Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes

Abstract

Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA) supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR) in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect-propionic acid (PRO) and protocatechuic acid (PCA)-has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination) and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action.

Keywords: docosahexaenoic acid; hepatocytes; lipidomics; metabolomics; nuclear magnetic resonance (NMR); propionic acid; protocatechuic acid.

Figure 1

Lipidomic phenotyping by nuclear magnetic resonance (NMR) analysis of cells not supplemented and supplemented with docosahexaenoic acid (DHA), alone or in combination with protocatechuic acid (PCA) or propionic acid (PRO), for (a) 6 h and (b) 24 h. Principal component analysis and canonical analysis (PCA-CA) score plots: PC1 vs. PC2. In each group, five samples derived from three independent experiments were analyzed. Each symbol represents a different sample. Blue symbols = NS; purple symbols = PCA; cyan symbols = PRO; orange symbols = DHA; green symbols = DHA + PCA; red symbols = DHA + PRO. For each PCA-CA model, the confusion matrix and the discrimination accuracy are reported; A: actual classes; P: predicted classes.

Figure 2

Fatty acid phenotyping by gas chromatography (GC) analysis of cells not supplemented (NS) and supplemented with docosahexaenoic acid (DHA), alone or in combination with protocatechuic acid (PCA) or propionic acid (PRO), after (a) 6 h and (b) 24 h supplementation. Principal component analysis and canonical analysis (PCA-CA) score plots: PC1 vs. PC2. In each group, four samples derived from three independent experiments were analyzed. Each symbol represents a different sample. Blue symbols = NS; purple symbols = PCA; cyan symbols = PRO; orange symbols = DHA; green symbols = DHA + PCA; red symbols = DHA + PRO. For each PCA-CA model, the confusion matrix and the discrimination accuracy are reported; A: actual classes; P: predicted classes.

Figure 3

Lipid droplet accumulation in not supplemented (NS) and supplemented cells with docosahexaenoic acid (DHA), protocatechuic acid (PCA) and propionic acid (PRO), after (a) 6 h and (b) 24 h supplementation. Data in each group are means ± standard deviation (SD) of five samples derived from two independent experiments. Neutral lipid content is expressed as the percentage of the value obtained in NS cells (assigned as 100%). Statistical analysis was carried out by one-way ANOVA (panels (a,b) p < 0.001) using Dunnett’s post-test to compare NS and supplemented cells (** p < 0.01; *** p < 0.001).

Figure 4

Intracellular cholesterol concentration in not supplemented (NS) and supplemented cells with docosahexaenoic acid (DHA), protocatechuic acid (PCA) and propionic acid (PRO) after (a) 6 h and (b) 24 h supplementation. Data are expressed in µM cholesterol per well, and are means ± standard deviation (SD) of five samples derived from three independent experiments. Statistical analysis was carried out by the one-way ANOVA (panels (a,b) p < 0.05) using Dunnett’s post-test to compare NS and supplemented cells (* p < 0.05).

Figure 5

Cytoplasmic metabolomic phenotyping by nuclear magnetic resonance (NMR) analysis of cells not supplemented and supplemented cells with docosahexaenoic acid (DHA), alone or in combination with protocatechuic acid (PCA) or propionic acid (PRO), after (a) 6 h and (b) 24 h supplementation. Principal component analysis and canonical analysis (PCA-CA) score plots: PC1 vs. PC2. In each group, five samples derived from three independent experiments were analyzed. Each symbol represents a different sample. Blue symbols = NS; purple symbols = PCA; cyan symbols = PRO; orange symbols = DHA; green symbols = DHA + PCA; red symbols = DHA + PRO. For each PCA-CA model, the confusion matrix and the discrimination accuracy are reported; A: actual classes; P: predicted classes.

Figure 6

Box plots of the metabolites differentially concentrated in “no-DHA” (not supplemented (NS), protocatechuic acid (PCA), propionic acid (PRO), blue symbols) and “DHA” group (docosahexaenoic acid (DHA), DHA + PCA, DHA + PRO, yellow symbols) according to NMR analysis after (a) 6 h and (b) 24 h supplementation. For each comparison, the p-value (by the Wilcoxon test) is also reported.

Figure 6

Box plots of the metabolites differentially concentrated in “no-DHA” (not supplemented (NS), protocatechuic acid (PCA), propionic acid (PRO), blue symbols) and “DHA” group (docosahexaenoic acid (DHA), DHA + PCA, DHA + PRO, yellow symbols) according to NMR analysis after (a) 6 h and (b) 24 h supplementation. For each comparison, the p-value (by the Wilcoxon test) is also reported.