Acs Jproteome 8b00663

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Article
Integrated metabolomics and lipidomics analyses reveal
metabolic reprogramming in human glioma with IDH1 mutation
Lina Zhou, Zhichao Wang, Chunxiu Hu, Chaoqi Zhang, Petia Kovatcheva-Datchary, Di Yu, Shasha
Liu, Feifei Ren, Xiaolin Wang, Yanli Li, Xiaoli Hou, Hailong Piao, Xin Lu, Yi Zhang, and Guowang Xu
J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00663 • Publication Date (Web): 31 Dec 2018
Downloaded from http://pubs.acs.org on January 1, 2019
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Page 1 of 32 Journal of Proteome Research
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Journal of Proteome Research Page 2 of 32
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Integrated metabolomics and lipidomics analyses reveal metabolic
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6 reprogramming in human glioma with IDH1 mutation
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11 Lina Zhou1, ▲, Zhichao Wang1,3, ▲, Chunxiu Hu1, Chaoqi Zhang2, Petia
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13 Kovatcheva-Datchary 1, Di Yu1,3, Shasha Liu2, Feifei Ren2, Xiaolin Wang1,Yanli Li1,
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16 Xiaoli Hou1, Hailong Piao1, Xin Lu1, Yi Zhang2,*, Guowang Xu1,*
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20 1 CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian
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Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
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2 Biotherapy Center and Cancer Center,The First Affiliated Hospital of Zhengzhou
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27 University, Zhengzhou 450052, China
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29 3 University of Chinese Academy of Sciences, Beijing 100049, China.
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34 ▲: contributed equally to this paper.
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37 Corresponding Authors:
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39 *Guowang Xu, CAS Key Laboratory of Separation Science for Analytical
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41 Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,
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44 Dalian 116023, China. Telephone: +86-411-84379530; Fax: +86-411-84379559;
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46 E-mail: xugw@dicp.ac.cn；
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Yi Zhang, Biotherapy Center and Cancer Center, The First Affiliated Hospital of
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51 Zhengzhou University, Zhengzhou 450052, China. E-mail: yizhang@zzu.edu.cn.
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ABSTRACT
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6 Mutations in isocitrate dehydrogenase (IDH) 1 are high-frequency events in low grade
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8 glioma and secondary glioblastoma and IDH1 mutant glioma are vulnerable to
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11 interventions. Metabolic reprogramming is a hallmark of cancer. In this study,
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13 comprehensive metabolism investigation of clinical IDH1 mutant glioma specimens
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15 was performed to explore its specific metabolic reprogramming in real
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microenviroment. Massive metabolic alterations from glycolysis to lipid metabolism
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20 were identified in the IDH1 mutant glioma tissue when compared to IDH1 wild-type
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22 glioma. Of note, TCA cycle intermediates were in similar levels in both groups,
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having more pyruvate found entering TCA cycle in IDH1 mutant glioma. The pool of
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27 fatty acyl chains was also reduced, displaying as decreased triglycerides and
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29 sphingolipids, though membrane phophatidy lipids were not changed. The lower fatty
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31 acyl pool may be mediated by the lower protein expression levels of long-chain
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34 acyl-CoA synthetase 1 (ACSL1), ACSL4 and very long-chain acyl-CoA synthetase 3
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36 (ACSVL3) in IDH1 mutant glioma. Lower ACSL1 was further found contributing to
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38 the better survival of IDH1 mutant glioma patients based on the The Cancer Genome
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Atlas (TCGA) RNA sequencing data. Our research provides valuable insights in the
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43 tissue metabolism of human IDH1 mutant glioma and unravels new lipid-related
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45 targets.
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50 KEY WORDS: glioma, IDH1, metabolomics, lipidomics, metabolic
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reprogramming
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INTRODUCTION
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6 Glioma is the most common malignant brain tumors with significant mortality and
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8 morbidity 1. It has been reported that isocitrate dehydrogenase (IDH) 1 mutation on
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11 amino acid 132 occurs in more than 70% of the low grade gliomas and secondary
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13 glioblastomas on the basis of histopathological and clinical criteria established by the
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15 World Health Organization (WHO) 2-4. Wild-type IDH catalyzes the oxidative
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decarboxylation of isocitrate to alpha-ketoglutarate (2-KG), producing nicotinamide
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20 adenine dinucleotide phosphate (NADPH) 5, a cofactor whose function is to maintain
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22 normal levels of reduced glutathione and rescue the cell from reactive oxygen species
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(ROS) 6. Mutant IDH loses the wild-type enzyme function, but acquires a new ability
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27 to catalyze the reduction of 2-KG to 2-hydroglutarate (2-HG), consuming NADPH 7-9.
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29 Thus, with the significant production of 2-HG, IDH mutation may present an
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31 increased oxidative stress and great metabolic stress in maintaining tricarboxylic acid
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34 cycle (TCA) and molecular synthesis for cell proliferation 10. In the recent years, IDH
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36 mutation has been found closely related to oncogenesis either through induction of
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38 hypoxia inducible factor-1 pathway 11, or contributing to tumorigenesis and tumor
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progression through producing the oncometabolite 2-HG, resulting in histone and
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DNA hypermethylation 7-9. However, patients with IDH1 or IDH2 mutations were
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45 reported to have a longer survival than those without IDH mutation 4. The exact roles
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of IDH mutations in the occurrence and the progression of glioma are still not clear.
