Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5 -/- mice, decreases plasma cholesterol and epididymal fat deposition.

We previously showed that Fmo5 -/- mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5 -/- and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5 -/- mice. Antibiotic-treatment of Fmo5 -/- mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5 -/- mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5 -/- to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5 -/- mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5 -/- mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

Metabolic Biomarkers of Ageing in C57BL/6J Wild-Type and Flavin-Containing Monooxygenase 5 (FMO5)-Knockout Mice.

It was recently demonstrated in mice that knockout of the flavin-containing monooxygenase 5 gene, Fmo5, slows metabolic ageing via pleiotropic effects. We have now used an NMR-based metabonomics approach to study the effects of ageing directly on the metabolic profiles of urine and plasma from male, wild-type C57BL/6J and Fmo5-/- (FMO5 KO) mice back-crossed onto the C57BL/6J background. The aim of this study was to identify metabolic signatures that are associated with ageing in both these mouse lines and to characterize the age-related differences in the metabolite profiles between the FMO5 KO mice and their wild-type counterparts at equivalent time points. We identified a range of age-related biomarkers in both urine and plasma. Some metabolites, including urinary 6-hydroxy-6-methylheptan-3-one (6H6MH3O), a mouse sex pheromone, showed similar patterns of changes with age, regardless of genetic background. Others, however, were altered only in the FMO5 KO, or only in the wild-type mice, indicating the impact of genetic modifications on mouse ageing. Elevated concentrations of urinary taurine represent a distinctive, ageing-related change observed only in wild-type mice.

Effect of Flavin-Containing Monooxygenase Genotype, Mouse Strain, and Gender on Trimethylamine N-oxide Production, Plasma Cholesterol Concentration, and an Index of Atherosclerosis.

The objectives of the study were to determine the contribution, in mice, of members of the flavin-containing monooxygenase (FMO) family to the production of trimethylamine (TMA) N-oxide (TMAO), a potential proatherogenic molecule, and whether under normal dietary conditions differences in TMAO production were associated with changes in plasma cholesterol concentration or with an index of atherosclerosis (Als). Concentrations of urinary TMA and TMAO and plasma cholesterol were measured in 10-week-old male and female C57BL/6J and CD-1 mice and in mouse lines deficient in various Fmo genes (Fmo1-/- , 2-/- , 4-/- , and Fmo5-/- ). In female mice most TMA N-oxygenation was catalyzed by FMO3, but in both genders 11%-12% of TMA was converted to TMAO by FMO1. Gender-, Fmo genotype-, and strain-related differences in TMAO production were accompanied by opposite effects on plasma cholesterol concentration. Plasma cholesterol was negatively, but weakly, correlated with TMAO production and urinary TMAO concentration. Fmo genotype had no effect on Als. There was no correlation between Als and either TMAO production or urinary TMAO concentration. Our results indicate that under normal dietary conditions TMAO does not increase plasma cholesterol or act as a proatherogenic molecule.

Identification of Flavin-Containing Monooxygenase 5 (FMO5) as a Regulator of Glucose Homeostasis and a Potential Sensor of Gut Bacteria.

We have previously identified flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic aging. The aim of the present study was to investigate the role of FMO5 in glucose homeostasis and the impact of diet and gut flora on the phenotype of mice in which the Fmo5 gene has been disrupted (Fmo5-/- mice). In comparison with wild-type (WT) counterparts, Fmo5-/- mice are resistant to age-related changes in glucose homeostasis and maintain the higher glucose tolerance and insulin sensitivity characteristic of young animals. When fed a high-fat diet, they are protected against weight gain and reduction of insulin sensitivity. The phenotype of Fmo5-/- mice is independent of diet and the gut microbiome and is determined solely by the host genotype. Fmo5-/- mice have metabolic characteristics similar to those of germ-free mice, indicating that FMO5 plays a role in sensing or responding to gut bacteria. In WT mice, FMO5 is present in the mucosal epithelium of the gastrointestinal tract where it is induced in response to a high-fat diet. In comparison with WT mice, Fmo5-/- mice have fewer colonic goblet cells, and they differ in the production of the colonic hormone resistin-like molecule ßFmo5-/- mice have lower concentrations of tumor necrosis factor α in plasma and of complement component 3 in epididymal white adipose tissue, indicative of improved inflammatory tone. Our results implicate FMO5 as a regulator of body weight and of glucose disposal and insulin sensitivity and, thus, identify FMO5 as a potential novel therapeutic target for obesity and insulin resistance.

