Aurélia Bruneau

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.

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.

Absence of the gut microbiota enhances anxiety-like behavior and neuroendocrine response to acute stress in rats

BACKGROUND AND AIMS Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety. METHODS Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not. RESULTS GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats. CONCLUSIONS In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior.

Microbial Community Development in a Dynamic Gut Model Is Reproducible, Colon Region Specific, and Selective for Bacteroidetes and Clostridium Cluster IX

ABSTRACT Dynamic, multicompartment in vitro gastrointestinal simulators are often used to monitor gut microbial dynamics and activity. These reactors need to harbor a microbial community that is stable upon inoculation, colon region specific, and relevant to in vivo conditions. Together with the reproducibility of the colonization process, these criteria are often overlooked when the modulatory properties from different treatments are compared. We therefore investigated the microbial colonization process in two identical simulators of the human intestinal microbial ecosystem (SHIME), simultaneously inoculated with the same human fecal microbiota with a high-resolution phylogenetic microarray: the human intestinal tract chip (HITChip). Following inoculation of the in vitro colon compartments, microbial community composition reached steady state after 2 weeks, whereas 3 weeks were required to reach functional stability. This dynamic colonization process was reproducible in both SHIME units and resulted in highly diverse microbial communities which were colon region specific, with the proximal regions harboring saccharolytic microbes (e.g., Bacteroides spp. and Eubacterium spp.) and the distal regions harboring mucin-degrading microbes (e.g., Akkermansia spp.). Importantly, the shift from an in vivo to an in vitro environment resulted in an increased Bacteroidetes/Firmicutes ratio, whereas Clostridium cluster IX (propionate producers) was enriched compared to clusters IV and XIVa (butyrate producers). This was supported by proportionally higher in vitro propionate concentrations. In conclusion, high-resolution analysis of in vitro-cultured gut microbiota offers new insight on the microbial colonization process and indicates the importance of digestive parameters that may be crucial in the development of new in vitro models.

Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease

Objective There is substantial inter-individual diversity in the susceptibility of alcoholics to liver injury. Alterations of intestinal microbiota (IM) have been reported in alcoholic liver disease (ALD), but the extent to which they are merely a consequence or a cause is unknown. We aimed to demonstrate that a specific dysbiosis contributes to the development of alcoholic hepatitis (AH). Design We humanised germ-free and conventional mice using human IM transplant from alcoholic patients with or without AH. The consequences on alcohol-fed recipient mice were studied. Results A specific dysbiosis was associated with ALD severity in patients. Mice harbouring the IM from a patient with severe AH (sAH) developed more severe liver inflammation with an increased number of liver T lymphocyte subsets and Natural Killer T (NKT) lymphocytes, higher liver necrosis, greater intestinal permeability and higher translocation of bacteria than mice harbouring the IM from an alcoholic patient without AH (noAH). Similarly, CD45+ lymphocyte subsets were increased in visceral adipose tissue, and CD4+T and NKT lymphocytes in mesenteric lymph nodes. The IM associated with sAH and noAH could be distinguished by differences in bacterial abundance and composition. Key deleterious species were associated with sAH while the Faecalibacterium genus was associated with noAH. Ursodeoxycholic acid was more abundant in faeces from noAH mice. Additionally, in conventional mice humanised with the IM from an sAH patient, a second subsequent transfer of IM from an noAH patient improved alcohol-induced liver lesions. Conclusions Individual susceptibility to ALD is substantially driven by IM. It may, therefore, be possible to prevent and manage ALD by IM manipulation.

Drastic changes in fecal and mucosa-associated microbiota in adult patients with short bowel syndrome

Short bowel syndrome (SBS) is observed in Humans after a large resection of gut. Since the remnant colon and its associated microbiota play a major role in the outcome of patients with SBS, we studied the overall qualitative and quantitative microbiota composition of SBS adult patients compared to controls. The population was composed of 11 SBS type II patients (with a jejuno-colonic anastomosis) and 8 controls without intestinal pathology. SBS patients had 38 +/- 30 cm remnant small bowel length and 66 +/- 19% of residual colon. The repartition of proteins, lipids, carbohydrates and fibres was expressed as % of total oral intake in patients and controls. The microbiota was profiled from stool and biopsy samples with temporal temperature gradient gel electrophoresis and quantitative PCR. We show here that microbiota of SBS patients is unbalanced with a high prevalence of Lactobacillus along with a sub-dominant presence and poor diversity of Clostridium leptum, Clostridium coccoides and Bacteroidetes. In addition, Lactobacillus mucosae was detected within the fecal and mucosa-associated microbiota of SBS patients, whereas it remained undetectable in controls. Thus, in SBS the microbial composition was deeply altered in fecal and mucosal samples, with a shift between dominant and sub-dominant microbial groups and the prevalence of L. mucosae.

Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats.

In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 x 10(10) colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species- and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.

Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2-dimethylhydrazine

Propionibacterium freudenreichii, a food-grade bacterium able to kill colon cancer cell lines in vitro by apoptosis, may exert an anticarcinogenic effect in vivo. To assess this hypothesis, we administered daily 2 x 10(10) colony-forming units (CFU) of P. freudenreichii TL133 to human microbiota-associated (HMA) rats for 18 d. Either saline or 1,2-dimethylhydrazine (DMH) was also administered on days 13 and 17 and rats were killed on day 19. The levels of apoptosis and proliferation in the mid and distal colon were assessed by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunolabelling, respectively. The administration of P. freudenreichii TL133 significantly increased the number of apoptotic cells in DMH-treated rats compared to those given DMH only (P < 0.01). Furthermore, propionibacteria were able to decrease the proliferation index in the distal colon after treatment with DMH (P < 0.01). Conversely, propionibacteria alone did not exert such an effect on healthy colonic mucosa. P. freudenreichii TL133 thus facilitated the elimination of damaged cells by apoptosis in the rat colon after genotoxic insult and may play a protective role against colon cancer.

Short-chain fructo-oligosaccharides modulate intestinal microbiota and metabolic parameters of humanized gnotobiotic diet induced obesity mice.

Prebiotic fibres like short-chain fructo-oligosaccharides (scFOS) are known to selectively modulate the composition of the intestinal microbiota and especially to stimulate Bifidobacteria. In parallel, the involvement of intestinal microbiota in host metabolic regulation has been recently highlighted. The objective of the study was to evaluate the effect of scFOS on the composition of the faecal microbiota and on metabolic parameters in an animal model of diet-induced obesity harbouring a human-type microbiota. Forty eight axenic C57BL/6J mice were inoculated with a sample of faecal human microbiota and randomly assigned to one of 3 diets for 7 weeks: a control diet, a high fat diet (HF, 60% of energy derived from fat)) or an isocaloric HF diet containing 10% of scFOS (HF-scFOS). Mice fed with the two HF gained at least 21% more weight than mice from the control group. Addition of scFOS partially abolished the deposition of fat mass but significantly increased the weight of the caecum. The analysis of the taxonomic composition of the faecal microbiota by FISH technique revealed that the addition of scFOS induced a significant increase of faecal Bifidobacteria and the Clostridium coccoides group whereas it decreased the Clostridium leptum group. In addition to modifying the composition of the faecal microbiota, scFOS most prominently affected the faecal metabolome (e.g. bile acids derivatives, hydroxyl monoenoic fatty acids) as well as urine, plasma hydrophilic and plasma lipid metabolomes. The increase in C. coccoides and the decrease in C. leptum, were highly correlated to these metabolic changes, including insulinaemia, as well as to the weight of the caecum (empty and full) but not the increase in Bifidobacteria. In conclusion scFOS induce profound metabolic changes by modulating the composition and the activity of the intestinal microbiota, that may partly explain their effect on the reduction of insulinaemia.

Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat

Previous studies have suggested that intestinal microbiota modulates colonic epithelium renewal. The objective of our work was to study the effects of microbiota on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic crypts and amount of cell cycle-related proteins were compared between germ-free (GF), conventional (CV), and conventionalized rats by histochemistry and Western blot. Ki67 and proliferating cell nuclear antigen (PCNA) were used as surrogates for proliferative cells; p21(cip1) and p27(kip1) were markers of cell cycle arrest; anti- and proapoptotic proteins, Bcl2 and Bax, respectively, were also studied. We observed 40% increase of the crypt proliferative area 2 days after inoculation of GF rats with a complex microbiota. This recruitment of proliferative cells may account for the 30% increase of crypt depth observed between CV and GF rats. The hyperproliferative boost induced by microbiota was compensated by a fourfold increase of p21(cip1) and p27(kip1) involved in cell cycle arrest and a 30% drop of antiapoptotic Bcl2 protein while Bax was unchanged. Inductions of p21(cip1), p27(kip1), and PCNA protein were not paralleled by an increase of the corresponding mRNA. We also showed that p21(cip1) induction by microbiota was partially restored by Bacteroides thetaiotaomicron, Ruminococcus gnavus, and Clostridium paraputrificum. Colonization of the colon by a complex microbiota increases the crypt depth of colon epithelium. This event takes place in conjunction with a multistep process: a hyperproliferative boost accompanied by compensatory events as induction of p21(cip1) and p27(kip1) and decrease of Bcl2.