Isotopologue distributions of fatty acid oxidation inhibition in U87 glioma cells

Glioblastoma (GBM) metabolism has traditionally been characterized by a primary dependence on aerobic glycolysis, prompting the use of the ketogenic diet (KD) as a potential therapy. In this study we evaluated the effectiveness of the KD in GBM and assessed the role of fatty acid oxidation (FAO) in promoting GBM propagation. In vitro assays revealed FA utilization throughout the GBM metabolome, and growth inhibition in nearly every cell line in a broad spectrum of patient-derived glioma cells treated with FAO inhibitors. In vivo assessments revealed that knockdown of carnitine palmitoyltransferase 1A (CPT1A), the rate limiting enzyme for FAO, reduced the rate of tumor growth and increased survival. However, the unrestricted ketogenic diet did not reduce tumor growth, and for some models significantly reduced survival. Altogether, these data highlight important roles for FA and ketone body metabolism that could serve to improve targeted therapies in GBM.

Methods
Gliomaspheres were dissociated into single cells with Accumax™ and 2x105 cells were cultured for 48 hours in 2.5 mM glucose media in triplicate for each sample. Cells were then rinsed with PBS and re-plated in 2.5 mM glucose media, either unlabeled or containing 200 μM fully-labeled 13C-palmitic acid for an additional 48 hours. Cells were then centrifuged and rinsed with 1ml ice-cold 150 mM ammonium acetate (pH 7.3). Centrifugation was performed again and 1ml of ice-cold 80% methanol was added. Cells were transferred to an Eppendorf tube, and 10 nmol norvaline (Sigma-Aldrich, N7502) was added to each sample. Samples were centrifuged for 5 min at top speed and the supernatant was transferred into a glass vial. Samples were resuspended in 200 μL cold 80% methanol, followed again by centrifugation, after which the supernatant was added to the glass vial. Samples were dried in an EZ-2Elite evaporator. The remaining pellet was resuspended in RIPA buffer and a Bradford assay was performed to quantify total protein concentration for sample normalization. Dried metabolites were resuspended in 50% ACN and 5 μL loaded onto a Luna 3 μm NH2 100 A (150 × 2.0 mm) column (Phenomenex). The chromatographic separation was performed on an UltiMate 3000 RSLC (Thermo Scientific) with mobile phases A (5 mM NH4AcO pH 9.9) and B (ACN) and a flow rate of 200 μL/min. The gradient from 15% A to 95% A over 18 min was followed by 9 min isocratic flow at 95% A and re-equilibration. Metabolite detection was achieved with a Thermo Scientific Q Exactive mass spectrometer run in polarity switching mode (+3.5 kV/− 3.5 kV). TraceFinder 4.1 (Thermo Scientific) was used to quantify the area under the curve for metabolites by using accurate mass measurements (< 3 ppm) and the retention time of purchased reference standards. Relative amounts of metabolites were calculated by summing up all isotopologues of a given metabolite and normalized to cell number. Correction for naturally occurring 13C as well as calculation of fractional contributions and clustering analyses were done in R. Fractional contribution was calculated as , where n is the number of carbons in the metabolite,  is the iteration of each possible 13C-labeled carbon, and  is the relative abundance of the  isotopologue. The relative amount is calculated as the sum of all isotopologues of each metabolite normalized to total protein.

胶质母细胞瘤（Glioblastoma, GBM）的代谢历来被认为主要依赖有氧糖酵解，这促使生酮饮食（Ketogenic Diet, KD）被作为潜在治疗手段加以探索。本研究评估了生酮饮食在胶质母细胞瘤治疗中的有效性，并探究了脂肪酸氧化（Fatty Acid Oxidation, FAO）在促进胶质母细胞瘤增殖中的作用。体外实验显示，胶质母细胞瘤代谢组可利用脂肪酸，且用脂肪酸氧化抑制剂处理的一系列患者来源胶质瘤细胞系中，几乎所有细胞系的增殖均受到抑制。体内实验表明，敲除脂肪酸氧化的限速酶肉碱棕榈酰转移酶1A（Carnitine Palmitoyltransferase 1A, CPT1A）可减慢肿瘤生长速率并延长模型动物的生存期。但无限制的生酮饮食并未抑制肿瘤生长，在部分模型中甚至显著缩短了动物生存期。综上，本研究数据揭示了脂肪酸与酮体代谢在胶质母细胞瘤中的关键作用，可为优化胶质母细胞瘤靶向治疗提供新思路。

实验方法
使用Accumax™试剂将神经胶质瘤球（Gliomaspheres）解离为单细胞，每例样本取2×10⁵个细胞，于2.5 mM葡萄糖培养基中进行三次重复培养48小时。随后用磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤细胞，重悬接种于2.5 mM葡萄糖培养基中，分为未标记组与添加200 μM全标记¹³C-棕榈酸组，继续培养48小时。随后离心收集细胞，用1 mL预冷的150 mM乙酸铵溶液（pH 7.3）洗涤。再次离心后，加入1 mL预冷的80%甲醇溶液。将细胞转移至Eppendorf管中，每例样本加入10 nmol 正缬氨酸（Sigma-Aldrich, N7502）。将样本以最高转速离心5分钟，取上清液转移至玻璃进样瓶中。随后用200 μL预冷的80%甲醇重悬沉淀，再次离心后将上清液合并至上述玻璃进样瓶中。将样本置于EZ-2Elite型蒸发仪中干燥。剩余沉淀用放射免疫沉淀缓冲液（Radio Immunoprecipitation Assay buffer, RIPA）重悬，通过布拉德福德蛋白定量实验（Bradford assay）测定总蛋白浓度，用于样本归一化处理。将干燥后的代谢物用50%乙腈（Acetonitrile, ACN）溶液重悬，取5 μL上样至Luna 3 μm NH₂ 100 Å（150 × 2.0 mm）色谱柱（Phenomenex）。色谱分离采用UltiMate 3000 RSLC型超高效液相色谱仪（Thermo Scientific），流动相A为5 mM乙酸铵（Ammonium Acetate, NH₄AcO）溶液（pH 9.9），流动相B为乙腈（ACN），流速设置为200 μL/min。洗脱梯度设置为：18分钟内流动相A比例从15%升至95%，随后以95%流动相A等度洗脱9分钟，最后进行色谱柱平衡。代谢物检测采用赛默飞世尔科技Q Exactive型质谱仪，以极性切换模式（+3.5 kV / -3.5 kV）运行。采用赛默飞世尔科技TraceFinder 4.1软件，通过精确质量测定（误差<3 ppm）与外购标准品的保留时间，对代谢物的峰面积进行定量分析。代谢物的相对含量通过累加某一代谢物所有同位素异构体的峰面积之和，并以细胞数进行归一化。针对天然同位素¹³C的校正、同位素标记贡献率的计算以及聚类分析均通过R语言完成。同位素标记贡献率的计算公式为：，其中n为代谢物中的碳原子数，为每一种可能的¹³C标记碳原子的迭代项，为对应同位素异构体的相对丰度。代谢物的相对含量则通过累加各代谢物所有同位素异构体的峰面积之和，并以总蛋白浓度进行归一化得到。