Nanjing 3 2013 Lecture "Nutrigenomics part 3"


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From healthy to too much
"The role of Muscle, White Adipose Tissue, Liver for metabolic flexibility"

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  • Inflammation has been associated with many disease phenotypes including steatohepatitis or diabetes. This relationship is in particular when inflammation is chronic or non-resolving. There is an interaction between metabolism and inflammation with positive or negative consequences with respect to organ and systemic health.In my talk I will briefly discuss two unpublished studies, one investigating the important interaction of WAT and liver in particular under conditions of diet-induced obesity. Organ-specific macrophages in WAT and liver play an crucial role in progressing organ-specific inflammatory phenotypes. In the second study we found very interesting interaction between dietary fat and macrophages in mesenteric lymph nodes that are exposed postprandially to very high concentrations of chylomicrons. We used a k.o. mouse for ANGPTL4 and could show that chronic consumption of saturated fat can be deadly.
  • 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.
  • Dietary amino acids are firstly used for protein synthesis; however, this can only happen to a limited extent. Subsequently, carbon skeletons can be utilised for gluconeogenesis in a very limited amount. An overload of the liver with dietary amino acids promotes catabolism to acetyl-CoA. Synthesised acetyl-CoA is either channelled into the TCA cycle or used for BHB production. With increasing ingestion of protein, amino acid oxidation and production of BHB from acetyl-CoA becomes more important in relation to gluconeogenesis and protein synthesis.
  • Nanjing 3 2013 Lecture "Nutrigenomics part 3"

