Microbiota influence on behavior: Integrative analysis of serotonin metabolism and behavioral profile in germ-free mice.

Previous studies on germ-free (GF) animals have described altered anxiety-like and social behaviors together with dysregulations in brain serotonin (5-HT) metabolism. Alterations in circulating 5-HT levels and gut 5-HT metabolism have also been reported in GF mice. In this study, we conducted an integrative analysis of various behaviors as well as markers of 5-HT metabolism in the brain and along the GI tract of GF male mice compared with conventional (CV) ones. We found a strong decrease in locomotor activity, accompanied by some signs of increased anxiety-like behavior in GF mice compared with CV mice. Brain gene expression analysis showed no differences in HTR1A and TPH2 genes. In the gut, we found decreased TPH1 expression in the colon of GF mice, while it was increased in the cecum. HTR1A expression was dramatically decreased in the colon, while HTR4 expression was increased both in the cecum and colon of GF mice compared with CV mice. Finally, SLC6A4 expression was increased in the ileum and colon of GF mice compared with CV mice. Our results add to the evidence that the microbiota is involved in regulation of behavior, although heterogeneity among studies suggests a strong impact of genetic and environmental factors on this microbiota-mediated regulation. While no impact of GF status on brain 5-HT was observed, substantial differences in gut 5-HT metabolism were noted, with tissue-dependent results indicating a varying role of microbiota along the GI tract.

Gut Microbiome Integration in Drug Discovery and Development of Small Molecules.

Human microbiomes, particularly in the gut, could have a major impact on the efficacy and toxicity of drugs. However, gut microbial metabolism is often neglected in the drug discovery and development process. Medicen, a Paris-based human health innovation cluster, has gathered more than 30 international leading experts from pharma, academia, biotech, clinical research organizations, and regulatory science to develop proposals to facilitate the integration of microbiome science into drug discovery and development. Seven subteams were formed to cover the complementary expertise areas of 1) pharma experience and case studies, 2) in silico microbiome-drug interaction, 3) in vitro microbial stability screening, 4) gut fermentation models, 5) animal models, 6) microbiome integration in clinical and regulatory aspects, and 7) microbiome ecosystems and models. Each expert team produced a state-of-the-art report of their respective field highlighting existing microbiome-related tools at every stage of drug discovery and development. The most critical limitations are the growing, but still limited, drug-microbiome interaction data to produce predictive models and the lack of agreed-upon standards despite recent progress. In this paper we will report on and share proposals covering 1) how microbiome tools can support moving a compound from drug discovery to clinical proof-of-concept studies and alert early on potential undesired properties stemming from microbiome-induced drug metabolism and 2) how microbiome data can be generated and integrated in pharmacokinetic models that are predictive of the human situation. Examples of drugs metabolized by the microbiome will be discussed in detail to support recommendations from the working group. SIGNIFICANCE STATEMENT: Gut microbial metabolism is often neglected in the drug discovery and development process despite growing evidence of drugs' efficacy and safety impacted by their interaction with the microbiome. This paper will detail existing microbiome-related tools covering every stage of drug discovery and development, current progress, and limitations, as well as recommendations to integrate them into the drug discovery and development process.

Male mice engaging differently in emotional eating present distinct plasmatic and neurological profiles.

Objective: Stressed individuals tend to turn to calorie-rich food, also known as 'comfort food' for the temporary relief it provides. The emotional eating drive is highly variable among subjects. Using a rodent model, we explored the plasmatic and neurobiological differences between 'high and low emotional eaters' (HEE and LEE).Methods: 40 male mice were exposed for 5 weeks to a protocol of unpredictable chronic mild stress. Every 3 or 4 days, they were submitted to a 1-h restraint stress, immediately followed by a 3-h period during which a choice between chow and chocolate sweet cereals was proposed. The dietary intake was measured by weighing. Plasmatic and neurobiological characteristics were compared in mice displaying high vs low intakes.Results: Out of 40 mice, 8 were considered as HEE because of their high post-stress eating score, and 8 as LEE because of their consistent low intake. LEE displayed higher plasma corticosterone and lower levels of NPY than HEE, but acylated and total ghrelin were similar in both groups. In the brain, the abundance of NPY neurons in the arcuate nucleus of the hypothalamus was similar in both groups, but was higher in the ventral hippocampus and the basal lateral amygdala of LEE. The lateral hypothalamus LEE had also more orexin (OX) positive neurons. Both NPY and OX are orexigenic peptides and mood regulators.Discussion: Emotional eating difference was reflected in plasma and brain structures implicated in emotion and eating regulation. These results concur with the psychological side of food consumption.

