Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells

Abstract

Recent studies have highlighted the role of palmitoleic acid [16:1n-7 (cis-9-hexadecenoic acid)] as a lipid hormone that coordinates cross-talk between liver and adipose tissue and exerts anti-inflammatory protective effects on hepatic steatosis and insulin signaling in murine models of metabolic disease. More recently, a 16:1n-7 isomer, cis-7-hexadecenoic acid (16:1n-9), that also possesses marked anti-inflammatory effects, has been described in human circulating monocytes and monocyte-derived macrophages. By using gas chromatographic/mass spectrometric analyses of dimethyl disulfide derivatives of fatty acyl methyl esters, we describe in this study the presence of a third 16:1 isomer, sapienic acid [16:1n-10 (6-cis-hexadecenoic acid)], in phagocytic cells. Cellular levels of 16:1n-10 appear to depend not only on the cellular content of linoleic acid, but also on the expression level of fatty acid desaturase 2, thus revealing a complex regulation both at the enzyme level, via fatty acid substrate competition, and directly at the gene level. However, unlike 16:1n-7 and 16:1n-9, 16:1n-10 levels are not regulated by the activation state of the cell. Moreover, while 16:1n-7 and 16:1n-9 manifest strong anti-inflammatory activity when added to the cells at low concentrations (10 μM), notably higher concentrations of 16:1n-10 are required to observe a comparable effect. Collectively, these results suggest the presence in phagocytic cells of an unexpected variety of 16:1 isomers, which can be distinguished on the basis of their biological activity and cellular regulation.

Keywords: inflammation; lipid mediators; monocytes/macrophages.

Fig. 1.

GC/MS analyses of 16:1 isomers. Authentic 16:1 fatty acid standards were methylated and transformed into DMDS adducts as described in the Materials and Methods. A: Chromatographic separation of 16:1 fatty acid isomers. Mass spectra corresponding to the n-10 (B), n-9 (C), n-7 (D), and n-5 (E) isomers showing the molecular ion (M⁺) and the three diagnostic peaks (fragment corresponding to the terminal methyl part of the molecule, fragment containing the carboxyl group, and the latter with the loss of methanol).

Fig. 2.

Analysis of 16:1 isomers in human cells. After sample collection and lipid extraction, total lipids were transmethylated. The resulting fatty acid methyl esters were derivatized with DMDS and DMDS adducts of 16:1 isomers measured by GC/MS. Chromatographic region showing the main 16:1 fatty acid isomers present in human monocytes (A), human monocyte-derived macrophages (B), and human serum (C). The mass spectra corresponding to the n-10 (D, H), n-9 (E, I), n-7 (F, J), and n-5 (G, K) isomers, indicating the molecular ion (M⁺) and the three diagnostic peaks (fragment corresponding to the terminal methyl part of the molecule, fragment containing the carboxyl group, and the latter with the loss of methanol) are also shown.

Fig. 3.

Analysis of 16:1 fatty acids in activated cells. Human monocytes were treated with 10 μM arachidonic acid (A), 100 ng/ml LPS (B), 1 mg/ml zymosan (C), or 2 μM calcium ionophore A23187 (D) for the times indicated. Afterward, the cellular content of 16:1n-10 (gray), 16:1n-9 (dark red), and 16:1n-7 (green) was measured by GC/MS as described in the Materials and Methods. Results are shown as the mean ± SE of three independent experiments with incubations in duplicate. *P < 0.05, significantly different from the corresponding fatty acid in the unstimulated state (zero time). Note that in the experiments utilizing zymosan, no data on 16:1n-7 can be shown because zymosan particles contain relatively high amounts of this fatty acid.

Fig. 4.

Phagocytic cells of murine origin show elevated levels of 16:1n-10. After sample collection and lipid extraction, total lipids were transmethylated. The resulting fatty acid methyl esters were derivatized with DMDS and DMDS adducts of 16:1 isomers measured by GC/MS. Chromatographic region showing the main 16:1 fatty acid isomers present in mouse peritoneal macrophages (A), RAW264.7 cells (B), P388D₁ cells (C), and THP1 cells (D). The mass spectra corresponding to the n-10 (E, I), n-9 (F, J), n-7 (G, K), and n-5 (H, L) isomers, indicating the molecular ion (M⁺) and the three diagnostic peaks (fragment corresponding to the terminal methyl part of the molecule, fragment containing the carboxyl group, and the latter with the loss of methanol) are also shown.

Fig. 5.

Distribution of 16:1 fatty acids between phospholipids. A: Total content of 16:1 fatty acids in phospholipids. B: Profile of 16:1-containing PC (red), PE (light green), PI (yellow), and PS (pink) species in unstimulated RAW264.7 cells, as determined by LC/MS. Data in A are the sum of values of the various molecular species of each subclass shown in B. C–F: Identification of 16:1 isomer composition of the different phospholipid subclasses (see ordinate axes) of RAW264.7 cells, as determined by GC/MS. The data are given as percentage of the total 16:1 molar mass levels determined for each phospholipid subclass. Results are shown as the mean ± SE of three independent experiments with incubations in duplicate.