    1. 1. Lecture 3From healthy to too muchThe role of Muscle, WAT, Liver for metabolic flexibilityMichael MüllerNutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University
    2. 2. Adipocytes at the crossroads of energy homeostasis
    3. 3. Liver functions related to nutrition1. Bile formation2. Gluconeogenesis3. Glycogen-synthesis4. Lipogenesis (new fat, TG)5. VLDL formation6. LDL uptake7. Cholesterol synthesis8. Bile acid synthesis (from cholesterol)
    4. 4. Liver
    5. 5. Hepatocyte
    6. 6. Liver dysfunctions /diseasesrelated to nutrition1. Hepatic steatosis(fatty liver)2. Liver inflammation3. NASH (non-alcoholicsteatohepatitis)4. Fibrosis5. Cirrhosis6. Cancer
    7. 7. Metabolic defects leading to thedevelopment of hepatic steatosis
    8. 8. White (WAT)Brown (BAT)Adipose tissue
    9. 9. Adipose tissue
    10. 10. Adipocyte
    11. 11. WAT Functions related to Nutrition1. Lipolysis2. Lipogenesis (TG)3. Maintaining triglyceride and free fatty acid levels& determining insulin resistance4. Protecting other organs from lipotoxicity
    12. 12. WAT dysfunctions related to nutrition1. Overweight, Obesity, Metabolicsyndrome, Diabetes, CVD, Cancer….2. Abdominal fat has a different metabolicprofile & being more prone to induceinsulin resistance.3. Central obesity is a marker of impairedglucose tolerance & is an independentrisk factor for cardiovascular disease
    13. 13. de Wit NJ, Afman LA, Mensink M, Müller MPhenotyping the effect of diet on non-alcoholicfatty liver disease J Hepatol 2012.
    14. 14. Communication between liver and adiposetissue essential for adequate lipid storage
    15. 15. Healthy (Homeostasis)
    16. 16. Unhealthy (Type 2 Diabetes)
    17. 17. Balance between insulin and glucagonSREBP-1cLXRChREBPGRFOXA2CREBPPARaFed state Fasted stateGlucose FFA
    18. 18. Metabolism & Inflammation
    19. 19. Liver, FAT & NASH/NAFLD Nonalcoholic Fatty Liver Diseases (NAFLD):Liver component of Metabolic Syndrome Different stages in NAFLD progression: Molecular events involved in NASH pathogenesis: Role of PPARa (Endocrinology 2008 & Hepatology 2010) Role Kupffer cells (Hepatology 2010) Role of macrophages in lipid metabolism (JBC 2008; Cell Metabolism 2010)hepatic steatosis steatohepatitis (NASH) & fibrosis cirrhosis
    20. 20. Interaction between WAT and liver tissueessential for NASH/NAFLD in C57Bl/6 miceObjective:– Nonalcoholic fatty liver disease (NAFLD) isstrongly linked to obesity and diabetes,suggesting an important role of adipose tissuein the pathogenesis of NAFLD.– Here we aimed to investigate the interactionbetween adipose tissue and liver in NAFLD,and identify potential early plasma markersthat predict NASH.
    21. 21. Experimental Design• stratificationon body weight• liver• plasma collectionmultiple protein assaysRNA extraction: Affx microarraystissue collectionrun-in diet 20 weeks diet interventionfrozen sections: histological feat.• ep. white adipose tissue10% lowfat diet(palm oil)10 LFD10 HFD45% highfat diet(palm oil)20 LFDRNA extraction: real-time PCRparaffin sections: histological feat.lipid contentquality control &data analysispipelineMousegenome430 2.00 2 4 8 12 16 20 weeks-3
    22. 22. High fat diet-induced obesity05101520250 2 4 8 12 16 20weeks under diet interventionBWgain(g)****** ** *LFLLFHHFLHFH******Liver TG content04080120160200mgTG/gliverALTactivity(UI)ALT plasma activityRatioLW/BW(%)Hepatomegaly**0246810***020406080100* *LFL LFH HFL HFH
    23. 23. A subpopulation of mice fed HFD develops NASH
    24. 24. Immunohistochemical staining confirms enhanced liverinflammation and early fibrosis in HFH miceMacrophage CD68CollagenStellate cell GFAP
    25. 25. Upregulation of inflammatory and fibroticgene expression in HFH responder mice
    26. 26. Adipose dysfunction in HFH mice
    27. 27. Change in adipose gene expressionindicate adipose tissue dysfunction
    28. 28. Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 mice
    29. 29. Plasma proteins as early predictivebiomarker for NASH in C57Bl/6 miceMultivariate analysis of association of proteinplasma concentrations with final livertriglyceride content
    30. 30. Conclusions• The data support the existence of a tightrelationship between adipose tissuedysfunction and NASH pathogenesis.• It points to several novel potentialpredictive biomarkers for NASH.
    31. 31. PeripheralbloodProtein turnoverAmino acidmetabolismGlycogenogenesisGlyconeogenesisGlycolysisLipogenesis,oxidationGI-tractMacronutrientcompositionof the dietLiverGut peptidesNutrientsBacterialderivedcomponentsInfluence of dietary protein on gene expression andmetabolic phenotype in the gut-liver axis
    32. 32. Objective Investigating the effect of a high protein diet onhepatic lipid accumulation. Unravel mechanisms which are responsible forthe reduced liver fat.
    33. 33. Design & diets1 week 12 weeksAcute effectof a high fat /high protein dietLong term diet effecton the developmentof liver steatosis2 weeksRun-in:control dietExperimental diets Carbohydrate (en%) Fat (en%) Protein (en%)Two low fat diet – normal or high proteinLF-NP 75 10 15LF-HP 40 10 50Two high fat diet – normal or high proteinHF-NP 50 35 15HF-HP 15 35 50
    34. 34. Body composition and food intakeSchwarz, J. et al., PLoS ONE 2012.
    35. 35. Hepatic steatosis50 µmSchwarz, J. et al., PLoS ONE 2012.
    36. 36. Fasting and postprandial plasma triglyceridesSchwarz, J. et al., PLoS ONE 2012.
    37. 37. Microarray analysis to study geneexpression
    38. 38. Enrichment map for HP vs. NP feedingto identify biological functionsSchwarz, J. et al., PLoS ONE 2012.
    39. 39. Changes in liver amino acid metabolic pathways induced byincreasing dietary proteinSchwarz, J. et al., PLoS ONE 2012.
    40. 40. Skeletal muscle functions & disorders• Force production =>Movement• Heat production• Protein storage• Glucose & lipidhomeostasis• Myopathies (muscularweakness)• Atrophy• Sarcopenia of aging• Ectopic fat disposition• Insulin resistance
    41. 41. Skeletal muscle
    42. 42. Myocyte
    43. 43. Interplay between adipokines andmyokines represent a yin–yang balancePedersen, B. K. & Febbraio, M. A. (2012) Muscles, exercise and obesity: skeletal muscle as a secretory organNat. Rev. Endocrinol. doi:10.1038/nrendo.2012.49
    44. 44. Skeletal muscle is a secretory organ
    45. 45. Link of physical activity to protectionagainst premature mortality
    46. 46. The Molecular Basis of Adaptation to Exercise
    47. 47. Transcriptional Regulators of Metabolism and Adaptation in Skeletal Muscle
    48. 48. The timeline of the study (A) and set-upof the endurance exercise bout (B)
    49. 49. Exercise increases heart rate and plasma levelsof FFA, insulin, cortisol and noradrenaline
    50. 50. Exercise mainly causes upregulation of gene expressionin both the exercising and non-exercising leg
    51. 51. Top 20 of most highly induced genes inexercising and non-exercising leg
    52. 52. Induction of transcription factorpathways by exercise
    53. 53. Nutrition, Metabolism & Genomics Group
    54. 54. Wageningen University• Founded in 1918• ~3000 employees• ~7500 students• ~220 PhD graduations per year