A platelet-activating factor (PAF) receptor deficiency exacerbates diet-induced obesity but PAF/PAF receptor signaling does not contribute to the development of obesity-induced chronic inflammation
Graphical abstract
Introduction
Obesity is an emerging global medical issue that has long been considered as one of the most prominent risk factors for metabolic syndrome, including insulin resistance, hypertension, and hyperlipidemia [1]. Recent extensive studies have shown that insulin resistance in obesity can be attributed to chronic low-grade inflammation in adipose tissue [2]. As obesity develops, neutrophils, macrophages, and T cells infiltrate into expanding adipose tissues, and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β are released from these infiltrating immune cells [3], [4], [5]. These secreted pro-inflammatory cytokines have been shown to suppress insulin signaling and evoke insulin resistance [6]. Therefore, many factors that are associated with inflammatory responses are known to regulate chronic inflammation and insulin resistance [7].
Signaling lipids such as prostaglandins, eicosanoids, phosphoinositides, sphingolipids, and fatty acids control cellular processes, and also play important roles in immune responses. These lipid mediators are produced by lipid-modifying enzymes and exert their biological effects by binding to cognate receptors. Recent studies demonstrated that these lipid-associated enzymes and receptors regulated obesity-induced insulin resistance and indicated that signaling lipids also play crucial roles in insulin resistance [8], [9]. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a bioactive phospholipid that was first identified as a substance that aggregates platelets during anaphylactic shock in rabbits [10]. However, recent studies found that PAF mediated a broad range of biological actions including immune and non-immune responses. PAF is enzymatically synthesized by two distinct pathways: a de novo pathway and remodeling pathway. PAF is synthesized from 1-O-alkyl-2-acetyl-sn-glycerol in the de novo pathway. In the remodeling pathway, lyso-PAF (1-O-alkyl-sn-glycero-3-phosphocholine) is generated from 1-O-alkyl-2 acyl-sn-glycero-3-phosphocholine by phospholipase A2 (PLA2), and cytosolic PLA2α (cPLA2α) plays a major role in the production of PAF by inflammatory cells [11]. PAF is synthesized through the remodeling pathway under inflammatory conditions, and PAF is then biosynthesized from lyso-PAF by lysophosphatidylcholine acyltransferase 2 (LPCAT2) [12]. PAF was previously shown to exert its bioactive effects by binding to its G-protein-coupled seven-transmembrane receptor [13], [14]. The PAF receptor couples with Gαi and Gαq proteins, which regulate the concentrations of cyclic AMP and Ca2+, respectively. PAF has well-characterized biological actions, such as the stimulation of inflammatory cells, including platelets and neutrophils, promotion of leukocyte chemotaxis, regulation of pro-inflammatory cytokines, and smooth muscle contraction, and has been implicated in many inflammatory diseases [15], [16], [17], [18], [19], [20].
We previously reported that adiposity and weight gain were increased with age in PAF receptor-knockout (PAFR-KO) mice, and, although these mice developed severe obesity, abnormal liver weights were not observed [21]. The epididymal white adipose tissue (WAT) of PAFR-KO mice at 24 weeks was susceptible to chronic inflammation (the infiltration of macrophages and production of proinflammatory adipokines) earlier than that of the wild-type (WT) littermates; however, PAFR-KO mice did not exhibit metabolic disorders such as hyperglycemia [21]. We here investigated the role of PAF receptor signaling in obesity-induced chronic inflammation in WAT using high-fat diet (HFD)-fed mice. Inflammatory cytokine mRNA levels and the infiltration of classically activated macrophages in the WAT were markedly higher in HFD-fed PAFR-KO mice than in HFD-fed WT mice in vivo, even though PAF receptor signaling promoted inflammatory phenotypes in the in vitro experiments. Furthermore, fasting serum levels of glucose were higher and glucose tolerance was more severely impaired in HFD-fed PAFR-KO mice than in HFD-fed WT mice. In the present study, we demonstrated for the first time that PAFR-KO mice fed a HFD developed chronic inflammation and glucose metabolism disorders in vivo.
Section snippets
Reagents
Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was purchased from Difco Laboratories (Detroit, MI). 1-O-Hexadecyl-2-(N-methylcarbamyl)-sn-glycero-3-phosphocholine (c-PAF) was purchased from Sigma–Aldrich (St. Louis, MO). IL-4 was purchased from R&D Systems (Minneapolis, MN).
Animals
PAFR-KO and WT mice were originally obtained from Drs. Satoshi Ishii and Takao Shimizu, as described previously [22]. Mice were housed under specific pathogen-free conditions at the University of Shizuoka and given ad
The PAF receptor, cPLA2α, and LPCAT2 were highly expressed in WAT
In the first series of experiments, we evaluated the expression of genes encoding the PAF receptor (Ptafr), cPLA2α, LPCAT2, and LPCAT1 in adipose tissue. Whole lung tissue was used as a positive control [11]. The expression levels of PAF receptor mRNA were similar between brown adipose tissue and whole lung tissue or were higher in WAT. PAF receptor mRNA levels were markedly upregulated in epididymal WAT (Fig. 1A). The PAF synthetic enzymes, cPLA2α and LPCAT2, were also highly expressed in both
Discussion
In the present study, we generated obesity model mice at a young age with glucose metabolism disorders; a deficiency in the PAF receptor in 20-week-old mice fed a HFD for 12 weeks resulted in more severe obesity, higher levels of serum glucose, and less glucose tolerance than WT mice (Fig. 3, Fig. 9). Body weight with age and adiposity were higher in ND-fed PAFR-KO mice than in ND-fed WT littermates, and PAFR-KO mice did not exhibit metabolic disorders such as mild hyperglycemia or fatty liver
Acknowledgments
This work was supported in part by a Grant-in-Aid for Young Scientists (B) (26860041, to MY) and Grant-in-Aid for challenging Exploratory Research (26670032, to JS) from the JSPS KAKENHI and by the Sasakawa Scientific Research Grant from The Japan Science Society (to MY). We gratefully acknowledge Ryu Shou, Yoshiki Hattori, and Masayuki Miyatake for their excellent technical assistance.
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Masakazu Matsui and Ryoko Higa contributed equally to this study.