Fig. 6.

Fatty acid composition of human and murine phagocytic cells. The total fatty acid profile cells were determined by GC/MS after converting the fatty acid glyceryl esters into fatty acid methyl esters. Approximately 10⁷ cells were utilized for these determinations. Results are shown as the mean ± SE of three independent experiments with incubations in duplicate.

Fig. 7.

Levels of 16:1n-10 are influenced by the cellular content of 18:2n-6. A: Effect of the FADS2 inhibitor, SC-26196, on 16:1 fatty acid levels. RAW264.7 cells were incubated with the indicated concentrations of the inhibitor for 24 h. Afterward, the content of 16:1n-10 (gray circles), 16:1n-9 (dark red circles), and 16:1n-7 (green circles) was determined by GC/MS. Results are referred to the amount of fatty acid determined in the absence of inhibitor (11.8 ± 1.8 nmol/mg, 2.6 ± 0.4 nmol/mg, and 16.6 ± 2.1 nmol/mg for 16:1n-10, 16:1n-9, and 16:1n-7, respectively). B: The 16:0 to 18:2n-6 molar ratio in different phagocytic cells. Total lipids were transmethylated and fatty acid methyl esters were measured by GC/MS. C: Enrichment of cells with linoleic acid decreases the content of 16:1n-10 in RAW264.7 cells. Cells were incubated without (cyan bars) or with 200 μM linoleic acid (complexed with albumin at a 2:1 ratio) (pink bars) for 48 h. Lipid extracts were transmethylated and fatty acid methyl esters were measured by GC/MS. D: Expression of the FADS2 gene in different cell types. Results are shown as the mean ± SE of three independent experiments with incubations in duplicate (A, C) or triplicate (B, D).

Fig. 8.

Incorporation of exogenous 16:1 fatty acids into cellular lipids. RAW264.7 cells were incubated with 10 μM of either 16:1n-10 (gray bars), 16:1n-9 (red bars), or 16:1n-7 (green bars) for 14 h. Afterward, various cellular lipid fractions were isolated and the 16:1 fatty acid content was determined by GC/MS (A). The phospholipid fraction was further separated into subclasses and the 16:1 content was determined by GC/MS (B). Results are shown as the mean ± SE of three independent experiments with incubations in duplicate. GPL, glycerophospholipids; GL, glycerolipids; CE, cholesterol esters.

Fig. 9.

Anti-inflammatory effect of 16:1 fatty acids. A–C: The cells were incubated with 10 μM of 16:1n-10 (gray bar), 16:1n-9 (dark red bar), 16:1n-7 (green bar), or neither (black bar) for 14 h. Afterward, the cells were stimulated by 100 ng/ml LPS for 6 h and expression of Il6 (A), Ptgs2 (B), and Ccl2 (C) was measured by qPCR. D: Dose-response determinations. The cells incubated with the indicated fatty acid concentrations (16:1n-9, dark red bars; 16:1n-10, gray bars) were stimulated by 100 ng/ml LPS and gene expression (Nos2) was measured by qPCR. Responses of control incubations receiving the different fatty acid concentrations, but not LPS, were no different than control unstimulated cells (open bar) and are omitted for clarity. E: The 16:1n-10 does not alter the effect of 16:1n-9. The cells, incubated with 10 μM 16:1n-10 (gray bar), 16:1n-9 (dark red bar), both (pink bar), or neither (black bar), were stimulated by 100 ng/ml LPS for 6 h and gene expression (Ccl2) was measured by qPCR. F: Effect of 16:1 fatty acids on zymosan-induced gene expression. The cells were incubated with 10 μM of 16:1n-10 (gray bar), 16:1n-9 (dark red bar), 16:1n-7 (green bar), or neither (black bar) for 14 h. Afterward, the cells were stimulated by 200 μg/ml zymosan for 6 h and gene expression (Ccl2) was measured by qPCR. Results are shown as the mean ± SE of three independent experiments with incubations in triplicate. *P < 0.05, significantly different from incubations with LPS without fatty acids.

Fig. 10.

Effect of 16:1 fatty acids on LPS-induced signaling. RAW264.7 cells were incubated with 10 μM of 16:1n-9 (A), 16:1n-10 (B), or neither as indicated (vehicle) for 14 h. Afterward, the cells were stimulated by 100 ng/ml LPS for the indicated times and the phosphorylation of extracellular-regulated kinases p44 and p42 was studied by immunoblot. Blots shown are representative of three independent experiments and the figures below the blots show the relative quantifications of p44 and p42 phosphorylation with respect to β-actin. Note that the blots corresponding to total p44 and p42 are from the same samples as phosphorylated p44 and p42, but from a different gel run in parallel.