The gut microbiota has been implicated as an environmental factor that modulates obesity, and recent evidence suggests that microbiota-mediated changes in bile acid profiles and signalling through the bile acid nuclear receptor farnesoid X receptor (FXR) contribute to impaired host metabolism. Here we investigated if the gut microbiota modulates obesity and associated phenotypes through FXR.

Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.

Intermittent hypoxia (IH)-induces alterations in tumor-associated macrophages (TAMs) that are associated with adverse cancer outcomes, as reported in patients suffering from sleep apnea. Adipose tissues (AT) and bone-marrow (BM)-derived cells are the inferred sources of macrophages infiltrating malignant tumors. Here, the sources of TAMs and the phenotypic changes induced by IH in the ipsilateral and contralateral AT were investigated by using a syngeneic murine solid tumor model (TC1). C57/B6 male mice were exposed to either IH or room air (RA) for 6 weeks, with TC1 cells being inoculated in the 2nd week. Macrophage content, phenotype and tissue origin were assessed in tumors, and ipsilateral and contralateral AT. IH induced a ~2.2-fold increase in TAM tumor infiltration. However, differential responses in the tumor ipsilateral and contralateral AT emerged: IH increased infiltration of preferentially M1 macrophages in contralateral AT, while reductions in macrophages emerged in ipsilateral AT and primarily consisted of the M2 phenotype. These changes were accompanied by reciprocal increases in resident and BM-derived TAMs in the tumor. IH-induced phenotypic alterations in AT macrophages surrounding the tumor and their increased infiltration within the tumor may contribute to the accelerated tumor progression associated with IH.

Obesity is associated with a cluster of metabolic disorders, low-grade inflammation and altered gut microbiota. Whether host metabolism is controlled by intestinal innate immune system and the gut microbiota is unknown. Here we report that inducible intestinal epithelial cell-specific deletion of MyD88 partially protects against diet-induced obesity, diabetes and inflammation. This is associated with increased energy expenditure, an improved glucose homeostasis, reduced hepatic steatosis, fat mass and inflammation. Protection is transferred following gut microbiota transplantation to germ-free recipients. We also demonstrate that intestinal epithelial MyD88 deletion increases anti-inflammatory endocannabinoids, restores antimicrobial peptides production and increases intestinal regulatory T cells during diet-induced obesity. Targeting MyD88 after the onset of obesity reduces fat mass and inflammation. Our work thus identifies intestinal epithelial MyD88 as a sensor changing host metabolism according to the nutritional status and we show that targeting intestinal epithelial MyD88 constitutes a putative therapeutic target for obesity and related disorders.

The gut microbiota is central to human health, but its establishment in early life has not been quantitatively and functionally examined. Applying metagenomic analysis on fecal samples from a large cohort of Swedish infants and their mothers, we characterized the gut microbiome during the first year of life and assessed the impact of mode of delivery and feeding on its establishment. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota. Microbiota composition and ecological network had distinctive features at each sampled stage, in accordance with functional maturation of the microbiome. Our findings establish a framework for understanding the interplay between the gut microbiome and the human body in early life.

Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD.

Both fibroblast growth factors (FGFs), by binding to FGF receptors (FGFRs), and activation of the gravitostat, by artificial loading, decrease the body weight (BW). Previous studies demonstrate that both the FGF system and loading have the capacity to regulate BW independently of leptin. The aim of the current study was to determine the possible interactions between the effect of increased loading and the FGF system for the regulation of BW. We observed that the BW-reducing effect of increased loading was abolished in mice treated with a monoclonal antibody directed against FGFR1c, suggesting interactions between the two systems. As serum levels of endocrine FGF21 and hepatic FGF21 mRNA were increased in the loaded mice compared with the control mice, we first evaluated the loading response in FGF21 over expressing mice with constant high FGF21 levels. Leptin treatment, but not increased loading, decreased the BW in the FGF21-overexpressing mice, demonstrating that specifically the loading effect is attenuated in the presence of high activity in the FGF system. However, as FGF21 knockout mice displayed a normal loading response on BW, FGF21 is neither mediating nor essential for the loading response. In conclusion, the BW-reducing effect of increased loading but not of leptin treatment is blocked by high activity in the FGF system. We propose that both the gravitostat and the FGF system regulate BW independently of leptin and that pharmacologically enhanced activity in the FGF system reduces the sensitivity of the gravitostat.

