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