Download Supplementary information

Supplementary information
A top-down systems biology view of microbiome-mammalian metabolic
interactions in a mouse model
François-Pierre J. Martin, Marc-Emmanuel Dumas, Yulan Wang, Cristina
Legido-Quigley, Ivan K. S. Yap, Huiru Tang, Séverine Zirah, Gerard M.
Murphy, Olivier Cloarec, John C. Lindon, Norbert Sprenger, Laurent B. Fay,
Sunil Kochhar, Elaine Holmes, and Jeremy K. Nicholson
Markers of increased caloric recovery
Our results demonstrated the essential microbial role on modulating calorie
recovery efficiency from the diet, even if no significant differences in body mass were
observed between the groups over the course of this experiment (four weeks,
Supplementary Figure 2). These observations are however in agreement with previous
reports stating that small and persistent microbial-induced changes in energy
metabolism may lead in significant changes in body weight over the course of a year
(Flegal & Troiano, 2000; Turnbaugh et al, 2006).
There is growing awareness of the critical involvement of the gut microbiota
in complex metabolic disease traits through controlling host metabolic regulations
involved in insulin resistance, type 2 diabetes and caloric recovery (Backhed et al,
2004; Dumas et al, 2006; Ley et al, 2006; Ozcan et al, 2006; Turnbaugh et al, 2006).
Here, higher production of SCFAs was observed in conventional animals with
different composition when compared to HBF mice (Table 2) which illustrates the
differences in the yield of dietary resources processing (including starches). In
particular, HBF mice showed lower acetate/propionate ratio (2.7) than reconventionalized (5.2) and conventional mice (7.3). Acetate has been shown to
increase cholesterol synthesis, whilst propionate, a gluconeogenic substrate, has been
shown to inhibit cholesterol synthesis. Therefore, it has been proposed that decreasing
the acetate/propionate ratio may reduce serum lipids and possibly cardiovascular
disease risk (Pereira & Gibson, 2002; Wong et al, 2006), which strongly supports our
mechanistic model (Figure 8). Furthermore, n-butyrate is a major metabolic substrate
for the colonic mucosa and plays an important role in decreasing the transformation of
primary to secondary bile acids as a result of colonic acidification (Wong et al, 2006).
Hence, higher n-butyrate production in conventional and re-conventionalized mice
might contribute to the differences in bile acid pool observed in ileal flushes.
In addition, a higher bacterial production and diversity of SCFAs was
observed in the caecum from conventional mice (Table 2), which might be
metabolized further by the host organism to produce energy. Previous studies of
colonization of germ-free mice with known microbes and by comparisons of the
genomes of members of the intestinal microbiota have revealed the critical
involvement of the microbiome in supplying host calorific requirement (Backhed et al,
2004). In particular, conventional animals require 30% less caloric intake to maintain
their body weight than their germ-free counterparts (Hooper et al, 2002). This is due
to the gut microbiota’s ability to regulate fat de novo hepatic synthesis and storage, as
well as increase the calorific recovery from the diet (Backhed et al, 2004; Dumas et al,
2006). The liver receives blood draining from the small intestine through the hepatic
portal vein, and elevation of glucose and lactate inflow generally leads to glycogen
storage. Here, the liver of conventional mice showed higher concentrations of
glycogen and several amino acids when compared to HBF mice (Figure 3). Our
observations suggest that exogenous metabolites derived from the gut bacteria
metabolism are stored or converted in the liver, which are related to increased
glycogenesis (Backhed et al, 2004; Metges, 2000; Metges et al, 2006; Xu et al, 2003).
Further disruption of other key liver functions by gut microbiota
Investigation of the urinary metabolic profiles of conventional mice showed
significant increases in the microbial co-metabolite phenylacetylglycine (Goodwin et
al, 1994) (PAG) which is formed via glycine conjugation of the phenylacetyl-CoA
intermediate in the liver. In the present study, we show that urinary PAG levels can be
attributed to variability in the gut microflora as suggested earlier by Nicholls et al.
(Nicholls et al, 2003). Fermentation of dietary and endogenous proteins is one
possible source of phenylacetic acid and is consistent with the different abilities of
specific microbiota to process dietary lipids (Goodwin et al, 1994; Smith &
Macfarlane, 1996). In addition, the microbiota play a role in tryptophan metabolism
via production of indoleacetic acid (IAA) (Shantz, 1966) and tryptamine (Figure 3)
(Donaldson, Jr., 1962). Specific anaerobic fecal bacteria are responsible for the
production of IAA by protein fermentation (Weissbach et al, 1959), including
Bacteroides, Clostridium and E. coli. (Xu et al, 2002). IAA is detoxified in the liver
mitochondria by conjugation with glycine to produce indoleacetylglycine (IAG)
(Shantz, 1966). The conversion of tryptamine and hepatic tryptophan to IAA occurs in
mammalian liver, and is independent of the effects of the gut microbiome (Gordon et
al, 1972). Moreover, it has been reported that fecal bacteria cannot generally
metabolize tryptamine to IAA (Chung et al, 1975). In the present study, HBF mice
showed higher excretion of IAG and lower levels of tryptamine in urine associated
with higher populations of Bacteroides, Clostridium and Enterobacteria when
compared to conventional mice, which agrees with previous reports.
