Abstract
The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.
Highlights
The mitochondrial amidoxime reducing component is the fourth mammalian molybdenum containing enzyme besides sulfite oxidase, xanthine oxidase and aldehyde oxidase [1], [2]
Gene expressions for mARC1, mARC2, cytochrome b5 reductase (CYB5R) and CYB5B were clearly enhanced in HepG2 cells that were grown either in 1.0 g/l or 4.5 g/l glucose
Transcripts changes for HepG2 cell line were mirrored in protein abundances where mARC1, CYB5R and CYB5B protein levels measured with Western Blot were higher in cells cultured with glucose. mARC2 was not detectable in any cell lysates
Summary
The mitochondrial amidoxime reducing component (mARC) is the fourth mammalian molybdenum containing enzyme besides sulfite oxidase, xanthine oxidase and aldehyde oxidase [1], [2]. High abundances of the enzyme after differentiation of 3T3-cells to adipocytes were shown [4], [14] and recently Neve et al provided evidence on its function in lipid synthesis [4] As the latter findings indicate an involvement in energy metabolism, we studied how changes in energy supply influence the mARC containing enzyme system. HFD and leptin-receptor-deficiency results in an obese habitus with most of the symptoms of the metabolic syndrome [16] In both cell culture and mice experiments, we focused on mRNA and protein levels such as the in vitro activity of the enzyme complex. We have been able to dispel these concerns during this study
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