A guide to the identification of metabolites in NMR-based metabonomics/metabolomics experiments.

Metabonomics/metabolomics is an important science for the understanding of biological systems and the prediction of their behaviour, through the profiling of metabolites. Two technologies are routinely used in order to analyse metabolite profiles in biological fluids: nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), the latter typically with hyphenation to a chromatography system such as liquid chromatography (LC), in a configuration known as LC-MS. With both NMR and MS-based detection technologies, the identification of the metabolites in the biological sample remains a significant obstacle and bottleneck. This article provides guidance on methods for metabolite identification in biological fluids using NMR spectroscopy, and is illustrated with examples from recent studies on mice.

The phenotype of a knockout mouse identifies flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic ageing.

We report the production and metabolic phenotype of a mouse line in which the Fmo5 gene is disrupted. In comparison with wild-type (WT) mice, Fmo5(-/-) mice exhibit a lean phenotype, which is age-related, becoming apparent after 20 weeks of age. Despite greater food intake, Fmo5(-/-) mice weigh less, store less fat in white adipose tissue (WAT), have lower plasma glucose and cholesterol concentrations and enhanced whole-body energy expenditure, due mostly to increased resting energy expenditure, with no increase in physical activity. An increase in respiratory exchange ratio during the dark phase, the period in which the mice are active, indicates a switch from fat to carbohydrate oxidation. In comparison with WT mice, the rate of fatty acid oxidation in Fmo5(-/-) mice is higher in WAT, which would contribute to depletion of lipid stores in this tissue, and lower in skeletal muscle. Five proteins were down regulated in the liver of Fmo5(-/-) mice: aldolase B, ketohexokinase and cytosolic glycerol 3-phosphate dehydrogenase (GPD1) are involved in glucose or fructose metabolism and GPD1 also in production of glycerol 3-phosphate, a precursor of triglyceride biosynthesis; HMG-CoA synthase 1 is involved in cholesterol biosynthesis; and malic enzyme 1 catalyzes the oxidative decarboxylation of malate to pyruvate, in the process producing NADPH for use in lipid and cholesterol biosynthesis. Down regulation of these proteins provides a potential explanation for the reduced fat deposits and lower plasma cholesterol characteristic of Fmo5(-/-) mice. Our results indicate that disruption of the Fmo5 gene slows metabolic ageing via pleiotropic effects.

The phenotype of a flavin-containing monooyxgenase knockout mouse implicates the drug-metabolizing enzyme FMO1 as a novel regulator of energy balance.

Flavin-containing monooxygenases (FMOs) of mammals are thought to be involved exclusively in the metabolism of foreign chemicals. Here, we report the unexpected finding that mice lacking Fmos 1, 2 and 4 exhibit a lean phenotype and, despite similar food intake, weigh less and store less triglyceride in white adipose tissue (WAT) than wild-type mice. This is a consequence of enhanced whole-body energy expenditure, due mostly to increased resting energy expenditure (REE). This is fuelled, in part, by increased fatty acid ß-oxidation in skeletal muscle, which would contribute to depletion of lipid stores in WAT. The enhanced energy expenditure is attributed, in part, to an increased capacity for exercise. There is no evidence that the enhanced REE is due to increased adaptive thermogenesis; instead, our results are consistent with the operation in WAT of a futile energy cycle. In contrast to FMO2 and FMO4, FMO1 is highly expressed in metabolic tissues, including liver, kidney, WAT and BAT. This and other evidence implicates FMO1 as underlying the phenotype. The identification of a novel, previously unsuspected, role for FMO1 as a regulator of energy homeostasis establishes, for the first time, a role for a mammalian FMO in endogenous metabolism. Thus, FMO1 can no longer be considered to function exclusively as a xenobiotic-metabolizing enzyme. Consequently, chronic administration of drugs that are substrates for FMO1 would be expected to affect energy homeostasis, via competition for endogenous substrates, and, thus, have important implications for the general health of patients and their response to drug therapy.