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Metabolic reprogramming is one of the hallmarks of cancer 12. Except for IDH
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52 mutation, somatic mutations in other two metabolic enzymes, succinate
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54 dehydrogenase (SDH) and fumarate hydratase (FH), have been found associated with
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57 tumorigenesis 13. Also, metabolic reprogramming is regulated by oncogenic signaling
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3 to aid cell growth and proliferation 14-15. Thus, except for 2-HG production, other
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6 metabolic changes may also be related to tumor progression. To investigate the
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8 metabolic characteristics of IDH1 mutant glioma, some researches have been
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10 performed by investigating isogenic IDH1-R132H mutant cell models, cultured under
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normoxia 16-20. Most of the differential amino acids were accumulated, but glutamate,
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15 aspartate and intermediates in the late TCA cycle were decreased in IDH1-R132
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17 mutant cells and 2-HG treated cells, together with increased glycerol phosphocholine,
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but decreased phosphocholine 16. Decreased glutamate and phosphocholine were also
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reported in IDH1 mutant cells, together with lactate 17. Lactate dehydrogenase
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24 (LDHA) activity was found lower in IDH1 mutant glioma cells, and silencing of the
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26 LDHA was associated with increased methylation of the LDHA promoter 20. Glucose
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29 flux to TCA cycle was reduced through pyruvate dehydrogenase (PDH) activity in
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31 U87 glioblastoma and NHA-IDH1 mutant cells, which was associated with increased
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33 PDH inhibitory phosphorylation regulated by pyruvate dehydrogenase kinase-3 18-19.
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Further, CE-MS based metabolomics analysis of human glioma tissue revealed that
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38 N-acetylated amino acids, glutamine and glutamate were decreased in 13 IDH1
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40 wild-type glioma tissues and 20 IDH1-R132H mutant glioma tissues 21. Another study
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on animal and human gliomas also reported lower level of phosphocholine and higher
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glycerol phosphocholine in IDH1-R132H mutant glioma monitored by 31P high
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47 resolution magic angle spinning spectroscopy 22. Recently, using mass spectrometry
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49 imaging on patient-derived xenografts, the metabolic landscape in IDH-mutant glioma
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52 versus wild type glioma has been shown to have phospholipid, energy, and oxidative
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stress pathways affected 23. Collectively, increasing metabolic features have been
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56 provided, which will increase the understanding of IDH1 mutant glioma malignant
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progression. But still more systematic investigations of the metabolic consequences of
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3 IDH1 mutation need to be performed in human glioma tissues, which reflect the
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6 metabolic reprogramming in response to the real tissue microenvironment.
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8 Metabolomics enables a comprehensive monitoring of the responses of
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10 endogenous metabolites towards pathophysiological stimuli including genetic
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12 24.
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alterations and disturbances in metabolic activities Here we performed gas
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15 chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC)-MS
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17 based metabolomics and LC-MS based lipidomics analyses to identify important
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metabolic alterations in glioma tissues with and without IDH1 mutations. Key related
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22 proteins were also analyzed to explore important targets responsible for the key
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24 metabolic alterations in human glioma tissues with IDH1 mutation.
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29 EXPERIMENTAL SECTION
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31 Subjects
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34 Seventy-five glioma patients in the first affiliated hospital of Zhengzhou University
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36 were recruited for the retrospective study. Brain tumor tissue from all glioma patients
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38 and adjacent tissue from 41 of the patients were collected during surgery. Samples of
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grossly normal adjacent brain tissues were removed as far from the tumor as the
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43 operative field permitted. Tissue samples were immediately kept on ice within one
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45 hour and then stored at -80 ˚C until analysis. All subjects gave their informed consent
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for inclusion before they participated in the study. The study was conducted in
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50 accordance with the Declaration of Helsinki, and the protocol was approved by the
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52 Scientific Research and Clinical Trial Ethics Committee of the First Affiliated
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54 Hospital of Zhengzhou University.
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3 Glioma histopathological and molecular classification
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6 All glioma tissues were subjected to consensus review by at least two
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8 neuropathologists and graded according to histopathological and clinical criteria
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10 established by the WHO. Genomic DNA was isolated from the brain tumor samples
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using the Animal Tissues/Cells Genomic DNA Extraction kit (Solarbio, Beijing,
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15 China), following the manufacture protocol. The assessments of IDH1 and IDH2
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17 mutations in the glioma tissues were performed by Tsingke Biotech Co., Ltd.
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(Beijing, China) with a 2720 Thermal cycler (Applied Biosystems, USA) using the
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22 following primer sets: IDH1-F: 5`-AAT GAG CTC TAT ATG CCA TCA CTG-3`;
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24 IDH1-R:5`-TTC ATA CCT TGC TTA ATG GGT GT-3`; IDH2-F: 5`- CCC GTC
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26 TGG CTG TGT TGT T-3`; IDH2-R: 5`-TCC TTG ACA CCA CTG CCAT-3`.
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31 Metabolomics and lipidomics analysis
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33 The tissue samples were not thawed until sample preparation for metabolomics and
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lipidomics analysis. The procedure for GC-MS and LC-MS based metabolomics,
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38 LC-MS based lipidomics analysis, including sample preparation, data acquisition and
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40 the following data preprocessing are provided in the supplemental materials.
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45 Statistical analyses
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47 The multivariate partial least-squares discriminant analysis (PLS-DA) was performed
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49 employing SIMCA-P 11.0 (Umetrics, Sweden) with all variables unit variance scaled.
