Abstract
The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet-induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.
Highlights
The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme
The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes
Among the tested substrates, only amitriptyline N-oxide was reduced in murine KO samples to the same extent as WT samples (Fig. 6B). It is well-accepted, that MARC plays a crucial role in N-reductive xenobiotic and drug metabolism [11, 12]
Summary
An apparent phenotypical feature of MARC2 KO mice is a higher body temperature by, on average, one to two degrees (WT, 24.2 °C Ϯ 0.3; KO, 26.0 °C Ϯ 0.7) and a lower body weight. A previous study indicated that MARC1 and MARC2 protein abundance is increased in livers of animals on an HFD, suggesting a functional link between the MARC complex and lipid metabolism [16] In this study, both WT and MARC2 KO mice were fed an HFD or a normal diet (ND) for 23 weeks with weekly body weight measurements. 7-Fold increased levels of BAO in KO mice demonstrate a substantial decrease in N-reductive activity (Fig. 3). The expression pattern of MARC1/2 and CYB5B is well in agreement with high benzamidoxime reductase activity in the kidneys or liver and low reductase activity in the lungs (Fig. 4A). To investigate the influence of MARC2 on reductive metabolism, tissue from KO and WT mice was used for a reductase assay with several known substrates (Fig. 6A) and Western blot analysis. Among the tested substrates, only amitriptyline N-oxide was reduced in murine KO samples to the same extent as WT samples (Fig. 6B)
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