(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.
(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.
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DOI：
10.1016/j.neuint.2015.12.008
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发表时间：
2016-02
影响因子：
4.2
通讯作者：
Jekabsons MB
中科院分区：
文献类型：
--
作者：
Gebril HM;Avula B;Wang YH;Khan IA;Jekabsons MB
糖酵解、线粒体底物氧化和磷酸戊糖途径 (PPP) 对于神经元生物能学和氧化还原稳态至关重要，但量化它们的通量仍然具有挑战性，特别是当考虑 PPP 中的磷酸己糖（即葡萄糖/6-磷酸果糖）回收等过程时。开发了磷酸己糖回收模型，该模型利用葡萄糖消耗、乳酸产生和线粒体呼吸的速率，通过复制[1,2-13C2]葡萄糖代谢中的[13C]乳酸标记来推断通过贴壁小脑颗粒神经元的主要葡萄糖消耗途径的通量。通量计算基于稳态系统，其反应具有已知的化学计量和碳原子跃迁。非氧化性 PPP 活性和随后的磷酸己糖回收，以及细胞质苹果酸酶产生的丙酮酸，均通过该模型进行了优化，并发现分别占磷酸己糖和丙酮酸标记的 28±2% 和 7.7±0.2%。从产生的通量中，52±6％的葡萄糖通过糖酵解代谢，相比之下，19±2％的葡萄糖通过允许己糖磷酸循环的组合氧化/非氧化戊糖循环代谢，29±8％的葡萄糖通过组合的氧化PPP/从头核苷酸合成反应代谢。通过扩展，62±6%的葡萄糖转化为丙酮酸，丙酮酸的代谢导致16±1%的葡萄糖被线粒体氧化，46±6%作为乳酸输出。结果表明，戊糖循环和合成核苷酸的反应利用了令人惊讶的高比例的葡萄糖，并以乳酸的形式输出。虽然神经元暴露的体外条件（高葡萄糖、无乳酸或其他外源底物）限制了将这些结果外推到体内状态，但该方法提供了一种从最小的测量集评估 PPP 中磷酸己糖循环背景下的许多代谢通量的方法。
Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [13C]lactate labeling from metabolism of [1,2-13C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28±2 % and 7.7±0.2 % of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52±6 % of glucose was metabolized by glycolysis, compared to 19±2 % by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29±8 % by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62±6 % of glucose was converted to pyruvate, the metabolism of which resulted in 16±1 % of glucose oxidized by mitochondria and 46±6 % exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements.