Characterization of the serum and liver proteomes in gut-microbiota-lacking mice.
ABSTRACT: Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help study gut bacteria and disease prevention.
Project description:An increasing number of studies have recently indicated the important effects of gut microbes on various functions of the central nervous system. However, the underlying mechanisms by which gut microbiota regulate brain functions and behavioral phenotypes remain largely unknown. We therefore used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to obtain proteomic profiles of the hippocampus in germ-free (GF), colonized GF, and specific pathogen-free (SPF) mice. We then integrated the resulting proteomic data with previously reported mRNA microarray data, to further explore the effects of gut microbes on host brain functions. We identified that 61 proteins were upregulated and 242 proteins were downregulated in GF mice compared with SPF mice. Of these, 124 proteins were significantly restored following gut microbiota colonization. Bioinformatic analysis of these significant proteins indicated that the glucocorticoid receptor signaling pathway and inflammation-related pathways were the most enriched disrupted pathways. This study provides new insights into the pathological mechanisms of gut microbiota-regulated diseases.
Project description:There is an interaction and bidirectional selection between dietary intake and gut microbiota due to the different efficiency of nutrients in the gut. The nutritional composition of germ-free (GF) diets differs significantly from specific pathogen-free (SPF) diets. There is, however, no data revealing how SPF animals from the same microbial background respond to them and if they affect the host. We examined the growth of SPF mice on the GF diet and found that it reduced body weight, intestinal length and intestinal morphology. Interestingly, the GF diet increased the level of pro-inflammatory bacteria in the gut of SPF mice, including Proteobacteria, <i>Burkholderiaceae, Alloprevotella</i> and <i>Parasutterella</i>. Furthermore, GF diets caused significant increases in malondialdehyde (MDA), IL-1β, IL-6, and D-lactate levels in the serum of SPF mice and significantly altered their serum metabolic profile, especially amino acid metabolism. In conclusion, GF diets are not suitable for the growth and development of SPF mice. These findings, based on the role of gut microbiota in diet selection, provide new insights into the scientific and rational use of experimental animal diets.
Project description:During the past decade, there has been a substantial rise in the knowledge about the effects of gut microbiota on host physiology and behavior, including depressive behavior. Initial studies determined that gut microbiota can regulate host tryptophan levels, which is a main serotonin precursor. A dysfunctional serotonergic system is considered to be one of the main factors contributing to the development of depression. Therefore, we hypothesized that regulation of brain tryptophan and serotonin can explain, at least partly, the effects of microbiota on depressive behavior. To test this hypothesis, we examined depressive-like behavior and brain levels of serotonin and tryptophan, of germ free (GF) and specific-pathogen free (SPF) mice under basal conditions, or after acute tryptophan depletion (ATD) procedure, which is a method to decrease tryptophan and serotonin levels in the brain. In basal conditions, GF mice exhibited less depressive-like behavior in sucrose preference, tail-suspension and forced swim tests, compared to SPF mice. In addition, in mice that were not subjected to ATD, GF mice displayed higher levels of tryptophan, serotonin and 5-hydroxyindoleacetic acid (the main degradation product of serotonin) in medial prefrontal cortex (mPFC) and hippocampus (HIPPO), compared to SPF mice. Interestingly, ATD increased depressive-like behavior of GF, but not of SPF mice. These behavioral changes were accompanied by a stronger reduction of tryptophan, serotonin and 5-hydroxyindoleacetic acid in mPFC and HIPPO in GF mice after ATD, when compared to SPF mice. Therefore, the serotonergic system of GF mice is more vulnerable to the acute challenge of tryptophan reduction, and GF mice after tryptophan reduction behave more similarly to SPF mice. These data provide functional evidence that microbiota affects depression-like behavior through influencing brain tryptophan accessibility and the serotonergic system.
Project description:Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.
Project description:<h4>Objective</h4>Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice.<h4>Methods</h4>GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing.<h4>Results</h4>GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17?-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order <i>Clostridiales</i> (phylum <i>Firmicutes</i>) as a characteristic feature of normal SPF mice fed lard.<h4>Conclusions</h4>In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism.
