A lecture I gave in the Charite Berlin on April 16, 2013.

1. "Nutrition, metabolism and inflammation:
an essential but dangerous crosstalk"
Michael Müller
Netherlands Nutrigenomics Centre
& Nutrition, Metabolism and Genomics Group
Division of Human Nutrition, Wageningen University

2. Our “paleolithic” genes + modern diets
Paleolithic era Modern Times
1.200.000 Generations 2-3 Generations
between feast en famine in energy abundance
% Energy % Energy
100 Low-fat meat 100 Grain
Milk/-products
Chicken Isolated Carboh.
Eggs Isolated Fat/Oil
Fish Alcohol
50 Fruit 50 Meat
Chicken
Vegetables (carrots) Fish
Nuts
Honey
Fruit
Vegetables
0 0 Beans
“Unsafe” foods = Many ligands of NRs “Safe” foods = Less ligands of NRs

3. Phenotype plasticity
Phenotypic plasticity is the ability of an organism to
change its phenotype in response to changes in the
environment (e.g. nutrition or exercise).
CYP4A10
14
12
10
FC vs WT ctrl
8
6
4
2
0
WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO
ctrl WY feno C10:0TG C18:1TG C18:2TG C18:3TG C20:5TG C22:6TG

4. Genome plasticity

5. “We are what we eat and have eaten”
Received, Recorded, Remembered & Revealed

6. Transcription-factor pathways
mediating nutrient-gene interaction
, RXRs
TLR4

7. Understanding Nutrition
How nutrients regulate our genes: via sensing molecular switches
Changed
organ
metabolic
capacity
J Clin Invest. 2004;114:94-103 BMC Genomics 2008; 9:262 Physiol. Genomics 2009 PLoS One. 2012;7(8):e43260.
J Biol Chem. 2006;28:934-44 J Biol Chem. 2008;283:22620-7 Circulation 2010 J Hepatol. 2012 Dec;57(6):1370-3.
Endocrinology. 2006;147:1508-16 Arterioscler Thromb Vasc Biol. 2009;29:969-74.
Diabetes 2010 Am J Physiol Gastrointest Liver Physiol. 2012
Physiol Genomics. 2007;30:192-204 Plos One 2009;4(8):e6796 Cell Metabolism 2010 Physiol Genomics. 2012 Mar 19;44(6):352-61.
Endocrinology. 2007;148:2753-63 Hepatology 2010;51:511-522 Physiol Genomics. 2011;43(23):1307-18. Am J Physiol Endocrinol Metab. 2012
BMC Genomics 2007; 8:267 Am J Clin Nutr. 2009; 90:415-24 PLoS One. 2011;6(4):e19145. Prog Lipid Res. 2012 Jan;51(1):63-70.
Arterioscler Thromb Vasc Biol. 2007;27:2420-7 Am J Clin Nutr. 2009;90:1656-64 Nature 2011 May 22 Mol Cell Biol. 2013 Jan 22.
Am J Clin Nutr. 2007;86(5):1515-23 Mol Cell Biology 2009;29:6257-67 PLoS One. 2012;7(12):e49868. Hepatology. 2013 Jan 21.
PLOS ONE 2008;3(2):e1681 Am J Clin Nutr. 2010;91:208-17 PLoS One. 2012;7(11):e51066. J Nutr. 2013 Jan 16.
BMC Genomics 2008; 9:231 BMC Genomics 2009 PLoS One. 2012;7(10):e47303. Carcinogenesis. 2013 March
BMC Med Genomics. 2012 Aug 28

8. Nutrigenomics & molecular nutrition allows
us to define the mechanistic framework
Blood
triglycerides

9. “2 hits” in Metabolic Syndrome
Too much metabolic & inflammatory stress
Nature Medicine 2012

10. de Wit NJ, Afman LA, Mensink M, Müller M
Phenotyping the effect of diet on non-alcoholic
fatty liver disease J Hepatol 2012
.

12. Proximity of metabolic and immune cells
in adipose tissue and liver

14. Depletion of
Kupffer Cells

15. Conclusion
• The data point toward important crosstalk
between Kupffer cells and hepatocytes in the
regulation of hepatic triglyceride storage.
• The effect of Kupffer cells on liver triglycerides is
at least partially mediated by IL-1b, which
potently suppresses PPARa expression and
activity via NF-kB dependent inhibition of PPARa
promoter activity.

16. Control of inflammatory responses in adipose tissue and
liver by alternatively activated M2 macrophages

17. Energy homeostasis & obesity

18. 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

19. 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

20. A subpopulation of mice fed HFD develops NASH

21. Immunohistochemical staining confirms enhanced liver
inflammation and early fibrosis in HFH mice
HFL HFH
Macrophage CD68
Collagen
Stellate cell GFAP

22. Upregulation of inflammatory and fibrotic
gene expression in HFH responder mice

23. Adipose dysfunction in HFH mice

24. Change in adipose gene expression
indicate adipose tissue dysfunction

25. Conclusions
• The data support the existence of a
tight relationship between adipose
tissue dysfunction and NASH
pathogenesis.
Duval et al. Diabetes 2010

26. Too much saturated fat & macrophages

27. Differential regulation between saturated
and unsaturated fatty acids
Protective Toxic
60 5.0
50 4.0
Fold change
Fold change
40
3.0
30
2.0
20
10 1.0
0 0.0

