Individualized microbiotas dictate the impact of dietary fiber on colitis sensitivity.

BACKGROUND: The observation that the intestinal microbiota is  central in the development of IBD suggests that dietary fiber, the microbiota's primary source of nourishment, could play a central role in these diseases. Accordingly, enriching diets with specific soluble fibers remodels microbiota and modulates colitis sensitivity. In humans, a recent study suggests that the microbiota of select IBD patients might influence the impacts they would experience upon fiber exposure. We sought here to define the extent to which individual microbiotas varied in their responsiveness to purified soluble fiber inulin and psyllium. Moreover, the extent to which such variance might impact proneness to colitis. RESULTS: We observed a high level of inter-individual variation in microbiota responsiveness to fiber inulin and psyllium: while microbiotas from select donors exhibited stark fiber-induced modulation in composition, pro-inflammatory potential, and metabolomic profile, others were only minimally impacted. Mice transplanted with fiber-sensitive microbiomes exhibited colitis highly modulated by soluble fiber consumption, while mice receiving fiber-resistant microbiotas displayed colitis severity irrespective of fiber exposure. CONCLUSION: The extent to which select soluble fibers alter proneness to colitis is highly influenced by an individual's microbiota composition and further investigation of individual microbiota responsiveness toward specific dietary fiber could pave the way to personalized fiber-based intervention, both in IBD patients and healthy individuals. Video Abstract.

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.

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.

Psyllium Fiber Protects Against Colitis Via Activation of Bile Acid Sensor Farnesoid X Receptor.

BACKGROUND & AIMS: Fiber-rich foods promote health, but mechanisms by which they do so remain poorly defined. Screening fiber types, in mice, revealed psyllium had unique ability to ameliorate 2 chronic inflammatory states, namely, metabolic syndrome and colitis. We sought to determine the mechanism of action of the latter. METHODS: Mice were fed grain-based chow, which is naturally rich in fiber or compositionally defined diets enriched with semi-purified fibers. Mice were studied basally and in models of chemical-induced and T-cell transfer colitis. RESULTS: Relative to all diets tested, mice consuming psyllium-enriched compositionally defined diets were markedly protected against both dextran sulfate sodium- and T-cell transfer-induced colitis, as revealed by clinical-type, histopathologic, morphologic, and immunologic parameters. Such protection associated with stark basal changes in the gut microbiome but was independent of fermentation and, moreover, maintained in mice harboring a minimal microbiota (ie, Altered Schaedler Flora). Transcriptomic analysis revealed psyllium induced expression of genes mediating bile acids (BA) secretion, suggesting that psyllium's known ability to bind BA might contribute to its ability to prevent colitis. As expected, psyllium resulted in elevated level of fecal BA, reflecting their removal from enterohepatic circulation but, in stark contrast to the BA sequestrant cholestyramine, increased serum BA levels. Moreover, the use of BA mimetics that activate the farnesoid X receptor (FXR), as well as the use of FXR-knockout mice, suggested that activation of FXR plays a central role in psyllium's protection against colitis. CONCLUSIONS: Psyllium protects against colitis via altering BA metabolism resulting in activation of FXR, which suppresses pro-inflammatory signaling.

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.

Selenium-dependent metabolic reprogramming during inflammation and resolution.

Trace element selenium (Se) is incorporated as the 21st amino acid, selenocysteine, into selenoproteins through tRNA[Ser]Sec. Selenoproteins act as gatekeepers of redox homeostasis and modulate immune function to effect anti-inflammation and resolution. However, mechanistic underpinnings involving metabolic reprogramming during inflammation and resolution remain poorly understood. Bacterial endotoxin lipopolysaccharide (LPS) activation of murine bone marrow-derived macrophages cultured in the presence or absence of Se (as selenite) was used to examine temporal changes in the proteome and metabolome by multiplexed tandem mass tag-quantitative proteomics, metabolomics, and machine-learning approaches. Kinetic deltagram and clustering analysis indicated that addition of Se led to extensive reprogramming of cellular metabolism upon stimulation with LPS enhancing the pentose phosphate pathway, tricarboxylic acid cycle, and oxidative phosphorylation, to aid in the phenotypic transition toward alternatively activated macrophages, synonymous with resolution of inflammation. Remodeling of metabolic pathways and consequent metabolic adaptation toward proresolving phenotypes began with Se treatment at 0 h and became most prominent around 8 h after LPS stimulation that included succinate dehydrogenase complex, pyruvate kinase, and sedoheptulokinase. Se-dependent modulation of these pathways predisposed bone marrow-derived macrophages to preferentially increase oxidative phosphorylation to efficiently regulate inflammation and its timely resolution. The use of macrophages lacking selenoproteins indicated that all three metabolic nodes were sensitive to selenoproteome expression. Furthermore, inhibition of succinate dehydrogenase complex with dimethylmalonate affected the proresolving effects of Se by increasing the resolution interval in a murine peritonitis model. In summary, our studies provide novel insights into the role of cellular Se via metabolic reprograming to facilitate anti-inflammation and proresolution.

