Glutamate-Glutamine Metabolism in Primary Human Brain Tumors

描述(申请人提供)：这项R21申请的主要目标是确定原发人类神经胶质瘤(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运输谷氨酸，通过合成谷氨酰胺(通过谷氨酰胺合成酶(GS))来解毒氨，并通过特定的谷氨酰胺转运体将谷氨酰胺输出到细胞外间隙和血液中。神经元运输谷氨酰胺，并通过磷酸激活的谷氨酰胺酶(PAG)将其转化回谷氨酸，以进行突触神经传递。这种谷氨酸-谷氨酰胺循环在胶质瘤中发生了根本性的改变。胶质瘤不是清除细胞外的谷氨酸，而是在足够高的浓度下通过特定的共转运蛋白释放谷氨酰胺和谷氨酸，从而对周围组织造成兴奋性损伤。周围正常神经元和神经胶质细胞的死亡使胶质瘤侵袭到周围的神经束。然而，在胶质瘤细胞中，谷氨酰胺合成谷氨酸的具体途径(S)还不完全清楚。有人假设，化学计量学的变化将允许GS反向运行。然而，GS是一种依赖于ATP的酶，虽然它在一些胶质瘤中高度表达，但热力学和酶动力学的考虑表明GS不是谷氨酸产生的来源。我们的试点数据显示，在较高级别的肿瘤中，GS活性显著降低。因此，我们推测胶质瘤表达的谷氨酰胺酶能将谷氨酰胺转化为谷氨酸和氨。在具体目标1中，我们将在两个系统中检验这一假设：急性分离的人类胶质瘤和生长在小鼠皮质中的人胶质瘤细胞系。如果这一假说得到证实，将为药物治疗提供一个新的靶点。还将进行蛋白质印迹和免疫组织化学研究，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，同时减少葡萄糖/糖原的可获得性，将通过增加谷氨酸氧化来减少细胞外谷氨酸的释放，从而降低肿瘤细胞的增殖能力。这些研究将在两种准备工作中进行。我们将主要使用暴露于13C标记化合物的体外切片来测量人类和小鼠组织中的谷氨酸和谷氨酰胺代谢。将使用液-质联用(LC/MS/MS)来确定同位素富集型式。这种方法的优势在于，它使我们不仅可以研究谷氨酸代谢，而且还可以研究一个由神经元、星形胶质细胞和胶质瘤细胞组成的系统中的谷氨酸外流。作为这个项目的一部分，我们将解决人类原发胶质瘤的代谢异质性，因为描述那些可以通过针对谷氨酸代谢和释放的治疗来控制的胶质瘤的标志物是很重要的。与健康相关：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，并可能允许开发方法，快速生成单个胶质瘤的功能代谢谱，从而可能导致对这些毁灭性肿瘤进行更好的个体化治疗。与公共健康相关：脑瘤释放的神经递质谷氨酸浓度非常高，可以杀死周围组织，使肿瘤在有限的颅骨空间内生长。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。在这项拨款中，我们将把切除的原发人脑肿瘤样本暴露于各种稳定的潜在谷氨酸前体同位素，以阐明1)胶质瘤产生释放到细胞外空间的谷氨酸的代谢途径；2)不同的代谢燃料是否可以限制谷氨酸的合成和释放。在荷瘤小鼠身上也将进行平行研究，这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的生长。最后，作为这项提议的一部分，我们将为那些可能对饮食治疗有反应的肿瘤确定代谢生物标记物。

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.