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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).