Top-down systems biology integration of conditional prebiotic modulated transgenomic interactions in a humanized microbiome mouse model

Francois-Pierre J Martin^1,2, Yulan Wang¹, Norbert Sprenger², Ivan K S Yap¹, Serge Rezzi², Ziad Ramadan², Emma Peré-Trepat², Florence Rochat², Christine Cherbut², Peter van Bladeren², Laurent B Fay², Sunil Kochhar², John C Lindon¹, Elaine Holmes¹ & Jeremy K Nicholson¹

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Synopsis

Adult humans carry ca. 1.5 kg of gut microbial symbiotic and commensal organisms that are in intimate communication with the host metabolic and immune systems (Nicholson et al, 2005; Dethlefsen et al, 2007). This symbiosis is the result of a long period of co-evolution and co-adaptation between the host genotype and the complex and variable microbiome (Gill et al, 2006). The effects of consuming live microbial supplements (probiotics) on the microbial ecology, and on human health and nutritional status have been investigated extensively over many years (Collins and Gibson, 1999; Rastall, 2005; Sonnenburg et al, 2006; Martin et al, 2007b). In addition, there is now considerable evidence that manipulation of the gut microbiota by prebiotics can beneficially influence the health of the host (Gibson and Roberfroid, 1995; Roberfroid, 1998; Delzenne and Kok, 2001; Sartor, 2004; Lim et al, 2005; Rastall, 2005; Parracho et al, 2007). For example, oligosaccharides have been suggested to represent the most important prebiotic dietary factor in human milk, promoting the development of a beneficial intestinal microbiota (Kunz et al, 2000; Bode, 2006).

Nowadays, clinical trials support the claims of efficacy of pro- and prebiotic nutritional intervention with regard to various proposed beneficial health effects, which has raised the requirement for providing additional evidence and for elucidation of the molecular bases of their action. This can be captured effectively only by studying the global system response of an organism to an intervention using top-down systems biology approaches. Recently, we have described that germ-free mice re-inoculated with a model of human baby microbiota (HBM mice) offer a simplified microbiome mouse model well adapted to assess the impact of nutritional intervention on gut microbial functional ecosystem and subsequent effects on host metabolism (Martin et al, 2008). The aim of the present study is to extend our previous investigations and to compare the effects of consumption of a synthetic galactosyl-oligosaccharide (Pre1) with those due to consumption of an in-house preparation of galactosyl-oligosaccharides (Pre2). We have assessed the impact of prebiotics on the microbial balance and the mammalian metabolism of HBM mice supplemented with a probiotic, Lactobacillus paracasei or L. rhamnosus (Figure 1).

Figure 1

Schematic diagram of the experimental study design.

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Here, we show specific effects of two prebiotics on the microbial populations of HBM mice when co-administered with two probiotics. These microbial changes were associated with specific host metabolic phenotypes, for example, variations in the fecal carbohydrate content, reduction in the level of hepatic triglyceride content and increased hepatic concentrations of PUFAs and hepatic glucogenic amino acids. These data provide further evidence for the critical involvement of prebiotics in host metabolism through modulation of the gut microbiome.

Our data provide additional evidence that the populations of beneficial bacteria in the gastrointestinal tract can, to some extent, be controlled with dietary interventions, here based on supplementation with galactosyl-oligosaccharides. Here, increases in the fecal populations of beneficial bacteria, namely Bifidobacterium longum and B. breve (Table I), were specifically associated with supplementation of prebiotics, Pre2 offering a greater ability to modulate the gut microbiota in HBM mice compared with Pre1. Carbohydrate fermentation may result in inhibition of the growth of pathogens by acidification of the environment through production of large quantities of carboxylic acids (Kikuchi et al, 1992; Ito et al, 1993; Rowland, 1993; Gibson and Roberfroid, 1995; Djouzi and Andrieux, 1997), which may explain the observation of a greater reduction in Escherichia coli and Clostridium perfringens bacterial counts in the feces (Table I).

Table 1: Microbial species counts in mouse fecal and jejunal contents

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Interestingly, in HBM mice supplemented with L. paracasei, populations of lactobacilli were slightly reduced with both prebiotic supplementations, and bifidobacteria showed only upward trends, which suggested a competition for the prebiotics between bifidobacteria and L. paracasei. Moreover, the observation of higher fecal content of oligosaccharides O1 and O3 specific to HBM mice fed with prebiotics and L. paracasei indicated that the microbiota may use these substrates poorly when compared with other groups.

Furthermore, ingestion of galacto-oligosaccharides or fructo-oligosaccharides is known to specifically induce bacterial hydrolysis of the substrate (Djouzi and Andrieux, 1997), as well as to modulate some bacterial activities, including glycolytic properties, hydrolysis of oligosaccharides, and formation of phenols and indoles (Mitsuoka et al, 1987; Ito et al, 1993; Rowland, 1993). In the current study, investigation of the metabolite changes in urinary excretion suggests that prebiotics intervention may reduce proteolytic activities previously ascribed to the basal metabolism of L. paracasei on casein medium (Martin et al, 2008), which is in agreement with reduced number of these bacteria observed in this study.

In parallel to gut microbial changes, relative reduction of hepatic triglycerides and increased concentrations of PUFAs were observed with mice supplemented with prebiotics. Non-digestible but fermentable carbohydrates were reported to decrease triglycerides in both serum and liver via modulation of the activity and gene expression of the lipogenic enzymes (Delzenne and Kok, 1998, 2001; Roberfroid and Delzenne, 1998; Pereira and Gibson, 2002). Moreover, fructans-type feeding may reduce the ability of isolated hepatocytes to synthesize and secrete triglycerides by 54% (Kok et al, 1996), as well as their ability to esterify fatty acids into triacylglycerols (Fiordaliso et al, 1995), which suggest a similar mechanism with galactosyl-oligosaccharides that may explain the relative increase in the NMR signals of PUFA-containing phospholipids in the current study. PUFAs can act by directing fatty acids away from triglyceride storage and toward oxidation, and can also enhance glucose flux to glycogen (Kliewer et al, 1997).

Notably, animals fed with L. paracasei in combination with prebiotics showed the most significant hepatic reduction in triglycerides, which was associated with a high fecal content of oligosaccharides. Previous studies showed that some prebiotics induce changes in lipogenic enzyme activities by reducing postprandial insulinemia and glycemia (Kok et al, 1998; Delzenne and Kok, 2001), via stimulation of the intestinal release of hormonal mediators (Morgan, 1996), or modification of the intestinal absorption of carbohydrates (Stanley and Newsholme, 1985) and shortening small intestinal transit time (Roberfroid and Delzenne, 1998). Our results suggest that a similar mechanism may be involved in mice supplemented with prebiotics co-administered with L. paracasei, as the higher concentrations of fecal oligosaccharides may reflect poorer digestion and absorption resulting in lower energy generation from carbohydrates, with a consequent switch to fat metabolism. However, further work is needed to understand the functional link between the residual fecal carbohydrate and the digestion of prebiotics by the gut microbiota, for instance by assessing experimentally the metabolic abilities of the bacterial species to utilize the prebiotics in future studies.

In conclusion, integrative systemic metabolic and microbiome profiling demonstrated the importance of nutritional intervention based on prebiotics and probiotic combinations in determining the host metabolic status and the levels of a diverse range of compounds in multiple pathways. Our data highlight that digestion of dietary carbohydrates by gut microbial species is a key factor in determining the resulting host metabolic phenotypes and use of prebiotics is highly promising to address personalized nutrition. The perspective of inducing unique changes in the host metabolism triggered by unique combinations of prebiotics and probiotics establishes an important step forward in the efforts to develop tailored nutritional solutions at an individual level.

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