Vancomycin prevents fermentable fiber-induced liver cancer in mice with dysbiotic gut microbiota.

Owing to their health benefits, dietary fermentable fibers, such as refined inulin, are increasingly fortified in processed foods to enhance their nutritional value. However, we previously demonstrated that when inulin was fed to Toll-like receptor 5 deficient (T5KO) mice susceptible to dysbiosis, a subset of them developed cholestasis and subsequently liver cancer in a gut microbiota-dependent manner. Therefore, we hypothesized that clearance of bacterial taxa, and thereby gut metabolites, involved in the onset and progression to liver cancer could abate the disease in these mice. Such a reshaping of microbiota by vancomycin treatment was sufficient to halt the development of liver cancer in inulin-fed T5KO mice; however, this intervention did not remedy disease penetrance for cholestatic liver injury and its sequelae, including hyperbilirubinemia, hypolipidemia, cholemia and liver fibrosis. Selective depletion of gut bacterial communities was observed in vancomycin-treated mice, including Gram-positive Lachnospiraceae and Ruminococcaceae belonging to the phylum Firmicutes, Bifidobacteria of the phylum Actinobacteria, which ferment fibers, and Clostridium cluster XIVa, which produce secondary bile acids. Lack of liver cancer in vancomycin-treated mice strongly correlated with the substantial loss of secondary bile acids in circulation. Although cholemia was unabated by vancomycin, the composition of serum bile acids shifted toward an abundance of hydrophilic primary bile acids, denoted by the increase in conjugated-to-unconjugated bile acid ratio. Taken together, the present study suggests that microbiotal regulation of bile acid metabolism is one of the critical mediators of fermentable fiber-induced liver cancer in dysbiotic mice.

Gender Differences in Phytoestrogens and the Relationship with Speed of Processing in Older Adults: A Cross-Sectional Analysis of NHANES, 1999-2002.

Sex hormone changes in adults are known to play a part in aging, including cognitive aging. Dietary intake of phytoestrogens can mimic estrogenic effects on brain function. Since sex hormones differ between genders, it is important to examine gender differences in the phytoestrogen-cognition association. Therefore, the goal of this study is to examine the relationship between urinary phytoestrogens and speed of processing (SOP) and the variation of the association between genders in older adults. Participants were drawn from the 1999-2002 National Health and Nutrition Examination Survey and included 354 individuals aged 65-85 years old. General linear models (GLMs) were used to test for significant gender differences in the relationship between phytoestrogens and SOP. Results from the GLMs showed significant gender differences in the relationship between genistein and SOP. Higher levels of genistein were associated with better SOP in women. This relationship was reversed in men: higher genistein levels were associated with worse performance. Results indicate that there are distinct gender differences in the relationship between genistein and SOP. These results emphasize the importance of considering gender differences when devising dietary and pharmacologic interventions that target phytoestrogens to improve brain health.

Isolation and Identification of Aryl Hydrocarbon Receptor Modulators in White Button Mushrooms (Agaricus bisporus).

Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional and germ-free mice. High-performance liquid chromatography (HPLC) fractionation and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) combined with an AHR-responsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by the cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cells by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated HPLC fractionation and UHPLC-MS/MS combined with a cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.

Lipid Emulsion Added to a Liquid High-Carbohydrate Diet and Voluntary Running Exercise Reduce Lipogenesis and Ameliorate Early-Stage Hepatic Steatosis in Mice.

