Keystone Symposia on Systems Biology of Lipid Metabolism, Feb 9 - Feb 13, 2015, Beaver Run Resort in Breckenridge, Colorado

1. .
Feb. 10, 2015
Natal van Riel, Maaike Oosterveer, Christian Tiemann,
Brenda Hijmans, Aldo Grefhorst, Yared Paalvast,
Yvonne Rozendaal, Jan Albert Kuivenhoven, Albert
Groen
Eindhoven University of Technology, the Netherlands
Dept. of Biomedical Engineering
Systems Biology and Metabolic Diseases
n.a.w.v.riel@tue.nl
@nvanriel

2. Liver X Receptor
• Liver X Receptor (LXR, nuclear receptor),
induces transcription of multiple genes
modulating metabolism of fatty acids,
triglycerides, and lipoproteins
• LXR agonists increase plasma high density
lipoprotein cholesterol (HDLc)
• LXR as target for anti-
atherosclerotic therapy?
/ biomedical engineering PAGE 211-2-2015
Levin et al, (2005) Arterioscler
Thromb Vasc Biol. 25(1):135-42
LDLR-/-
RXR: retinoid X receptor Calkin & Tontonoz 2012

3. Hypothesis 1: increase in HDLc is the result of
increased peripheral cholesterol efflux to HDL
• C57Bl/6J mice
• control, treated with T0901317 for 1, 2, 4, 7, 14, and 21 days
/ biomedical engineering PAGE 311-2-2015
Grefhorst et al. Atherosclerosis, 2012, 222: 382– 389
0 10 20
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Hepatic TG
Time [days]
[umol/g]
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Hepatic CE
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Hepatic FC
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Hepatic TG
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Hepatic CE
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Plasma CE
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HDL-CE
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Plasma TG
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VLDL clearance
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ratio TG/CE
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VLDL diameter
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DNL
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4. Mechanism-based model of lipid and
lipoprotein metabolism
• Differential equations
• Mathematical parameters inferred from data
/ biomedical engineering PAGE 411-2-2015

5. Data integration
• Estimation of unobserved metabolic parameters
• At unobserved time points
/ biomedical engineering PAGE 511-2-2015
1. Metabolite concentrations
-Hepatic free cholesterol (FC)
-Hepatic cholesteryl ester (CE)
-Hepatic triglyceride (TG)
-Plasma free fatty acids (FFA)
-Plasma TG
-Plasma total cholesterol
-HDL cholesterol
-VLDL (very low density lipoprotein) TG/C ratio
-Nascent VLDL particle diameter
2. Fluxes
-VLDL-TG production
-Hepatic cholesterol synthesis
-VLDL catabolism/clearance from the plasma

6. ADAPT: Analysis of Dynamic Adaptations in
Parameter Trajectories
ADAPT model connects and describes the data accurately
/ biomedical engineering PAGE 611-2-2015
• Data: black bars and
white dots
• Model: the darker the
more likely
• Variability in
data 
differences in
accuracy of
mathematical
parameters 
quantification
of uncertainty
in predictions

7. Analysis: HDL cholesterol
/ biomedical engineering PAGE 711-2-2015
Analysis: increased excretion of cholesterol
Observation: increased HDLc

8. • SR-B1 (Scavenger Receptor-B1)
• Protein activity:
Reduced presence of SR-B1 in liver
membranes contributes to induction of HDLc
• HDL excretion and uptake flux
are increased
• Transcription:
/ biomedical engineering PAGE 811-2-2015
mRNA of cholesterol efflux transporters
Tiemann et al., PLOS Comput Biol 2013
SR-B1 protein content is decreased in
hepatic membranes
Sr-b1 mRNA
expression not
changed
model: decreased
hepatic capacity to
clear cholesterol

9. Hepatic steatosis
• Hypothesis 2: LXR-induced hepatic steatosis is caused by an
increase in de novo lipogenesis (DNL)
/ biomedical engineering PAGE 911-2-2015
Liver section of mice
treated 4 days with LXR
agonist T0901317
Oil-Red-O staining for
neutral fat
hepatic steatosis
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Hepatic CE
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Hepatic FC
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Hepatic TG
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Hepatic CE
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Hepatic FC
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Plasma CE
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HDL-CE
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Plasma TG
Time [days]
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8
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12
VLDL clearance
Time [days]
[-]
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200
300
400
ratio TG/CE
Time [days]
[-]
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5
10
15
VLDL diameter
Time [days]
[nm]
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1
2
3
VLDL-TG production
Time [days]
[umol/h]
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1
2
3
Hepatic mass
Time [days]
[gram]
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0.2
0.4
DNL
Time [days]
[-]

10. Increased hepatic FFA influx is the initial
contributor to hepatic TG accumulation
• [13C]16-palmitate infusion
• FA mass isotopomer distributions
in hepatic and plasma FAs by GCMS
/ biomedical engineering PAGE 1011-2-2015
SFA = saturated fatty acid
C16:0 palmitate
C18:0 stearate
MUFA = monounsaturated fatty acid
C16:1 palmitoleate
C18:1 oleate
Hijmans et al. (2014) FASEB J.


11. Conclusions
• LXR activation in C57Bl/6J mice leads to complex time-dependent
perturbations in cholesterol and triglyceride metabolism
HDL cholesterol metabolism
• Peripheral cholesterol efflux to HDL and hepatic HDLc uptake increase over
time
• Reduced presence of SR-B1 in liver membranes despite an increment in
hepatic HDLc uptake
Hepatic triglyceride metabolism
• Input and output fluxes to liver TG are massively upregulated and a minor
imbalance between input and output fluxes causes steatosis
• Increased hepatic FFA influx is the initial contributor to hepatic TG
accumulation
/ biomedical engineering PAGE 1111-2-2015

12. Acknowledgements
• Peter Hilbers
• Christian Tiemann
• Joep Vanlier
• Yvonne Rozendaal
• Fianne Sips
• Bert Groen
• Jan Albert Kuivenhoven
• Maaike Oosterveer
• Brenda Hijmans
• Yared Paalvast
/ biomedical engineering PAGE 1211-2-2015
Systems Biology of Disease Progression -
ADAPT modeling
http://www.youtube.com/watch?v=x54ysJDS7i8