Original ContributionPalmitate promotes monocyte atherogenicity via de novo ceramide synthesis
Graphical abstract
Highlights
► Palmitate but not oleate induces a proatherogenic increase in CD11b and CD36 expression in monocytes. ► Elevated CD11b in palmitate-treated monocytes associates with increased adhesion to endothelial bifurcations. ► Increased CD36 expression promotes the uptake of oxidized LDL in palmitate-treated monocytes. ► Mitochondrial reactive oxygen species (ROS), intracellular C16 ceramide, and sphingomyelin are increased by palmitate but not oleate. ► Inhibition of de novo ceramide synthesis but not ROS mitigated the phenotypic changes in monocytes induced by palmitate.
Introduction
Monocyte recruitment and adhesion to the endothelium are early events in atherogenesis: these events depend upon expression and activation of integrins/adhesion molecules [1]. CD11b is the most abundant α integrin subunit expressed by monocytes and associates with β2 integrin (CD18) to form the heterodimer, Mac1/CR3 [2], one of the ligands responsible for the firm adhesion of monocytes, binding to endothelial intercellular adhesion molecule-1 (ICAM-1) [3]. After migration into the subendothelial space, monocytes differentiate into macrophages under the influence of proinflammatory cytokines [4]. Uptake of oxidized low-density lipoproteins (LDL) into monocyte-derived macrophages is facilitated by scavenger receptors, including CD36 [5], typically giving rise to foam cells.
Epidemiological evidence supports a proatherogenic effect of diets rich in saturated fatty acids (FAs) such as palmitate, whereas monounsaturated FAs such as oleate appear protective [6], [7]. Dietary FAs are resynthesized into triacylglycerol, with stereospecific orientation of saturated and monounsaturated FAs within the enterocyte and released into the blood as chylomicrons [8]. In health, chylomicrons are rapidly cleared by target organs including liver, adipocytes, and skeletal muscle; during fasting or insulin insensitivity, free fatty acids (FFAs) are released into the circulation by lipolysis.
Elevated plasma FFA levels represent an increased risk for cardiovascular disease (CVD) [9], [10], [11]. Raised fasting plasma FFA levels are associated with increased all-cause and cardiovascular mortality, constituting an independent risk factor for sudden death in patients after coronary angiography [10]. Raised FFA levels increase the risk of myocardial infarction and stroke [11]. Also, increased circulating FFA levels frequently accompany increased blood pressure, impaired endothelial function, and increased inflammatory markers in subjects with and without diabetes, all being associated with increased risk of CVD [12], [13]. FFAs can increase the cellular concentrations of bioactive lipids, including ceramides, which associate with significant tissue dysfunction [14], and raised FFAs are also reported to increase mitochondrial reactive oxygen species (ROS) production, which associates with insulin resistance [15]. The underlying mechanisms linking elevated concentrations of FFA to an atherogenic phenotype of monocytes are unclear; little is known about the effects of the saturated FA palmitate and the unsaturated FA oleate on monocyte metabolism and function [16].
To investigate whether palmitate, the most abundant saturated FA in human plasma, may alter monocyte phenotype and function, this study has examined palmitate-induced CD11b and CD36 expression in U937 monocytes, monocyte adhesion to aortic endothelium, and monocyte uptake of oxidized LDL. The study has also assessed the potential roles of mitochondrial reactive oxygen species, cellular redox state, and ceramide, a sphingolipid metabolite generated from palmitate, as mediators of the altered monocyte phenotype.
Section snippets
Conjugation of fatty acids to bovine serum albumin (BSA)
Stock solutions of palmitate (200 mM) or oleate (100 mM) were prepared by dissolving sodium palmitate or sodium oleate into 0.1 M NaOH in 70% ethanol as previously described [17]. FAs were then complexed with 5% FA-free BSA to a concentration of 5 mM at 37 °C, stirred for 4 h, and adjusted to pH 7.4 to give a fatty acid:BSA ratio of 2:1. After sterilizing through a 0.2-μm filter, solutions were stored at 4 °C for no longer than 2 weeks. A control solution comprised 5% BSA with 0.1 M NaOH in 70%
Cell proliferation is reduced by palmitate
Our studies of FA effects on monocytes have addressed the effects of physiological FFA concentrations. Plasma palmitate ≤600 μM and oleate ≤300 μM are reported in subjects at risk for CVD; therefore 300 μM was the maximal FA concentration studied [11], [12]. Experiments were undertaken using cell lines rather than in vivo lipid infusion studies as animal models of lipid metabolism are not representative of human metabolic effects [27].
Incubation of U937 and THP-1 monocytes with palmitate (100 μM
Discussion
This study has shown that the saturated FA palmitate, but not the monounsaturated FA oleate, increases the expression of the integrin CD11b and scavenger receptor CD36 by viable human monocytes in a mitochondrial-ROS-independent but ceramide-dependent manner. The highest concentration of palmitate investigated here (300 μM) induced a small but significant loss of viability as has been reported for other cells [28] and our present findings suggest that mitochondrial-derived ROS play a role in
Acknowledgments
Dr. D. Gao acknowledges funding from Aston University and the ORSAS for her Ph.D. studies. Mr. C. Pararasa is funded by the BBSRC via a targeted priority studentship and further supported by Aston Research Centre for Healthy Ageing.
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These authors have contributed equally to this work.