Background: The use of parenteral nutrition formulas is often associated with the development of hepatic steatosis. We have shown previously that the addition of a lipid emulsion (LE) rich in n-6 (ω-6) fatty acids (FAs) ameliorated triglyceride (TG) accumulation in the livers of nonobese mice fed a high-carbohydrate diet (HCD) for 5 wk. However, it remains unclear how rapidly this condition develops and whether it can be prevented by LE with or without a running wheel for voluntary exercise (Exe).Objective: We investigated in an 8-d study whether mice develop steatosis and whether the administration of LE with or without Exe reduces the concentration of total FAs and prevents an increase in the expression of genes in the liver associated with lipogenesis.Methods: Male C57BL/6 mice aged 5 wk were randomized into 5 groups: standard feed pellet (SFP); a liquid HCD (77% of total energy from carbohydrates and 0.5% from fat); HCD + Exe; HCD + 13.5% LE (67% carbohydrates and 13.5% fat); or HCD + 13.5% LE + Exe. Hepatic TG concentration, lipogenic genes, and total FAs were measured on day 8.Results: Oil Red O staining and TG quantification showed hepatic TG accumulation on day 8; the addition of 13.5% LE either with or without Exe suppressed the TG accumulation compared with HCD (P < 0.005). With the use of quantitative reverse transcriptase-polymerase chain reaction analysis, the expression concentrations of lipogenic genes [ATP-citrate lyase, acetyl coenzyme A carboxylase 1, FA synthase (Fasn), and stearoyl coenzyme A desaturase 1 (Scd1)] in the HCD + 13.5% LE group were 26-60% of HCD (P < 0.01) and 11-38% of HCD in the HCD + 13.5% LE + Exe group (P < 0.001), with interactions for Fasn and Scd1 (P < 0.05). With the use of gas chromatography-mass spectrometry analysis, the HCD + 13.5% LE group had lower monounsaturated fatty acids (38.7% of HCD) but higher polyunsaturated fatty acids (164% of HCD) (P < 0.001).Conclusions: In short-term studies designed to resemble the early dynamic stage of the development of hepatic steatosis, the addition of 13.5% LE to a liquid HCD reduced hepatic lipogenesis. Exe exerted an independent protective effect and interacted with LE to further reduce the expression of Scd1.

Modulation of urinary siderophores by the diet, gut microbiota and inflammation in mice.

Mammalian siderophores are believed to play a critical role in maintaining iron homeostasis. However, the properties and functions of mammalian siderophores have not been fully clarified. In this study, we have employed Chrome Azurol S (CAS) assay which is a well-established method for bacterial siderophores study, to detect and quantify mammalian siderophores in urine samples. Our study demonstrates that siderophores in urine can be altered by diet, gut microbiota and inflammation. C57BL/6 mice, fed on plant-based chow diets which contain numerous phytochemicals, have more siderophores in the urine compared to those fed on purified diets. Urinary siderophores were up-regulated in iron overload conditions, but not altered by other tested nutrients status. Further, germ-free mice displayed 50% reduced urinary siderophores, in comparison to conventional mice, indicating microbiota biotransformation is critical in generating or stimulating host metabolism to create more siderophores. Altered urinary siderophores levels during inflammation suggest that host health conditions influence systemic siderophores level. This is the first report to measure urinary siderophores as a whole, describing how siderophores levels are modulated under different physiological conditions. We believe that our study opens up a new field in mammalian siderophores research and the technique we used in a novel manner has the potential to be applied to clinical purpose.

Editor's Highlight: Perfluorooctane Sulfonate-Choline Ion Pair Formation: A Potential Mechanism Modulating Hepatic Steatosis and Oxidative Stress in Mice.

The mechanisms underlying perfluorooctane sulfonate (PFOS)-induced steatosis remain unclear. The hypothesis that PFOS causes steatosis and other hepatic effects by forming an ion pair with choline was examined. C57BL/6 mice were fed either a control diet or a marginal methionine/choline-deficient (mMCD) diet, with and without 0.003, 0.006, or 0.012% potassium PFOS. Dietary PFOS caused a dose-dependent decrease in body weight, and increases in the relative liver weight, hepatic triglyceride concentration and serum markers of liver toxicity and oxidative stress. Some of these effects were exacerbated in mice fed the mMCD diet supplemented with 0.012% PFOS compared with those fed the control diet supplemented with 0.012% PFOS. Surprisingly, serum PFOS concentrations were higher while liver PFOS concentrations were lower in mMCD-fed mice compared with corresponding control-fed mice. To determine if supplemental dietary choline could prevent PFOS-induced hepatic effects, C57BL/6 mice were fed a control diet, or a choline supplemental diet (1.2%) with or without 0.003% PFOS. Lipidomic analysis demonstrated that PFOS caused alterations in hepatic lipid metabolism in the PFOS-fed mice compared with controls, and supplemental dietary choline prevented these PFOS-induced changes. Interestingly, dietary choline supplementation also prevented PFOS-induced oxidative damage. These studies are the first to suggest that PFOS may cause hepatic steatosis and oxidative stress by effectively reducing the choline required for hepatic VLDL production and export by forming an ion pair with choline, and suggest that choline supplementation may prevent and/or treat PFOS-induced hepatic steatosis and oxidative stress.

Modulation of colon cancer by nutmeg.