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52 And permutation test was exerted 200 times to see whether the constructed model was
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54 overfitted. Paired sample t-test was performed for comparison of paired cancer tissue
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56 and adjacent tissue. Pathway analysis was performed on metaboAnalyst 4.0
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(http://www.metaboanalyst.ca), the important pathways were defined as having
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3 -log(p)>3 and pathway impact factor>0.1. To explore the important metabolic
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6 differences of glioma tissues with and without IDH1 mutations, Mann-Whitney U
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8 tests, significance analysis of microarrays (SAM) and heat map analysis of significant
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10 metabolites were performed using the Multi Experiment Viewer
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(http://www.tm4.org). The level significance for the univariate analysis was set at
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15 p<0.05. The metabolite alterations projected in the pathways were drawn with the
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17 help of Vanted software, referring metabolic pathways in KEGG Pathway Database
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(http://www.genome.jp/kegg/pathway.html) and Lipidmaps
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22 (http://www.lipidmaps.org/). GraphPad Prism 7 was used for the box & whiskers plot
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24 analysis.
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29 Western blot analysis
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31 Western blotting was performed on fresh frozen tissue from all 75 glioma tissues as
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33 previously described 25. Top 24 samples, ranked by actin intensity, in each IDH1
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wild-type and mutant groups, were chosen for next analysis. Antibodies against
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38 pyruvate dehydrogenesa kinase isozyme 1 (PDK1, 10026-1-AP), pyruvate
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40 carboxylase (PCE, 16588-1-AP), long-chain acyl-CoA synthetase isoforms (ACSL1,
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13989-1-AP), (ACSL3, 20710-1-AP), (ACSL4, 22401-1-AP) and (ACSL5,
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45 15708-1-AP), carnitine palmitoyltransferase 1a and 1c (CPT1a and 1c, 15184-1-AP
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47 and 12969-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies
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49 against phosphor-acetyl-CoA carboxylase (pACC, #3661), acetyl-CoA carboxylase
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52 (ACC, #3662), fatty acids synthases (FASN, #3180) were purchased from Cell
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RESULTS AND DISCUSSION
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6 IDH mutation detection and grouping
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8 Sequencing analysis of IDH1 and IDH2 in the 75 glioma samples revealed 30 IDH1
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11 mutations at residue R132H and no IDH2 mutations. The clinical parameters of the
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13 enrolled patients, including age, gender (basically matched) and WHO grading are
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15 summarized in Table 1. Among these patients, following-up information of 16 IDH1
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mutant and 17 wild-type patients was available. The time interval between the last
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20 patient enrolled and the latest following-up was 35 months.
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Table 1 Clinical characteristics of the recruited glioma patients in this study.
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27 Glioma without IDH1 mutation Glioma with IDH1 mutation
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29 (n=45) (n=30)
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Gender, Male/Female 23/22 15/15
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34 Age, range (mean±SD) 20~76 （51.1±13.9) 24~62（42.6±9.5）
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WHO grading (II/III/IV) 7/16/22 16/13/1
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39 IDH2 mutation 0 0
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41 Having follow-up
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44 information
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48 Metabolic disruptions in glioma tissues compared to paired adjacent tissues
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Comprehensive metabolic profiling of cancer tissues and adjacent tissues from glioma
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53 patients was performed combining GC-MS and LC-MS based metabolomics analyses
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55 and LC-MS based lipidomics analysis. The samples in each platform were analyzed
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within one batch and the coefficient of variation (CV) for each metabolite in QC
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3 samples was calculated. The proportion of metabolites having CV<30% was 86.9%
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6 (detected by LC-MS and GC-MS metabolomics platform, shown in Figure S1A) and
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8 the proportion of lipids having CV<30% was 99.8% (detected by lipidomics platform,
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10 shown in Figure S1B). This indicates that the data are reliable for the following
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statistical analysis. In total, 619 metabolites were identified in both types of tissues
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15 (Table S1). Initially, compared to their paired adjacent tissues, the common tumor
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17 metabolic disruptions of IDH1 mutant and wild type were found, including 18 amino
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acids, 10 organic acids and 268 lipids (Table S2). Most amino acids, short-chain
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22 (C2~C5) acylcarnitines, the ratio of acetylcarnitine (CN-C2) versus free carnitine, and
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24 the ratio of propanoylcarnitine (CN-C3) versus free carnitine were significantly
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26 elevated in glioma tissues (Figure S2a and Table S2). Lipid synthesis precursors,
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29 glycerol-3-phosphate was also elevated in the glioma tissues, while others such as
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31 glycerol and myo-inositol phosphate were significantly decreased (Figure S2a and
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33 Table S2). Likewise, total fatty acids and total phosphatidyl lipids including
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phosphatidylcholine (PC), phosphatidylethanolamine (PE), ceramide (Cer) and
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38 cardiolipin (CL) were significantly decreased in glioma tissues (Figure S2a). Further,
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40 pathway analysis on the differential metabolites, confirmed a profound disruption of
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amino acids metabolism (including glycine, serine and threonine metabolism, arginine
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45 and proline metabolism, alanine, aspartate and glutamate metabolism),
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47 aminoacyl-tRNA biosynthesis and glycerolphospholipid metabolism in the glioma
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49 tissues (Figure S2b). As we focused on the IDH1 mutation associated metabolic
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52 disruptions, here we did not go further to explore more detailed metabolic disruptions
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54 related to glioma subtypes or tumor infiltration. Differences in lipid and metabolite
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56 profiles of glioma and brain parenchyma have been previously studied by desorption
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electrospray ionization-MS 26.
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6 Altered metabolism related to IDH1 mutation when compared with IDH1
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8 wild-type
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10 To further investigate metabolic alterations in IDH1 mutant human glioma, we
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compared the metabolome and lipidome of IDH1 mutatnt glioma tissues to that of
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15 IDH1 wild-type. Partial least squares discriminative analysis (PLS-DA) revealed a
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17 good separation trend between the two groups on the score plot of the first two
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principal components, when using the overall metabolome and lipidome information
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22 for model construction. The PLS-DA model had accumulative R2Y=0.58 and Q2=0.37
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24 (Figure 1a). The model was not overfitted when permutated 200 times for validation,
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26 having the intercepts of R2 and Q2 equaling to 0.38 and -0.13, respectively (Figure
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29 1b).
