Glutamate-Glutamine Metabolism in Primary Human Brain Tumors

描述（由申请人提供）：本R21申请的主要目标是确定原发性人类神经胶质瘤（神经胶质瘤）释放的细胞外谷氨酸的来源。在正常大脑中，神经胶质细胞贪婪地转运谷氨酸，通过合成谷氨酰胺（通过谷氨酰胺合成酶（GS））将其用于氨解毒，并通过特定的谷氨酰胺转运蛋白将谷氨酰胺输出到细胞外空间和血液中。神经元转运谷氨酰胺，并通过磷酸活化的谷氨酰胺酶（PAG）将其转化为谷氨酸，用于突触神经传递。这种谷氨酸-谷氨酰胺循环在神经胶质瘤中发生了根本性的改变。胶质瘤不是清除细胞外谷氨酸，而是摄取谷氨酰胺并通过特异性共转运蛋白以足够高的浓度释放氨和谷氨酸，以诱导对周围组织的兴奋性毒性损伤。周围正常神经元和胶质细胞的死亡使得胶质瘤能够侵入周围的神经元。然而，神经胶质瘤细胞中谷氨酸由谷氨酰胺合成的具体途径尚未完全了解。据推测，化学计量的变化将允许GS反向运行。然而，GS是一种ATP依赖性酶，虽然它在一些神经胶质瘤中高度表达，但热力学和酶动力学方面的考虑表明GS不是谷氨酸产生的来源。我们的试验数据显示GS活性在更高级别的肿瘤中显著降低。因此，我们假设谷氨酰胺通过胶质瘤表达的谷氨酰胺酶转化为谷氨酸和氨。在具体目标1中，我们将在两个系统中测试这一假设：急性分离的人脑胶质瘤和在小鼠皮层中生长的人脑胶质瘤细胞系。如果这一假设得到证实，将为药物治疗提供新的靶点。还将进行蛋白质印迹和免疫组织化学，以确认功能研究。在具体目标2中，我们提出将燃料平衡改变为有利于脂肪酸和酮体的燃料平衡，同时降低葡萄糖/糖原的可用性，这将通过增加谷氨酸氧化来减少细胞外谷氨酸的释放，从而降低肿瘤细胞增殖的能力。这些研究将在两种制备液中进行。我们将主要使用暴露于13 C标记化合物的离体切片测量人和鼠组织中的谷氨酸和谷氨酰胺代谢。将使用液相色谱串联质谱法（LC/MS/MS）测定同位素富集模式。这种方法的优势在于，它使我们能够在具有神经元、星形胶质细胞和胶质瘤细胞的系统中研究谷氨酸代谢和谷氨酸流出。作为该项目的一部分，我们将解决原发性人类神经胶质瘤的代谢异质性，因为重要的是要描述那些可以通过靶向谷氨酸代谢和释放的治疗来控制的神经胶质瘤的标志物。健康相关性：我们正在使用一种创新的技术组合来表征我们假设发生在人类胶质瘤和动物模型中的谷氨酸代谢的复杂变化。我们预计，这些数据将有助于开发新的治疗方法，也可能允许开发快速生成单个胶质瘤的功能代谢谱的方法，这可能导致对这些破坏性肿瘤进行更好的个性化治疗。公共卫生相关性：脑肿瘤以非常高的浓度释放神经递质谷氨酸盐，这可以杀死周围的组织，使肿瘤在头骨的有限空间内生长。这些肿瘤用于产生释放的谷氨酸的特定代谢途径尚未确定，并提出了一种可能的治疗途径。在这项研究中，我们将把切除的原发性人脑肿瘤样本暴露于潜在谷氨酸前体的各种稳定同位素中，以阐明1）神经胶质瘤产生谷氨酸并释放到细胞外空间的代谢途径; 2）如果不同的代谢燃料可以限制谷氨酸的合成和释放。在荷瘤小鼠中的平行研究也将进行，并将使我们能够确定低碳水化合物饮食是否可以改变谷氨酸代谢，从而潜在地改变肿瘤生长。最后，作为这项提议的一部分，我们将确定那些可能对饮食疗法有反应的肿瘤的代谢生物标志物。

DESCRIPTION (provided by applicant): The principal goal of this R21 application is to determine the source of extracellular glutamate released by primary human glial tumors (gliomas). In normal brain, glial cells avidly transport glutamate, use it to detoxify ammonia by synthesizing glutamine (via glutamine synthetase (GS)), and export glutamine into the extracellular space and blood via specific glutamine transporters. Neurons transport glutamine and convert it back to glutamate via phosphate activated glutaminase (PAG) for synaptic neurotransmission. This glutamate-glutamine cycling is radically altered in gliomas. Instead of clearing extracellular glutamate, gliomas take up glutamine and release ammonia and glutamate through specific co-transporters in sufficiently high concentrations to induce excitotoxic damage to the surrounding tissue. The death of surrounding normal neurons and glia allows the glioma to invade into the surrounding neuropil. However, the specific pathway(s) by which glutamate is synthesized from glutamine in glioma cells are not fully understood. It has been hypothesized that the changes in stoichiometry would allow GS to run in reverse. However, GS is an ATP-dependent enzyme, and while it is highly expressed in some gliomas, thermodynamic and enzyme kinetic considerations suggest that GS is not a source for glutamate production. Our pilot data show that GS activity is dramatically reduced in higher grade tumors. Therefore, we hypothesize that glutamine is converted to