Furthermore, HBF mice showed higher urinary levels of 3-hydroxyisovalerate when compared to conventional animals. Increased urinary excretion of 3-
hydroxy-isovalerate has been characterized as an early biomarker for biotin deficiency
(Bender, 1999). In biotin deficiency, reduced hepatic activity of the biotin-dependent
enzyme methylcrotonyl-CoA carboxylase causes the enzyme's substrate 3methylcrotonyl-CoA to be shunted via an alternate pathway to 3-hydroxyisovaleric
acid, which is excreted in the urine (Mock & Mock, 1992). The diet composition was
controlled during this experiment and did not indicate any deficiency in biotin.
Moreover, biotin generally originates from diets, intestinal bacterial synthesis and is
actively recycled by the host organism (Bender, 1999). This minor deficiency might
be a consequence of the formerly germ-free status of the animals. However, such
changes were not observed in urine from re-conventionalized mice, which suggested
that HBF has reduced ability to normalize the absorption and/or bacterial production
of biotin.
Reference List
Bender, D. A. (1999). Optimum nutrition: thiamin, biotin and pantothenate. Proc.
Nutr. Soc. 58(2), 427-433.
Chung, K. T., Anderson, G. M., and Fulk, G. E. (1975). Formation of indoleacetic
acid by intestinal anaerobes. J. Bacteriol. 124(1), 573-575.
Donaldson, R. M., Jr. (1962). Excretion of tryptamine and indole-3-acetic acid in
urine of rats with intestinal diverticula. Am. J. Physiol. 202, 289-292.
Goodwin, B. L., Ruthven, C. R., and Sandler, M. (1994). Gut flora and the origin of
some urinary aromatic phenolic compounds. Biochem. Pharmacol. 47(12), 2294-2297.
Gordon, S. A., Fry, R. J., and Barr, S. (1972). Origin of urinary auxin in the germfree
and conventional mouse. Am. J. Physiol 222(2), 399-403.
Mock, N. I., and Mock, D. M. (1992). Biotin deficiency in rats: disturbances of
leucine metabolism are detectable early. J. Nutr. 122(7), 1493-1499.
Nicholls, A. W., Mortishire-Smith, R. J., and Nicholson, J. K. (2003). NMR
spectroscopic-based metabonomic studies of urinary metabolite variation in
acclimatizing germ-free rats. Chem. Res. Toxicol. 16(11), 1395-1404.
Shantz, E. M. (1966). Chemistry of naturally-occurring growth-regulating substances.
Annu. Rev. Plant. Physiol. 17(1), 409-438.
Smith, E. A., and Macfarlane, G. T. (1996). Enumeration of human colonic bacteria
producing phenolic and indolic compounds: effects of pH, carbohydrate availability
and retention time on dissimilatory aromatic amino acid metabolism. J. Appl.
Bacteriol. 81(3), 288-302.
Weissbach, H., Udenfriend, S., Sjoerdsma, A., and King, W. (1959). Formation of
indole-3-acetic acid and tryptamine in animals: a method for estimation of indole-3acetic acid in tissues. J. Biol. Chem. 234(1), 81-86.
Xu, Z. R., Hu, C. H., and Wang, M. Q. (2002). Effects of fructooligosaccharide on
conversion of L-tryptophan to skatole and indole by mixed populations of pig fecal
bacteria. J. Gen. Appl. Microbiol. 48(2), 83-90.
Supplementary Figure 1: Comparison of area normalized (A, B) and nonnormalized (C, D) intensities of the methyl (δ0.84) and methylene (δ1.27) signals of
lipoproteins obtained from
1
H CPMG NMR plasma metabolic profiles of
conventional, re-conventionalized and human baby flora (HBF) mice displayed using
box-and-whisker plots (n=9,10 and 7 /group respectively). Comparison of area
normalized (E,F) intensities of the methyl (δ1.84) and methylene (δ1.27) signals of
lipids obtained from 1H CPMG MAS NMR metabolic profiles of intact liver tissue for
the three groups of mice.
Supplementary Figure 2: Comparison of animal weights in grams obtained from 10
week old conventional, re-conventionalized and human baby flora (HBF) mice
displayed using box-and-whisker plots (n=10/group).