Colon cancer is the most common cancer and the third leading cause of cancer mortality in humans. Using mass spectrometry-based metabolomics, the current study revealed the accumulation of four uremic toxins (cresol sulfate, cresol glucuronide, indoxyl sulfate, and phenyl sulfate) in the serum of mice harboring adenomatous polyposis coli (APC) gene mutation-induced colon cancer. These uremic toxins, likely generated from the gut microbiota, were associated with an increase in the expression of the proinflammatory cytokine IL-6 and a disorder of lipid metabolism. Nutmeg, which exhibits antimicrobial activity, attenuated the levels of uremic toxins and decreased intestinal tumorigenesis in Apc(min/+) mice. Nutmeg-treated Apc(min/+) mice had decreased IL-6 levels and normalized dysregulated lipid metabolism, suggesting that uremic toxins are responsible, in part, for the metabolic disorders that occur during tumorigenesis. These studies demonstrate a potential biochemical link among gut microbial metabolism, inflammation, and metabolic disorders and suggest that modulation of gut microbiota and lipid metabolism using dietary intervention or drugs may be effective in colon cancer chemoprevention strategies.

Crucial role of macrophage selenoproteins in experimental colitis.

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trsp(fl/fl)LysM(Cre)) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

Novel metabolites and roles for α-tocopherol in humans and mice discovered by mass spectrometry-based metabolomics.

BACKGROUND: Contradictory results from clinical trials that examined the role of vitamin E in chronic disease could be a consequence of interindividual variation, caused by factors such as xenobiotic use. Cometabolism of vitamin E with other pharmaceutical products could affect the bioavailability of the drug. Thus, it is necessary to understand fully the metabolic routes and biological endpoints of vitamin E. OBJECTIVE: The objective was to uncover novel metabolites and roles of vitamin E in humans and mouse models. DESIGN: Human volunteers (n = 10) were fed almonds for 7 d and then an α-tocopherol dietary supplement for 14 d. Urine and serum samples were collected before and after dosing. C57BL/6 mice (n = 10) were also fed α-tocopherol-deficient and -enriched diets for 14 d. Urine, serum, and feces were collected before and after dosing, and liver samples were collected after euthanization. Ultraperformance liquid chromatography electrospray ionization time-of-flight mass spectrometry and multivariate data analysis tools were used to analyze the samples. RESULTS: Three novel urinary metabolites of α-tocopherol were discovered in humans and mice: α-carboxyethylhydroxychroman (α-CEHC) glycine, α-CEHC glycine glucuronide, and α-CEHC taurine. Another urinary metabolite, α-CEHC glutamine, was discovered in mice after α-CEHC gavage. Increases in liver fatty acids and decreases in serum and liver cholesterol were observed in mice fed the α-tocopherol-enriched diet. CONCLUSION: Novel metabolites and metabolic pathways of vitamin E were identified by mass spectrometry-based metabolomics and will aid in understanding the disposition and roles of vitamin E in vivo.

Disruption of phospholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can develop into cirrhosis, hepatic failure, and hepatocellular carcinoma. Although several metabolic pathways are disrupted and endogenous metabolites may change in NASH, the alterations in serum metabolites during NASH development remain unclear. To gain insight into the disease mechanism, serum metabolite changes were assessed using metabolomics with ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and a conventional mouse NASH model induced by a methionine- and choline-deficient (MCD) diet. Significant decreases in serum palmitoyl-, stearoyl-, and oleoyl-lysophosphatidylcholine (LPC) and marked increases in tauro-ß-muricholate, taurocholate and 12-hydroxyeicosatetraenoic acid (12-HETE) were detected in mice with NASH. In agreement with these metabolite changes, hepatic mRNAs encoding enzymes and proteins involved in LPC degradation (lysophosphatidylcholine acyltransferase [Lpcat] 1-4), basolateral bile acid excretion (ATP-binding cassette subfamily C member [Abcc] 1/4/5 and organic solute transporter ß), and 12-HETE synthesis (arachidonate 12-lipoxygenase) were significantly up-regulated. In contrast, the expression of solute carrier family 10 member 1 (Slc10a1) and solute carrier organic anion transporter family member (Slco) 1a1 and 1b2, responsible for transporting bile acids into hepatocytes, were markedly suppressed. Supplementation of the MCD diet with methionine revealed that the changes in serum metabolites and the related gene expression were derived from steatohepatitis, but not dietary choline deficiency or steatosis. Furthermore, tumor necrosis factor-α and transforming growth factor-ß1 induced the expression of Lpcat2/4 and Abcc1/4 and down-regulated Slc10a1 and Slco1a1 in primary hepatocytes, suggesting an association between the changes in serum LPC and bile acids and proinflammatory cytokines. Finally, induction of hepatitis in ob/ob mice by D-galactosamine injection led to similar changes in serum metabolites and related gene expression. CONCLUSION: Phospholipid and bile acid metabolism is disrupted in NASH, likely due to enhanced hepatic inflammatory signaling.

Xenobiotic metabolism: a view through the metabolometer.

The combination of advanced ultraperformance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here, a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Last, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse and can be adapted to study xenobiotic metabolism in any animal species, including humans. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic, thereby enhancing knowledge of their modes of toxic action.