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31 To identify the detailed metabolic differences between glioma tissues with IDH1
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33 mutation and wild-type, Mann-Whitney U test was next performed on all detected
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metabolites, detected lipids and the following calculated indexes, including total
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38 amounts of lipids in each class and ratios among lipids or metabolites. After using
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40 Benjamini-Hochberg correction to control the false positive rate (FDR, set as FDR
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smaller than 0.05), there were 248 detected metabolites and lipids significantly altered
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45 in IDH1 mutant glioma tissues compared to the wild-type glioma tissues, having 63
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47 metabolites elevated and 185 decreased in IDH1 mutant glioma tissues. (Figure 1c).
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49 Further, pathway analysis on those differential metabolites, confirmed profound
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52 disruptions of the TCA cycle, glycolysis and gluconeogenesis, amino acids
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54 metabolism (including glycine, serine and threonine metabolism, arginine and proline
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56 metabolism, alanine, aspartate and glutamate metabolism, taurine and hypotaurine
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metabolism, beta-alanine metabolism), lipid metabolism (including glycerolipid
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3 metabolism, glycerophospholipid metabolism and sphingolipid metabolism) and
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6 pantothenate and Coenzyme A biosynthesis in the glioma tissues with IDH1 mutation
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8 (Figure 1d).
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10 From the 284 significantly altered variables (including detected metabolites,
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detected lipids and calculated indexes), 158 were kept after further SAM analysis to
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15 study the importance of the differential variables. Compared to IDH1 wild-type,
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17 2-hydroxyglutaric acid (2-HG) was the most notably elevated metabolite in the IDH1
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mutant glioma, and there was no 2-HG elevation in wild-type when compared to their
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22 paired adjacent tissues. All detected triglycerides (TG) were strongly decreased in the
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24 IDH1 mutant glioma (Figure 2), which was also noticeable.
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29 Reprogramming of core metabolic pathways related to IDH1 mutation
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31 KEGG pathway-based mapping that considers all differentially expressed metabolites
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33 between glioma tissues with IDH1 mutation and IDH1 wild-type were performed to
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study the central carbon metabolism in IDH1 mutant glioma (Figure 3).
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38 Glucose is the main energy source for the cells in the brain. The tissue levels of
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40 glucose and its glycolysis end product, lactate, were similar in the two types of
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glioma. All significantly altered intermediates in glycolysis were reduced in the
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45 glioma tissues with IDH1 mutation compared to the IDH1 wild-type (Figure 3 and
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47 Table S3). The glycolysis intermediates, including 6-phosphogluconic acid,
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49 3-phosphoglyceraldehyde and phosphoenolpyruvic acid, together with serine and
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52 glycine (decreased) can feed nucleotide synthesis (Figure 3). Uridine and inosine
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54 were elevated (Figure 3, Table S3) in the glioma with IDH1 mutation compared to
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56 IDH1 wild-type, with the degradation product of uracil decreased. Though a large
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amount of 2-ketoglutarate was converted into 2-HG in the glioma with IDH1
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3 mutation, most of the intermediates in the TCA cycle were not influenced, except for
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6 2-ketoglutarate and isocitric acid (which are adjacent to 2-HG) decreased in the IDH1
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8 mutant glioma compared to the IDH1 wild-type (Figure 3 and Table S3). Similar
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2-KG, malate, fumarate, succinate, or isocitrate were reported in a smaller cohort of
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15 patients, where IDH1R132 mutant glioma tissues were compared to those of IDH1
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17 wild-type glioma tissues 7.
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Pyruvate, an intermediate of glucose entering into TCA cycle, was reduced in the
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22 glioma with IDH1 mutation, but the ratio of pyruvate to lactate did not differ between
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24 the glioma tissues with and without IDH1 mutation. To investigate how pyruvate
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26 enters in the TCA cycle, we measured protein expression level of PDK1 regulating
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29 the conversion of pyruvate to acetyl-CoA and pyruvate carboxylase (PCE) that
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31 carboxylates it to oxaloacetate 28. The levels of PDK1 was reduced and PCE increased
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33 in the glioma tissues with IDH1 mutation compared to IDH1 wild-type (Figure S3).
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This suggested that conversion of pyruvate prior entering into the TCA was activated
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38 possibly through both pyruvate dehydrogenation and carboxylation. Collectively,
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40 more glucose was consumed for nucleotide synthesis and more pyruvate can enter and
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feed TCA in IDH1 mutant glioma. Glutaminolysis has been reported as an important
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anaplerosis pathway in glioma cells with IDH1 mutation 29-31. Here, glutamine and
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47 glutamate did not differ between the two types of glioma tissues (Figure 3).
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52 Lipid metabolism reprogramming related to IDH1 mutation
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54 To have an overview of lipid metabolism alterations in IDH1 mutant glioma,
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56 differential total lipids in each class, lipid precursors and individual lipid molecules
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were projected on the lipid synthesis and remodeling pathways (Figure 4). There
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3 were no differences in the total free fatty acids or the total amount of membrane
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6 phospholipids between the IDH1 mutant glioma and IDH1 wild-type glioma (Figure
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8 4). The levels of total fatty acids had no significant alterations, whereas fatty acid 18:0
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10 (FA 18:0), FA 20:3, FA 22:5 and FA 20:5 were maintained at a relatively higher
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levels in the IDH1 mutant glioma, although the saturated very-long-chain fatty acids
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15 (SVLFA) decreased (Table S3). All detected differential phosphatidylserine (PS)
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17 molecules were increased in the IDH1 mutant glioma. But we measured great
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decrease in TG and intermediates in sphingolipid pathways, indicating that the total
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22 fatty acid constituents stored in TGs were decreased (Figure 4, Table S4).
