Effects of Omega‐6 Fatty Acids on Interleukin‐6, Phospholipase A2, and Prostaglandin‐Endoperoxide Synthase 2 Gene Expression in Lipopolysaccharide‐Challenged Murine Macrophages and Adipocytes

Abstract

Obese adipose tissue (AT) has been described as an organ involved in meta‐inflammation‐a chronic low‐grade inflammation linked to the predisposition to chronic diseases. Both adipocytes and macrophages are found in AT, with obese AT containing a higher percentage of macrophages compared to lean AT. Adipocytes and macrophages can produce pro‐inflammatory molecules (adipokines), with research showing that the macrophage is the primary contributor to inflammatory adipokine release by obese AT. Our lab has described differential effects of omega‐6 (n‐6) fatty acids (FAs) on the production of adipokines by 3T3‐L1 murine adipocytes and RAW 264.7 murine macrophages. The objective of this study was to compare the effects of n‐6 FAs on gene expression of inflammation‐related molecules in 3T3‐L1 and RAW264.7. Gene expression of interleukin‐6 (IL‐6), prostaglandin‐endoperoxide synthase‐2 (PTGS2), and phospholipase A2 (PLA2) were quantified in both of these cell lines after incubation with n‐6 FAs and a lipopolysaccharide (LPS) challenge. PGTS2 and PLA2 are enzymes involved in the production of inflammatory eicosanoids, while the IL‐6 gene codes for the IL‐6 protein. Macrophages were pre‐incubated with 100 μM of linoleic acid (LA; n‐6) or arachidonic acid (ARA; n‐6) for 24 h, followed by a 6 h, 0.01 μg/ml LPS challenge. Fibroblasts (3T3‐L1) were differentiated into adipocytes with the addition of 100 μM of one of the n‐6 FAs throughout the differentiation period, followed by a 6 h, 1 μg/ml LPS challenge once they were fully differentiated. After LPS challenge, RNA was extracted for quantitative PCR analysis. Fatty acid treatments had no effect on expression of the target genes (IL‐6, PTGS2, PLA2) in 3T3‐L1 cells (two‐way ANOVA). PTGS2 expression was downregulated in LA (0.24 ± 0.13 2ΔΔCt) and ARA (0.16 ± 0.13 2ΔΔCt)‐treated macrophages challenged with LPS compared to control (1.07 ± 0.13 2ΔΔCt) and LPS (1.70 ± 0.13 2ΔΔCt)‐challenged macrophages (two‐way ANOVA; p<0.0001). There were no significant LPS or LPS+n‐6 FA effects on gene expression of IL‐6 and PLA2in macrophages (two‐way ANOVA). Time‐response experimentation is necessary to determine the kinetic model of gene expression of these target genes in both cell lines.