The document discusses the interplay between non-resolving metabolic stress and pro-inflammatory stress in the context of nutrition and its impact on liver health and obesity-related conditions. It emphasizes the relationship between adipose tissue dysfunction and non-alcoholic steatohepatitis (NASH), highlighting key research findings on inflammatory markers and dietary influences. The conclusions stress the need for better understanding early inflammatory processes and the development of specific biomarkers for evaluating organ function in nutritional studies.

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. I will not talk about
Anti-inflammatory foods

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. A consideration of biomarkers to be used for evaluation of inflammation in human
nutritional studies ILSI working group BJN 2012 submitted

5. The two hits
Non-resolving metabolic & proinflammatory stress
2003

6. “2 hits” in Metabolic Syndrome
Too much metabolic & inflammatory stress
2012

7. PPARa
THE LIVER & THE 2 HITS

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. 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. 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 diet
MCP-1 (CCL2) 18 29 37 75
Mip1α (CCL3) 260 260 200 160
Mip-1β (CCL4) 28 12 43 69
MCP-3 (CCL7) 52 54 52 70
Eotaxin (CCL11) 793 780 753 879
MCP-5(CCL12) 23 22 25 36
Mip-1γ (CCL15) 8.6 9.8 14 14
Mip-3β (CCL19) 140 110 110 160
MDC (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 18
IP-10 (CXCL10) 28 31 46 109

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. OBESITY & NASH
INTERACTION OF WAT & LIVER

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. 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. A subpopulation of mice fed HFD develops NASH

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

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

18. Adipose dysfunction in HFH mice

19. Change in adipose gene expression
indicate adipose tissue dysfunction

20. Plasma proteins as early predictive
biomarker for NASH in C57Bl/6 mice

21. Plasma proteins as early predictive
biomarker for NASH in C57Bl/6 mice
Multivariate analysis of association of
protein plasma concentrations with final
liver triglyceride content

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

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

24. TOO MUCH SATURATED FAT
& MACROPHAGES

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

26. Examination of the role of Angptl4 under
conditions of lipid overload
Low fat
Angptl4 +/+ High fat
Low fat
Angptl4 -/- High fat

27. Angptl4-- mice on HFD become very ill

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

29. Inflammatory response independent of microbiota

30. No effect of medium chain or PUFA TGs

31. Massive enlargement of mesenteric lymph
nodes in Angptl4-/- mice fed HFD

32. Angptl4 protects against lipolysis and
subsequent foam cell formation

33. Angptl4 protects against lipolysis and
subsequent foam cell formation

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. Nutritional regulation of immune capacity
Veldhoen M, Brucklacher-Waldert V.
Dietary influences on intestinal immunity.
Nat Rev Immunol. 2012; 12 (10):696-708

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. We are different
“Personal” systemic inflammatory states
.PLoS One. 2010 Dec 23;5(12):e14422

39. Human nutrigenomics study
Dietary 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)

40. Design of the SFA vs MUFA-rich
intervention study
T=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

41. “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

42. 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

43. 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).

44. Sander Kersten
Rinke Stienstra
Lydia Afman
Guido Hooiveld
Mark Boekschoten
Laetitia Lichtenstein
Caroline Duval
& NMG group
Christian Trautwein
Folkert Kuipers
Ben van Ommen
& many more