Activation of Peroxisome Proliferator-Activated Receptor-ß/δ (PPARß/δ) in Keratinocytes by Endogenous Fatty Acids.

Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and chromatin. The present studies examined the hypothesis that endogenous ligands activate peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in keratinocytes. The phorbol ester treatment or HRAS infection of primary keratinocytes increased fatty acids that were associated with enhanced PPARß/δ activity. Fatty acids caused PPARß/δ-dependent increases in chromatin occupancy and the expression of angiopoietin-like protein 4 (Angptl4) mRNA. Analyses demonstrated that stearoyl Co-A desaturase 1 (Scd1) mediates an increase in intracellular monounsaturated fatty acids in keratinocytes that act as PPARß/δ ligands. The activation of PPARß/δ with palmitoleic or oleic acid causes arrest at the G2/M phase of the cell cycle of HRAS-expressing keratinocytes that is not found in similarly treated HRAS-expressing Pparb/d-null keratinocytes. HRAS-expressing Scd1-null mouse keratinocytes exhibit enhanced cell proliferation, an effect that is mitigated by treatment with palmitoleic or oleic acid. Consistent with these findings, the ligand activation of PPARß/δ with GW0742 or oleic acid prevented UVB-induced non-melanoma skin carcinogenesis, an effect that required PPARß/δ. The results from these studies demonstrate that PPARß/δ has endogenous roles in keratinocytes and can be activated by lipids found in diet and cellular components.

Surgery for Crohn's Disease Is Associated With a Dysbiotic Microbiome and Metabolome: Results From Two Prospective Cohorts.

BACKGROUND & AIMS: Crohn's disease is associated with alterations in the gut microbiome and metabolome described as dysbiosis. We characterized the microbial and metabolic consequences of ileal resection, the most common Crohn's disease surgery. METHODS: Patients with and without intestinal resection were identified from the Diet to Induce Remission in Crohn's Disease and Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease studies. Stool samples were analyzed with shotgun metagenomics sequencing. Fecal butyrate was measured with 1H nuclear magnetic resonance spectroscopy. Fecal bile acids and plasma 7α-hydroxy-4-cholesten-3-one (C4) was measured with mass spectrometry. RESULTS: Intestinal resection was associated with reduced alpha diversity and altered beta diversity with increased Proteobacteria and reduced Bacteroidetes and Firmicutes. Surgery was associated with higher representation of genes in the KEGG pathway for ABC transporters and reduction in genes related to bacterial metabolism. Surgery was associated with reduced concentration of the But gene but this did not translate to reduced fecal butyrate concentration. Surgery was associated with decreased abundance of bai operon genes, with increased plasma C4 concentration, increased primary bile acids and reduced secondary bile acids, including isoLCA. Additionally, Egerthella lenta, Adlercreutzia equalofaciens, and Gordonibacter pamelaeae were lower in abundance among patients with prior surgery in both cohorts. CONCLUSIONS: In 2 different populations, prior surgery in Crohn's disease is associated with altered fecal microbiome. Patients who had undergone ileal resection had reduction in the potentially beneficial bacteria E lenta and related actinobacteria and secondary bile acids, including isoLCA, suggesting that these could be biomarkers of patients at higher risk for disease progression.

Immune cell-intrinsic Ah receptor facilitates the expression of antimicrobial REG3G in the small intestine.

The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.

Sex Dimorphic Effects of Bile Acid Metabolism in Liver Cancer in Mice.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a male-dominant disease, but targeted sex hormone therapies have not been successful. Bile acids are a potential liver carcinogen and are biomolecules with hormone-like effects. A few studies highlight their potential sex dimorphism in physiology and disease. We hypothesized that bile acids could be a potential molecular signature that explains sex disparity in HCC. METHODS & RESULTS: We used the farnesoid X receptor knockout (FxrKO) mouse model to study bile acid-dependent HCC. Temporal tracking of circulating bile acids determined more than 80% of FxrKO females developed spontaneous cholemia (ie, serum total bile acids ≥40 µmol/L) as early as 8 weeks old. Opposingly, FxrKO males were highly resistant to cholemia, with ∼23% incidence even when 26 weeks old. However, FxrKO males demonstrated higher levels of deoxycholate than females. Compared with males, FxrKO females had more severe cholestatic liver injury and further aberrancies in bile acid metabolism. Yet, FxrKO females expressed more detoxification transcripts and had greater renal excretion of bile acids. Intervention with CYP7A1 (rate limiting enzyme for bile acid biosynthesis) deficiency or taurine supplementation either completely or partially normalized bile acid levels and liver injury in FxrKO females. Despite higher cholemia prevalence in FxrKO females, their tumor burden was less compared with FxrKO males. An exception to this sex-dimorphic pattern was found in a subset of male and female FxrKO mice born with congenital cholemia due to portosystemic shunt, where both sexes had comparable robust HCC. CONCLUSIONS: Our study highlights bile acids as sex-dimorphic metabolites in HCC except in the case of portosystemic shunt.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Comparative urine metabolomics of mice treated with non-toxic and toxic oral doses of (-)-epigallocatechin-3-gallate.

