Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, DK2200 Copenhagen, Denmark.
Xlab, Center for Healthy Aging, Department of Biomedical Sciences, University of Copenhagen, DK2200 Copenhagen, Denmark.
J Biol Chem. 2019 Sep 6;294(36):13304-13326. doi: 10.1074/jbc.RA118.006756. Epub 2019 Jul 18.
Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD levels, but the metabolic phenotype of deficient hepatocytes in mice is unknown. Here, we assessed role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific knockout (HNKO) mice, having a 50% reduction of liver NAD levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD, suggesting that the mitochondrial NAD pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [N]glutamine, HNKO hepatocytes had a higher [N]NAD enrichment than WT hepatocytes, indicating that HNKO mice compensate through NAD synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD levels and that the knockout has only minor consequences for mitochondrial function in the mouse liver.
补充烟酰胺核糖（NR）等 NAD 前体已被证明可以增强肝脏中的线粒体功能，并防止高脂肪饮食（HFD）喂养的啮齿动物肝脂质积累。NAD 合成酶烟酰胺磷酸核糖转移酶（NAMPT）的肝特异性敲除会降低肝脏 NAD 水平，但缺乏 NAMPT 的肝细胞在小鼠中的代谢表型尚不清楚。在这里，我们评估了它在维持小鼠肝脏线粒体和代谢功能中的作用。我们使用 Cre-LoxP 系统生成了肝特异性敲除（HNKO）小鼠，其肝脏 NAD 水平降低了 50%。我们对 HFD 喂养 20 周（±NR 补充）后 HNF4A 敲除小鼠的代谢功能障碍迹象进行了筛选，发现 NR 增加了肝 NAD 水平，但不影响 HNKO 或 WT 动物的脂肪量或葡萄糖耐量。高分辨率呼吸测定显示，NR 补充并未增加 HNKO 小鼠的状态 III 呼吸，而在 NR 补充的 WT 小鼠中观察到了这种情况。HNKO 原代肝细胞的线粒体耗氧量和脂肪酸氧化没有改变。从整个 HNF4A 敲除肝脏中分离出的线粒体 NAD 减少了 20%，这表明与整个组织 NAD 池相比，HNKO 对线粒体 NAD 池的影响较小。当在存在[N]谷氨酰胺的情况下用色氨酸刺激时，HNKO 肝细胞的[N]NAD 富集高于 WT 肝细胞，这表明 HNF4A 敲除小鼠通过补救途径合成 NAD 来进行代偿。我们得出结论，NAMPT 缺陷型肝细胞可以维持相当高的 NAD 水平，而 HNF4A 敲除对小鼠肝脏线粒体功能的影响很小。