Sex-dependent impact of microbiota status on cerebral µ-opioid receptor density in fischer rats.

µ-opioid receptors (MOPr) play a critical role in social play, reward and pain, in a sex- and age-dependent manner. There is evidence to suggest that sex and age differences in brain MOPr density may be responsible for this variability; however, little is known about the factors driving these differences in cerebral MOPr density. Emerging evidence highlights gut microbiota's critical influence and its bidirectional interaction with the brain on neurodevelopment. Therefore, we aimed to determine the impact of gut microbiota on MOPr density in male and female brains at different developmental stages. Quantitative [3 H]DAMGO autoradiographic binding was carried out in the forebrain of male and female conventional (CON) and germ-free (GF) rats at postnatal days (PND) 8, 22 and 116-150. Significant 'microbiota status X sex', 'age X brain region' interactions and microbiota status- and age-dependent effects on MOPr binding were uncovered. Microbiota status influenced MOPr levels in males but not females, with higher MOPr levels observed in GF versus CON rats overall regions and age groups. In contrast, no overall sex differences were observed in GF or CON rats. Interestingly, within-age planned comparison analysis conducted in frontal cortical and brain regions associated with reward revealed that this microbiota effect was restricted only to PND22 rats. Thus, this pilot study uncovers the critical sex-dependent role of gut microbiota in regulating cerebral MOPr density, which is restricted to the sensitive developmental period of weaning. This may have implications in understanding the importance of microbiota during early development on opioid signalling and associated behaviours.

Colorectal cancer-associated microbiota contributes to oncogenic epigenetic signatures.

Sporadic colorectal cancer (CRC) is a result of complex interactions between the host and its environment. Environmental stressors act by causing host cell DNA alterations implicated in the onset of cancer. Here we investigate the stressor ability of CRC-associated gut dysbiosis as causal agent of host DNA alterations. The epigenetic nature of these alterations was investigated in humans and in mice. Germ-free mice receiving fecal samples from subjects with normal colonoscopy or from CRC patients were monitored for 7 or 14 wk. Aberrant crypt foci, luminal microbiota, and DNA alterations (colonic exome sequencing and methylation patterns) were monitored following human feces transfer. CRC-associated microbiota induced higher numbers of hypermethylated genes in murine colonic mucosa (vs. healthy controls' microbiota recipients). Several gene promoters including SFRP1,2,3, PENK, NPY, ALX4, SEPT9, and WIF1 promoters were found hypermethylated in CRC but not in normal tissues or effluents from fecal donors. In a pilot study (n = 266), the blood methylation levels of 3 genes (Wif1, PENK, and NPY) were shown closely associated with CRC dysbiosis. In a validation study (n = 1,000), the cumulative methylation index (CMI) of these genes was significantly higher in CRCs than in controls. Further, CMI appeared as an independent risk factor for CRC diagnosis as shown by multivariate analysis that included fecal immunochemical blood test. Consequently, fecal bacterial species in individuals with higher CMI in blood were identified by whole metagenomic analysis. Thus, CRC-related dysbiosis induces methylation of host genes, and corresponding CMIs together with associated bacteria are potential biomarkers for CRC.

Depressive symptoms, fruit and vegetables consumption and urinary 3-indoxylsulfate concentration: a nested case-control study in the French Nutrinet-Sante cohort.

