COUNTERSTRIKE kick-off seminar

April 22, 2015 Copenhagen

Natal van RielEindhoven University of Technology, the Netherlands

Dept. of Biomedical Engineering

Systems Biology and Metabolic Diseases

n.a.w.v.riel@tue.nl

@nvanriel

Lipoprotein metabolism

• 3 types of lipoproteins

• Chylomicrons

• Very low density lipoproteins

(VLDL), apoB

• High density lipoproteins (HDL),

apoA

• A continuum of particles of

different size, different

composition of TG,

cholesterol and CE

• With distinct apo-

lipoproteins

• Lipoprotein distribution (LPD)

codetermines metabolic and

cardiovascular disease risks

0 10 20 30 40 50FP

LC

(a

rbit

rary

un

its)

Fraction number

VLDL

IDL/LDL

HDL

Plasma lipoprotein cholesterol profiles

• Fast protein liquid chromatography

Rigotti et al, 1997, PNAS 94: 12610-12615 PLTP

Jiang et al, J. Clin. Invest.

103:907–914 (1999). scavenger receptor class B type I (SR-B1)

plasma phospholipid transfer protein (PLTP)

Concept

• Fasted condition, no chylomicrons

Concept

• Particle size and heterogeneity

• Fasted condition, no chylomicrons

selective uptake

CE index

Triglycerides

Cholesteryl ester

Sips, et al. (2014) PLoS Comput Biol 10(5): e1003579.

Relating TG and CE content (model) to particle

diameter

• …and diameter to fraction number (FPLC)

Processes in the model

• ApoB-containing lipoprotein

metabolism (VLDL, LDL)

• ApoA-containing lipoprotein

metabolism (HDL)

CETP

PLTP

PLTP: phospholipid transfer protein

CETP: cholesteryl ester transport protein

Model-based data analysis

• Integration of model and data

• Inference of model parameters

(parameter estimation)

• Dealing with imperfect data

(noisy, missing, inconsistent)

• Data for model development

and calibration

• Independent data to validate

model predictions

In silico cholesterol FPLC profiles of

transgenic mice

SR-B1 -/-

PLTP -/-

WT

PLTP +/-

PLTP -/-

LDLr -/-WT LDLr -/-

Modelling and monitoring of intervention

• 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?

Levin et al, (2005) Arterioscler

Thromb Vasc Biol. 25(1):135-42

LDLR-/-

RXR: retinoid X receptor Calkin & Tontonoz 2012

Pharmaceutical intervention

• Extending the model

• Hypotheses

• E1: extra cholesterol accumulation in

HDL

• E2: extra HDL lipoprotein uptake

• E3: additional large nascent HDL

/ biomedical engineering PAGE 1122-4-2015

Grefhorst et al. (2002) J Biol

Chem 277(37):34182

Quantitative distinction between hypotheses:

fluxes

• Although the three different models yield equivalent lipoprotein

profiles, there are clear differences in the predictions of lipid

fluxes and lipoprotein metabolism

Sips, et al. (2014) PLoS Comput Biol 10(5): e1003579.

Modelling progressive changes in

metabolic fluxes

Modelling and monitoring of intervention

• Treated with T0901317 for 1, 2, 4, 7, 14, and 21 days

• Hypothesis 1: increase in HDLc is the result of increased

peripheral cholesterol efflux to HDL

Grefhorst et al. Atherosclerosis, 2012, 222: 382– 389

0 10 200

100

200Hepatic TG

Time [days]

[um

ol/g]

0 10 200

1

2

3Hepatic CE

Time [days]

[um

ol/g]

0 10 200

2

4

6Hepatic FC

Time [days]

[um

ol/g]

0 10 200

50

100Hepatic TG

Time [days]

[um

ol]

0 10 200

0.5

1

1.5Hepatic CE

Time [days]

[um

ol]

0 10 200

2

4Hepatic FC

Time [days]

[um

ol]

0 10 200

1000

2000

3000Plasma CE

Time [days]

[um

ol/L]

0 10 200

1000

2000

3000HDL-CE

Time [days]

[um

ol/L]

0 10 200

500

1000

1500Plasma TG

Time [days]

[um

ol/L]

0 10 206

8

10

12VLDL clearance

Time [days]

[-]

0 10 20100

200

300

400ratio TG/CE

Time [days]

[-]

0 10 200

5

10

15VLDL diameter

Time [days]

[nm

]

0 10 200

1

2

3VLDL-TG production

Time [days]

[um

ol/h]

0 10 201

2

3Hepatic mass

Time [days]

[gra

m]

0 10 200

0.2

0.4DNL

Time [days]

[-]

Mechanism-based model

• Differential equations

• Interconnected system (network)

• Model parameters have biological meaning

Data integration

• Estimation of unobserved metabolic parameters

• At unobserved time points

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

Tiemann et al. (2013) PLOS Comput Biol. 9(8):e1003166

ADAPT: Analysis of Dynamic Adaptations in Parameter Trajectories

• Model parameters inferred from data

• Mathematical model + ADAPT computation connects and

describes the data accurately

• 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

Analysis: HDL cholesterol

Analysis: increased excretion of cholesterol

Observation: increased HDLc

• 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

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

Hepatic steatosis

• Hypothesis 2: LXR-induced hepatic steatosis is caused by an

increase in de novo lipogenesis (DNL)

Liver section of mice

treated 4 days with LXR

agonist T0901317

Oil-Red-O staining for

neutral fat

hepatic steatosis

0 10 200

100

200Hepatic TG

Time [days]

[um

ol/g]

0 10 200

1

2

3Hepatic CE

Time [days]

[um

ol/g]

0 10 200

2

4

6Hepatic FC

Time [days]

[um

ol/g]

0 10 200

50

100Hepatic TG

Time [days]

[um

ol]

0 10 200

0.5

1

1.5Hepatic CE

Time [days]

[um

ol]

0 10 200

2

4Hepatic FC

Time [days]

[um

ol]

0 10 200

1000

2000

3000Plasma CE

Time [days]

[um

ol/L]

0 10 200

1000

2000

3000HDL-CE

Time [days]

[um

ol/L]

0 10 200

500

1000

1500Plasma TG

Time [days]

[um

ol/L]

0 10 206

8

10

12VLDL clearance

Time [days]

[-]

0 10 20100

200

300

400ratio TG/CE

Time [days]

[-]

0 10 200

5

10

15VLDL diameter

Time [days]

[nm

]

0 10 200

1

2

3VLDL-TG production

Time [days]

[um

ol/h]

0 10 201

2

3Hepatic mass

Time [days]

[gra

m]

0 10 200

0.2

0.4DNL

Time [days]

[-]

Increased hepatic FFA influx is the initial

contributor to hepatic TG accumulation

• [13C]16-palmitate infusion

Hijmans et al. (2015) FASEB J. 29(4):1153-64

C16:0 palmitate

C18:0 stearate

C16:1 palmitoleate

C18:1 oleate

saturated fatty acid monounsaturated fatty

acid

Conclusions (2)

• 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

Acknowledgements

• Peter Hilbers

• Christian Tiemann

• Joep Vanlier

• Yvonne Rozendaal

• Fianne Sips

• Bert Groen

• Jan Albert Kuivenhoven

• Maaike Oosterveer

• Brenda Hijmans

Systems Biology of Disease Progression -

ADAPT modeling

http://www.youtube.com/watch?v=x54ysJDS7i8

/ biomedical engineering