Research article
Obesity activates toll-like receptor-mediated proinflammatory signaling cascades in the adipose tissue of mice

https://doi.org/10.1016/j.jnutbio.2010.10.012Get rights and content

Abstract

Obesity is characterized by low-grade and chronic inflammation, a phenomenon explained with a new term, metaflammation. Recent studies suggest that adipocytes may play an important role in the physiological regulation of immune responses in fat deposits via toll-like receptor (TLR) signaling cascades. This study investigates the role of the visceral as well as subcutaneous adipose tissues in the development of metaflammation by characterizing the tissue-specific expression profiles of TLRs and downstream signaling molecules and explores the differential responsiveness of TLR-mediated proinflammatory signaling cascades to diet-induced obesity (DIO) and obesity induced by a leptin gene deficiency. The obesity that was induced by a high-fat diet or leptin deficiency up-regulated the expression of TLR1–9 and TLR11–13 in murine adipose tissues, a phenomenon linked with downstream nuclear factor κB, interferon regulatory factors, and STAT-1 activation, and up-regulated the expression of cytokines and chemokines via MyD88-dependent and MyD88-independent cascades. The extent of the obesity-induced up-regulation of most TLR genes and related proinflammatory signaling cascades was much greater in the epididymal adipose tissues than in the subcutaneous fat tissues of the mice with DIO. Furthermore, the magnitudes of the obesity-induced up-regulation of the TLR1, TLR4, TLR5, TLR8, TLR9, and TLR12 genes and most of the downstream signaling molecules and target cytokine genes in the visceral adipose tissue were greater in the DIO mice than in the ob/ob mice. These results suggest that TLRs and related proinflammatory signaling molecules that are overexpressed in enlarged adipose tissues may play an important role in the obesity-associated phenomenon of metaflammation.

Introduction

Adipose tissue had been regarded as a silent and inert organ that stores excess energy as triglycerides and releases energy as fatty acid. Beginning with the discovery of leptin in 1995, however, the view of adipose tissue has changed considerably: it is now recognized as an endocrine organ that secretes a wide variety of hormones, cytokines, chemokines, and growth factors that influence metabolism, vascular and endothelial functions, appetite, satiety, immunity, inflammation, tumor growth, and many other physiological processes [1]. In particular, obesity is increasingly being associated with a phenomenon recently termed metaflammation (metabolically triggered inflammation), which describes low-grade and chronic activation of the inflammatory response [2]. The physiological mechanisms that link obesity to metaflammation include the production of various adipocytokines (e.g., adiponectin, resistin, visfatin, and leptin) and proinflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-1 by the expanded adipose tissues [3], [4], [5], [6]. Furthermore, elevated free fatty acids (FFAs) in the serum and visceral fat tissues in obese humans, as well as in animal models of obesity, have been shown to induce proinflammatory signaling and insulin resistance in peripheral tissues [7], [8].

Toll-like receptors (TLRs) are a family of pattern-recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microorganisms [9]. Until recently, 10 distinct TLRs have been identified in humans [10], and 13, in mice [11]. Mesenchymal stem cells that were isolated from human adipose tissue were reported to express TLR1 to TLR6 and TLR9 [12], whereas mature adipocytes of human subcutaneous and visceral adipose tissues were reported to express TLR1 to TLR4 and TLR6 [13]. The TLRs recognize the conserved pathogen-associated molecular patterns (PAMPs) of invading microbial pathogens, the structures of which include lipids, carbohydrates, nucleic acid, and various proteins [10], [14], [15]. The TLRs occur as dimers from the PAMPs [16]. For example, TLR1 and TLR2 heterodimerize, and the resulting dimmer recognizes bacterial triacylated lipopeptides, whereas TLR2, which heterodimerizes with TLR6, senses bacterial diacylated lipopeptides. Homodimerized TLRs include TLR4, a receptor for the Gram-negative bacterial product lipopolysaccharide; TLR9, a receptor for the unmethylated CpG-containing DNA motifs that occur in bacterial and viral DNAs; TLR3, which senses synthetic and viral double-stranded RNAs and TLR5, which binds flagellin from bacteria [10], [14], [17], [18], [19]. TLR8, which binds viral single-stranded RNAs, heterodimerizes with TLR7 or TLR9. TLR11 responds specifically to uropathogenic bacteria [20], [21], whereas the ligands for TLR10, TLR12 and TLR13 have not yet been identified [17].

