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

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Abstract

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 β-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.

Introduction

Flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) catalyze the NADPH-dependent oxidative metabolism of many structurally diverse foreign chemicals, including therapeutic drugs, pesticides and dietary-derived compounds [1]. The human genome contains 11 FMO genes, five of which, FMO1, 2, 3, 4 and 5, are functional [2], [3]. FMOs 1–4 are clustered on chromosome 1, in the region q24.3, and FMO5 is located at 1q21.1. In mouse, the Fmo1–4 genes are clustered on Chromosome 1 and Fmo5 is on Chromosome 3 [2].

Drug substrates of FMOs include anti-depressants, anti-tuberculars, inhibitors of monoamine oxidase A and B and the anti-oestrogen tamoxifen [1]. FMOs are categorized, therefore, as drug-metabolizing enzymes and, next to the cytochromes P450, are the second largest protein family involved in Phase-I drug metabolism.

A small number of potential endogenous substrates, cysteamine [4], lipoic acid and lipoamide [5], have been identified for FMO1. However, a direct link between FMO1 and the metabolism of these chemicals in vivo has not been established. For most mammalian FMOs an endogenous role appears unlikely. The majority of humans do not express FMO2 because of a nonsense mutation, which results in the production of a non-functional protein [6], [7]. Mutations in FMO3 cause the disorder trimethylaminuria [8], which is characterized by an unpleasant body odour, due to a defect in the ability to convert trimethylamine to trimethylamine N-oxide, but has no overt physiological symptoms [9]. Little is known of FMO4 and the protein has yet to be detected in vivo. A physiological role for FMO1 is, however, suspected. The coding sequence of the human FMO1 gene contains few single-nucleotide polymorphisms, each of which is present at low frequency [3], [10] and does not significantly affect catalytic activity [11]. In mouse, expression of the Fmo1 gene is subject to a circadian rhythm [12].

To investigate the roles of FMOs in multi-pathway drug metabolism we developed a mouse line that lacks Fmo1, Fmo2 and Fmo4 genes and, thus, whose FMO content in liver reflects that of adult humans [13], [14]. The mouse line has been used to identify the role of FMO1 in the metabolism of the anti-depressant imipramine [14], [15] and of FMO2 in the metabolism of the anti-tubercular ethionamide [16]. The line also provides a valuable experimental model for investigation of the endogenous role of FMO. Here, we report the metabolic phenotype of the mouse line, which identifies, for the first time, a role for a mammalian FMO in endogenous metabolism. The phenotype indicates that FMO1 is a novel regulator of energy balance. This unexpected role means that FMO1 can no longer be considered to function solely as a xenobiotic-metabolizing enzyme and, importantly, that treatment of individuals with drugs that are substrates of FMO1 might affect energy metabolism.

Section snippets

Animal maintenance, body parameter and food measurements

Production of the knockout (KO) mouse line was as previously described [13], [14]. Heterozygous KO mice were backcrossed with wild-type (WT) C57BL/6 mice for eight generations to produce the congenic KO mouse line used in this study. WT C57BL/6 mice were used as controls. Mice were bred at UCL and fed a standard chow diet (Teklad Global 18% Protein Rodent Diet, Harlan Laboratories, Inc., Madison, WI). Experiments were done on 10-week-old male mice, unless stated otherwise. Body weight and food

Fmo knockout mice exhibit a lean phenotype, weigh less and have smaller fat depots than wild-type mice

Fmo1−/−, 2−/−, 4−/− mice were apparently healthy. However, when fed a standard diet, the KO mice weighed less than WT mice (Fig. 1A). The weight difference was maintained throughout the period studied (6–25 weeks of age) and occurred despite a similar intake of food (Fig. 1B). To investigate the basis of the difference in body weight, we focused on 10-week-old male animals. At this age KO mice (24.20 ± 0.40 g, n = 17) weighed 14% less than WT mice (28.14 ± 0.46 g, n = 24) (P < 0.0001). The nasal-to-anal

Discussion

The phenotype of a knockout mouse line, produced for investigation of the role of FMOs in the metabolism of drugs in vivo, has provided a valuable insight into the endogenous role of mammalian FMO. Mice that lack genes for FMO1, 2 and 4 appeared healthy, but were leaner than WT mice and stored less TG in WAT. The reduction in fat was secondary to enhanced whole-body energy expenditure, due mostly to increased REE. The RER indicates that both lipid and carbohydrate oxidation were higher in KO

Acknowledgements

We thank D. Saggerson and Y. Tsuchiya for advice on the β-oxidation assay and M. Sugden and M. Holness for comments on the typescript. Work was supported by scholarships or studentships from: ORS and UCL graduate School (SV), Drummond Trust (LH), BBSRC CASE (MH), CONACYT, Mexico (SGGM), The Wellcome Trust (FS) and Primer Design (SGGM, FS).

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