The green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), has been studied for its potential positive health effects, but human and animal model studies have reported potential toxicity at high oral bolus doses. This study used liquid chromatography-mass spectrometry-based metabolomics to compare the urinary EGCG metabolite profile after administration of a single non-toxic (100 mg kg-1) or toxic (750 mg kg-1) oral bolus dose to male C57BL6/J mice to better understand how EGCG metabolism varies with dose. EGCG metabolites, including methyl, glucuronide, sulfate, and glucoside conjugates, were tentatively identified based on their mass to charge (m/z) ratio and fragment ion patterns. Partial least squares discriminant analysis (PLS-DA) results showed clear separation of the urine metabolite profiles between treatment groups. The most differentiating metabolites in the negative and positive ion modes were provisionally identified as di-glucuronidated EGCG quinone and di-glucuronidated EGCG, respectively. The presence of EGCG oxidation products at toxic dose is consistent with studies showing that EGCG toxicity is associated with oxidative stress. Relative amounts of methylated metabolites increased with dose to a lesser extent than glucuronide and sulfate metabolites, indicating that methylation is more prominent at low doses, whereas glucuronidation and sulfation may be more important at higher doses. One limitation of the current work is that the lack of commercially-available EGCG metabolite standards prevented absolute metabolite quantification and identification. Despite this limitation, these findings provide a basis for better understanding the dose-dependent changes in EGCG metabolism and advance studies on how these differences may contribute to the toxicity of high doses of EGCG.

Changes in fecal lipidome after treatment with ivacaftor without changes in microbiome or bile acids.

BACKGROUND: Alterations in gastrointestinal health are prominent manifestations of cystic fibrosis (CF) and can independently impact pulmonary function. Ivacaftor has been associated with robust improvements in pulmonary function and weight gain, but less is known about the impact of ivacaftor on the fecal microbiome, lipidome, and bile acids. METHODS: Stool samples from 18 patients with CF and gating mutations (ages 6-61 years, 13 pancreatic insufficient) were analyzed for fecal microbiome and lipidome composition as well as bile acid concentrations at baseline and after 3 months of treatment with ivacaftor. Microbiome composition was also assessed in a healthy reference cohort. RESULTS: Alpha and beta diversity of the microbiome were different between CF and reference cohort at baseline, but no treatment effect was seen in the CF cohort between baseline and 3 months. Seven lipids increased with treatment. No differences were seen in bile acid concentrations after treatment in CF. At baseline, 403 lipids and unconjugated bile acids were different between pancreatic insufficient (PI-CF) and sufficient (PS-CF) groups and 107 lipids were different between PI-CF and PS-CF after 3 months of treatment. CONCLUSIONS: The composition and diversity of the fecal microbiome were different in CF as compared to a healthy reference, and did not change after 3 months of ivacaftor. We detected modest differences in the fecal lipidome with treatment. Differences in lipid and bile acid profiles between PS-CF and PI-CF were attenuated after 3 months of treatment.

Induction of AHR Signaling in Response to the Indolimine Class of Microbial Stress Metabolites.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays an important role in gastrointestinal barrier function, tumorigenesis, and is an emerging drug target. The resident microbiota is capable of metabolizing tryptophan to metabolites that are AHR ligands (e.g., indole-3-acetate). Recently, a novel set of mutagenic tryptophan metabolites named indolimines have been identified that are produced by M. morganii in the gastrointestinal tract. Here, we determined that indolimine-200, -214, and -248 are direct AHR ligands that can induce Cyp1a1 transcription and subsequent CYP1A1 enzymatic activity capable of metabolizing the carcinogen benzo(a)pyrene in microsomal assays. In addition, indolimines enhance IL6 expression in a colonic tumor cell line in combination with cytokine treatment. The concentration of indolimine-248 that induces AHR transcriptional activity failed to increase DNA damage. These observations reveal an additional aspect of how indolimines may alter colonic tumorigenesis beyond mutagenic activity.

Accumulation of Linoleic Acid by Altered Peroxisome Proliferator-Activated Receptor-α Signaling Is Associated with Age-Dependent Hepatocarcinogenesis in Ppara Transgenic Mice.

