Metabolic and transcriptional patterns accompanying glutamine depletion and repletion in mouse hepatoma cells: a model for physiological regulatory networks
Metabolic and transcriptional patterns accompanying glutamine depletion and repletion in mouse hepatoma cells: a model for physiological regulatory networks
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DOI：
10.1152/physiolgenomics.00088.2003
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发表时间：
2004-01-15
影响因子：
4.6
通讯作者：
Kelleher, JK
中科院分区：
文献类型：
--
作者：
Wong, MS;Raab, RM;Kelleher, JK
后基因组生物学的一个重要目标是通过开发包括来自基因组和功能水平的数据的生理网络来将基因表达与功能联系起来。在这里，我们开发了一个模型，用于分析肝脏模型系统中代谢物、通量和基因表达的时间依赖变化。实验框架为细胞外谷氨酰胺对小鼠肝细胞系Hepa1-6融合培养的调节作用。谷氨酰胺在哺乳动物细胞培养中的重要性已被证实，它通过谷氨酰胺耗竭和再耗尽而加速代谢转变。我们的方法是将谷氨酰胺从培养液中去除24小时，然后将其返回第二个24小时。糖酵解、三羧酸(TCA)循环和脂肪生成的流量分析以特定的间隔进行。在没有谷氨酰胺的情况下，所有这些通量都下降了，当补充谷氨酰胺时，这些通量又恢复了。同质异构体光谱分析确定葡萄糖和谷氨酰胺是生脂碳的同等来源。对有机酸和氨基酸的代谢物测定表明，大多数代谢物随通量的变化而变化。放线菌素D的实验表明，在谷氨酰胺耗竭/再耗竭过程中，观察到的通量变化需要从头合成mRNA。DNA微阵列的基因表达数据分析显示，与糖酵解通量和谷氨酰胺水平反相关的基因比与这些指标相关的基因要多得多。总之，这个模型可能是有用的原型生理调节网络，其中基因表达谱分析与细胞功能的变化相一致。
An important objective in postgenomic biology is to link gene expression to function by developing physiological networks that include data from the genomic and functional levels. Here, we develop a model for the analysis of time-dependent changes in metabolites, fluxes, and gene expression in a hepatic model system. The experimental framework chosen was modulation of extracellular glutamine in confluent cultures of mouse Hepa1-6 cells. The importance of glutamine has been demonstrated previously in mammalian cell culture by precipitating metabolic shifts with glutamine depletion and repletion. Our protocol removed glutamine from the medium for 24 h and returned it for a second 24 h. Flux assays of glycolysis, the tricarboxylic acid (TCA) cycle, and lipogenesis were used at specified intervals. All of these fluxes declined in the absence of glutamine and were restored when glutamine was repleted. Isotopomer spectral analysis identified glucose and glutamine as equal sources of lipogenic carbon. Metabolite measurements of organic acids and amino acids indicated that most metabolites changed in parallel with the fluxes. Experiments with actinomycin D indicated that de novo mRNA synthesis was required for observed flux changes during the depletion/repletion of glutamine. Analysis of gene expression data from DNA microarrays revealed that many more genes were anticorrelated with the glycolytic flux and glutamine level than were correlated with these indicators. In conclusion, this model may be useful as a prototype physiological regulatory network where gene expression profiles are analyzed in concert with changes in cell function.