Objective- To investigate the effect of gut microbiota and diet on atherogenesis. Approach and Results- Here, we investigated the interaction between the gut microbiota and diet on atherosclerosis by feeding germ-free or conventionally raised Apoe-/- mice chow or Western diet alone or supplemented with choline (which is metabolized by the gut microbiota and host enzymes to trimethylamine N-oxide) for 12 weeks. We observed smaller aortic lesions and lower plasma cholesterol levels in conventionally raised mice compared with germ-free mice on a chow diet; these differences were not observed in mice on a Western diet. Choline supplementation increased plasma trimethylamine N-oxide levels in conventionally raised mice but not in germ-free mice. However, this treatment did not affect the size of aortic lesions or plasma cholesterol levels. Gut microbiota composition was analyzed by sequencing of 16S rRNA genes. As expected, the global community structure and relative abundance of many taxa differed between mice fed chow or a Western diet. Choline supplementation had minor effects on the community structure although the relative abundance of some taxa belonging to Clostridiales was altered. Conclusions- In conclusion, the impact of the gut microbiota on atherosclerosis is dietary dependent and is associated with plasma cholesterol levels. Furthermore, the microbiota was required for trimethylamine N-oxide production from dietary choline, but this process could not be linked to increased atherosclerosis in this model.

A functional mucus layer is a key requirement for gastrointestinal health as it serves as a barrier against bacterial invasion and subsequent inflammation. Recent findings suggest that mucus composition may pose an important selection pressure on the gut microbiota and that altered mucus thickness or properties such as glycosylation lead to intestinal inflammation dependent on bacteria. Here we used TM-IEC C1galt (-/-) mice, which carry an inducible deficiency of core 1-derived O-glycans in intestinal epithelial cells, to investigate the effects of mucus glycosylation on susceptibility to intestinal inflammation, gut microbial ecology and host physiology. We found that TM-IEC C1galt (-/-) mice did not develop spontaneous colitis, but they were more susceptible to dextran sodium sulphate-induced colitis. Furthermore, loss of core 1-derived O-glycans induced inverse shifts in the abundance of the phyla Bacteroidetes and Firmicutes. We also found that mucus glycosylation impacts intestinal architecture as TM-IEC C1galt(-/-) mice had an elongated gastrointestinal tract with deeper ileal crypts, a small increase in the number of proliferative epithelial cells and thicker circular muscle layers in both the ileum and colon. Alterations in the length of the gastrointestinal tract were partly dependent on the microbiota. Thus, the mucus layer plays a role in the regulation of gut microbiota composition, balancing intestinal inflammation, and affects gut architecture.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.

Humans and mice differ substantially in their bile acid profiles as mice in addition to cholic acid (CA) predominantly synthesize 6β-hydroxylated muricholic acids (MCAs) whereas humans produces chenodeoxycholic acid (CDCA) and CA as primary bile acids. Identifying the gene performing 6β-hydroxylation would be useful for 'humanizing' the bile acid profile in mice for studies of the interaction between bile acids, gut microbiota, and host metabolism. We investigated the formation of MCAs in primary murine hepatocytes and found that αMCA is synthesized from CDCA and βMCA from UDCA. It is commonly assumed that the P450-enzyme CYP3A11 catalyzes 6β-hydroxylation of bile acids, thus we hypothesized that mice without the Cyp3a11 gene would lack MCAs. To test this hypothesis, we analyzed bile acid profiles in Cyp3a deficient mice, which lack 7 genes in the Cyp3a gene cluster including Cyp3a11, and compared them with wild-type littermate controls. Bile acid composition in liver, gallbladder, caecum and serum from Cyp3a knock out mice and wild-type littermate controls was analyzed with UPLC-MS/MS and revealed no major differences in bile acid composition. We conclude that Cyp3a11 is not necessary for 6β-hydroxylation and the formation of MCAs.

