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. 2019 Dec 20;9(1):19593.
doi: 10.1038/s41598-019-55985-z.

Lipid accumulation and oxidation in glioblastoma multiforme

Bouchra Taïb  1 , Amine M Aboussalah  2 , Mohammed Moniruzzaman  3 , Suming Chen  3 , Norman J Haughey  3 , Sangwon F Kim  1   4 , Rexford S Ahima  5
Affiliations

Lipid accumulation and oxidation in glioblastoma multiforme

Bouchra Taïb et al. Sci Rep. .

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor in adults. Despite the multimodal standard treatments for GBM, the median survival is still about one year. Analysis of brain tissues from GBM patients shows that lipid droplets are highly enriched in tumor tissues while undetectable in normal brain tissues, yet the identity and functions of lipid species in GBM are not well understood. The aims of the present work are to determine how GBM utilizes fatty acids, and assess their roles in GBM proliferation. Treatment of U138 GBM cells with a monounsaturated fatty acid, oleic acid, induces accumulation of perilipin 2-coated lipid droplets containing triglycerides enriched in C18:1 fatty acid, and increases fatty acid oxidation. Interestingly, oleic acid also increases glucose utilization and proliferation of GBM cells. In contrast, pharmacologic inhibition of monoacylglycerol lipase attenuates GBM proliferation. Our findings demonstrate that monounsaturated fatty acids promote GBM proliferation via triglyceride metabolism, suggesting a novel lipid droplet-mediated pathway which may be targeted for GBM treatment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effect of oleic acid treatment on LDs and TAGs accumulation in U138 human GBM cells and CRL 8621 human astrocytes. (A) LD accumulation in response to oleic acid (OA, 100 μM) was revealed using Bodipy staining, DAPI (blue) and fluorescence (green). (B) Lipid levels were determined by thin layer chromatography (TLC) and quantified using Image J software. Relative levels of fatty acids were normalized by protein content. Values were means ± SE, N = 3 independent experiments for TLC. N.D: not detected. Statistical analyses were performed with unpaired Student’s t-test. ****p < 0.0001 versus control.
Figure 2
Figure 2
Oleic acid stimulates U138-MG GBM cells but not CRL 8621 astrocytes proliferation. (A) EdU proliferation test was performed in the presence of oleic acid (OA, 100 μM). Proliferating cells were detected via EdU (red). DAPI stained nuclei in blue. Merged view of EdU (red) and DAPI (blue). The images are representative of the data obtained. Quantification was performed using Image J software. Values were means ± SE. N = 3 independent experiments. Statistical analyses were performed with unpaired student’s t-test. ****p < 0.0001 versus control. (Scale bar = 0.1 mm).
Figure 3
Figure 3
Oleic acid modifies lipid composition in U138 human GBM cells. (A) Hierarchical clustering heatmap analysis of lipids in U138-MG glioma cells in response to serum starvation and oleic acid treatment. The columns represent sample’s conditions and the rows indicate lipids. Each colored cell on the map corresponds to a concentration value. The distances among the clusters are presented by dendrograms between the clusters and indicate similarity between lipids species. (B) Reconstructed Lipid species spectra in response to oleic acid treatment. Intensity of lipids species is presented as peaks in (µg/mg). The differences between control and oleic acid treatment are presented as a red spread. (C) Triacylglycerols. (D) Sphingomyelins. (E) Phosphatidylcholine. (F) Non-esterified Fatty acid composition in U138 GBM cells in response to 24 hrs oleic acid treatment versus control. Relative levels of fatty acids were normalized by protein content. Values were means ± SE. N = 3 for mass spectrometry. Results are presented in (µg/mg). Statistical analyses were performed with unpaired Student’s t-test. *, **, and ****p < 0.05, 0.01, 0.0001 versus control.
Figure 4
Figure 4
Oleic acid increases β oxidation, glucose utilization and expression of genes involved in LCFA metabolism in U138 GBM cells. (A) Gene expression was determined by qPCR and mRNA levels were normalized to 18 S levels. Perilipin (PLIN), Diacylglycerol O-acyl-transferase (DGAT), Carnitine acyltransferase1 (CPT-1), Sterol regulatory element-binding proteins (SREBP), Fatty acid synthase (FASN). (B) Cropped western blot images and quantitation of PLIN2 and GAPDH protein levels from cell lysates. Human hepatoma (VL17A) cell line was used as a positive control (+control). Full length blots are in Supplementary Fig. 1. N = 3 independent experiments in triplicate. (C) Oleic acid oxidation, and (D) glucose utilization were measured using a 14C-oleic acid and 3H-glucose). N = 3 independent experiments in triplicate. Values were means ± SE. Statistical analyses were performed with unpaired Student t-test *, **, ***, and ****p < 0.05, 0.01, 0.001, and 0.0001 versus control. OA: Oleic acid.
Figure 5
Figure 5
DGAT1 inhibitor decreases slightly TAGs accumulation without abolishing the effect of oleic acid on U138 GBM cell proliferation. (A) LD accumulation in response to DGAT1 inhibitor (DGAT1i,  PF-04620110, 3 µM) was revealed using Bodipy staining, DAPI (blue) and fluorescence (green). (B) Lipid levels were determined by thin layer chromatography (TLC) and quantified using Image J software. (C) EDU proliferation test was performed in presence of oleic acid, (OA, 100 µM), in response to (PF-04620110, 3 µM). Proliferating cells were detected via EDU (red). DAPI stained nuclei in blue. Merged view of EdU (red) and DAPI (blue). N = 3 independent experiments. Statistical analyses were performed with unpaired Student’s t-test *p < 0.05 versus control.
Figure 6
Figure 6
General lipases inhibition attenuates the effect of oleic acid on GBM cell proliferation. EDU proliferation test was performed in presence of oleic acid (OA, 100 µM), in response to different concentrations of the general lipase inhibitor diethyl-p-nitrophenylphosphate (20 µM, 100 µM). Proliferating cells were detected via EDU (red). DAPI stained nuclei in blue. Merged view of EdU (red) and DAPI (blue). N = 3 independent experiments. Statistical analyses were performed with unpaired Student’s t-test **, and ***p < 0.01 and 0.001 versus control. (Scale bar = 0.1 mm).
Figure 7
Figure 7
MAGL inhibition attenuates the effect of oleic acid on GBM cell proliferation. (A) EDU proliferation test was performed in presence of oleic acid (OA, 100μM ), in response to different concentrations of JZL184 (1, 5, and 10 µM). Proliferating cells were detected via EDU (red). DAPI stained nuclei in blue. Merged view of EdU (red) and DAPI (blue). N = 3 independent experiments. Statistical analyses were performed with unpaired Student’s t-test **, and *** p < 0.01 and 0.001 versus control. (Scale bar = 0.1 mm). (B) U138 GBM cells show elevations in MAG species in presence of JZL184 (5 and 10 µM) N = 3 for mass spectrometry. Results are presented in (µg/mg). Statistical analyses were performed with unpaired Student’s t-test. *, **, and ***p < 0.05, 0.01, 0.001 versus control.
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