1. NCSB/TIFN Short-chain fatty acid project TNO, UMCG, WUR
Barbara Bakker
Albert A. de Graaf
Vitor Martins dos Santos

2. Transorgan SCFA metabolism
In vitro and animal models to probe SCFA metabolism
Colonic metabolism
Liver metabolism
Whole body metabolism
e.g. 50 mM
butyrate
bacterial production
portal
vein
hepatic
vein
SCFA
SCFA
SCFA
SCFA
e.g. 10 uM
butyrate
colon
cells
liver
colon
lumen
arterial
pool ?
SCFA
substrate
SCFA project - WP2

3. Structure of the project
Structure of project (From the proposal’)
 The project is divided into 4 work packages that address different aspects of the project (see Figure 1). WP1 focuses on the quantification and analysis of the microbial population dynamics in relation to exogenous factors (diet) and endogenous factors (host response). WP2 aims at quantifying the metabolic activities in the gut microbiota using stable isotope labeling and metabolomics. WP3 looks at the analysis of, and intervention in, liver metabolism. In WP4, data from the other work packages and the flanking projects will be combined and used to develop mathematical models of the host response, which will be compared to other systems, notably pig and human. Close interactions between the different work packages are foreseen. Moreover, strong interactions are foreseen with NBIC and the Modelling and Data Analysis Group from the NISB on modeling and data analysis issues, software availability and data storage and management.
SCFA project – WP1

4. WP1 - Gut microbiomics of SCFA metabolism & metagenome-scale metabolic models Vitor 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

5. WP1 – Metagenome pathway analysis of gut microbial consortia, for SCFA production
Here an example of how we model and validate, just stress that it’s an example, not necessarily to be used as such. The link to dynamic models is through zooming in in parts of the networks that are imporant and couple to the dynamic models developed I WP2_WP4
SCFA project – WP1

6. 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

7. WP2 Data type overview – TIM-2 in vitro model
e.g. 50 mM
butyrate
HITchip data
[U-13C] starch, inulin, lactose
bacterial production
SCFA
RNA-SIP profiles
colon
lumen
SCFA profiles
Various other unlabeled carbohydrate substrates
substrate
SCFA kinetics
SCFA isotopomers
SCFA project - WP2

8. WP2 Data type overview - mouse
[1-13C]butyrate
Colonic metabolism
Liver metabolism
Whole body metabolism
portal
vein
hepatic
vein
SCFA
SCFA
SCFA
SCFA
e.g. 10 uM
butyrate
colon
cells
liver
colon
lumen
arterial
pool ?
SCFA
substrate
SCFA isotopomers
Amino acid isotopomers
+ kinetics
SCFA project - WP2

9. Potential isotopic markers of colonocytes TCA cycle activity2
[1-13C] butyrate
1-13C aspartate
& 1
4-13C aspartate 4 1
5-13C glutamate 5 1 3
1-13C glutamate
&
SCFA project - WP2
13CO2

10. WP2 Data type overview – pig
[1-13C]butyrate
Different infusion rates
Colonic metabolism
Liver metabolism
Whole body metabolism
portal
vein
hepatic
vein
SCFA
SCFA
SCFA
SCFA
e.g. 10 uM
butyrate
colon
cells
liver
colon
lumen
arterial
pool ?
SCFA
Levels of [1-13C]butyrate
SCFA project - WP2

11. 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

12. WP 3 + 4 (Groningen) The role of SCFA in mouse metabolism
Short-chain
fatty acids
SCFA metabolism?
Regulation?
CO2 +ATP/
Elongation, storage
Carbohydrate and
(long-chain)
fatty-acid fluxes

13. 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

14. Glucose accessible [1-13C]-acetate Intestine Glucose
Peripheral disposal
blood
[1-13C]-acetate
Glucose
Glucose
Glucose-6-P
Glycogen
glycerol
Pyruvate
Intestine
CHOL
alanine
Acetyl-CoA
lactate
FFA

15. Calculations