Genetic Control of Obesity and Gut Microbiota Composition in Response to High-Fat,High-Sucrose Diet in Mice

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Highlights? Detailed analysis of diet-induced obesity in more than 100 inbred mouse strains ? Identification of 11 genetic loci associated with obesity and dietary responsiveness ? Significant overlap between mouse loci with human GWAS loci for obesity ? Strain-specific shifts in gut microbiota composition in response to dietary intervention     

The Gut Microbiota and Developmental Programming of the Testis in Mice

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.     

The Gut Microbiota Modulates Glycaemic Control and Serum Metabolite Profiles in Non-Obese Diabetic Mice

Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.     

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

     

Gut Microbiota of the Tick Vector Ixodes scapularis Modulate Colonization of the Lyme Disease Spirochete

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Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota

Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota.     

Colonic Lesions,Cytokine Profiles,and Gut Microbiota in Plasminogen-Deficient Mice

Plasminogen-deficient (FVB/NPan-plg^tm1Jld, plg^tm1Jld) mice, which are widely used as a wound-healing model, are prone to spontaneous rectal prolapses. The aims of this study were 1) to evaluate the fecal microbiome of plg^tm1Jld mice for features that might contribute to the development of rectal prolapses and colonic inflammation and 2) to assess the relevance of the inflammatory phenotype to the variability in wound healing in this model. The plg^tm1Jld mice exhibited delayed wound healing, and they could be divided into 3 distinct groups that differed according to the time until wound closure. Colonic lesions in plg^tm1Jld mice, which were characterized by necrotizing ulcerations and cystically dilated glands, were restricted to the intermediate and distal parts of the colon. The cytokine profile was indicative of chronic tissue damage, but the genetic modification did not change the composition of the gut microbiota, and none of the clinical or biochemical parameters correlated with the gut microbiota composition.Several studies using plasminogen-deficient (plg^tmJld) mice have demonstrated that plasminogen, the proenzyme of plasmin, can degrade fibrin and other extracellular matrix proteins.⁴⁴ Plasminogen is essential for wound healing in skin,⁴⁰ which begins with inflammation, followed by epithelial proliferation, and thereafter tissue remodeling. Because the migrating keratinocytes of plg^tm1Jld mice have a decreased ability to dissect the platelet-rich fibrin matrix, they exhibit severely impaired wound healing.^15,40 In addition, plasmin mediates various pathologic processes, such as tumor growth and cancer metastasis,⁸ and therapeutic intervention related to plasminogen has shown encouraging results in experimental tumors.³¹ Therefore, one important application of these mice is the induction of wound healing to study basic mechanistic functions of plasmin, such as the clearance of the extracellular matrix and activation of tumor growth factors.³¹Spontaneous rectal prolapse and colonic ulceration in plg^tm1Jld mice compromise studies using these mice by leading to loss of body weight (wasting disease)⁶ and wellbeing-related, early study termination.⁶ Like other inflammatory conditions, rectal prolapse and chronic colonic inflammation might affect wound healing and contribute to the wide interindividual variation in the wound-healing processes of plg^tm1Jld mice.^28,40The development of rectal prolapses and colonic ulcerations in plg^tm1Jld mice reportedly is due to vascular occlusion.⁶ This pathologic condition is alleviated by superimposing fibrinogen deficiency on plasminogen deficiency, suggesting that fibrin is the primary substrate for plasmin.^7,15 The wide variation in effective tissue remodeling during the wound healing of plasminogen-deficient mice remains unexplained.Wound healing depends to a large extent on cells and factors of the immune system.^3,53 We previously have shown that disease development in mouse models for various inflammatory conditions, including type 1 diabetes,^17-19,35 type 2 diabetes,^4,13,42 atopic dermatitis³⁰ and inflammatory bowel disease,²⁰ is influenced by the composition of gut microbiota. Therefore, gut inflammation can be presumed to interfere with wound healing and thus may increase the uncontrolled interindividual variation in these models. In addition, gut inflammatory conditions in humans, such as inflammatory bowel disease⁴³ and irritable bowel syndrome,²³ are linked to dysbiosis in the intestine. In mice deficient in IL10 or IL2 and in rats carrying HLA-B27,⁵² inflammatory bowel disease can be alleviated by germ-free status^10,49,52 or ampicillin.²⁰ However, the possible role of the gut microbiome in rectal prolapse, colonic lesions, and wound healing in plasminogen-deficient mice has not previously been assessed.The aims of the current study were 1) to evaluate the fecal microbiome of plg^tm1Jld mice and their unaffected WT littermates for features that might contribute to their rectal prolapse and colonic inflammation phenotypes and 2) to assess the relevance of the inflammatory phenotype to the variability in wound healing in this model.     

