TNFα is a potent inducer of platelet-activating factor synthesis in adipocytes but not in preadipocytes. Differential regulation by PI3K

Abstract

Tumour necrosis factor alpha (TNFα) induces platelet-activating factor (PAF) synthesis in many inflammatory cells. Here, we investigate the possibility that TNFα stimulates PAF synthesis in rat adipocytes and preadipocytes and that phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) are implicated in this process. Primary cultures were incubated with [³H]lyso-PAF and stimulated by TNFα in the presence or absence of wortmannin. We found that, although both cultures synthesized PAF at a similar basal rate, TNFα-induced PAF synthesis in adipocytes was 7-fold higher than in preadipocytes. This suggested a maturation of PAF–TNFα interrelationship during adipocyte differentiation. Wortmannin enhanced TNFα-dependent PAF synthesis in adipocytes but not in preadipocytes, indicating the negative control by PI3K in mature cells. PAF increase was due to the regulation of its biosynthesis since PAF-acetylhydrolase (PAF-AH) activity was TNFα- and wortmannin-independent. Our hypothesis is that PAF mediates TNFα inflammatory effects in both adipocytes and preadipocytes and that this pathway is enhanced during adipocyte differentiation, a mechanism which is highly active during the development of obesity.

Introduction

White Adipose Tissue (WAT),¹ besides its traditional role in energy storage, is now well documented as a secretory and endocrine organ, which actively participates in the regulation of various physiological and pathological processes including immunity and inflammation [1]. Apart from adipocytes, which comprise the main cell type, WAT also contains stromovascular and immune cells, as well as connective tissue matrix and nerve tissue. WAT secretes biologically active proteins, collectively known as adipokines, derived from both adipocytes and the non-adipocyte fraction of the adipose tissue [2]. Several of these adipokines are known to be associated with inflammation as well as with obesity or metabolic syndrome [1], [3], [4]. Among them, tumour necrosis factor α (TNFα), which is oversecreted from WAT of obese individuals [5], has been suggested to be responsible for metabolic disturbances associated with obesity, mainly with insulin resistance [6].

TNFα is a proinflammatory cytokine implicated in a number of cell functions such as differentiation, proliferation and apoptosis [7]. Many cell types, including adipocytes, express and secrete TNFα, which acts in a paracrine, autocrine and even endocrine manner via two distinct receptors: TNFR1 and TNFR2 [8], [9], [10]. The downstream targets of TNFα, however, are not fully elucidated, although TNFα has been shown to activate phosphoinositide 3-kinase (PI3K) [11], [12], [13] as well as members of the mitogen-activated protein kinase (MAPK) family [14], [15], [16]. In addition, the implication of lipid mediators, such as platelet-activating factor (PAF), has been reported [17], [18], [19]. PAF, which is also a mediator of inflammation, is synthesized in many cell types by de novo or remodeling pathways [20]. Upon stimulation, PAF is synthesized mainly through the remodeling pathway which involves phospholipase A₂ (PLA₂) action on membrane glyceryl ethers and the consequent acetylation of the lyso-product at the sn-2 position by PAF-acetyltransferase [21]. Interestingly, in this pathway, extracellular signal-regulated kinase (ERK) is implicated; it has been shown that, in human neutrophils, ERK participates in PLA₂ activation which is the initial step of PAF synthesis [22]. PAF mediates a number of biological activities of TNFα, including TNFα-induced lethal hepatitis and the impairment of skeletal muscle contractility [17], [18].

In adipocytes, TNFα has multiple effects, such as regulation of gene expression [12], [23], downregulation of adipocyte specific proteins [24], regulation of adipocyte number and size [8], [25], induction of insulin resistance [26] and stimulation of lipolysis [14], [27], [28]. There is evidence that PI3K as well as ERK1/2 are involved in some of these effects [11], [12], [14], [28].

Our research group has already shown the ability of mature adipocytes to synthesize PAF under stimulation and the presence of PAF-acetylhydrolase (PAF-AH) activity in these cells. This enzyme catalyzes the hydrolysis of the acetyl ester at the sn-2 position of PAF and converts it into its inactive metabolite, lyso-PAF, thus regulating PAF levels in a number of cells [29]. In the present study we investigate the possibility that TNFα stimulates PAF biosynthesis in adipocytes. We are also interested in whether a similar effect exists in preadipocytes. This would suggest that PAF, a potent inducer of inflammation, is recruited by TNFα to exert its inflammatory effects in adipose tissue. Our interest is also focused on the possible implication of PI3K and ERK1/2 in this process.