Nuclear receptor subfamily 1, group H, member 4 (Nr1h4, FXR) is a bile acid activated nuclear receptor mainly expressed in the liver, intestine, kidney and adrenal glands. Upon activation, the primary function is to suppress cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classic or neutral bile acid synthesis pathway. In the present study, a novel Fxr deficient mouse line was created and studied with respect to metabolism and liver function in ageing mice fed chow diet. The Fxr deficient mice were similar to wild type mice in terms of body weight, body composition, energy intake and expenditure as well as behaviours at a young age. However, from 15 weeks of age and onwards, the Fxr deficient mice had almost no body weight increase up to 39 weeks of age mainly because of lower body fat mass. The lower body weight gain was associated with increased energy expenditure that was not compensated by increased food intake. Fasting levels of glucose and insulin were lower and glucose tolerance was improved in old and lean Fxr deficient mice. However, the Fxr deficient mice displayed significantly increased liver weight, steatosis, hepatocyte ballooning degeneration and lobular inflammation together with elevated plasma levels of ALT, bilirubin and bile acids, findings compatible with non-alcoholic steatohepatitis (NASH) and cholestasis. In conclusion, ageing Fxr deficient mice display late onset leanness associated with elevated energy expenditure and improved glucose control but develop severe NASH-like liver pathology.

The gut microbiota has been proposed as an environmental factor that affects the development of metabolic and inflammatory diseases in mammals. Recent reports indicate that gut bacteria-derived lipopolysaccharide (LPS) can initiate obesity and insulin resistance in mice; however, the molecular interactions responsible for microbial regulation of host metabolism and mediators of inflammation have not been studied in detail. Hepatic serum amyloid A (SAA) proteins are markers and proposed mediators of inflammation that exhibit increased levels in serum of insulin-resistant mice. Adipose tissue-derived SAA3 displays monocyte chemotactic activity and may play a role in metabolic inflammation associated with obesity and insulin resistance. To investigate a potential mechanistic link between the intestinal microbiota and induction of proinflammatory host factors, we performed molecular analyses of germ-free, conventionally raised and genetically modified Myd88-/- mouse models. SAA3 expression was determined to be significantly augmented in adipose (9.9+/-1.9-fold; P<0.001) and colonic tissue (7.0+/-2.3-fold; P<0.05) by the presence of intestinal microbes. In the colon, we provided evidence that SAA3 is partially regulated through the Toll-like receptor (TLR)/MyD88/NF-kappaB signaling axis. We identified epithelial cells and macrophages as cellular sources of SAA3 in the colon and found that colonic epithelial expression of SAA3 may be part of an NF-kappaB-dependent response to LPS from gut bacteria. In vitro experiments showed that LPS treatments of both epithelial cells and macrophages induced SAA3 expression (27.1+/-2.5-fold vs. 1.6+/-0.1-fold, respectively). Our data suggest that LPS, and potentially other products of the indigenous gut microbiota, might elevate cytokine expression in tissues and thus exacerbate chronic low-grade inflammation observed in obesity.

We have investigated the interrelationship between diet, gut microbial ecology, and energy balance using a mouse model of obesity produced by consumption of a prototypic Western diet. Diet-induced obesity (DIO) produced a bloom in a single uncultured clade within the Mollicutes class of the Firmicutes, which was diminished by subsequent dietary manipulations that limit weight gain. Microbiota transplantation from mice with DIO to lean germ-free recipients promoted greater fat deposition than transplants from lean donors. Metagenomic and biochemical analysis of the gut microbiome together with sequencing and metabolic reconstructions of a related human gut-associated Mollicute (Eubacterium dolichum) revealed features that may provide a competitive advantage to members of the bloom in the Western diet nutrient milieu, including import and processing of simple sugars. Our study illustrates how combining comparative metagenomics with gnotobiotic mouse models and specific dietary manipulations can disclose the niches of previously uncharacterized members of the gut microbiota.