PURPOSE: Previous epidemiologic studies have provided some evidence of an inverse association between fruit and vegetables consumption and risk of developing recurrent depressive symptoms. This association could possibly be explained by the role of such dietary factors on the gut microbiota. Especially, indole, a metabolite of tryptophan produced by gut bacteria, may be associated with the development of mood disorders. Thus, the purpose of this study was to investigate relationships between fruit and vegetables intake, recurrent depressive symptoms and indole, using measurement of its main urinary excretion form, i.e., 3-indoxylsulfate, as a biomarker. METHODS: A nested case-control study was conducted in 891 women (aged 45-65) participating to the web-based NutriNet-Santé cohort with available dietary data and biological samples. Cases (individuals with recurrent depressive symptoms, n = 297) were defined as having two Center for Epidemiologic Studies-Depression Scale (CES-D) scores ≥ 16 during the follow-up and were matched with 2 controls having two CES-D scores < 16. Urinary 3-indoxylsulfate concentration was measured as a biomarker of indole production by the gut microbiota. Multivariable conditional logistic regression models were used to test the association of both fruit and vegetables consumption and urine 3-indoxylsulfate measurements with recurrent depressive symptoms. We also tested the association between fruit and vegetables consumption and urinary 3-indoxylsulfate levels using multivariate analysis of variance models. RESULTS: We found a significant inverse association between fruit and vegetables consumption and the risk of having recurrent depressive symptoms over a 2-year period. Fruit and vegetables consumption was inversely associated to urinary 3-indoxylsulfate concentration. However, no significant association was observed between urinary 3-indoxylsulfate levels and recurrent depressive symptoms within this sample. CONCLUSIONS: Our results confirm that low fruit and vegetables consumption could be associated with recurrent depressive symptoms. We also found an inverse association between fruit and vegetable intake and urinary levels of 3-indoxylsulfate. However, it is not possible to conclude to a possible mediation role of the indole produced by the gut microbiota from tryptophan, since there was no relationship between 3-indoxylsulfate and recurrent depressive symptoms.

The Impact of Gut Microbiota-Derived Metabolites in Autism Spectrum Disorders.

Autism Spectrum Disorder (ASD) is a set of neurodevelopmental disorders characterised by behavioural impairment and deficiencies in social interaction and communication. A recent study estimated that 1 in 89 children have developed some form of ASD in European countries. Moreover, there is no specific treatment and since ASD is not a single clinical entity, the identification of molecular biomarkers for diagnosis remains challenging. Besides behavioural deficiencies, individuals with ASD often develop comorbid medical conditions including intestinal problems, which may reflect aberrations in the bidirectional communication between the brain and the gut. The impact of faecal microbial composition in brain development and behavioural functions has been repeatedly linked to ASD, as well as changes in the metabolic profile of individuals affected by ASD. Since metabolism is one of the major drivers of microbiome-host interactions, this review aims to report emerging literature showing shifts in gut microbiota metabolic function in ASD. Additionally, we discuss how these changes may be involved in and/or perpetuate ASD pathology. These valuable insights can help us to better comprehend ASD pathogenesis and may provide relevant biomarkers for improving diagnosis and identifying new therapeutic targets.

Commensal microbiota influence systemic autoimmune responses.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.

Gut microbiota richness promotes its stability upon increased dietary fibre intake in healthy adults.

Gut microbiota richness and stability are important parameters in host-microbe symbiosis. Diet modification, notably using dietary fibres, might be a way to restore a high richness and stability in the gut microbiota. In this work, during a 6-week nutritional trial, 19 healthy adults consumed a basal diet supplemented with 10 or 40 g dietary fibre per day for 5 days, followed by 15-day washout periods. Fecal samples were analysed by a combination of 16S rRNA gene pyrosequencing, intestinal cell genotoxicity assay, metatranscriptomics sequencing approach and short-chain fatty analysis. This short-term change in the dietary fibre level did not have the same impact for all individuals but remained significant within each individual gut microbiota at genus level. Higher microbiota richness was associated with higher microbiota stability upon increased dietary fibre intake. Increasing fibre modulated the expression of numerous microbiota metabolic pathways such as glycan metabolism, with genes encoding carbohydrate-active enzymes active on fibre or host glycans. High microbial richness was also associated with high proportions of Prevotella and Coprococcus species and high levels of caproate and valerate. This study provides new insights on the role of gut microbial richness in healthy adults upon dietary changes and host microbes' interaction.

