Linoleic acid suppresses cholesterol efflux and ATP-binding cassette transporters in murine bone marrow-derived macrophages

Abstract

Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease (CVD), possibly associated with elevated plasma free fatty acid concentrations. Paradoxically, evidence suggests that unsaturated, compared to saturated fatty acids, suppress macrophage cholesterol efflux, favoring cholesterol accumulation in the artery wall. Murine bone marrow-derived macrophages (BMDM) were used to further explore the relationship between saturated and unsaturated fatty acids, and cholesterol efflux mediated by ATP-binding cassette transporters (ABCA1 and ABCG1) through transcription factors liver-x-receptor-alpha (LXR-α) and sterol receptor element binding protein (SREBP)-1. BMDM isolated from C57BL/6 mice were exposed to 100 μM linoleic acid (18:2) or palmitic acid (16:0) for 16 h, and 25 μg/mL oxidized low density lipoprotein for an additional 24 h. ABCA1 and ABCG1 mRNA expression was suppressed to a greater extent by 18:2 (60 % and 54 %, respectively) than 16:0 (30 % and 29 %, respectively) relative to the control (all p < 0.01). 18:2 decreased ABCA1 protein levels by 94 % and high density lipoprotein (HDL) mediated cholesterol efflux by 53 % (both p < 0.05), and had no significant effect on ABCG1, LXR-α or SREBP-1 protein levels. 16:0 had no effect on ABCA1, ABCG1, LXR-α or SREBP-1 protein expression or HDL-mediated cholesterol efflux. These results suggest that 18:2, relative to 16:0, attenuated macrophage HDL-mediated cholesterol efflux through down regulation of ABCA1 mRNA and protein levels but not through changes in LXR-α or SREBP-1 expression. The effect of 18:2 relative to 16:0 on macrophages cholesterol homeostasis may exacerbate the predisposition of individuals with T2DM to increased CVD risk.

Fig. 1

Effect of (A) oxLDL (B) oxLDL +/− FFA on lipid accumulation in BMDM. Lipids were first extracted from cells. Triacylglycerol (TAG) was determined using a colorimetric TAG Accumulation Kit and total and free cholesterol (TC and FC) were determined by gas chromatography, cholesteryl esters (CE) was determined by the difference between TC and FC. Values are representative of 3 independent experiments performed in triplicate. §p < 0.0001 for the comparison of BSA versus BSA + oxLDL.

Fig. 2

Effect of 18:2 and 16:0 on cholesterol efflux in BMDM. OxLDL- and [³H] cholesterol-loaded BMDM were stimulated with 50 ug/mL HDL for 4 hours. Fraction cholesterol efflux was estimated from the ratio of the radiotracer in the medium to the total (medium + cells) and normalized for cellular protein. Values are representative of 3 independent experiments in triplicate. Bars without a common letter are significantly different (p<0.05).

Fig. 3

Effect of 18:2 and 16:0 on ABCA1, ABCG1, SR-B1 and LXR-α mRNA and protein expression in BMDM. (A) ABCA1, ABCG1, SR-B1 and LXR-α mRNA expression was determined by real-time PCR. β-actin was used as a standard housekeeping gene. (B) ABCA1, ABCG1, SR-B1, LXR-α and β-actin protein expression was determined by Western blotting of BMDM cell lysates. β-actin was used to control equal loading. (C) Representative Western blot. Values are the mean +/− SD 3 independent experiments performed in triplicate. Bars without a common letter are significantly different (p<0.05).

Fig. 4

Effect of 18:2 and 16:0 on SREBP-1a, 1c, 2, SCD1, and ACC mRNA and SREBP-1 nuclear protein expression in BMDM. (A) SREBP-1a, 1c, 2, SCD1, and ACC mRNA expression was determined by real-time PCR. β-actin was used as a standard housekeeping gene. (B) SREBP-1 nuclear protein expression was determined by Western blotting of BMDM nuclear extracts. β-actin was used to control equal loading. (C) Representative Western blot. Values represent the mean +/−SD of 3 independent experiments performed in triplicate. Bars without a common letter are significantly different (p<0.05).