Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.,PLOS ONE

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Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.
PLOS ONE ( IF 2.9 ) Pub Date : 2019-03-15 , DOI: 10.1371/journal.pone.0214000
Nobumasa Hara ₁ , Harumi Osago ₁ , Mineyoshi Hiyoshi ₁ , Mikiko Kobayashi-Miura ₁ , Mikako Tsuchiya ₁

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NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (μM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 μM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown.

中文翻译：

定量分析烟酰胺磷酸核糖基转移酶诱导对哺乳动物细胞中NAD +合成和分解的速率的影响，方法是使用稳定的同位素标记和质谱联用。

NAD +主要由限速酶Nam磷酸核糖基转移酶（Nampt）由烟酰胺（Nam）合成，并在哺乳动物中被NAD +降解酶降解为Nam。大量研究报告说，在衰老和与年龄有关的疾病中，组织中NAD +的含量会下降，并表明补充NAD +可以促进健康的衰老。尽管增加Nampt的表达可能是健康衰老的有前途的干预手段，但据报道，强迫表达Nampt基因导致酶蛋白水平增加了10倍以上，据报道仅能使哺乳动物细胞中NAD +的水平提高40-60％。NAD +水平有限增加的潜在机制仍有待确定。在这里，我们显示Nampt在细胞中被抑制，并且Nampt的增强表达激活NAD +分解。结合对每个细胞体积的测量，我们使用带有2H（D）标记的Nam的通量测定法确定了NAD +合成（RS）和分解（RB）速率的绝对值（μM/ h），以及包括原代培养物在内的各种哺乳动物细胞中的NAD +绝对浓度心肌细胞在生理条件下，研究了总细胞Nampt活性，RS，RB和NAD +浓度之间的关系。细胞中的NAD +浓度保持在狭窄范围内（400-700μM）。RS比总的Nampt活性小得多，表明细胞中Nam的NAD +合成受到抑制。强迫表达的Nampt导致总Nampt活性增加6倍，仅诱导细胞NAD +浓度增加1.6倍。在这种条件下，RS增加了2倍，而RB也增加了2倍。细胞中NAD +浓度的小幅增加可能是由于NAD +合成的抑制增加及其分解的激活所致。我们的发现表明，细胞内NAD +的浓度不会因Nampt表达的生理波动而发生显着变化，并且表明NAD +合成与其分解之间存在紧密联系。

更新日期：2019-03-17

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