Mitochondrial amidoxime-reducing component 2 (MARC2) has a significant role in N-reductive activity and energy metabolism

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.

Keywords: N reduction; drug metabolism; energy metabolism; enzyme catalysis; gene KO; lipid metabolism; mitochondrial amideoxime-reducing component (mARC); molybdenum; transgenic mice; xenobiotic metabolism.

Figure 1.

Schematic of the MARC enzyme system. Shown is the N-reductive enzyme system consisting of CYB5R, CYB5B, and MARC as well as their cofactors FAD, heme, and the molybdenum cofactor (Moco). Catalysis is demonstrated by means of the reduction of an amidoxime.

Figure 2.

MARC2 KO mice are resistant to HFD-induced obesity. A, mean body weights of mice in each treatment group throughout a period of 23 weeks. Two groups (WT and MARC2 KO) were fed an ND (10% of calories from fat), and the other two (WT and MARC2 KO) were fed an HFD (60% of calories from fat). Differences were tested at the end of the experiment with a U test (n = 8). Whiskers indicate S.D. B, representative liver histology of WT and MARC2 KO mice on the HFD at the end of the 23-weeks experiment, stained with hematoxylin and eosin (magnification, ×200). C, serum biochemical readouts. The differences in pairwise comparisons were tested with a t test (n = 8). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Figure 3.

Serum concentration of BAO in WT and KO mice. BAO serum concentrations of six WT and six KO mice. Statistical significance was proven by U test. The limit of detection was at 2.5 μmol of BAO/liter of serum. **, p < 0.01.

Figure 4.

Correlation of reductase activity and protein expression. A, N-reductive activity was determined by reduction of BAO in multiple incubations (N ≥ 4) of different tissue homogenates of four individual WT mice over a time period of 20 min. B, homogenates were examined via Western blot analysis. C, expression levels of POI were measured and normalized to the loading control (GAPDH) per lane. Validation of the GAPDH signal for normalization and full original western blots are shown in Figs. S1 and S7.

Figure 5.

Protein expression of tissues from KO and WT mice. A, protein expression of six individual MARC2 KO mice and six individual WT mice was examined immunologically by SDS-PAGE and subsequent Western blot analysis. Representative COX4 loading control lanes are shown (for details, see Figs. S3–S6). B, quantification of expression levels. The ration of POI signal to loading control signal was calculated per lane. Validation of the COX4 signal for normalization and full original western blots are shown in Figs. S2–S6. Significance was proven by t and U test. *, p < 0.05; ***, p < 0.001; n.s., not significant.

Figure 6.

Reductase activity of WT and KO tissue homogenates. A and B, tested N-oxygenated substrates (A) and N-reductive activity (B) of tissue homogenates (liver, kidneys, and lungs) of six individual WT and MARC2 KO mice. Every homogenate was incubated separately in multiple incubations (N ≥ 4) over a time range of up to 90 min. The limit of quantification was 0.1 nmol·mg⁻¹·min⁻¹ for BAO and guanoxabenz and 0.02 nmol·mg⁻¹·min⁻¹ for N⁴-hydroxycytidine and amitriptyline-N-oxide. Statistical significance was proven by U test and t test. Negative controls without NADH were performed with each substrate, and none of the respective metabolites could be detected in these samples. In case of guanoxabenz, the detected residual N-reductive activity of KO samples (2.4 ± 0.1 nmol·mg⁻¹·min⁻¹) meets the value of the negative control without NADH (2.1 ± 0.1 nmol·mg⁻¹·min) and was corrected by this value. #, below the limit of quantification. **, p < 0.01; ***, p < 0.001; n.s., not significant.