NCSB/TIFN Short-chain fatty acid project

TNO, UMCG, WUR

Barbara BakkerAlbert A. de GraafVitor Martins dos Santos

SCFA project - WP2

Transorgan SCFA metabolism

• In vitro and animal models to probe SCFA metabolism

colonlumen

coloncells

liver arterial pool

portalvein

SCFA

hepaticvein

e.g. 10 uM butyrate

Liver metabolism

Colonic metabolism

SCFA SCFA SCFA

?

bacterial production

substrate

SCFA

e.g. 50 mM butyrate

Whole body metabolism

SCFA project – WP1

Structure of the project

gut microbiota liver

epithelial cell layer

SCFA

feedback signals

diet probioticsprebiotics 1

2 3

1

2

Workpackage 1: population dynamics of gut microbiota in relation to exogenous and endogenous factors

Workpackage 2: quantification of metabolic activities of gut microbiota

3 Workpackage 3: analysis of host response, and intervention in liver metabolism

4 Workpackage 4: models of the host, comparison with pig and human situation

mouse

pighuman

4

SCFA project – WP1

WP1 - Gut microbiomics of SCFA metabolism & metagenome-scale metabolic modelsVitor dos Santos

Purpose

The construction of genome-scale metabolic models of the gut

microbiota, focusing on SCFA metabolism and the metabolic

reconstruction and subsequent classification of metabolism of

all sequenced species found in the gut, allowing for a

reconstruction of the gut food web

SCFA project – WP1

WP1 – Metagenome pathway analysis of gut microbial consortia, for SCFA production

Glucose

G6P

F6P

G3P

PEP

Pyr

AcCoA

Ribu5P

Rib5P

SucCoASuc

Xyl5P

a-KG

IsoCit

Cit

Fum

Mal

OaA Biomass

Biomass

Biomass

Biomass

NH4

Lactate

GAP

NADPH NADH

r1

r2

r3r4r6

r7

r8

r1+r9r10

r11r12

r17

r18

r21

r22

r23

r24

r25r26r27 Glx

r30r31

r33r34

Acetate

NADH

FADH

O2 ATP

r37r38

r39

Formiate

DHAP

r20

MG r42

r43+r44+r46

4CSal

r1

4ClCat

3CisMuc

Prot

r2

r3

r4

cisAcry

Lav

r5

Glucose

G6P

F6P

G3P

PEP

Pyr

AcCoA

SucCoASuc

a-KG

IsoCit

Cit

Fum

Mal

OaA Biomass

Biomass

Biomass

NH4

Lactate

GAP

NADPH NADH

r1

r2

r3r4r6

r7

r8

r1+r9r10

r11r12

r17

r18

r21

r22

r23

r24

r25r26r27 Glx

r30r31

r33r34

Acetate

NADH

FADH

O2 ATP

Formiate

DHAP

r20

MG r42

r43+r44+r46

4ClCat

cislac

r3

r4

Mal

Oxo

r5

3CisMuc

Prot

r3

r4

cisAcry

Lav

r5

r6

r7

Ribu5P

Rib5PXyl5P

Biomass

r37r38

r39

Glucose

G6P

F6P

G3P

PEP

Pyr

AcCoA

SucCoASuc

a-KG

IsoCit

Cit

Fum

Mal

OaA Biomass

Biomass

Biomass

NH4

Lactate

GAP

NADPH NADH

r1

r2

r3r4r6

r7

r8

r1+r9r10

r11r12

r17

r18

r21

r22

r23

r24

r25r26r27 Glx

r30r31

r33r34

Acetate

NADH

FADH

ATP

Formiate

DHAP

r20

MG r42

r43+r44+r46

Ribu5P

Rib5PXyl5P

Biomass

r37r38

r39

MT4

MT1

MT3

MT4

MT1

MT3

94

67

43

30

20

14

3 4 5 6 7 8 9 10

12 3

4

56

7

8

9

1 outer membrane protein(root adhesin)MW34900 pI 4,63

2 ketol-acid reductoisomeraseMW36370 pI 5,48

3 putrescine-bindingperiplasmatic proteinMW

4 succinyl-CoA (beta-chain)MW 41240 pI 5,83

5 translation elongation factorMW 30534 pI 5,08

6 DNAse

7 succinyl-CoA-synthetase (alphachain)MW 30110 pI 5,89

8 iron superoxide dismutaseMW 21939 pI 5,5

9 outer membrane proteinMW 19314 pI 5,9

MT1 Fed-Batch 1 mM

Time [min]

