Original Contribution
Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

https://doi.org/10.1016/j.freeradbiomed.2012.05.026Get rights and content

Abstract

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24 h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300 μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300 μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.

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.

References (43)

  • H.R. Griffiths et al.

    Homocysteine from endothelial cells promotes LDL nitration and scavenger receptor uptake

    Free Radic. Biol. Med.

    (2006)
  • J. Bielawski et al.

    Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography–tandem mass spectrometry

    Methods

    (2006)
  • S. Gao et al.

    Mechanisms underlying different responses of plasma triglyceride to high-fat diets in hamsters and mice: roles of hepatic MTP and triglyceride secretion

    Biochem. Biophys. Res. Commun

    (2010)
  • R.H. Unger et al.

    Lipoapoptosis: its mechanism and its diseases

    Biochim. Biophys. Acta

    (2002)
  • B. Fuhrman et al.

    Ox-LDL induces monocyte-to-macrophage differentiation in vivo: possible role for the macrophage colony stimulating factor receptor (M-CSF-R)

    Atherosclerosis

    (2008)
  • M. Febbraio et al.

    A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism

    J. Biol. Chem.

    (1999)
  • M.J. Thubrikar et al.

    Pressure-induced arterial wall stress and atherosclerosis

    Ann. Thorac. Surg.

    (1995)
  • H.M. Chen et al.

    The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity

    J. Biol. Chem.

    (1993)
  • J.C. Mazzei et al.

    Suppression of intestinal inflammation and inflammation-driven colon cancer in mice by dietary sphingomyelin: importance of peroxisome proliferator-activated receptor γ expression

    J. Nutr. Biochem.

    (2011)
  • R. Ghidoni et al.

    Use of sphingolipid analogs: benefits and risks

    Biochim. Biophys. Acta

    (1999)
  • J. Sot et al.

    Different effects of long- and short-chain ceramides on the gel-fluid and lamellar-hexagonal transitions of phospholipids: a calorimetric, NMR, and X-ray diffraction study

    Biophys. J.

    (2005)
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    These authors have contributed equally to this work.

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