Metabolic Pathway of Monounsaturated Lipids Revealed by In-Depth Structural Lipidomics by Mass Spectrometry

Abstract

The study of lipid metabolism relies on the characterization of the lipidome, which is quite complex due to the structure variations of the lipid species. New analytical tools have been developed recently for characterizing fine structures of lipids, with C=C location identification as one of the major improvements. In this study, we studied the lipid metabolism reprograming by analyzing glycerol phospholipid compositions in breast cancer cell lines with structural specification extended to the C=C location level. Inhibition of the lipid desaturase, stearoyl-CoA desaturase 1, increased the proportion of n-10 isomers that are produced via an alternative fatty acid desaturase 2 pathway. However, there were different variations of the ratio of n-9/n-7 isomers in C18:1-containing glycerol phospholipids after stearoyl-CoA desaturase 1 inhibition, showing increased tendency in MCF-7 cells, MDA-MB-468 cells, and BT-474 cells, but decreased tendency in MDA-MB-231 cells. No consistent change of the ratio of n-9/n-7 isomers was observed in SK-BR-3 cells. This type of heterogeneity in reprogrammed lipid metabolism can be rationalized by considering both lipid desaturation and fatty acid oxidation, highlighting the critical roles of comprehensive lipid analysis in both fundamental and biomedical applications.

Fig. 1.

Deep GP structure analysis workflow by LC-MS coupling with online PB reaction to reveal C=C location isomer metabolic pathway variations and FAO activity.

Fig. 2.

Lipid unsaturation decreased upon SCD1 inhibition. (A) Biosynthetic pathways of monounsaturated FA. SCD1 and SCD5, stearoyl-CoA desaturase 1 and 5; FADS2, fatty acid desaturase 2; FAO, fatty acid oxidation; Elovl, fatty acid elongase. (B) Mass spectra of PC profile before and after SCD1 inhibition in MCF-7 cells. (C and D) The relative ratios of PC 32:1/PC 32:0 and PE 34:1/PE 34:0. Differences between CAY10566-treated group samples and control group samples were evaluated for statistical significance using the 2-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bar represents the standard deviation, n = 3.

Fig. 3.

The relative amount of n-10 C=C location isomers increased significantly after SCD1 inhibition. (A to C) Relative increase of n-10 isomers in C16:1- and C18:1-containing GPs after SCD1 inhibition. (D) PB-MS/MS spectra of PC 36:1 before and after SCD1 inhibition in BT-474 cells and the corresponding structures. (E) Compositional increase of n-10 C=C location isomers in BT-474 cells after SCD1 inhibition. Differences between the 2 groups were evaluated for statistical significance using the 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bar represents the standard deviation, n = 3.

Fig. 4.

Isomer-resolved lipidomics analysis of C18:1-containing GPs in MCF-7 cells. (A) PB-MS/MS analysis of PC 34:1 and PC 36:1 before and after SCD1 inhibition. (B to D) Compositional variations of C=C location isomers in C18:1 fatty acyl in PCs, PEs, and PSs after SCD1 inhibition. (E) Compositional variations of C=C location isomers in C18:1-containing PCs after siRNA-mediated gene silencing of SCD1. Differences between the 2 groups were evaluated for statistical significance using the 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bar represents the standard deviation, n = 3.

Fig. 5.

Human breast cancer cell lines responded differently to SCD1 inhibition. (A) Hierarchical cluster analysis discriminated the 5 subtypes of human breast cancer cells by quantitative analysis of n-9/n-7 isomers in C18:1-containing GPs. (B) Fold change of n-9/n-7 isomeric ratio in C18:1-containing GPs after SCD1 inhibition. (C) Compositional variations of C=C location isomers in FA 18:1 after SCD1 inhibition. Differences between the 2 groups were evaluated for statistical significance using the 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bar represents the standard deviation, n = 3.

Fig. 6.

The measurement of FAO reveals its potential relationship with breast cancer cell line invasiveness. (A) PB-MS/MS spectra of PC 16:0_16:1 in MCF-7 cells before and after SCD1 inhibition. (B to D) Compositional variations of C=C location isomers in C16:1-containing GPs after small-molecule inhibitor or siRNA-mediated gene silencing of SCD1. (E) The inhibition of FAO by etomoxir compensates for the decrease of n-9/n-7 isomers in C18:1-containing GPs caused by SCD1 inhibition in MDA-MB-231 cells. (F) Schematic illustration of the indicators to measure FAO activity, and the relationship between FAO and metastasis. The indicators of C16:1 n-9/n-7 ratio can be used to infer FAO activity and cancer cell invasiveness. Differences between the group samples were evaluated for statistical significance using the 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bar represents the standard deviation, n = 3.