Long-term ligand activation of PPARα in mice causes hepatocarcinogenesis through a mechanism that requires functional PPARα. However, hepatocarcinogenesis is diminished in both Ppara-null and PPARA-humanized mice, yet both lines develop age-related liver cancer independently of treatment with a PPARα agonist. Since PPARα is a master regulator of liver lipid metabolism in the liver, lipidomic analyses were carried out in wild-type, Ppara-null, and PPARA-humanized mice treated with and without the potent agonist GW7647. The levels of hepatic linoleic acid in Ppara-null and PPARA-humanized mice were markedly higher compared to wild-type controls, along with overall fatty liver. The number of liver CD4+ T cells was also lower in Ppara-null and PPARA-humanized mice and was negatively correlated with the elevated linoleic acid. Moreover, more senescent hepatocytes and lower serum TNFα and IFNγ levels were observed in Ppara-null and PPARA-humanized mice with age. These studies suggest a new role for PPARα in age-associated hepatocarcinogenesis due to altered lipid metabolism in Ppara-null and PPARA-humanized mice and the accumulation of linoleic acid as part of an overall fatty liver that is associated with loss of CD4+ T cells in the liver in both transgenic models. Since fatty liver is a known causal risk factor for liver cancer, Ppara-null and PPARA-humanized mice are valuable models for examining the mechanisms of PPARα and age-dependent hepatocarcinogenesis.

Refined fiber inulin promotes inflammation-associated colon tumorigenesis by modulating microbial succinate production.

BACKGROUND AND AIM: There is an increased risk of colon cancer associated with inflammatory bowel disease (IBD). Dietary fibers (DFs) naturally present in vegetables and whole grains offer numerous beneficial effects on intestinal health. However, the effects of refined DFs on intestinal health remain unclear. Therefore, we elucidated the impact of the refined DF inulin on colonic inflammation and tumorigenesis. METHODS: Four-week-old wild-type (WT) mice were fed diets containing insoluble DF cellulose (control) or refined DF inulin for 4 weeks. A subgroup of mice was then switched to drinking water containing dextran sulfate sodium (DSS, 1.4% wt/vol) for colitis induction. In another subgroup of mice, colitis-associated colorectal cancer (CRC) was initiated with three 7-day alternate cycles of DSS following an initial dose of mutagenic substance azoxymethane (AOM; 7.5 mg/kg body weight; i.p.). Post 7 weeks of AOM treatment, mice were euthanized and examined for CRC development. RESULTS: Mice consuming inulin-containing diet exhibited severe colitis upon DSS administration, as evidenced by more body weight loss, rectal bleeding, and increased colonic inflammation than the DSS-treated control group. Correspondingly, histological analysis revealed extensive disruption of colon architecture and massive infiltration of immune cells in the inulin-fed group. We next examined the effect of inulin on CRC development. Surprisingly, significant mortality (~50%) was observed in the inulin-fed but not in the control group during the DSS cycle. Consequently, the remaining inulin-fed mice, which completed the study exhibited extensive colon tumorigenesis. Immunohistochemical characterization showed comparatively high expression of the cell proliferation marker Ki67 and activation of the Wnt signaling in tumor sections obtained from the inulin-fed group. Gut microbiota and metabolite analysis revealed expansion of succinate producers and elevated cecal succinate in inulin-fed mice. Human colorectal carcinoma cells (HCT116) proliferated more rapidly when supplemented with succinate in an inflamed environment, suggesting that elevated luminal succinate may contribute to tumorigenesis. CONCLUSIONS: Our study uncovers that supplementation of diet with refined inulin induces abnormal succinate accumulation in the intestinal lumen, which in part contributes to promoting colon inflammation and tumorigenesis.

Aryl Hydrocarbon Receptor Activation Coordinates Mouse Small Intestinal Epithelial Cell Programming.

In the face of mechanical, chemical, microbial, and immunologic pressure, intestinal homeostasis is maintained through balanced cellular turnover, proliferation, differentiation, and self-renewal. Here, we present evidence supporting the role of the aryl hydrocarbon receptor (AHR) in the adaptive reprogramming of small intestinal gene expression, leading to altered proliferation, lineage commitment, and remodeling of the cellular repertoire that comprises the intestinal epithelium to promote intestinal resilience. Ahr gene/protein expression and transcriptional activity exhibit marked proximalHI to distalLO and cryptHI to villiLO gradients. Genetic ablation of Ahr impairs commitment/differentiation of the secretory Paneth and goblet cell lineages and associated mucin production, restricts expression of secretory/enterocyte differentiation markers, and increases crypt-associated proliferation and villi-associated enterocyte luminal exfoliation. Ahr-/- mice display a decrease in intestinal barrier function. Ahr+/+ mice that maintain a diet devoid of AHR ligands intestinally phenocopy Ahr-/- mice. In contrast, Ahr+/+ mice exposed to AHR ligands reverse these phenotypes. Ligand-induced AHR transcriptional activity positively correlates with gene expression (Math1, Klf4, Tff3) associated with differentiation of the goblet cell secretory lineage. Math1 was identified as a direct target gene of AHR, a transcription factor critical to the development of goblet cells. These data suggest that dietary cues, relayed through the transcriptional activity of AHR, can reshape the cellular repertoire of the gastrointestinal tract.