Intestinal CD103+ dendritic cells are key players in the innate immune control of Cryptosporidium parvum infection in neonatal mice.

Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.

Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is prevalent among obese people and is considered the hepatic manifestation of metabolic syndrome. However, not all obese individuals develop NAFLD. Our objective was to demonstrate the role of the gut microbiota in NAFLD development using transplantation experiments in mice. DESIGN: Two donor C57BL/6J mice were selected on the basis of their responses to a high-fat diet (HFD). Although both mice displayed similar body weight gain, one mouse, called the 'responder', developed hyperglycaemia and had a high plasma concentration of pro-inflammatory cytokines. The other, called a 'non-responder', was normoglycaemic and had a lower level of systemic inflammation. Germ-free mice were colonised with intestinal microbiota from either the responder or the non-responder and then fed the same HFD. RESULTS: Mice that received microbiota from different donors developed comparable obesity on the HFD. The responder-receiver (RR) group developed fasting hyperglycaemia and insulinaemia, whereas the non-responder-receiver (NRR) group remained normoglycaemic. In contrast to NRR mice, RR mice developed hepatic macrovesicular steatosis, which was confirmed by a higher liver concentration of triglycerides and increased expression of genes involved in de-novo lipogenesis. Pyrosequencing of the 16S ribosomal RNA genes revealed that RR and NRR mice had distinct gut microbiota including differences at the phylum, genera and species levels. CONCLUSIONS: Differences in microbiota composition can determine response to a HFD in mice. These results further demonstrate that the gut microbiota contributes to the development of NAFLD independently of obesity.

tHIS way to cognitive development.

The effect of the microbiota-gut-brain axis on cognitive development in infancy is increasingly being scrutinized. In this issue of Cell Host & Microbe, Cerdó, Ruiz, and colleagues skillfully combine clinical and preclinical analyses, including a fecal transplantation experiment, to reveal associations between microbiota composition, cognitive scores, and histidine metabolism.

Effects of a Lacticaseibacillus Mix on Behavioural, Biochemical, and Gut Microbial Outcomes of Male Mice following Chronic Restraint Stress.

The effect of supplementation with Lactobacillus strains to prevent the consequences of chronic stress on anxiety in mouse strains sensitive to stress and the consequences on gut microbiota have been relatively unexplored. Thus, we administered a Lacticaseibacillus casei LA205 and Lacticaseibacillus paracasei LA903 mix to male BALB/cByJrj mice two weeks before and during 21-day chronic restraint stress (CRS) (non-stressed/solvent (NS-PBS), non-stressed/probiotics (NS-Probio), CRS/solvent (S-PBS), CRS/probiotics (S-Probio)). CRS resulted in lower body weight and coat state alteration, which were attenuated by the probiotic mix. S-Probio mice showed less stress-associated anxiety-like behaviours than their NS counterpart, while no difference was seen in PBS mice. Serum corticosterone levels were significantly higher in the S-Probio group than in other groups. In the hippocampus, mRNA expression of dopamine and serotonin transporters was lower in S-Probio than in S-PBS mice. Few differences in bacterial genera proportions were detected, with a lower relative abundance of Alistipes in S-Probio vs. S-PBS. CRS was accompanied by a decrease in the proportion of caecal acetate in S-PBS mice vs. NS-PBS, but not in the intervention groups. These data show that the probiotic mix could contribute to better coping with chronic stress, although the precise bacterial mechanism is still under investigation.

Neonatal necrotizing enterocolitis: Clostridium butyricum and Clostridium neonatale fermentation metabolism and enteropathogenicity.