L cells are an important class of enteroendocrine cells secreting hormones such as glucagon like peptide-1 and peptide YY that have several metabolic and physiological effects. The gut is home to trillions of bacteria affecting host physiology, but there has been limited understanding about how the microbiota affects gene expression in L cells. Thus, we rederived the reporter mouse strain, GLU-Venus expressing yellow fluorescent protein under the control of the proglucagon gene, as germ-free (GF). Lpos cells from ileum and colon of GF and conventionally raised (CONV-R) GLU-Venus mice were isolated and subjected to transcriptomic profiling. We observed that the microbiota exerted major effects on ileal L cells. Gene Ontology enrichment analysis revealed that microbiota suppressed biological processes related to vesicle localization and synaptic vesicle cycling in Lpos cells from ileum. This finding was corroborated by electron microscopy of Lpos cells showing reduced numbers of vesicles as well as by demonstrating decreased intracellular GLP-1 content in primary cultures from ileum of CONV-R compared with GF GLU-Venus mice. By analysing Lpos cells following colonization of GF mice we observed that the greatest transcriptional regulation was evident within 1 day of colonization. Thus, the microbiota has a rapid and pronounced effect on the L cell transcriptome, predominantly in the ileum.

An altered intestinal microbiota composition has been implicated in the pathogenesis of metabolic disease including obesity and type 2 diabetes mellitus (T2DM). Low grade inflammation, potentially initiated by the intestinal microbiota, has been suggested to be a driving force in the development of insulin resistance in obesity. Here, we report that bacterial DNA is present in mesenteric adipose tissue of obese but otherwise healthy human subjects. Pyrosequencing of bacterial 16S rRNA genes revealed that DNA from the Gram-negative species Ralstonia was most prevalent. Interestingly, fecal abundance of Ralstonia pickettii was increased in obese subjects with pre-diabetes and T2DM. To assess if R. pickettii was causally involved in development of obesity and T2DM, we performed a proof-of-concept study in diet-induced obese (DIO) mice. Compared to vehicle-treated control mice, R. pickettii-treated DIO mice had reduced glucose tolerance. In addition, circulating levels of endotoxin were increased in R. pickettii-treated mice. In conclusion, this study suggests that intestinal Ralstonia is increased in obese human subjects with T2DM and reciprocally worsens glucose tolerance in DIO mice.

Gut microbiota modulates adiposity and glucose metabolism in humans and mice. Here we investigated how colonization of germ-free (GF) mice affects kinetics of adiposity and glucose metabolism.

Diet strongly affects gut microbiota composition, and gut bacteria can influence the colonic mucus layer, a physical barrier that separates trillions of gut bacteria from the host. However, the interplay between a Western style diet (WSD), gut microbiota composition, and the intestinal mucus layer is less clear. Here we show that mice fed a WSD have an altered colonic microbiota composition that causes increased penetrability and a reduced growth rate of the inner mucus layer. Both barrier defects can be prevented by transplanting microbiota from chow-fed mice. In addition, we found that administration of Bifidobacterium longum was sufficient to restore mucus growth, whereas administration of the fiber inulin prevented increased mucus penetrability in WSD-fed mice. We hypothesize that the presence of distinct bacteria is crucial for proper mucus function. If confirmed in humans, these findings may help to better understand diseases with an affected mucus layer, such as ulcerative colitis.

An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies and immune cell profiling, complemented with gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.

Preliminary evidence from animal and human studies shows that gut microbiota composition and levels of microbiota-derived metabolites, including short-chain fatty acids (SCFAs), are associated with blood pressure (BP). We hypothesized that faecal microbiota composition and derived metabolites may be differently associated with BP across ethnic groups.