NMR based metabolomics study of Y2 receptor activation by neuropeptide Y in the SK-N-BE2 human neuroblastoma cell line

Neuropeptide Y (NPY) and its Y2 receptors (Y2R) play an important role in regulating growth of various tumors and are highly expressed in human neuroblastoma brain tumors. Cell lines derived from neuroblastoma brain tumors provide a good model to study the consequences of NPY activation of Y2R in tumor metabolism. In this study, the metabolic response to activation and inhibition of Y2R was examined in the human neuroblastoma cell line SK-N-BE2 using high-resolution nuclear magnetic resonance spectroscopy (NMR). Three treatments were evaluated: (1) activation of Y2R with NPY, (2) blocking Y2R with BIIE0246, and (3) treatment of cells with both NPY and BIIE. The largest metabolic changes were observed for NPY activation of Y2R. The principal response to NPY activation of Y2R was increased glycolysis (Warburg effect). Our results also showed depleted intracellular nutrients in NPY activated cells, which may have been due to the high rate of conversion of glucose to lactate, glycine and alanine. All amino acids except glutamine were increased in response to Y2R activation, implying increased autophagic degradation of proteins. TCA cycle intermediates were not detected, possibly due to low steady-state concentrations, but the increase of glutamate and its high correlation with glucose provided evidence of increased TCA activity. The changes of most metabolites observed upon NPY treatment were reversed with BIIE treatment. In summary, our study indicates that NPY activation of Y2R in human neuroblastoma cells stimulates glycolysis, glutaminolysis and possibly TCA activity.

神经肽Y（Neuropeptide Y, NPY）及其Y2受体（Y2 receptor, Y2R）在调控多种肿瘤的生长进程中发挥关键作用，且在人神经母细胞瘤脑肿瘤中呈高表达状态。源自神经母细胞瘤脑肿瘤的细胞系，是探究NPY激活Y2R对肿瘤代谢影响的理想模型。本研究借助高分辨率核磁共振波谱法（high-resolution nuclear magnetic resonance spectroscopy, NMR），对人神经母细胞瘤细胞系SK-N-BE2中Y2R激活与抑制后的代谢响应进行了检测。

本研究共评估了三种处理方案：（1）以NPY激活Y2R；（2）以BIIE0246阻断Y2R；（3）联合使用NPY与BIIE处理细胞。其中，NPY激活Y2R所引发的代谢变化幅度最大。NPY激活Y2R后的主要代谢响应为糖酵解增强（瓦博格效应，Warburg effect）。

研究结果同时显示，经NPY激活的细胞内营养物质出现耗竭，这大概率源于葡萄糖向乳酸、甘氨酸与丙氨酸的高转化率。Y2R激活后，除谷氨酰胺外的所有氨基酸水平均有所升高，提示蛋白质发生了增强的自噬降解。三羧酸循环（tricarboxylic acid cycle, TCA cycle）中间体未被检出，这可能与其稳态浓度偏低有关；但谷氨酸水平升高及其与葡萄糖的高度相关性，为三羧酸循环活性增强提供了佐证。经NPY处理后出现的多数代谢物变化，可被BIIE处理逆转。

综上，本研究证实：人神经母细胞瘤细胞中Y2R被NPY激活后，可刺激糖酵解、谷氨酰胺分解及潜在的三羧酸循环活性。