Elsevier

Atherosclerosis

Volume 202, Issue 2, February 2009, Pages 382-393
Atherosclerosis

Induction of proinflammatory cytokines by long-chain saturated fatty acids in human macrophages

https://doi.org/10.1016/j.atherosclerosis.2008.05.033Get rights and content

Abstract

Increased circulating free fatty acids in subjects with type 2 diabetes may contribute to activation of macrophages, and thus the development of atherosclerosis. In this study, we investigated the effect of the saturated fatty acids (SFA) palmitate, stearate, myristate and laurate, and the unsaturated fatty acid linoleate, on the production of proinflammatory cytokines in phorbol ester-differentiated THP-1 cells, a model of human macrophages. Palmitate induced secretion and mRNA expression of TNF-α, IL-8 and IL-1β, and enhanced lipopolysaccharide (LPS)-induced IL-1β secretion. Proinflammatory cytokine secretion was also induced by stearate, but not by the shorter chain SFA, myristate and laurate, or linoleate. Triacsin C abolished the palmitate-induced cytokine secretion, suggesting that palmitate activation to palmitoyl-CoA is required for its effect. Palmitate-induced cytokine secretion was decreased by knockdown of serine palmitoyltransferase and mimicked by C2-ceramide, indicating that ceramide is involved in palmitate-induced cytokine secretion. Palmitate phosphorylated p38 and JNK kinases, and blocking of these kinases with specific inhibitors diminished the palmitate-induced cytokine secretion. Palmitate also activated the AP-1 (c-Jun) transcription factor. Knockdown of MyD88 reduced the palmitate-induced IL-8, but not TNF-α or IL-1β secretion. In conclusion, our data suggest that the long-chain SFA induce proinflammatory cytokines in human macrophages via pathways involving de novo ceramide synthesis. This might contribute to the activation of macrophages in atherosclerotic plaques, especially in type 2 diabetes.

Introduction

It is well established that subjects with insulin resistance and type 2 diabetes have an increased risk of developing atherosclerosis [1]. Possible mechanisms involved in the acceleration of atherosclerosis in diabetes are numerous, complex and poorly characterized, but a chronic immune response has been suggested to play a role [2], [3], [4]. Proinflammatory cytokines such as IL-6 and TNF-α are elevated in the circulation of patients with diabetes [5], [6].

Macrophages are multipotent inflammatory cells with the capacity to synthesize and secrete proinflammatory cytokines, such as TNF-α, IL-1β, IL-8 and IL-6. They play a central role during the development of atherosclerosis, and are one of the main cellular components of the atherosclerotic plaque [7], [8]. Activated macrophages have been shown to be present in higher numbers in atherosclerotic plaques from patients with diabetes compared with plaques from non-diabetic subjects [8], [9], [10], [11].

Circulating levels of free fatty acids (FFA) are increased in diabetes [12], and it has been suggested that they are involved in modulation of the immune system (reviewed in [13]). Recent studies indicate a specific proinflammatory effect of saturated fatty acids (SFA) on several cell types [14], [15]. For example, in murine macrophages, SFA, but not unsaturated FFA, induce expression of cyclooxygenase 2 (COX-2), IL-1α and iNOS [14]. The most prevalent SFA in the human diet are palmitate (C16:0), stearate (C18:0), myristate (C14:0), and laurate (C12:0). Palmitate is the most abundant SFA in plasma, and together with stearate it constitutes 90% of circulating SFA [15]. Palmitate has been shown to induce the expression of IL-6 in human myotubes [15], endothelial cells [16] and adipocytes [17], and of TNF-α in adipocytes [17].

The proinflammatory cytokine genes are regulated at the transcriptional level by several transcription factors including activator protein 1 (AP-1), nuclear factor-κB (NF-κB) and c-AMP-responsive element (CREB) [18], [19][20], [21], [22], [23], [24]. The activity of AP-1 and CREB transcription factors is in turn regulated by several kinases such as JNK and p38 kinases, which activate the transcription factors by phosphorylation [25], [26], [27].

Ceramide is a specific metabolite of long-chain SFA, such as palmitate and stearate [28]. This metabolite can act as an inducer of the proinflammatory cytokines, activates p38 and JNK kinases and the transcription factor AP-1 [29], [30]. As such, it might be involved in any long-chain SFA-mediated proinflammatory response.

We hypothesized that palmitate induces a ceramide-dependent production of the proinflammatory cytokines TNF-α, IL-8, IL-1β and IL-6 in human macrophages. The effect of palmitate on cytokine secretion and expression, on activation of the kinases p38 and JNK, and the transcription factors AP-1, NF-κB and CREB was investigated. We also assessed the importance of the de novo ceramide synthesis pathway in palmitate-induced cytokine secretion. In addition, we investigated the effect of other SFA (laurate, myristate and stearate), and the unsaturated fatty acid linoleate on the proinflammatory cytokine secretion in macrophages.

Section snippets

Materials

Sodium salts of laurate, myristate, palmitate, stearate and linoleate, bovine serum albumin (BSA; fatty acid free and low endotoxin), phorbol 12-myristate 13-acetate (PMA), α-tocopherol (Vitamin E), β-mercaptoethanol, dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS) from Salmonella typhimurium, triacsin C and N-acetyl-d-sphingosine (C2-ceramide) were obtained from Sigma–Aldrich (Stockholm, Sweden). Synthetic diacylated lipoprotein (FSL-1) was obtained from Invivogen (San Diego, CA, USA).

Palmitate and stearate induce proinflammatory cytokine secretion from macrophages

We initially investigated the effect of the long-chain saturated fatty acid palmitate and the unsaturated fatty acid linoleate on proinflammatory cytokine secretion in phorbol ester-differentiated THP-1 cells. At a concentration of 100 μM, palmitate increased the secretion of TNF-α, IL-8 and IL-1β in a time-dependent manner (Fig. 1A–C). The levels of the secreted cytokines (mean ± S.E.M.) from cells treated with palmitate for 27 h were: 130.1 ± 81.4 pg TNF-α/ml, 30.1 ± 7.2 ng IL-8/ml and 204.9 ± 83 pg

Discussion

In this study we show that two long-chain SFA, palmitate and stearate, induced the secretion of TNF-α, IL-1β and IL-8 in human macrophages derived from THP-1 cells, as opposed to the unsaturated fatty acid linoleate and the shorter chain SFA, laurate and myristate. The effect of palmitate required activation of palmitate to palmitoyl-CoA and possibly further conversion involving the de novo ceramide synthesis pathway. The palmitate effect on cytokine secretion was also shown to be dependent of

Acknowledgments

We thank Christina Ullström for skillful technical assistance and Rosie Perkins for critical reading of the manuscript. This study was supported by grants from the Swedish Medical Research Council, the Swedish Heart and Lung foundation, Emelle foundation, Sahlgrenska University Hospital and Astra Zeneca (unrestricted grant).

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