BACKGROUND High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways. METHODS We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS). RESULTS AND CONCLUSIONS Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected. GENERAL SIGNIFICANCE By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome.

中文翻译：

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代谢指纹图谱显示了GLS多动症的广泛后果。

背景技术高谷氨酰胺酶（GLS； EC3.5.1.2）活性是在肿瘤发生和代谢疾病中的重要病理生理现象。深入了解高GLS活性的代谢后果有助于了解致癌途径和先天性谷氨酸代谢错误的病理生理学。谷氨酰胺酶催化谷氨酰胺向谷氨酸的转化，从而使许多代谢途径相互联系。方法我们开发了一种基于HEK293的细胞模型，该模型可通过将超形态GLS变体的表达与增量GLS抑制相结合来调节GLS活性。使用直接输注高分辨率质谱（DI-HRMS）通过代谢谱分析研究了增加GLS活性的代谢后果。结果与结论在12,437个检测到的特征[m / z]中，109个与内源性相关代谢产物相对应的特征受到高GLS活性的显着影响。如所预期的，这些包括谷氨酰胺的强烈降低和谷氨酸盐水平的升高。另外，还检测到三羧酸（TCA）中间体的水平增加，而柠檬酸水平下TCA循环被缩短，转氨反应，脯氨酸和鸟氨酸合成以及GABA代谢的代谢产物也增加。天冬酰胺和核苷酸代谢产物的水平与谷氨酰胺对GLS活性的依赖性相同。在这些核苷酸中，尤其是嘧啶胸腺嘧啶代谢的代谢产物受到高GLS活性的不利影响，这是显着的，因为它们的合成都依赖于天冬氨酸（谷氨酸的产物）和谷氨酰胺水平。合成γ-谷氨酰环的谷胱甘肽的代谢产物减少或不受影响。一般意义通过提供增加GLS活性的代谢指纹，这项研究显示出高谷氨酰胺酶活性对细胞代谢组的巨大影响。