There are several proofs of the involvement of TLR2 and TLR4 in metabolic functions as well as in innate immune responses in obesity. Shi et al. [22] suggested that TLR4 may be one mechanism by which fatty acids induce inflammation and insulin resistance in conventional insulin-target tissues, such as adipose tissue [22], [23], [24] and muscles [25], [26] of obese mice and human subjects. Mice with loss-of-function mutation in TLR4 (C3H/HeJ, a TLR4-deficient mouse strain) are protected against the development of diet-induced obesity, inflammation via nuclear factor κB (NFκB) activation and insulin resistance [25]. Recently, three different groups have demonstrated that the activation of a TLR2 signaling pathway is related to the development of insulin resistance in adipocytes or myotubes [27], [28], [29]. The adipocytes and preadipocytes that were isolated from the adipose tissues of the ob/ob and db/db mice, which are leptin and leptin receptor deficient, respectively, were characterized by more significant up-regulation of TLR1 to TLR9 expression than with wild-type cells [30], [31], [32]. Obesity-induced changes, however, in the expression patterns of TLRs and related signaling cascades in the adipose tissues remain widely unclear.

The metabolic and endocrine functions of adipose tissue from various depots differ in a way that may explain the association of visceral but not subcutaneous fat with obesity-related metabolic problems [33]. This study focuses on two aspects. First, it defines the role of the visceral and subcutaneous adipose tissues in the development of metaflammation through the characterization of the tissue-specific expression profiles of TLRs and downstream signaling molecules. Second, the degrees of responsiveness of TLR-mediated proinflammatory signaling cascades to obesity in enlarged adipose tissues between a mouse model with diet-induced obesity (DIO) and a leptin-deficient (ob/ob) obese mouse were compared.

Section snippets

Mice and experimental diets

Twenty-five 5-week-old male C57BL/6J mice and five 5-week-old ob/ob mice (Orient, Gyeonggi-do, Korea) were housed in a room with controlled temperature (21±2.0°C) and humidity (50±5%) and with a 12-hour light/12-hour dark cycle and were fed a commercial diet (Ralston-Purina, St. Louis, MO, USA) for a week. Twenty of the C57BL/6J mice were randomly divided into two groups and fed either the normal diet (ND) or a high-fat diet (HFD) for 12 weeks. The HFD contained 200 g of fat/kg (170 g of lard

Phenotypic characteristics of obese mice

The mice that were fed the HFD for 12 weeks had significantly greater final body weight (39% greater, P<.001) and body weight gains (188% greater, P<.001) and heavier relative weights of the visceral fat depots than the mice that were fed the ND. The retroperitoneal, epididymal, mesenteric, and perirenal fat pads were 215%, 151%, 64%,and 186% heavier, respectively, in the mice that were fed the HFD than in those that were fed the ND (P<.001). The HFD-fed mice exhibited significantly higher

Discussion

TLRs play a crucial role in host defense against invading pathogens by mediating innate and adaptive immunities. TLR signaling pathways are initiated by a conserved cytosolic domain called the TIR domain, which associates with intracellular TIR-domain-containing adaptors including MyD88, Tirap, TRIF and the TRIF-related adaptor molecule (TRAM) [17], [18], [19], [37]. Different TLRs trigger signals via different combinations of adaptors. The TLR4 activation recruits four major adaptors, MyD88,

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    This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (#A080020) and by the SRC program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (#2009-0063409).

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