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24 Next, the lipid synthesis precursors, including glycerol, glycerol-3-phosphate,
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26 ethanolamine and inositol were found increased in the IDH1 mutant tissues compared
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29 to the IDH1 wild-type tissues (Figure 4). Of note, glycerylphosphorycholine (GPC)
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31 and glycerylphosphorylethanolamine (GPE) were significantly elevated in the IDH1
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33 mutant glioma compared to the IDH1 wild-type (Figure 4). GPC elevation has also
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been previously reported in IDH1 mutant glioma cells, but in no agreement with the
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GPE levels 22, 32. The higher levels of lipid synthesis precursors, GPC and GPE may
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40 together indicate a relative shortage of fatty acyl chains in IDH1 mutant glioma.
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The primary fatty acyl chains of lipids in tumor are usually from de novo
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45 synthesis, and not from uptake of extracellular fatty acids, thus, cancer cells typically
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47 have increased rates of fatty acid synthesis 33. Further, tissues protein levels of fatty
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acids synthases (FASN), phosphorylated-acetyl-CoA carboxylase (pACC) and ACC,
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52 denoting the activity of de novo fatty acid synthesis pathway were measured. While
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54 there were no differences in the levels of FASN in the two type of gliomas, the ratio
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56 of pACC to ACC was significantly reduced (Figure S4a and b). Thus, indicating an
57
58
59 increased enzyme activity for de novo fatty acid synthesis in the IDH1 mutant glioma.
60 13

1
2
3 The ratios, monounsaturated fatty acid versus saturated fatty acid (MUFA/SFA) and
4
5
6 FA(18:1+16:1)/FA(18:0+16:0) (Table S4), reflecting the activity of stearoyl-CoA
7
8 desaturase (SCD) enzyme, were elevated in the IDH1 mutant tissue compared to the
9
10 IDH1 wild-type tissues. Lower TG may resulted from less de novo synthesis or more
11
12
13
TG lysis. In this study, considering the possible stress in providing carbon source for
14
15 de novo fatty acid synthesis in IDH1 mutant glioma, we further investigated the
16
17 differences in reprogramming of fatty acid flows for the lipid synthesis between IDH1
18
19
mutant and IDH1 wild-type glioma tissue.
20
21
22
23
24 Lower ACSL1 contributing to better survival of glioma patients having IDH1
25
26 mutation
27
28
29 Acyl-CoA synthetase (ACSL) isoforms are responsible for long-chain fatty acid
30
31 activation for both lipid synthesis and their beta-oxidation, with the locations and
32
33 roles of ACSL isoforms far from being defined 34-35. There were elevated tissue
34
35
36
protein levels of carnitine palmitoyltransferase 1a and 1c (CPT1a and CPT1c), which
37
38 were responsible for transferring fatty acids into mitochondrial for oxidation (Figure
39
40 S4c and d), in the IDH1 mutant glioma compared to the wild-type. However, there
41
42
were no differences in the levels of acylcarnitines or their ratios reflecting the enzyme
43
44
45 activities related to fatty acid oxidation. Further, to investigate the levels of fatty acids
46
47 that were activated for lipid synthesis in IDH1 mutant glioma, the protein levels of
48
49 ACSL1, ACSL3, ACSL4, ACSL5 and very long-chain acyl-CoA synthetase 3
50
51
52 (ACSVL3) were measured. The Western blot results showed ACSL1, ACSL4 and
53
54 ACSVL3 were significantly decreased, but ACSL5 level was increased in the IDH1
55
56 mutant glioma compared to IDH1 wild-type glioma (Figure S4e and f). Human
57
58
59
malignant glioma has abundant overexpression of ACSVL3, and depletion of this
60 14

1
2
3 enzyme decreases the malignant phenotype of glioma cells through disrupting Akt
4
5
6 function when studied in vitro and in mice glioma xenografts 36. Next, to explore the
7
8 possible relationships between the differential ACSLs and lipid metabolism,
9
10 correlation network analyses between the differential ACSLs and total lipids in each
11
12
13
class, proteins regulating de novo fatty acid synthesis and beta-oxidation were
14
15 performed (Figure 5). The derived networks showed much more interactions between
16
17 the ACSLs and total lipids in some classes and between the ACSLs and some proteins,
18
19
such as PCE, pACC and CPT1c, in the IDH1 mutant glioma compared to the
20
21
22 wild-type.
23
24 Among the 75 glioma patients, following-up information was obtained from 33
25
26 glioma patients (17 with IDH1 mutant glioma and 16 with wild-type glioma, Table 1).
27
28
29 IDH1 mutant patients had significant better survivals than the IDH1 wild-type
30
31 patients (Figure S5). However, because of the limited number of patients with
32
33 following-up information, survival analysis with differential metabolites or proteins
34
35
36
were limited. Therefore, to validate our hypothesis that ACSLs might be of
37
38 importance for the better survival of IDH1 mutant patients, we used The Cancer
39
40 Genome Atlas (TCGA) RNA sequencing data 37 from 489 low grade glioma patients,
41
42
whose prognosis information has been provided, to perform survival analysis.