Project description:BACKGROUND & AIMS:It is not clear how the complex interactions between diet and the intestinal microbiota affect development of mucosal inflammation or inflammatory bowel disease. We investigated interactions between dietary ingredients, nutrients, and the microbiota in specific pathogen-free (SPF) and germ-free (GF) mice given more than 40 unique diets; we quantified individual and synergistic effects of dietary macronutrients and the microbiota on intestinal health and development of colitis. METHODS:C56BL/6J SPF and GF mice were placed on custom diets containing different concentrations and sources of protein, fat, digestible carbohydrates, and indigestible carbohydrates (fiber). After 1 week, SPF and GF mice were given dextran sulfate sodium (DSS) to induce colitis. Disease severity was determined based on the percent weight change from baseline, and modeled as a function of the concentration of each macronutrient in the diet. In unchallenged mice, we measured intestinal permeability by feeding mice labeled dextran and measuring levels in blood. Feces were collected and microbiota were analyzed by 16S rDNA sequencing. We collected colons from mice and performed transcriptome analyses. RESULTS:Fecal microbiota varied with diet; the concentration of protein and fiber had the strongest effect on colitis development. Among 9 fiber sources tested, psyllium, pectin, and cellulose fiber reduced the severity of colitis in SPF mice, whereas methylcellulose increased severity. Increasing dietary protein increased the density of the fecal microbiota and the severity of colitis in SPF mice, but not in GF mice or mice given antibiotics. Psyllium fiber reduced the severity of colitis through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary casein protein and psyllium fiber in parallel accounted for most variation in gut microbial density and intestinal permeability in unchallenged mice, as well as the severity of DSS-induced colitis; changes in 1 ingredient could be offset by changes in another. CONCLUSIONS:In an analysis of the effects of different dietary components and the gut microbiota on mice with and without DSS-induced colitis, we found complex mixtures of nutrients affect intestinal permeability, gut microbial density, and development of intestinal inflammation.
Project description:The intestinal microbiota and its functions are intricately interwoven with host physiology. Colonizing rodents with donor microbiota provides insights into host-microbiota interactions characterization and the understanding of disease physiopathology. However, a better assessment of inoculation methods and recipient mouse models is needed. Here, we compare the engraftment at short and long term of genetically obese mice microbiota in germ-free (GF) mice and juvenile and adult specific pathogen free (SPF) mice. We also tested the effects of initial microbiota depletion before microbiota transfer. In the present work, donor microbiota engraftment was better in juvenile SPF mice than in adult SPF mice. In juvenile mice, initial microbiota depletion using laxatives or antibiotics improved donor microbiota engraftment 9 weeks but not 3 weeks after microbiota transfer. Microbiota-depleted juvenile mice performed better than GF mice 3 weeks after the microbiota transfer. However, 9 weeks after transfer, colonized GF mice microbiota had the lowest Unifrac distance to the donor microbiota. Colonized GF mice were also characterized by a chronic alteration in intestinal absorptive function. With these collective results, we show that the use of juvenile mice subjected to initial microbiota depletion constitutes a valid alternative to GF mice in microbiota transfer studies.
Project description:Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior.
Project description:The dysbiosis of gut microbiota is an important environmental factor that can induce mental disorders, such as depression, through the microbiota-gut-brain axis. However, the underlying pathogenic mechanisms are complex and not completely understood. Here we utilized mass spectrometry to identify the global phosphorylation dynamics in hippocampus tissue in germ-free mice and specific pathogen-free mice (GF vs SPF), fecal microbiota transplantation (FMT) model ("depression microbiota" and the "healthy microbiota" recipient mice). As a result, 327 phosphosites of 237 proteins in GF vs SPF, and 478 phosphosites of 334 proteins in "depression microbiota" vs "healthy microbiota" recipient mice were identified as significant. These phosphorylation dysregulations were consistently associated with glutamatergic neurotransmitter system disturbances. The FMT mice exhibited disturbances in lipid metabolism and amino acid metabolism in both the periphery and brain through integrating phosphoproteomic and metabolomic analysis. Moreover, CAMKII-CREB signaling pathway, in response to these disturbances, was the primary common perturbed cellular process. In addition, we demonstrated that the spliceosome, never directly implicated in mental disorders previously, was a substantially neuronal function disrupted by gut microbiota dysbiosis, and the NCBP1 phosphorylation was identified as a novel pathogenic target. These results present a new perspective to study the pathologic mechanisms of gut microbiota dysbiosis related depression and highlight potential gut-mediated therapies for depression.
Project description:Helicobacter pylori, is an invariably commensal resident of the gut microbiome associated with gastric ulcer in adults. In addition, these patients also suffered from a low grade inflammation that activates the immune system and thus increased shunting of energy to host defense mechanisms. To assess whether a H. pylori infection could affect growth in early life, we determined the expression levels of selected metabolic gut hormones in germ free (GF) and specific pathogen-free (SPF) mice with and without the presence of H. pylori. Despite H. pylori-infected (SPFH) mice display alteration in host metabolism (elevated levels of leptin, insulin and peptide YY) compared to non-infected SPF mice, their growth curves remained the same. SPFH mice also displayed increased level of eotaxin-1. Interestingly, GF mice infected with H. pylori (GFH) also displayed increased levels of ghrelin and PYY. However, in contrast to SPFH mice, GFH showed reduced weight gain and malnutrition. These preliminary findings show that exposure to H. pylori alters host metabolism early in life; but the commensal microbiota in SPF mice can attenuate the growth retarding effect from H. pylori observed in GF mice. Further investigations of possible additional side effects of H. pylori are highly warranted.