Role of leukotriene B4 receptor signaling in human preadipocyte differentiation

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Abstract

We investigated the role of leukotriene B4 (LTB4)–leukotriene receptor (BLT) signaling in preadipocyte differentiation into mature adipocytes. Blockade of BLT signaling by treatment with lipoxygenase inhibitors, a BLT antagonist, and small interfering RNAs for BLTs in human and mouse preadipocytes isolated from adipose tissues showed acceleration of differentiation into mature adipocytes. DNA microarray analysis revealed regulation of transforming growth factor, beta-induced 68 kDa (TGFBI) expression through the BLT signaling pathway during adipocyte differentiation. Knockdown of TGFBI also showed acceleration of preadipocyte differentiation. The LTB4–BLT signaling pathway may negatively regulate preadipocyte differentiation via induction of TGFBI expression as a rate-limiting system to control adipocyte differentiation.

Highlights

► There have been reported the involvement of various inflammatory mediators on obesity and insulin resistance. ► Blockade of BLT signaling accelerates human preadipocyte differentiation into mature adipocytes. ► LTB4–BLT signaling pathway negatively regulates excessive adipocyte differentiation as a brake system. ► BLT signaling pathway might be a potential target for control of adipocyte differentiation.

Introduction

The involvement of various inflammatory mediators such as tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) in obesity that is closely related to insulin resistance has been reported [1], [2], [3]. One of the most important organs on obesity and insulin resistance is thought to be adipose tissue, because adipocytes in adipose tissue generate various adipocytokines that play important roles in the onset of metabolic syndrome [1], [2], [3].

Leukotrienes (LTs) such as leukotriene B4, C4, and D4 (LTB4, LTC4, and LTD4, respectively) that are generated through lipoxygenase (LOX) pathways are well-known to induce various inflammatory and allergic reactions such as leukocyte activation, capillary permeability, and bronchial contraction [4], [5]. LTB4 binds to specific receptors, BLT1 and BLT2, to activate the signaling pathways [6], [7]. LTs have been reported to be involved in the proliferation of various cell types such as epithelial, endothelial and mesangial cells [8], [9]. In addition, we have reported that LTB4 controls immature neural stem cell proliferation and differentiation via the BLT signaling pathway [10]. Therefore, LTB4 and its signaling pathway could be involved in various cell proliferations and differentiations. However, there are few reports about the role of LTs in adipocyte differentiation. Furthermore, most of reported papers about the adipocyte differentiation have used mouse fibroblastic 3T3-L1 cells, but not human preadipocytes. Therefore, the exact role of the LTB4 signaling pathway in adipocyte differentiation of human preadipocytes is still unclear.

In this study, we investigated the role of LTB4 and its receptor signaling pathway in human adipocyte differentiation and the potential mechanisms.

Section snippets

Reagents and antibodies

Insulin (INS), dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX) and rosiglitazone (ROSI) were purchased from Sigma Japan (Tokyo, Japan). LT synthetase, lipoxygenase (LOX) inhibitor, nordehydroguaiaretic acid (NDGA), and 5-LOX specific inhibitor, AA-861, were also purchased from Sigma Japan (Tokyo, Japan). ONO-4057, a specific BLT antagonist, was a kind gift from ONO Pharmaceutical Co. Ltd. (Osaka, Japan). Anti-BLT1 and -BLT2 polyclonal antibodies were purchased from Cayman Chemicals (Ann

Effects of LOX inhibitors and the BLT antagonist on human preadipocyte differentiation

Preadipocytes can differentiate into mature adipocytes in the induction medium containing INS, DEX, IBMX, and ROSI (PPARγ-ligand) [11], [12]. We used this induction condition for differentiation of human preadipocytes derived from subcutaneous adipose tissues (Fig. 1A). NDGA, a pan-LOX inhibitor, enhanced the accumulation of lipids and the increase in TG contents, the index of human preadipocyte differentiation into mature adipocytes (Fig. 1B). No alteration of cell proliferation was observed

Discussion

In the present study, we clearly showed that the LTB4–BLT signaling pathway provides a potent regulatory signal that inhibits the differentiation of preadipocytes isolated from adipose tissues.

It is not fully understood whether inflammation-related lipid mediators, such as LTs and PGs, promote or inhibit the onset of metabolic syndrome. Several previous reports indicated that PGD2-derived 15-deoxy-Δ12,14-PGJ2 promoted adipocyte differentiation via activation of the PPARγ pathway [13], [14].

Acknowledgments

This work was supported in part by grants (21592357 and 24659823 to K.W.) from the Japan Society for the Promotion of Science.

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