The Ah Receptor from Toxicity to Therapeutics: Report from the 5th AHR Meeting at Penn State University, USA, June 2022.

The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

Bile salt hydrolase in non-enterotoxigenic Bacteroides potentiates colorectal cancer.

Bile salt hydrolase (BSH) in Bacteroides is considered a potential drug target for obesity-related metabolic diseases, but its involvement in colon tumorigenesis has not been explored. BSH-expressing Bacteroides is found at high abundance in the stools of colorectal cancer (CRC) patients  with overweight and in the feces of a high-fat diet (HFD)-induced CRC mouse model. Colonization of B. fragilis 638R, a strain with low BSH activity, overexpressing a recombinant bsh gene from B. fragilis NCTC9343 strain, results in increased unconjugated bile acids in the colon and accelerated progression of CRC under HFD treatment. In the presence of high BSH activity, the resultant elevation of unconjugated deoxycholic acid and lithocholic acid activates the G-protein-coupled bile acid receptor, resulting in increased ß-catenin-regulated chemokine (C-C motif) ligand 28 (CCL28) expression in colon tumors. Activation of the ß-catenin/CCL28 axis leads to elevated intra-tumoral immunosuppressive CD25+FOXP3+ Treg cells. Blockade of the ß-catenin/CCL28 axis releases the immunosuppression to enhance the intra-tumoral anti-tumor response, which decreases CRC progression under HFD treatment. Pharmacological inhibition of BSH reduces HFD-accelerated CRC progression, coincident with suppression of the ß-catenin/CCL28 pathway. These findings provide insights into the pro-carcinogenetic role of Bacteroides in obesity-related CRC progression and characterize BSH as a potential target for CRC prevention and treatment.

Contribution of circulating host and microbial tryptophan metabolites towards Ah receptor activation.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays an integral role in homeostatic maintenance by regulating cellular functions such as cellular differentiation, metabolism, barrier function, and immune response. An important but poorly understood class of AHR activators are compounds derived from host and bacterial metabolism of tryptophan. The commensal bacteria of the gut microbiome are major producers of tryptophan metabolites known to activate the AHR, while the host also produces AHR activators through tryptophan metabolism. We used targeted mass spectrometry-based metabolite profiling to determine the presence and metabolic source of these metabolites in the sera of conventional mice, germ-free mice, and humans. Surprisingly, sera concentrations of many tryptophan metabolites are comparable between germ-free and conventional mice. Therefore, many major AHR-activating tryptophan metabolites in mouse sera are produced by the host, despite their presence in feces and mouse cecal contents. AHR activation is rarely studied in the context of a mixture at relevant concentrations, as we present here. The AHR activation potentials of individual and pooled metabolites were explored using cell-based assays, while ligand binding competition assays and ligand docking simulations were used to assess the detected metabolites as AHR agonists. The physiological and biomedical relevance of the identified metabolites was investigated in the context of cell-based models for cancer and rheumatoid arthritis. We present data here that reframe AHR biology to include the presence of ubiquitous tryptophan metabolites, improving our understanding of homeostatic AHR activity and models of AHR-linked diseases.

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation.

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Bile acids and the gut microbiota: metabolic interactions and impacts on disease.

Despite decades of bile acid research, diverse biological roles for bile acids have been discovered recently due to developments in understanding the human microbiota. As additional bacterial enzymes are characterized, and the tools used for identifying new bile acids become increasingly more sensitive, the repertoire of bile acids metabolized and/or synthesized by bacteria continues to grow. Additionally, bile acids impact microbiome community structure and function. In this Review, we highlight how the bile acid pool is manipulated by the gut microbiota, how it is dependent on the metabolic capacity of the bacterial community and how external factors, such as antibiotics and diet, shape bile acid composition. It is increasingly important to understand how bile acid signalling networks are affected in distinct organs where the bile acid composition differs, and how these networks impact infectious, metabolic and neoplastic diseases. These advances have enabled the development of therapeutics that target imbalances in microbiota-associated bile acid profiles.