43
44
45 The gene expression levels of ACSLs and ACSVL3 were firstly investigated in
46
47 TCGA data (Figure S6). The changing trends of gene ACSL1, ACSL3 and ACSVL3
48
49 expressions are the same as those of their protein levels. Though RNA expression
50
51
52 levels of genes ACSL4 and ACSL5 were opposite to their protein expression levels,
53
54 their expression levels had no association with survival using the TCGA data. Patients
55
56 having lower expressions of ACSL1 and ACSVL3 had significant better survivals
57
58
59
(Figure S7a and b). Furthermore, in patients having IDH1 mutant glioma, lower
60 15

1
2
3 expression of ACSL1 (218 lower versus 177 higher) contributed to better survivals,
4
5
6 but not in the patients having no IDH1 mutation (26 lower versus 68 higher) (Figure
7
8 S7c and d). ACSVL3 was not related to survival in patients with IDH1 mutant glioma
9
10 (237 lower versus 158 higher). Most IDH1 wild-type patients had higher expression
11
12
13
levels of ACSVL3 (7 lower versus 87 higher). Finally, combination survival analyses
14
15 for IDH1 mutant patients showed that ACSVL3 levels had no effect on the
16
17 contribution of lower ACSL1 to better survival (Figure S7e). All the patients used for
18
19
survival analysis had tumor grade 2 (G2) or grade 3 (G3). To investigate whether the
20
21
22 correlation of ACSL1 expression level and survival in IDH1 mutant glioma patients
23
24 were confounded by tumor grade, survival analysis was performed separately in IDH1
25
26 mutant-G2 and IDH1 mutant-G3 glioma patients. It was found that glioma patients
27
28
29 having lower ACSL1 expression had better survivals only in the subgroup of IDH1
30
31 mutant-G3 glioma patients (Figure S7f). This indicated that when there was an IDH1
32
33 mutation, the ACSL1 expression level influenced the survival of patients having
34
35
36
relative higher tumor grade. Higher ACSL1 has been reported to inhibit cell apoptosis
37
38 by reducing fatty acid toxicity in Swann cells 38.
39
40
41
42
43 Conclusions
44
45 Limited systematic investigation of IDH1 mutant tumor metabolism has been
46
47
performed in real clinical tissue microenvironment, where IDH1 mutant glioma being
48
49
50 compared to IDH1 wild-type glioma. In this study we addressed this gap through
51
52 performing comprehensive analyses of the metabolome and the lipidome of IDH1
53
54 mutant human glioma tissues. The abundant production of 2-HG can lead to a large
55
56
57 metabolic stress for the TCA cycle and the de novo fatty acid synthesis. However,
58
59
60 16

1
2
3 most of the TCA cycle intermediates as well as the membrane phosphatidyl lipids
4
5
6 were not affected. Pyruvate was found supplementing TCA cycle in IDH1 mutant
7
8 glioma. Decrease in ACSL1, ACSL4 and ACSVL3 will activate less fatty acids for
9
10 lipid synthesis, which may explain the lower TGs in IDH1 mutant glioma. Still, due to
11
12
13
the scarcity of independent cohorts, the findings needed to be validated further,
14
15 especially more samples in the same grade need to be analyzed to remove background
16
17 bias from different patients. The cross talk between the ACSLs and related regulating
18
19
signals in IDH1 mutant cells should be further investigated, with the focus on the
20
21
22 ACSL1. Further investigations towards co-expression of IDH1 and other metabolic
23
24 enzymes in key interaction pathways should be performed to elucidate the inherent
25
26 interactions of metabolic blocks related to tumorigenesis and cancer progression.
27
28
29
30
31 ASSOCIATED CONTENT
32
33 Supporting Information
34
35
36
The following supporting information is available free of charge at ACS website
37
38 http://pubs.acs.org.
39
40 The supporting material 1 contains the procedure for GC-MS and LC-MS based
41
42
metabolomics, LC-MS based lipidomics analysis (including sample preparation, data
43
44
45 acquisition, data preprocessing and the following peak annotation) and the
46
47 supplemental figures S1-S7.
48
49 Figure S1 The distributions of the coefficient of variation for each metabolite in QC
50
51
52 samples.
53
54 Figure S2 Metabolic disruptions related to glioma.
55
56 Figure S3 Relative expression levels of pyruvate carboxylase (PCE) and pyruvate
57
58
59
dehydrogenesa kinase isozyme 1 (PDK1) key proteins related to pyruvate entering
60 17

1
2
3 into TCA cycle measured in human gliomas with IDH1 mutation (Mutant) and IDH1
4
5
6 wild-type (WT).
7
8 Figure S4 Relative expression levels of key proteins related to fatty acids metabolism
9
10 in human glioma tissues with IDH1 mutation (Mutant) versus IDH1 wild-type (WT).
11
12
13
Figure S5 Survival curve comparison of glioma patients having IDH1 mutation and
14
15 IDH1 wild-type.
16
17 Figure S6 Relative gene expression levels of ACSL1, ACSL3, ACSVL3, ACSL4 and
18
19
ACSL5 in IDH1 wild-type and mutant gliomas.
20
21
22 Figure S7 Survival curves of patients.
23
24 The supporting material 2 includes the supplemental Table S1-S4.
25
26 Table S1 The identification results of 619 metabolites detected in GC-MS, LC-MS
27
28
29 and lipidomics platforms, after deleting redundant detection.
30
31 Table S2 Differential metabolites identified in glioma compared to paired adjacent
32
33 tissues.
34
35
36
Table S3 Significantly altered metabolites in IDH1 mutant glioma compared to IDH1
37
38 wild type.
39
40 Table S4 Comparison of the alterations of lipids in total amount or ratios in IDH1
41
42
mutant glioma versus IDH1 wild type glioma.
