Background & Aims

The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice.

Methods

We generated MNADK knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate, and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines.

Results

The KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of nonalcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein-hepatocyte specific and peroxisome proliferator–activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, an NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase.

Conclusion

MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.

中文翻译：

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线粒体NAD激酶的缺乏导致应激诱导小鼠肝脂肪变性。

背景与目标

线粒体烟酰胺腺嘌呤二核苷酸（NAD）激酶（NADK2，也称为MNADK）催化NAD磷酸化以产生NADP。关于线粒体NADP和MNADK在肝脏生理学和病理学中的功能知之甚少。我们研究了通过删除小鼠中的MNADK来降低线粒体NADP的作用。

方法

我们在C57BL / 6NTac背景上产生了MNADK基因敲除（KO）小鼠；具有野生型Mnadk基因的小鼠用作对照。将一些小鼠置于致动脉粥样硬化的高脂肪饮食中（16％脂肪，41％碳水化合物和1.25％胆固醇补充0.5％胆酸钠）或腹膜内给予甲氨蝶呤。我们使用放射性标记的棕榈酸酯测量了原代肝细胞中脂肪酸氧化的速率，并使用间接量热法测量了小鼠中脂肪酸的氧化速率。我们测量了小鼠肝脏和原代肝细胞中活性氧的水平。代谢组学分析用于定量血清代谢产物，例如氨基酸和酰基肉碱。

结果

KO小鼠具有MNADK缺陷型患者的代谢特征，例如赖氨酸和C10：2肉碱的血清浓度升高。与对照小鼠相比，KO小鼠以致动脉粥样硬化的高脂饮食喂养时，会表现出非酒精性脂肪肝疾病的特征，并且肝脏和原代肝细胞中的活性氧水平升高。禁食期间，KO小鼠的脂肪酸氧化有缺陷。MNADK缺乏症减少了cAMP反应元件结合蛋白，肝细胞特异性和过氧化物酶体增殖物激活的受体α的激活，后者是介导空腹反应的转录激活因子。KO小鼠肝脏中线粒体sirtuins的活性降低。甲氨蝶呤抑制甲氨蝶呤注射液对小鼠肝细胞和肝脏中MNADK的催化活性。

结论

MNADK促进脂肪酸氧化，抵消氧化损伤，维持线粒体瑟土因活性，并防止小鼠因代谢应激而引起的非酒精性脂肪肝疾病。