glutamate and ammonia by glutaminase expressed by the gliomas. In Specific Aim 1, we will test this hypothesis in two systems: acutely isolated human glioma and human glioma cell lines grown in the mouse cortex. If this hypothesis is confirmed, it would provide a new target for pharmacologic therapy. Western blots and immunohistochemistry will also be done to confirm the functional studies. In Specific Aim 2, we propose that changing the fuel balance to one favoring fatty acids and ketone bodies with reduced glucose/glycogen availability will reduce extracellular glutamate release by increasing glutamate oxidation and thus reducing the ability of tumor cells to proliferate. These studies will be done in both preparations. We will primarily measure glutamate and glutamine metabolism in human and murine tissue using ex vivo slices exposed to 13C labeled compounds. The pattern of isotopic enrichment will be determined using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The strength of this is approach is that it allows us to study both glutamate metabolism but also glutamate efflux in a system that has neurons, astrocytes as well as glioma cells. As part of this project, we will address the metabolic heterogeneity of primary human gliomas as it is important to describe markers for those gliomas that can be controlled by therapies that target glutamate metabolism and release. Health Relevance: We are using an innovative combination of techniques to characterize the complex changes in glutamate metabolism that we hypothesize occur in human gliomas and in an animal model. We anticipate that these data will help to develop new approaches to therapy and may also allow for the development of methods to rapidly generate a functional metabolic profile of individual gliomas that may lead to better, individualized treatment of these devastating tumors. PUBLIC HEALTH RELEVANCE: Brain tumors release the neurotransmitter glutamate in very high concentrations which can kill the surrounding tissue, allowing the tumor to grow within the confined space of the skull. The specific metabolic pathway used by these tumors to generate the released glutamate has not been determined and presents a possible therapeutic avenue. In this grant, we will expose samples of resected primary human brain tumors to a variety of stable isotopes of potential glutamate precursors to elucidate 1) the metabolic pathway by which gliomas make the glutamate that is released into the extracellular space; and 2) if different metabolic fuels can limit the synthesis and release of glutamate. Parallel studies in tumor-bearing mice will also be done and will allow us to determine if a low carbohydrate diet can alter glutamate metabolism and thus potentially tumor growth. Finally, as part of this proposal, we will identify metabolic biomarkers for those tumors that might respond to dietary therapy.