43
44
45
46
47 Abbreviations
48
49 2-HG: 2-hydroglutarate; 2-KG, alpha-ketoglutarate; ACC: acetyl-CoA carboxylase;
50
51
52 ACSL1, 3, 4, 5: long-chain acyl-CoA synthetase isoforms; ACSVL3: very long-chain
53
54 acyl-CoA synthetase 3; CE-MS: capillary electrophoresis - mass spectrometry; Cer:
55
56 ceramide; CL: cardiolipin;CN: acylcarnitine; CN-C2: acetylcarnitine; CN-C3:
57
58
59
propanoylcarnitine; CN-C4: butanoylcarnitine; CPT1a and 1c: carnitine
60 18

1
2
3 palmitoyltransferase 1a and 1c; DG: diglycerides; ESI: electrospray ionization;
4
5
6 FASN: fatty acids synthases; FA: fatty acid; GC-MS: gas chromatography- mass
7
8 spectrometry; GPC: glycerylphosphorycholine; GPE:
9
10 glycerylphosphorylethanolamine; IDA: information-dependent acquisition; IDH:
11
12
13
isocitrate dehydrogenase; LC-MS: liquid chromatography- mass spectrometry; QC,
14
15 quality control; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanolamine;
16
17 MUFA: monounsaturated fatty acid; NADPH: nicotinamide adenine dinucleotide
18
19
phosphate; pACC: phosphor-acetyl-CoA carboxylase; PC: phosphatidylcholine;
20
21
22 PDK1: pyruvate dehydrogenesa kinase isozyme 1; PE: phosphatidylethanolamine;
23
24 PLS-DA: Partial Least Squares Discriminative Analysis; PS: phosphoserine; ROS:
25
26 reactive oxygen species; SAM: significance analysis of microarrays; SCD:
27
28
29 stearoyl-CoA desaturase; SVLFA: saturated very-long-chain fatty acid; TCA:
30
31 tricarboxylic acid cycle; TCGA: The Cancer Genome Atlas; TG: triglycerides; UPLC:
32
33 ultra-performance liquid chromatography; WHO: World Health Organization
34
35
36
37
38 AUTHOR INFORMATION
39
40 Corresponding Author
41
42
43
*Telephone: (86)-411-84379530; Fax: (86)-411-84379559; E-mail: xugw@dicp.ac.cn；
44
45 ORCID: 0000-0003-4298-3554
46
47
48
49
50 Notes
51
52 The authors declare no competing financial interests.
53
54
55
56
57
ACKNOWLEDGEMENTS
58
59
60 19

1
2
3 This research was supported by the foundations (81472374, 21505132) and key
4
5
6 foundation (21435006) from the National Natural Science Foundation of China and
7
8 the National Key Research and Development Program of China (2017YFC0906900).
9
10 And we thank Yongtao Liu for helping detecting IDH1 and IDH2 mutations.
11
12
13
14
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1
2
3
4 Figure legends
5
6 Figure 1. Metabolomics and lipidomics analyses identify metabolic changes associated with
7
8 IDH1 mutation in human glioma. PLS-DA score plot (A) and validation plot (B) of
9
10
metabolites in glioma tissues with and without IDH1 mutation. Each blue dot in (A)
11
12
13 represents a glioma tissue with IDH1 mutation and each green square represents a glioma
14
15 tissue without IDH1 mutation; (C) number of metabolites which were significantly increased,
16
17 or decreased, or had no changes in IDH1 mutation compared to that in IDH1 wild-type; (D)
18
19
Pathway analysis of differential metabolites related to IDH1 mutation in glioma. The
20
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22 important pathways were defined here as having -log(p)>3 and pathway impact factor>0.1
23
24
25
26
27 Figure 2. Important metabolites associated with IDH1 mutation in glioma. SAM analysis
28
29 based on 284 different metabolites or calculated variables. The variables were calculated
30
31 based on the detected metabolites or lipids as the total amount of lipids in each class, or as
32
33
ratios of individual lipids or metabolites.
34
35
36 Figure 3. Reprogramming of central carbon metabolism related to IDH1 mutation.
37
38 Differential metabolites (yellow columns stand for the IDH1 wild-type and blue columns for
39
40 the IDH1 mutant glioma) were mapped referring the KEGG metabolic pathway. Metabolites
41
42
detected, but having no significance are in black font, and metabolites not detected are in grey
43
44
45 font. G6P, Glucose 6-phosphate; F6P, Fructose 6-phosphate; F1,6BP, Fructose
46
47 1,6-bisphosphate; G3P, Glyceraldehyde 3-phosphate; 1,3BPG, 1,3-Bisphospho-glycerate;
48
49 2,3BPG, 2,3-Bisphospho-glycerate; 3PG, 3-Phosphoglycerate; PEP, Phosphoenolpyruvic
50
51
acid; LAC, Lactate; PYR, Pyruvic acid; Ala, Alanine; OAA, Oxaloacetate; FUM, Fumarate;
52
53
54 Asn, Asparagine; Asp, Asparate; ISC, Isocitric acid; 2-KG, 2-ketoglutaric acid; 2-HG,
55
56 2-Hydroxyglutaric acid; Gln, Glutamine; Glu, Glutamate; CIT, Citrate; AcCoA, Acetyl-CoA;
57
58 MCoA, Malonyl-CoA; FAs, Fatty acids; 6PG, 6-phosphogluconic acid; R5P, Ribose
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60 25

1
2
3 5-phosphate; PRPP, 5-Phosphoribosyl diphosphate; 3PHP, 3-Phosphonooxypyruvate; 3PSer,
4
5
3-Phosphoserine; Gly, Glycine; Ser, Serine; The proteins in yellow font were responsible for
6
7
8 the generation of downstream metabolites.
