DOI: 10.5068/d1r95w
title: Isotopologue distributions of fatty acid oxidation inhibition in U87 glioma cells

description: 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.
description: Gliomaspheres were dissociated into single cells with Accumax™
 and 2x10<sup>5</sup> 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 <sup>13</sup>C-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 (&lt; 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 <sup>13</sup>C 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 <sup>13</sup>C-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.
description: Description of Columns: Name: the name of
 compound KEGG.ID: KEGG ID Condition:
 experimental condition (sgRNA/sgRNA CNTL = Control guide RNA; ETOM = 100uM
 etomoxir; CPT1A CRISPR = guide RNA targeting CPT1A)
 Iso: isotopologue, the number of labeled carbons
 Nr.C: number of carbons in compound Exp1,
 Exp2, Exp3: techical replicate measurements Norm_Av:
 normalized average value of Exp1, Exp2, Exp3 Norm_Std:
 normalized standard deviation of Exp1, Exp2, Exp3 CV:
 coefficient of variation (ie. SD / mean) of Exp1, Exp2, Exp3
 Av: Average value of Exp1, Exp2, Exp3 MID1,
 MID2, MID3: mass isotopologue distribution (i.e. the percent contribution
 of each isotopologue to the pool of a given compound)
 ANOVA: ANOVA p-value compared across experimental conditions
 (sgRNA CNTL, sgRNA + ETOM, CPT1A CRISPR) Sig: degree of
 significance (" " = p&gt;0.05; "*" =
 p&lt;0.05; "**" = p&lt;0.01; "***" =
 p&lt;0.001).