)mg TG/g liver 160 8 ** *** 80 120 ** 6 60 * 80 4 40 * * 40 2 20 0 0 0 LFL LFH HFL HFH
    15. 15. A subpopulation of mice fed HFD develops NASH
    16. 16. Immunohistochemical staining confirms enhanced liver inflammation and early fibrosis in HFH mice HFL HFH Macrophage CD68 Collagen Stellate cell GFAP
    17. 17. Upregulation of inflammatory and fibrotic gene expression in HFH responder mice
    18. 18. Adipose dysfunction in HFH mice
    19. 19. Change in adipose gene expressionindicate adipose tissue dysfunction
    20. 20. Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 mice
    21. 21. Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 mice Multivariate analysis of association of protein plasma concentrations with final liver triglyceride content
    22. 22. Conclusions• The data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis. Duval et al. Diabetes 2010
    23. 23. How inflammation is initiated and developed in obesity Annu Rev Nutr. 2012 Mar 9.
    24. 24. TOO MUCH SATURATED FAT& MACROPHAGES
    25. 25. Chylomicron CE /TG Angptl4 LPL CE/TG FFA Chylomicron remnant
    26. 26. Examination of the role of Angptl4 under conditions of lipid overload Low fatAngptl4 +/+ High fat Low fatAngptl4 -/- High fat
    27. 27. Angptl4-- mice on HFD become very ill
    28. 28. Systemic inflammation in Angptl4-/- mice fed HFD Angptl4+/+ Angptl4-/-
    29. 29. Inflammatory response independent of microbiota
    30. 30. No effect of medium chain or PUFA TGs
    31. 31. Massive enlargement of mesenteric lymph nodes in Angptl4-/- mice fed HFD
    32. 32. Angptl4 protects against lipolysis and subsequent foam cell formation
    33. 33. Angptl4 protects against lipolysis and subsequent foam cell formation
    34. 34. Conclusion• A high saturated fat diet causes massive inflammation in Angptl4-/- mice originating in mesenteric lymph nodes.• In the absence of this protective mechanism, feeding a diet rich in saturated fat rapidly leads to enhanced lipid uptake into MLN-resident macrophages, triggering foam cell formation and a massive inflammatory response. Lichtenstein et al. Cell Metab. 2010
    35. 35. Nutritional regulation of immune capacity Veldhoen M, Brucklacher-Waldert V. Dietary influences on intestinal immunity. Nat Rev Immunol. 2012; 12 (10):696-708
    36. 36. Human study: Plasma Protein Profiling Reveals Protein Clusters Related to BMI and Insulin Levels in Middle-Aged Overweight Subjects AIM• Associate plasma protein profiles with BMI• Identify potential marker profile of early disease state . PLoS One. 2010 Dec 23;5(12):e14422
    37. 37. We are different“Personal” systemic inflammatory states .PLoS One. 2010 Dec 23;5(12):e14422
    38. 38. Human nutrigenomics studyDietary fat and inflammation in adipose tissue ? Change in diet composition Van Dijk et al. AJCN 2009 de Luca, C and Olefsky JM, Nature Medicine 12, 41 - 42 (2006)
    39. 39. Design of the SFA vs MUFA-rich intervention studyT=0 wks T=2 wks T=10 wks Run-in SFA-rich diet (n=10) SFA-rich diet (n=20) MUFA-rich diet (n=10) Baseline After intervention - Clamp - Clamp - Adipose tissue biopsy - Adipose tissue biopsy - Blood sampling - Blood sampling Van Dijk et al. AJCN 2009
    40. 40. “Obese-linked” pro-inflammatorygene expression profile by saturated fatSFA diet MUFA diet • The SFA-rich diet: • Induces a pro- inflammatory obese-linked gene expression profile • Decreases expression and plasma level of the anti- inflammatory cytokine adiponectin • “Personal Transcriptomes” Van Dijk et al. AJCN 2009
    41. 41. Fish-oil supplementation induces anti-inflammatory gene expression profiles in human blood mononuclear cells Less inflammation & decreased pro-arteriosclerosis markers = Anti-immuno-senescence Bouwens et al. Am J Clin Nutr. 2009
    42. 42. General conclusions• (Over)nutrition and inflammation are intimately linked => non-resolving metabolic and pro-inflammatory stress (the two hits).• It will be essential to get a better understanding of the very early events that lead to non-resolving organ inflammation and the precise role of nutrition (causal or preventive) in this pathophysiological development.• We need biomarkers specifically for organ function (“2 hit state”) to be able to specifically target and modulate.• The challenge will be the translation of the findings from mice studies to the human situation (“individual” health).
    43. 43. Sander KerstenRinke StienstraLydia AfmanGuido HooiveldMark BoekschotenLaetitia LichtensteinCaroline Duval& NMG groupChristian TrautweinFolkert KuipersBen van Ommen& many more

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