Effect of gut microbiota on host
metabolism in humans and mice
3rd NuGO workshop
“Nutritional Metabolomics”
Doris Jacobs
Unilever R&D
02 July 2010
• Gut-mediated metabolites from nondigestible food ingredients
• Kati Hanhineva
• Metabolomics to characterize
gastrointestinal disorders / diseases
• Daryl Rowan
• Effect of gut microbiota on host metabolism
in humans and mice
• Doris Jacobs
Understanding what’s happening...
Effect of
gut
microbiota
on
host
metabolism
Retrieved from ‘’Development of
functional ingredients for gut
health’’, R. Puupponen, Trends in
Food Science & Technology 13
(2002) 3–11
Gut microbiota and immune system
Evidence for the potential role of gut
microbiota in the development of
• Colon cancer and inflammatory bowel disease
• (Mazmanina, S.K. et al. Nature 2008, 453, 620-625.)
• Type 1 diabetes
• (Wen L. Nature, 2008, 455, 1109-1113.)
• obesity and related metabolic disorders
• Alteration in the gut microbiota that occur with obesity may
enhance energy extraction from the diet. (Turnbaugh, P.J. et al.
Nature 2006, 444, 1027-1031.)
• Increased blood levels of lipopolysaccharides, due to
impairment of the barrier function of the gut by altered gut
microbiota, might trigger obesity-associated inflammation. (Cani,
P.D. et al. Diabetes 2007, 56, 1761-1772.)
Host-microbe communication in systemic inflammation
Li, P. & Hotmaisligil, G., Nature 2010, 464, 1287-1288.
Microbiota
intestinal
lumen
intestinal
epithelium
circulation
microbial
particles/
metabolites?
immune /
non-immune cells
Inflammation
Metabolic disorders such as obesity are characterized by long-term, low-grade
inflammation. Under certain conditions, the resident microorganisms of the gut might
contribute to this inflammation, resulting in disease.
Effect of gut microflora on mammalian blood
metabolites
Wikoff, W.R. et al. PNAS 2009, 106(10), 3698-703 .
cinamoylglycine
phenylalanine
hippuric acid
phenylpropionyl
glycine
indole-3-propionic acid tryptophan
N-acetyl tryptophan
serotonin
indoxyl sulfate
phenylacetylglycine
Plasma extracts from germ-free mice compared to conventional animals:
• Amino acids, in particular indole-containing compounds derived from tryptophan
• Organic acids containing phenyl groups
Modulation of host energy and lipid
metabolism by gut microbiota in mice
Velagapudi, V. R. et al. J. Lipid Res. 2010, 106(10), 3698-703 .
Comparison between conv. and germ-free mice
in serum metabolome:
• Increased levels of energy metabolites e.g. pyruvic acid, citric acid, glumaric acid,
and malic acid
• Reduced levels of cholesterol and fatty acids
in lipidome of serum, adipose tissue and liver
• Increased lipid clearance (lower levels of triglyceride and higher levels in adipose
tissue and liver)
• increased levels of phosphatidylcholine (34:1) in serum and liver
Effect of Antibiotics
Romick-Rosendale, L. E. et al. Magn Reson Chem. 2009;47 Suppl 1:S36-46.
NMR-based metabonomics analysis of 15 mouse urine and fecal
extracts following oral treatment with the broad-spectrum
antibiotic enrofloxacin (Baytril).
In urine:
• decreased acetate due to loss of microbial catabolism of sugars
and polysaccharides,
• decreased trimethylamine-N-oxide due to loss of microbial
catabolism of choline,
• increased creatine and creatinine due to loss of microbial enzyme
degradation.
in fecal extracts:
• depletion of amino acids produced by microbial proteases,
• reduction in metabolites generated by lactate-utilizing bacteria,
• increased urea caused by loss of microbial ureases.
Multicompartmental effects of gut
microbiome on mouse metabolic phenotype
Claus, S. P.. et al. Mol. Sys. Biol. 2008, 4, 219 .