Site and Strain-Specific Variation in Gut Microbiota Profiles and Metabolism in Experimental Mice

Background

The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.

Methodology/Principal Findings

The GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial –specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.

Conclusion/Significance

Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions.     

Effect of Housing Condition and Diet on the Gut Microbiota of Weanling Immunocompromised Mice

Gastrointestinal microbiota are affected by a wide variety of extrinsic and intrinsic factors. In the husbandry of laboratory mice and design of experiments, controlling these factors where possible provides more reproducible results. However, the microbiome is dynamic, particularly in the weeks immediately after weaning. In this study, we characterized the baseline gastrointestinal microbiota of immunocompromised mice housed under standard conditions for our facility for 6 weeks after weaning, with housing either in an isolator or in individually ventilated cages and a common antibiotic diet (trimethoprim sulfamethoxazole). We compared these conditions to a group fed a standard diet and a group that was weaned to a standard diet then switched to antibiotic diet after 2 weeks. We found no clear effect of diet on richness and α diversity of the gastrointestinal microbiota. However, diet did affect which taxa were enriched at the end of the experiment. The change to antibiotic diet during the experiment did not convert the gastrointestinal microbiome to a state similar to mice consistently fed antibiotic diet, which may highlight the importance of the initial post-weaning period in the establishment of the gastrointestinal microbiome. We also observed a strong effect of housing type (isolator compared with individually ventilated cage) on the richness, α diversity, β diversity, and taxa enriched over the course of the experiment. Investigating whether the diet or microbiome affects a certain strain’s phenotype is warranted in some cases. However, our findings do not suggest that maintaining immunocompromised mice on antibiotic feed has a clinical benefit when potential pathogens are operationally excluded, nor does it result in a more consistent or controlled microbiome in the post-weaning period.

Gastrointestinal microbiota (GM) have significant effects on animal health and are affected by many factors including diet, antibiotic administration, age, sex, strain, and cage environment.^{4,5,7,8,11,16} When using mice to study human disease, particularly when studying immune response to disease, recognizing which portions of a study are or are not controlled is an important element.⁹ In the case of the GM, characterizing the populations of bacteria under defined housing conditions for commonly used strains is an important step in improving reproducibility and translatability of these models.⁹Feeding diets compounded with antibiotics is common practice in irradiated animals to prevent opportunistic infections in the postprocedural period, and that practice has been extended to other populations that are known to be immunocompromised, such as SCID or nude mice.^3,19 In our facility, standard practice is to maintain populations of known and presumed immunodeficient mice as well as immunologically-humanized mice, on trimethoprim-sulfamethoxazole (TMS) antibiotic chow after weaning. However, mice that consume TMS from ad libitum commercial diets or water do not achieve therapeutic plasma concentrations of antibiotic against most pathogens.^14,15 Non-judicious use of antibiotics also contributes to the growing problem of antimicrobial resistance.¹⁹ Because immunodeficient mice are used to study human disease, immunity, and autoimmunity, the use of an antibiotic that may affect immunity and be clinically irrelevant for preventing disease creates a significant confounding variable.^1,9,10,20,21 In addition, medicated chow is more expensive than standard rodent chow, so this policy represents a significant operational cost.This study had 2 primary goals. The first was to characterize the microbiome of our most common immunodeficient mouse model after weaning under 2 standard housing (individually ventilated cage (IVC) or open-top cage within a semirigid isolator (isolator)) and 3 diet (TMS diet, standard rodent chow, or a switch from standard to TMS diet) conditions. This information may be important for future model characterization and may assist in rescue of model phenotypes that depend on an inhouse microbiome that may drift over time. We hypothesized that housing would have a greater effect on the GM than would the type of chow because of the low concentration of TMS in the medicated chow. We also hypothesized that mice fed only a TMS diet would show lower GM species richness and α diversity as compared with groups fed standard rodent chow due to antibiotic dampening of certain bacterial populations.Concern about maintaining disease phenotypes that depend on diet is a major roadblock to changing our current practice of providing TMS diet. Therefore, our second goal was to determine the stability of the GM after a diet change in the post-weaning period. To assess this, we changed the diet of a subset of cages from standard diet back to TMS diet 2 weeks after weaning. We hypothesized that a change to the TMS diet after a 2-week period on standard chow would²⁴ negate any TMS-related effects on the microbiome. This outcome would be expressed as an alignment in richness, diversity, and relative populations with that found in the group fed TMS diet over the entire experiment under a given housing condition.     

