Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes
Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes
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DOI：
10.1074/jbc.m113.498626
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发表时间：
2014-01-24
影响因子：
4.8
通讯作者：
Lerche, Mathilde H.
中科院分区：
文献类型：
--
作者：
Christensen, Caspar Elo;Karlsson, Magnus;Lerche, Mathilde H.
越来越多的证据表明，吡啶核苷酸NAD具有比其在能量代谢中的经典作用更广泛的生物学功能。NAD被数百种催化底物氧化的酶使用，因此，它在各种生物过程中起着关键作用，如衰老，细胞死亡和氧化应激。已经表明游离胞质[NAD（+）]/[NADH]比率的变化反映了导致病理状态或与病理状态相关的代谢改变。我们设计了一种同位素标记的细胞内游离[NAD（+）]/[NADH]代谢生物探针，将磁增强技术（超极化）与细胞糖酵解活性相结合。生物探针基于动态测量的细胞内[丙酮酸]/[乳酸]比率报告游离胞质[NAD（+）]/[NADH]比率。我们证明了它在乳腺癌和前列腺癌细胞中的效用。在前列腺癌细胞中测定的游离胞质[NAD（+）]/[NADH]比比在乳腺癌细胞中高4倍。这一较高的比率反映了前列腺癌细胞的独特代谢表型，与先前报道的这些细胞能量代谢的改变一致。作为游离胞质[NAD（+）]/[NADH]比率的报告物，生物探针将能够更好地理解细胞的不同病理状态的起源以及监测疾病和/或治疗的细胞后果。
Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.