Bacterial colonization in the gut plays a pivotal role in neonatal necrotizing enterocolitis (NEC) development, but the relationship between bacteria and NEC remains unclear. In this study, we aimed to elucidate whether bacterial butyrate end-fermentation metabolites participate in the development of NEC lesions and confirm the enteropathogenicity of Clostridium butyricum and Clostridium neonatale in NEC. First, we produced C.butyricum and C.neonatale strains impaired in butyrate production by genetically inactivating the hbd gene encoding ß-hydroxybutyryl-CoA dehydrogenase that produces end-fermentation metabolites. Second, we evaluated the enteropathogenicty of the hbd-knockout strains in a gnotobiotic quail model of NEC. The analyses showed that animals harboring these strains had significantly fewer and less intense intestinal lesions than those harboring the respective wild-type strains. In the absence of specific biological markers of NEC, the data provide original and new mechanistic insights into the disease pathophysiology, a necessary step for developing potential novel therapies.

Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus in Physiological Conditions Mimicking the Human Digestive Ecosystem.

Metagenome analyses of the human microbiome suggest that horizontal gene transfer (HGT) is frequent in these rich and complex microbial communities. However, so far, only a few HGT studies have been conducted in vivo. In this work, three different systems mimicking the physiological conditions encountered in the human digestive tract were tested, including (i) the TNO gastro-Intestinal tract Model 1 (TIM-1) system (for the upper part of the intestine), (ii) the ARtificial COLon (ARCOL) system (to mimic the colon), and (iii) a mouse model. To increase the likelihood of transfer by conjugation of the integrative and conjugative element studied in the artificial digestive systems, bacteria were entrapped in alginate, agar, and chitosan beads before being placed in the different gut compartments. The number of transconjugants detected decreased, while the complexity of the ecosystem increased (many clones in TIM-1 but only one clone in ARCOL). No clone was obtained in a natural digestive environment (germfree mouse model). In the human gut, the richness and diversity of the bacterial community would offer more opportunities for HGT events to occur. In addition, several factors (SOS-inducing agents, microbiota-derived factors) that potentially increase in vivo HGT efficiency were not tested here. Even if HGT events are rare, expansion of the transconjugant clones can happen if ecological success is fostered by selecting conditions or by events that destabilize the microbial community. IMPORTANCE The human gut microbiota plays a key role in maintaining normal host physiology and health, but its homeostasis is fragile. During their transit in the gastrointestinal tract, bacteria conveyed by food can exchange genes with resident bacteria. New traits acquired by HGT (e.g., new catabolic properties, bacteriocins, antibiotic resistance) can impact the gut microbial composition and metabolic potential. We showed here that TIM-1, a system mimicking the upper digestive tract, is a useful tool to evaluate HGT events in conditions closer to the physiological ones. Another important fact pointed out in this work is that Enterococcus faecalis is a good candidate for foreign gene acquisition. Due to its high ability to colonize the gut and acquire mobile genetic elements, this commensal bacterium could serve as an intermediate for HGT in the human gut.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

Orally administered Odoribacter laneus improves glucose control and inflammatory profile in obese mice by depleting circulating succinate.

BACKGROUND: Succinate is produced by both human cells and by gut bacteria and couples metabolism to inflammation as an extracellular signaling transducer. Circulating succinate is elevated in patients with obesity and type 2 diabetes and is linked to numerous complications, yet no studies have specifically addressed the contribution of gut microbiota to systemic succinate or explored the consequences of reducing intestinal succinate levels in this setting. RESULTS: Using germ-free and microbiota-depleted mouse models, we show that the gut microbiota is a significant source of circulating succinate, which is elevated in obesity. We also show in vivo that therapeutic treatments with selected bacteria diminish the levels of circulating succinate in obese mice. Specifically, we demonstrate that Odoribacter laneus is a promising probiotic based on its ability to deplete succinate and improve glucose tolerance and the inflammatory profile in two independent models of obesity (db/db mice and diet-induced obese mice). Mechanistically, this is partly mediated by the succinate receptor 1. Supporting these preclinical findings, we demonstrate an inverse correlation between plasma and fecal levels of succinate in a cohort of patients with severe obesity. We also show that plasma succinate, which is associated with several components of metabolic syndrome including waist circumference, triglycerides, and uric acid, among others, is a primary determinant of insulin sensitivity evaluated by the euglycemic-hyperinsulinemic clamp. CONCLUSIONS: Overall, our work uncovers O. laneus as a promising next-generation probiotic to deplete succinate and improve glucose tolerance and obesity-related inflammation. Video Abstract.

Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats.

The endogenous gut microbiota affects the host in many ways. Prebiotics should favour beneficial intestinal microbes and thus improve host health. In this study, we investigated how a novel class of potential prebiotic long-chain arabinoxylans (LC-AX) and the well-established prebiotic inulin (IN) modulate the gut microbiota of humanized rats. Six weeks after axenic rats were inoculated with a human faecal microbiota, their colonic microbiota was similar to this inoculum (∼ 70%), whereas their caecal microbiota was enriched with Verrucomicrobia and Firmicutes concomitant with lower abundance of Bacteroidetes. Moreover, different Bifidobacterium species colonized the lumen (B. adolescentis) and mucus (B. longum and B. bifidum). Both LC-AX and IN increased SCFA levels and induced a shift from acetate towards health-promoting propionate and butyrate respectively. By applying a high-resolution phylogenetic micro-array (HITChip) at the site of fermentation (caecum), IN and LC-AX were shown to stimulate bacterial groups with known butyrate-producers (Roseburia intestinalis, Eubacterium rectale, Anaerostipes caccae) and bifidobacteria (B. longum) respectively. Prebiotic administration also resulted in lower caecal abundances of the mucin-degrading Akkermansia muciniphila and potentially more mucin production by the host. Both factors might explain the increased caecal mucin levels for LC-AX (threefold) and IN (sixfold). These mucins were degraded along the colon, resulting in high faecal abundances of Akkermansia muciniphila for LC-AX and especially IN-treated rats. Finally, the microbial changes caused an adaptation period for the host with less weight gain, after which the host fine-tuned the interaction with this altered microbiota. Our results demonstrate that next to IN, LC-AX are promising prebiotic compounds by stimulating production of health-promoting metabolites by specific microbes in the proximal regions. Further, prebiotic supplementation shifted mucin degradation to distal regions, where mucin-degraders may produce beneficial metabolites (e.g. propionate by Akkermansia muciniphila), so that prebiotics may potentially improve gut health along the entire length of the intestine.

Germ-free C57BL/6J mice are resistant to high-fat-diet-induced insulin resistance and have altered cholesterol metabolism.

Recent studies showed that germ-free (GF) mice are resistant to obesity when consuming a high-fat, high-carbohydrate Western diet. However, it remains unclear what mechanisms are involved in the antiobesity phenotype and whether GF mice develop insulin resistance and dyslipidemia with high-fat (HF) feeding. In the present study, we compared the metabolic consequences of HF feeding on GF and conventional (conv) C57BL/6J mice. GF mice consumed fewer calories, excreted more fecal lipids, and weighed significantly less than conv mice. GF/HF animals also showed enhanced insulin sensitivity with improved glucose tolerance, reduced fasting and nonfasting insulinemia, and increased phospho-Akt((Ser-473)) in adipose tissue. In association with enhanced insulin sensitivity, GF/HF mice had reduced plasma TNF-α and total serum amyloid A concentrations. Reduced hypercholesterolemia, a moderate accretion of hepatic cholesterol, and an increase in fecal cholesterol excretion suggest an altered cholesterol metabolism in GF/HF mice. Pronounced nucleus SREBP2 proteins and up-regulation of cholesterol biosynthesis genes indicate that enhanced cholesterol biosynthesis contributed to the cholesterol homeostasis in GF/HF mice. Our results demonstrate that fewer calorie consumption and increased lipid excretion contributed to the obesity-resistant phenotype of GF/HF mice and reveal that insulin sensitivity and cholesterol metabolism are metabolic targets influenced by the gut microbiota.