9
10
11
12
13 Figure 4. Reprogramming of lipid metabolism associated with IDH1 mutation. Differential
14
15 lipids (yellow columns stand for the IDH1 wild-type and blue columns for the IDH1 mutant
16
17 glioma) were mapped in simplified lipid synthesis and remodeling pathways. Lipids or
18
19
metabolites detected, but having no significance are in black font, and metabolites not
20
21
22 detected are in grey font. FA, Fatty acid; PA, phosphatidic acid; DG, diglycerides; PC,
23
24 phosphatidylcholine; LPC, lysophosphatidylcholine; GPC, glycerylphosphorycholine; GPE,
25
26 glycerylphosphorylethanolamine; LPE, lysophosphatidylethanolamine; PE,
27
28
phosphatidylethanolamine; PI, phosphatidylinsitol; CDP-DG,cytidinediphosphatediacy
29
30
31 glycerol; PG, phosphatidylglycerol; CL, cardiolipin; PS, phosphatidylserine; TG, triglycerides;
32
33 Cer, ceramide; GlcCer, galactosylceramide; CerG2, digalactosylceramide; SM, sphingomyelin.
34
35
36
37
38 Figure 5. Correlation network analyses of the differential ACSLs with lipid synthesis. Total
39
40 lipids in each class and proteins regulating de novo fatty acid synthesis and oxidation, such
41
42
as PCE, pACC and CPT1s, in the IDH1 wild-type (A) and mutant (B) gliomas are included.
43
44
45 Correlation coefficients between two variables larger than 0.5 were kept. No negative
46
47 correlation was obtained.
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39 Figure 1. Metabolomics and lipidomics analyses identify metabolic changes associated with IDH1 mutation in
40 human glioma. PLS-DA score plot (A) and validation plot (B) of metabolites in glioma tissues with and
41 without IDH1 mutation. Each blue dot in (A) represents a glioma tissue with IDH1 mutation and each green
42 square represents a glioma tissue without IDH1 mutation; (C) number of metabolites which were
43 significantly increased, or decreased, or had no changes in IDH1 mutation compared to that in IDH1 wild-
type; (D) Pathway analysis of differential metabolites related to IDH1 mutation in glioma. The important
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pathways were defined here as having -log(p)>3 and pathway impact factor>0.1.
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46 165x168mm (300 x 300 DPI)
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24 Figure 2. Important metabolites associated with IDH1 mutation in glioma. SAM analysis based on 284
different metabolites or calculated variables. The variables were calculated based on the detected
25
metabolites or lipids as the total amount of lipids in each class, or as ratios of individual lipids or
26 metabolites.
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28 175x91mm (600 x 600 DPI)
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45 Figure 3. Reprogramming of central carbon metabolism related to IDH1 mutation. Differential metabolites
46 (yellow columns stand for the IDH1 wild-type and blue columns for the IDH1 mutant glioma) were mapped
47 referring the KEGG metabolic pathway. Metabolites detected, but having no significance are in black font,
and metabolites not detected are in grey font. G6P, Glucose 6-phosphate; F6P, Fructose 6-phosphate;
48 F1,6BP, Fructose 1,6-bisphosphate; G3P, Glyceraldehyde 3-phosphate; 1,3BPG, 1,3-Bisphospho-glycerate;
49 2,3BPG, 2,3-Bisphospho-glycerate; 3PG, 3-Phosphoglycerate; PEP, Phosphoenolpyruvic acid; LAC, Lactate;
50 PYR, Pyruvic acid; Ala, Alanine; OAA, Oxaloacetate; FUM, Fumarate; Asn, Asparagine; Asp, Asparate; ISC,
51 Isocitric acid; 2-KG, 2-ketoglutaric acid; 2-HG, 2-Hydroxyglutaric acid; Gln, Glutamine; Glu, Glutamate; CIT,
52 Citrate; AcCoA, Acetyl-CoA; MCoA, Malonyl-CoA; FAs, Fatty acids; 6PG, 6-phosphogluconic acid; R5P,
53 Ribose 5-phosphate; PRPP, 5-Phosphoribosyl diphosphate; 3PHP, 3-Phosphonooxypyruvate; 3PSer, 3-
Phosphoserine; Gly, Glycine; Ser, Serine; The proteins in yellow font were responsible for the generation of
54
downstream metabolites.
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56 133x160mm (300 x 300 DPI)
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34 Figure 4. Reprogramming of lipid metabolism associated with IDH1 mutation. Differential lipids (yellow
35 columns stand for the IDH1 wild-type and blue columns for the IDH1 mutant glioma) were mapped in
36 simplified lipid synthesis and remodeling pathways. Lipids or metabolites detected, but having no
significance are in black font, and metabolites not detected are in grey font. FA, Fatty acid; PA, phosphatidic
37
acid; DG, diglycerides; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; GPC,
38 glycerylphosphorycholine; GPE, glycerylphosphorylethanolamine; LPE, lysophosphatidylethanolamine; PE,
39 phosphatidylethanolamine; PI, phosphatidylinsitol; CDP-DG,cytidinediphosphatediacy glycerol; PG,
40 phosphatidylglycerol; CL, cardiolipin; PS, phosphatidylserine; TG, triglycerides; Cer, ceramide; GlcCer,
41 galactosylceramide; CerG2, digalactosylceramide; SM, sphingomyelin.
42
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36 Figure 5. Correlation network analyses of the differential ACSLs with lipid synthesis. Total lipids in each class
37 and proteins regulating de novo fatty acid synthesis and oxidation, such as PCE, pACC and CPT1s, in the
38 IDH1 wild-type (A) and mutant (B) gliomas are included. Correlation coefficients between two variables
39 larger than 0.5 were kept. No negative correlation was obtained.
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21 Table of Contents
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23 82x36mm (300 x 300 DPI)
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