The conjugated linoleic acid isomer trans-9,trans-11 is a dietary occurring agonist of liver X receptor α

Abstract

Conjugated linoleic acid (CLA) isomers are dietary fatty acids that modulate gene expression in many cell types. We have previously reported that specifically trans-9,trans-11 (t9,t11)-CLA induces expression of genes involved in lipid metabolism of human macrophages. To elucidate the molecular mechanism underlying this transcriptional activation, we asked whether t9,t11-CLA affects activity of liver X receptor (LXR) α, a major regulator of macrophage lipid metabolism. Here we show that t9,t11-CLA is a regulator of LXRα. We further demonstrate that the CLA isomer induces expression of direct LXRα target genes in human primary macrophages. Knockdown of LXRα with RNA interference in THP-1 cells inhibited t9,t11-CLA mediated activation of LXRα including its target genes. To evaluate the effective concentration range of t9,t11-CLA, human primary macrophages were treated with various doses of CLA and well known natural and synthetic LXR agonists and mRNA expression of ABCA1 and ABCG1 was analyzed. Incubation of human macrophages with 10 μM t9,t11-CLA led to a significant modulation of ABCA1 and ABCG1 transcription and caused enhanced cholesterol efflux to high density lipoproteins and apolipoprotein AI. In summary, these data show that t9,t11-CLA is an agonist of LXRα in human macrophages and that its effects on macrophage lipid metabolism can be attributed to transcriptional regulations associated with this nuclear receptor.

Introduction

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometrical isomers of linoleic acid with conjugated double bonds. Depending on the position and geometry of the double bonds, several isomers of CLA have been identified. Most common CLA sources are beef, dairy products and vegetable oils [1]. The daily intake of CLA has been calculated for various countries and estimated at several hundred mg/day in a typical diet [2]. Whereas cis-9,trans-11 (c9,t11)-CLA can be found mainly in meat and dairy products [3], the trans-9,trans-11 (t9,t11)-CLA isomer is a major constituent of vegetable oils, because it is specifically generated during partial hydrogenation of vegetable oils and oil refining [4]. However, both CLA isomers can be formed from linoleic acid by ruminal bacteria (Butyrivibrio fibrisolvens and Clostridium proteoclasticum) [5], by lactic acid bacteria (Lactobacillus plantarum) and by human gastrointestinal bacteria (Bifidobacterium breve) [6], [7]. Interestingly, certain Bifidobacterium species convert c9,t11-CLA to t9,t11-CLA [7].

In recent years c9,t11-CLA has been reported to have various beneficial effects, such as anti-inflammatory and anti-atherogenic effects [8]. The underlying molecular mechanisms leading to most reported physiological effects are a result of peroxisome proliferator-activated receptor (PPAR) α, γ and δ activation. PPARs are well described nuclear transcription factors that play important roles in cellular differentiation, cancer, insulin sensitization, atherosclerosis and several metabolic diseases [9]. c9,t11-CLA is characterized as strong PPAR agonist [10].

We have previously reported that both CLA isomers have individual effects on gene expression in human macrophages [11]. t9,t11-, but not c9,t11-CLA activated the ATP-binding cassette (ABC) transporter G1 by a sterol regulatory element binding protein (SREBP)-1c dependent mechanism. ABCG1 belongs to the ABC-transporter family, is highly expressed in macrophages and mediates cholesterol efflux to mature high density lipoproteins (HDLs). Cholesterol efflux from macrophages to ApoAI is mediated by ABCA1 [12]. ABCA1 and ABCG1 have been shown to function cooperatively to remove cholesterol from cells, both transporters promote macrophage reverse cholesterol transport in vivo and are additive in their effects [13], [14]. SREBP-1c is a nuclear transcription factor controlling several genes involved in fatty acid biosynthesis and ABCG1 [9]. Its expression is mainly regulated at the transcriptional level, because it contains two liver X receptor (LXR) binding sites in its promoter [15].

Liver X receptors (LXRs) are ligand dependent transcription factors belonging to the nuclear receptor superfamily of proteins [16]. LXRs form heterodimers with retinoid X receptor (RXR) and then bind to LXR-responsive elements (LXR-Es) in the promoters of target genes. The liver X receptor subfamily consists of two members, LXRα and LXRβ, whereas LXRα is the major isoform expressed in macrophages [17]. LXRs control various genes of reverse cholesterol transport directly, such as ABCA1 and ABCG1, and several lipoprotein-remodeling enzymes, including cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) [18], [19], [20]. Consistent with their physiological roles, endogenous activators of LXR are oxidized cholesterol derivates (oxysterols). In addition to natural ligands, a number of synthetic LXR agonists have been developed. Remarkably, treatment of rodents with LXR agonists decreases atherosclerosis [21].

It is well accepted that specific isomers of CLA have distinct effects on gene expression. Although action of c9,t11-CLA is well described, action of t9,t11-CLA is poorly understood. Therefore, the aim of this study is to better understand the molecular mechanism underlying the biological activity of t9,t11-CLA. So far, the physiological effects of CLA isomers are attributed to transcriptional events associated with the nuclear receptor PPAR. This study is the first demonstrating that a CLA isomer is an agonist of the nuclear receptor LXRα.