Phylogenetic and Metabolic Tracking of Gut Microbiota during Perinatal Development

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. ¹H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota “core” of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.     

Lentinula edodes-Derived Polysaccharide Alters the Spatial Structure of Gut Microbiota in Mice

Lentinula edodes-derived polysaccharides possess many therapeutic characteristics, including anti-tumor and immuno-modulation. The gut microbes play a critical role in modulation of immune function. However, the impact of Lentinula edodes-derived polysaccharides on the gut microbes have not yet been explored. In this study, high-throughput pyrosequencing technique was employed to investigate the effects of a new heteropolysaccharide L2 from Lentinula edodes on microbiota diversity and composition of small intestine, cecum, colon and distal end of colon (feces) in mice. The results demonstrated that along mouse intestine the microbiota exhibit distinctly different space distribution. L2 treatment reduced the diversity and evenness of gut microbiota along the intestine, especially in the cecum and colon. In the fecal microbial communities, the decrease of Bacteroidetes by significantly increasing Proteobacteria were observed, which were characterized by the increased Helicobacteraceae and reduced S24-7 at family level. Some OTUs, corresponding to Bacteroides acidifaciens, Alistipes and Helicobacter suncus, were found to be significantly increased in L2 treated-mice. In particular, 4 phyla Chloroflexi, Gemmatimonadetes, Nitrospirae and Planctomycetes are exclusively present in L2-treated mice. This is helpful for further demonstrating healthy action mechanism of Lentinula edodes-derived polysaccharide L2.     

性激素变化对小鼠肠道菌群构成的影响

肠道菌群在维持宿主免疫和消化系统功能方面发挥着重要作用,肠道菌群的多样性和丰富度是衡量宿主健康状况的重要生理指标。性激素对动物生长发育发挥重要作用,但其对肠道菌群构成影响的相关实验研究相对较少。本研究以模式生物小鼠（Mus musculus）为对象,探究性激素的变化对小鼠肠道菌群构成的影响。采用外科手术方式建立小鼠去势模型,通过16S r RNA高通量测序技术,研究性激素对小鼠肠道菌群构成的影响。研究结果表明,雌性小鼠和雄性小鼠去势后,性激素水平显著下降。肠道菌群在门水平上,正常小鼠和去势小鼠肠道细菌群落均由拟杆菌门（Bacteroidetes）、厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）、埃普西隆杆菌门（Epsilonbacteraeota）、髌骨细菌门（Patescibacteria）、放线菌门（Actinobacteria）、软壁菌门（Tenericutes）、脱铁杆菌门（Deferribacteres）、酸杆菌门（Acidobacteria）和蓝藻细菌门（Cyanobacteria）组成,主要菌群为厚壁菌门和拟杆菌门物种,两门占物种相对丰度百分比...     

Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi

Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3) above 3.35 × 10⁷ CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP) and 84% (CAT). Embryogenesis was arrested in 16% (RHBP) and 20% (CAT) of laid eggs. Feeding females 10⁵ CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control.     

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.     

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

High Fat Diet-Induced Gut Microbiota Exacerbates Inflammation and Obesity in Mice via the TLR4 Signaling Pathway

Background & Aims

While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet.

Methods

C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high–fat (60 kcal % fat) diet (HFD) for 8 weeks.

Results

HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice.

Conclusions

HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.     

Response of Gut Microbiota to Fasting and Hibernation in Syrian Hamsters

Although hibernating mammals wake occasionally to eat during torpor, this period represents a state of fasting. Fasting is known to alter the gut microbiota in nonhibernating mammals; therefore, hibernation may also affect the gut microbiota. However, there are few reports of gut microbiota in hibernating mammals. The present study aimed to compare the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses. Hamsters were allocated to either torpid, fed active, or fasted active groups. Hibernation was successfully induced by maintaining darkness at 4°C. Flow cytometry analysis of cecal bacteria showed that 96-h fasting reduced the total gut bacteria. This period of fasting also reduced the concentrations of short chain fatty acids in the cecal contents. In contrast, total bacterial numbers and concentrations of short chain fatty acids were unaffected by hibernation. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments indicated that fasting and hibernation modulated the cecal microbiota. Analysis of 16S rRNA clone library and species-specific real-time quantitative PCR showed that the class Clostridia predominated in both active and torpid hamsters and that populations of Akkermansia muciniphila, a mucin degrader, were increased by fasting but not by hibernation. From these results, we conclude that the gut microbiota responds differently to fasting and hibernation in Syrian hamsters.Some mammalian species have evolved with the physiological phenomenon of hibernation to survive unfavorable winter environments (9). Hibernation is realized by entering torpor in order to eliminate the need to maintain a constant, high body temperature. During torpor, typical hibernating mammals, such as hamsters and ground squirrels, lower their body temperature to only a few degrees above ambient temperatures to reduce energy expenditure. Torpor is interrupted by periods of intense metabolic activity. During these interbout arousals, physiological parameters are restored rapidly to near-normal levels. Thus, hibernators alternate between hypothermic and euthermic states during hibernation.Some hibernating mammals awake to forage during torpor, while food-storing hibernators such as hamsters eat cached food during interbout arousals. However, hibernation essentially involves periods of fasting. Fasting is known to affect the gut microbiota in nonhibernating mammals such as mice (12); therefore, it is possible that hibernation also influences the gut microbiota. Given that the gut microbiota plays important roles in mammalian tissue development and homeostasis (28), it was of interest to investigate the changes in the gut microbiota that may take place during hibernation. To date, this issue has received little attention; to our knowledge, there are only two reports on the gut microbiota in hibernating mammals. Schmidt et al. showed that although the total counts of coliforms, streptococci, and psychrophilic organisms in the feces of arctic ground squirrels held in a cold room at 3°C remained constant the composition changed, with a decrease in coliform count and a 1,000-fold increase in the number of aerobic psychrophilic gram-negative bacteria (31). Barnes and Burton reported that although there was some reduction in total numbers of viable bacteria in the cecum during hibernation, composition of the microbiota remained stable (6). In terms of amphibians, Banas et al. and Gossling et al. reported a reduction and compositional changes of the gut microbiota in hibernating leopard frogs (4, 5, 18, 19).Only 20 to 40% of bacterial species from the mammalian intestinal tract can be cultured and identified using classical culture methods (22, 34, 36). In contrast, culture-independent methods based on the amplification of bacterial 16S rRNA genes by PCR have revealed a great diversity of microbiota in environmental samples (3, 37). The present study compared the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses.