The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice

PLoS One. 2014 Aug 21;9(8):e105371. doi: 10.1371/journal.pone.0105371. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzamidines / metabolism
  • Cell Line, Tumor
  • Cytochrome-B(5) Reductase / metabolism*
  • Cytochromes b5 / metabolism*
  • Diet, High-Fat*
  • Enzyme Activation
  • Fasting*
  • Humans
  • Hyperphagia / metabolism
  • Lipid Metabolism
  • Male
  • Mice
  • Mitochondrial Proteins / metabolism*
  • Oxidoreductases / metabolism*

Substances

  • Benzamidines
  • Mitochondrial Proteins
  • benzamidoxime
  • Cytochromes b5
  • Oxidoreductases
  • Cytochrome-B(5) Reductase
  • benzamidine

Grants and funding

This work was supported by N N401 017436 and N N401 532240 grants from Polish Ministry of Science and Higher Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.