0 500 1000 1500

4CS [

M]

-200

0

200

400

600

800

1000

Concentration [

M]

0

5

10

15

20

25

30

35

4CS4CC3CMcis-DLProtoanemonin

13C-flux analysis

Phenotyping

arrays

Proteomics

Transcriptomics

Metabolic profiling

Constraint-based of the microbial consortium as functional unit

Iterative model refinement

and validation

Genome-scale constraint-based

modelling of single strains

Genome

Microbial consortium

n

Experimental validation

Micr. 2 Micr. 3

Micr. 1

Dialysis membranes

SCFA project - WP2

WP2 - Purpose

Develop computational models that allow to predict the rates of

intestinal SCFA production and the rates of the main SCFA-

derived metabolic processes in the host, using knowledge on

the composition of the intestinal microbiota and the given

substrate

• Focus on processes associated with the proximal colon as this is the

principal site of SCFA production• Use available data (acquired in TIFN C-012 “Microbe-mediated gut

metabolism” project)

SCFA project - WP2

WP2 Data type overview – TIM-2 in vitro model

RNA-SIP profiles

SCFA profiles

SCFA kinetics

colonlumen

SCFA

bacterial production

substrate

e.g. 50 mM butyrate

HITchip data[U-13C] starch, inulin, lactose

Various other unlabeled carbohydrate substrates

SCFA isotopomers

SCFA project - WP2

WP2 Data type overview - mouse

colonlumen

coloncells

liver arterial pool

portalvein

SCFA

hepaticvein

e.g. 10 uM butyrate

Liver metabolism

Colonic metabolism

SCFA SCFA SCFA

?

substrate

SCFA

[1-13C]butyrateWhole body metabolism

SCFA isotopomers

Amino acid isotopomers

+ kinetics

SCFA project - WP2

Potential isotopic markers of colonocytes TCA cycle activity

[1-13C] butyrate

5-13C glutamate

1-13C aspartate

4-13C aspartate

1

2

31-13C glutamate

1

5

1

4

&

&13CO2

SCFA project - WP2

WP2 Data type overview – pig

colonlumen

coloncells

liver arterial pool

portalvein

SCFA

hepaticvein

e.g. 10 uM butyrate

Liver metabolism

Colonic metabolism

SCFA SCFA SCFA

?SCFA

Different infusion rates Whole body metabolism

[1-13C]butyrate

Levels of [1-13C]butyrate

WP2- The regulation of SCFA production

Experimental

• unlabeled substrates in TIM-2 SCFA profile analysis and HITchip analysis

• 13C labeled substrates in TIM-2 bacterial pathway kinetics and SIP analysis

13C labeled caecal bolus of butyrate butyrate metabolism

• 13C labeled caecal infusion of butyrate transorgan absorption/metabolism of butyrate in pig

Computational

• Multivariate “substrate characteristics SCFA profile” prediction model

• Correlation map of microbiota composition and SCFA profile

• Bottom-up ODE models of fatty acid production

• Extend visibility of bacterial & colonocyte fluxes from 13C experiments

• Regulation Analysis of interorgan butyrate metabolism

WP 3 + 4 (Groningen)The role of SCFA in mouse metabolism

Short-chainfatty acids

CO2 +ATP/Elongation, storage

SCFA metabolism?

Regulation?

Carbohydrate and(long-chain)

fatty-acid fluxes

The role of SCFA in mouse metabolism

Experimental

•13C labelled rectal infusion of acetate / propionate/ butyrate fate of SCFA

•13C labelled tracers (glucose, glycerol, acetate) infusion in blood regulatory effect of SCFA on central energy metabolism

Computational

• Extend visibility of fluxes from 13C experiments

• Stoichiometric map of mouse fatty acid metabolism, incl. SCFA

• Bottom-up ODE models of fatty acid oxidation and the regulatory role of SCFA

• Regulation Analysis

• Modular Control Analysis

Glucose-6-P GlycogenGlucose

Pyruvate

lactate

alanine

Glucose

Peripheral disposal

glycerol

Glucose

bloodaccessibl

e

Intestine

Glucose

Acetyl-CoA

FFA

CHOL

[1-13C]-acetate

Calculations