Genetic differences in nicotine sensitivity and metabolism in C57BL/6J and NOD/ShiLtJ mouse strains.

Genetic background impacts sensitivity to nicotine's rewarding and aversive effects and metabolism, which influences susceptibility to nicotine addiction. This is important because sensitivity to nicotine influences susceptibility to nicotine addiction. Thus, understanding genetic contribution to nicotine sensitivity can aid in identifying risk factors for nicotine addiction. Genetic variability in addiction phenotypes can be modeled in rodent systems, and comparisons of nicotine sensitivity in inbred mice can identify contributing genetic substrates. Our laboratory has identified differences in nicotine sensitivity in male mice from two inbred mouse strains, C57BL/6J and NOD/ShiLtJ. We found that the NOD/ShiLtJ strain experienced greater nicotine-induced locomotor depression and hypothermia than the C57BL/6J strain. To investigate possible differences in nicotine metabolism between strains, subjects were treated with acute nicotine and serum and urine samples were analyzed using LC-MS/MS to quantify nicotine and metabolites. This analysis revealed that NOD/ShiLtJ mice had similar serum nicotine but lower cotinine and 3'-hydroxycotinine levels after nicotine treatment when compared to C57BL/6J mice. Possible genetic factors mediating strain differences were identified by surveying nicotine sensitivity- and metabolism-related genes within the Mouse Phenome Database SNP retrieval tool. Polymorphisms were found in 15 of the 26 examined gene sequences. Liver expression levels of nicotine metabolism-related genes (Cyp2a5, Cyp2a4, and Aox1) were measured using qPCR. NOD/ShiLtJ mice showed lower expression of Cyp2a5 and Cyp2a4 and greater expression of Aox1 in liver tissue. These data demonstrate complex differences in nicotine sensitivity and metabolism driven by genetic differences between C57BL/6J and NOD/ShiLtJ inbred mouse strains.

Bile acid conjugation deficiency causes hypercholanemia, hyperphagia, islet dysfunction, and gut dysbiosis in mice.

Bile acid-CoA: amino acid N-acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, and fat-soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat-/- mice. The bile acid composition and hepatic gene expression were analyzed in 10-week-old Baat-/- mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat-/- mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild-type mice. Bile acid polyhydroxylation was markedly up-regulated to detoxify unconjugated bile acid accumulated in Baat-/- mice. Although the level of serum marker of bile acid synthesis, 7α-hydroxy-4-cholesten-3-one, was higher in Baat-/- mice, their bile acid pool size was smaller. When fed a WD, the Baat-/- mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat-/- mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine-conjugating enzyme. Its deletion protected the animals from diet-induced obesity, but caused glucose intolerance. The gut microbiome of the Baat-/- mice was altered to accommodate the unconjugated bile acid pool.

Metabolomic profiling of stool of two-year old children from the INSIGHT study reveals links between butyrate and child weight outcomes.

BACKGROUND: Metabolomic analysis is commonly used to understand the biological underpinning of diseases such as obesity. However, our knowledge of gut metabolites related to weight outcomes in young children is currently limited. OBJECTIVES: To (1) explore the relationships between metabolites and child weight outcomes, (2) determine the potential effect of covariates (e.g., child's diet, maternal health/habits during pregnancy, etc.) in the relationship between metabolites and child weight outcomes, and (3) explore the relationship between selected gut metabolites and gut microbiota abundance. METHODS: Using 1 H-NMR, we quantified 30 metabolites from stool samples of 170 two-year-old children. To identify metabolites and covariates associated with children's weight outcomes (BMI [weight/height2 ], BMI z-score [BMI adjusted for age and sex], and growth index [weight/height]), we analysed the 1 H-NMR data, along with 20 covariates recorded on children and mothers, using LASSO and best subset selection regression techniques. Previously characterized microbiota community information from the same stool samples was used to determine associations between selected gut metabolites and gut microbiota. RESULTS: At age 2 years, stool butyrate concentration had a significant positive association with child BMI (p-value = 3.58 × 10-4 ), BMI z-score (p-value = 3.47 × 10-4 ), and growth index (p-value = 7.73 × 10-4 ). Covariates such as maternal smoking during pregnancy are important to consider. Butyrate concentration was positively associated with the abundance of the bacterial genus Faecalibacterium (p-value = 9.61 × 10-3 ). CONCLUSIONS: Stool butyrate concentration is positively associated with increased child weight outcomes and should be investigated further as a factor affecting childhood obesity.

Reconstitution of the host holobiont in germ-free born male rats acutely increases bone growth and affects marrow cellular content.

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.