Atherogenic Trans Fatty Acid Alters Macrophage Lipid Metabolism with Sustained Dysregulation of Zinc Homeostasis

Atherosclerosis, through its sequelae of heart disease, stroke and peripheral vascular disease, underlies substantial human morbidity and mortality. The incidence of atherosclerosis increased markedly over the last century paralleling the increase in fat, particularly saturated fat, in the modern diet. Furthermore, artificial hydrogenation of saturated fats, originally introduced to provide a ‘healthier’ dietary substitute, resulted in consumption of non-physiological trans fatty acids that are themselves now strongly linked to the development of atherosclerosis. A major cellular target for the negative effects of fatty acids in the vasculature appears to be the macrophage, which through actions including the production of inflammatory cytokines, plays a central role of the pathophysiology of atherosclerosis. Despite the importance of these effects, our understanding of the fundamental molecular changes that underlie the negative effects of fatty acids, and especially trans fatty acids, remains fragmentary. In previous work we showed that prolonged exposure of human macrophages to the 18 carbon trans-monoenoic fatty acid elaidic acid impaired lipid metabolism, partially blocking beta-oxidation and thereby altering cellular lipid composition. To determine the functional consequences of this alteration in cellular lipids, we next examined changes in macrophage gene expression. Primary human macrophages derived from peripheral blood monocytes were treated with the cis fatty acid oleate or the trans fat elaidate for 44 hours, and gene expression was evaluated using the affymetrix gene array, with significant differences confirmed by real time quantitative PCR. Expression of genes linked directly to lipid metabolism differed, as expected, but the most striking differences between cis and trans fatty acid treated macrophages were in their expression of regulators of zinc homeostasis. In particular, there was a marked divergence in expression levels for metallothioneins, the zinc binding proteins which regulate intracellular zinc levels: metallothionein expression was consistently lower in the elaidate-treated macrophages. In contrast, the zinc transporter SLC39A10 was significantly upregulated in macrophages by exposure to the trans but not the cis fatty acid. SLC39A10 mediates uptake of extracellular zinc by cells and its release from organellar stores; its effects are opposed by SLC30, which export zinc from the cytosol but were not induced by exposure to elaidate. These results suggested that trans fats would increase zinc activity in macrophages. To test this we used the fluorescent zinc indicator FluoZin3 (N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl)amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)phenoxy]ethoxy]-4-methoxyphenyl]glycine). These studies confirmed that macrophages exposed to elaidate showed increased intracellular Zn2+, compared to both untreated or oleate-treated macrophages. Furthermore, the increase in zinc activity was associated with activation of the NFkB signaling pathway: macrophages treated with elaidic acid showed prominent nuclear translocation of NFkB p65, and the effect was abrogated by cotreatment with the zinc chelator TPEN. We next compared the effects of the trans fat with those of saturated fatty acids. Macrophages exposed to stearate or palmitate showed a milder and delayed increase in metallothionein expression, compared to those treated with oleate, but, like oleate, stearate and palmitate had no effect on the expression of zinc transporters. Studies using FluoZin3 showed a mild early increase in cytosolic Zn2+ following stearate or palmitate treatment, but at the later time points, when metallothionein expression was increased by the saturated fatty acids as well as by oleate, zinc activity returned to baseline. Only elaidic acid, which upregulated the zinc transporter, produced a sustained increase in intracellular macrophage Zn2+. Stearate and palmitate also stimulated early translocation of NFkB to the nucleus. These results suggest that trans fatty acids, while mimicking some activation pathways stimulated by the saturated fatty acids, exert distinct effects through sustained alterations in zinc regulatory proteins that promote elevated intracellular Zn2+.