Comparison between conv. (n=5) and germ-free mice (n=5)
• Gut microbiota modulate expression at both loacl (gut) and global (biofluid,
kidney, liver) system levels
• Reduced levels of hippurate in GF urine
• 5-aminovalerate in GF colon epithelium
• Raffinose in GF mice
• Elevated levels of betaine, choline, myo-inositol in GF kidney
Short-term intervention study:
Design
2 days
5 days
21 Males
Run-in
5 days
Wine/grape PP
Placebo
Placebo
Wine/grape PP
Intervention Wash-out
24h-Urine
Plasma t=0h
Plasma t=3h
Intervention Wash-out
24h-Urine
Plasma t=0h
Plasma t=3h
Population: 21 healthy males (18-69 years; non-smokers )
Treatment: wine / grape extract: mix of 870 mg Provinols red wine extract and
540 mg Mega-NaturalTM Rubired grape juice
extract corresponding to 800 mg GAE per day
placebo: microcrystalline cellulose
Diet control: low polyphenol diet during treatment period
same dietary intake during 36 hours before day 5 of each treatment period;
Sample collection: 24-hour urine after four days of polyphenol / placebo intake
plasma in fasted state (t=0h) on 5th treatment day
plasma 3 hours after polyphenol / placebo intake on 5th treatment day
Short-term intervention study:
Analysis
Metabolite profiling
Plasma
Urine
Protein Precipitation
Ultrafiltration
Creatinine determination
and sample adjustment
Separation into
Lipid and Polar Phase
Derivatisation
(Catecholamines)
Liquid/liquid extraction
(Steroids)
Sample adjustment to
creatinine
Sample adjustment to
creatinine
Urea degradation
Ultrafiltration
Derivatisation
GC-MS
Liquid/liquid extraction
(Steroids)
LC-MS/MS
Online-SPE-LC-MS/MS
Derivatisation
GC-MS
Derivatisation
(Catecholamines)
LC-MS/MS
Online-SPE-LC-MS/MS
Plasma/Urine: n = 251/256:
- 161/159 known metabolites, including
- 11/15 catecholamines and other monoamines,
- 8/7 steroids
- 90/97 "known unknown" analytes
Short-term intervention study:
Effects in plasma
Metabolites in plasma (0h)
locfdr
Tyrosine
0.456
Sphingomyelin
0.694
Oleic acid (C18:cis[9]1)
0.864
Testosterone-17-sulfate
0.909
minor dehydroepiandrosterone sulfate
Lysophosphatidylcholine (C18:2)
0.923
Phosphatidylcholine (C16:0,C20:4)
0.925
Phosphatidylcholine No 02
0.926
Threonine
0.936
Lysophosphatidylcholine (C20:4)
0.946
Phosphate, lipid fraction
0.946
Phosphatidylcholine (C18:0, C18:1)
0.962
Lysine
0.973
0
Metabolites in plasma (3h)
locfdr
3,4-Dihydroxyphenylacetic acid
0.064
Homovanillic acid
0.649
Hippuric acid
0.908
Taurine
0.965
Testosterone
0.967
1
2
c (wine and grape extract) / c (placebo)
Phenolic metabolites
0
Metabolites from amino acid / proteins
1
2
3
3
0
1
2
c (wine and grape extract) / c (placebo)
3
Short-term intervention study:
Effects in urine
Metabolites in 24h-urine
locfdr
3-Hydroxyhippuric acid
Pyrogallol
0.001
0.004
3-Hydroxyphenylacetic acid
Indole-3-lactic acid
Hippuric acid (NMR)
0.005
0.017
0.056
Catechol
4-Hydroxyhippuric acid
3,4-Dihydroxyphenylacetic acid
Vanillic acid
p-Cresol sulphate, minor o-cresol suphate
3,4-Dihydroxyphenylglycol
Glucose-1-phosphate, minor glucose
Sucrose
3-Indoxylsulfuric acid
trans-Ferulic acid
Nicotinic acid
1-Methylhistidine, minor 3-Methylhistidine
0.105
0.313
0.692
0.735
0.79
0.86
0.87
0.879
0.88
0.917
0.978
0.988
0
1
2
3
4
c (wine and grape extract) / c (placebo)
Phenolic metabolites
Metabolites from amino acid / proteins
5
Short-term intervention study:
Association
OH
OH
OH
OH
N
O
N
HO
HO
NH2
O
O
3,4-diOH-phenylacetate 3-OH-phenylacetate
CH3
1-Methylhistidine
OH
O
catechol
Gut Microbiota
Wine/grape
polyphenols
OH
N
Nicotinic acid
H
N
O
OCH3
OH
O
OH
O
vanillic acid
S
3-indoxylsulfate
O
H
N
O
OH
NH
OH
O
Indole-3-lactic acid
OH
hippurate
OH
O
CH3
O
OH
NH
NH
O
O
O
OH
O
OH
OH
S
O
3-hydroxyhippurate 4-hydroxyhippurate
p-cresolsulfate
O
Gut Microbiota / amino acid metabolism
OH
OH
Dietary
Protein /
Peptides
Questions
• How to generate causal relationships between the
gut microbiome and the host metabolic response?
• How to find evidence in humans?
• How to correlate the metabolome with the microbiome?
• How to deal with inter-individual variability caused
by the diverse gut microbial composition and the
host endogenous metabolism?
Expected outcome
• To compile a list of experimental factors that
are crucial when investigating microbiomemetabolome relationships
• To propose strategies / concepts for
systematic approaches to assess the effect
of the gut microbiome on the host
metabolome
Questions
• Can metabolomics help to deconvolute the
networks between gut microbiota or their
products and the host immune response?
Models of metabolic syndrome that arises as a result of altered
communication between the host immune system and the gut microbiota
(Vijay-Kumar, M. et al. Science (2010), 328, 228-231
Response and recovery in the plasma metabolome
tracks the acute LCMV-induced immune response
Wikoff et al. J Proteome Res. 2009 8(7):3578-87.
• Affected pathways during acute lymphocytic
choriomeningitis virus (LCMV) infection of mice at days
1, 3, 7, and 14 post infection:
•
•
•
•
•
TCA cycle intermediates (citrate, cis-aconitate, α-ketoglutarate)
γ-glutamyl dipeptides,
lysophosphatidyl cholines,
fatty acids,
kynurenine pathway (surprising because it is stimulated by IFNγ, which LCMV suppresses)