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

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.

线粒体脒肟还原组分（mitochondrial amidoxime reducing component, 下文简称mARC）是第四种哺乳动物钼酶。该蛋白能够还原N-氧化结构，但需要细胞色素b5（cytochrome b5）与细胞色素b5还原酶（cytochrome b5 reductase）参与电子传递，以催化此类反应。学界普遍认为，该酶参与N还原型药物代谢过程，例如脒肟类前体药物的活化。然而，该蛋白的内源性功能尚未完全阐明。在诸多潜在功能中，其被认为参与脂肪生成过程。为探究该蛋白在能量代谢中的潜在作用，我们检测了小鼠体内mARC蛋白及其互作蛋白是否会因禁食与高脂饮食发生表达调控变化。本研究采用qRT-PCR开展表达量分析、蛋白质印迹（Western Blot）实验检测蛋白水平，以及N还原型生物转化实验获取活性数据。实验结果证实，N还原系统的所有蛋白均受禁食调控，且其酶活性有所降低。为探究这些变化对体内前体药物活化的潜在影响，我们开展了另一项小鼠实验：向禁食处理的小鼠注射模型化合物苄脒肟（benzamidoxime），随后检测N还原反应的代谢产物苄脒（benzamidine）的含量。尽管体外实验中酶活性发生改变，但体内代谢产物浓度未出现可检测的变化，由此我们可以打消"禁食会改变动物模型中前体药物活化过程"的顾虑。针对高脂饮食情况，mARC蛋白会发生变化，进而导致N还原酶活性升高。本研究进一步证实，mARC蛋白的内源性功能与脂质代谢密切相关。