28. Differential regulation between saturated
and unsaturated fatty acids
Angptl4 CHOP
60 5.0
50 4.0
Fold change
Fold change
40
3.0
30
2.0
20
10 1.0
0 0.0

29. Chylomicron
CE /TG
Angptl4
LPL
CE/TG
FFA
Chylomicron
remnant

30. Examination of the role of Angptl4 under
conditions of lipid overload
Low fat
Angptl4 +/+ High fat
Low fat
Angptl4 -/- High fat
Lichtenstein et al. Cell Metab. 2010

31. Angptl4-- mice on HFD become very ill
Lichtenstein et al. Cell Metab. 2010

32. Altered tissue morphology in Angptl4-/- mice
fed HFD
Lichtenstein et al. Cell Metab. 2010

33. Systemic inflammation in Angptl4-/- mice fed HFD
Angptl4+/+
Angptl4-/-

34. Inflammatory response independent of microbiota
Lichtenstein et al. Cell Metab. 2010

35. No effect of medium chain or PUFA TGs

36. Massive enlargement of mesenteric lymph
nodes in Angptl4-/- mice fed HFD
Lichtenstein et al. Cell Metab. 2010

37. Angptl4 is highly induced by fatty acids
in macrophages
Lichtenstein et al. Cell Metab. 2010

38. Angptl4 prevents lipid uptake and
inflammation in peritoneal macrophages

39. Angptl4 inhibits lipolysis and subsequent foam
cell formation

40. Angptl4 protects against lipolysis and
subsequent foam cell formation

41. Conclusion
• A high saturated fat diet causes massive inflammation in Angptl4-/-
mice originating in mesenteric lymph nodes.
• MLN-resident macrophages are protected from the pro-inflammatory
effect of saturated fatty acids via expression of Angptl4, which is
strongly induced by chyle and fatty acids and which via inhibition of
LPL prevents lipolysis of chylomicron-TG.
• 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

42. How inflammation is initiated and
developed in obesity
Annu Rev Nutr. 2012 Mar 9.

43. The inflammasome: guardians of the
intracellular environment
Annu. Rev. Cell Dev. Biol. 2012. 28:7.1–7.25

44. The inflammasome controls caspase-1 activation
Signal 2 NLRP3
ASC
Caspase-1
Signal 1

45. Caspase-1 is activated in adipose tissue
during the development of obesity
Obesity
Relative gene expression
Diet-induced obesity Genetically-induced obesity
Stienstra et al. Cell Metab. 2010

46. Caspase-1 activation in adipose tissue
enhances cytokine production
Low Fat Diet High Fat Diet
Inactive
Active
Actin
Stienstra et al. Cell Metab. 2010

47. Caspase-1 activation contributes to the
development of adipose tissue inflammation
Wild-type HFD Casp-1-/-HFD
10x 10x
Stienstra et al. Cell Metab. 2010

48. Does inflammasome activation contribute to the
development of insulin resistance? Yes
Plasma Insulin levels Hyperinsulinemic euglycemic clamp study
in HFD-fed Wild-type and caspase-1-/- animals
Plasma concentration (pg/ml)
LFD HFD
90
8000
80
GIR (μl min-1 kg-1)
70
6000 ** * *
60
** 50
4000 **
40
30
2000 20
Casp1 -/-
10 WT
0 0
10 20 40 60 80
Time (minutes)

49. Why do immune cells infiltrate the adipose
tissue during the development of obesity ?
• Obesity promotes the presence of harmed (due to
hypoxia/lipotoxicity) adipocytes that need to be removed
• Cleaning up of dying/old cells: a highly regulated
mechanism
• Clearance of dying cells is an important fundamental
process serving multiple functions in the regulation of
normal tissue turnover and homeostasis
• The turnover rate in human adipose tissue has been
estimated to be ∼1 million cells per second each day.

50. Human adipose tissue gene expression levels after weight loss
Weight loss is accompanied by a reduction in NLRP3 gene expression and downstream target genes
Nat Med. 2011, 17(2):179-88
Mol Med. 2011, 17(7-8): 840–845
What drives inflammasome activation?

51. Visceral adipose tissue of mildly obese individuals is characterized
by enhanced caspase-1 activity levels and higher production of IL-
1 as compared to subcutaneous adipose tissue
Stienstra et al Cell Metabolism 2012

52. Summary
Summary
 Inflammasome-mediated caspase-1 is activated in adipose tissue
during obesity
 Inhibition of caspase-1 in obese animals improves adipose tissue
inflammation and insulin sensitivity
 Inhibition of the downstream target IL-1 improves glycaemic control
and insulin sensitivity in patients with Type 2 Diabetes
 Potential novel treatment options: CRIDs, IL-37, novel targets of
caspase-1 & loosing weight!

53. Human Nutrition & (anti)inflammory response

54. 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

55. “Obese-linked” pro-inflammatory
gene expression profile by saturated fat
SFA 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

56. 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 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).

57. Sander Kersten
Lydia Afman
Guido Hooiveld
Wilma Steegenga
Philip de Groot
Mark Boekschoten
Nicole de Wit
Rinke Stienstra
& many PhDs
Christian Trautwein
Folkert Kuipers
Ben van Ommen
Hannelore Daniel
Bart Staels
Edith Feskens
Leif Sander
Dirk Haller
Eline Slagboom
Daniel Thome
Mihai Nitea
& many more