*2.3.1. MCP-1 and CCR2*

(CLSs) [28]. However, the activation status of ATMs does not seem to be static. Whereas HFDinduced insulin resistance followed by a normal chow diet could reverse body insulin

macrophages no longer exhibited inflammatory pathway markers [29]. Furthermore, a class

identified in obese adipose tissues. These macrophages were likely transformed from M2 to M1 macrophages through lipid accumulation [27]. Therefore, there might be some mechanism

Classical activation of macrophages (M1) is induced by Toll-like receptor (TLR) ligands and IFN-γ, while alternative activation of macrophages (M2) is induced by IL-4/IL-13 (M2a), immune complexes (M2b), or anti-inflammatory cytokines IL-10 or TGF-β (M2c) to mediate Th1/Th2 immune responses [30]. In the classical activation of macrophages, adipose tissue secretes FFAs, which can activate TLR4 [31] by lipolysis and IFN-γ. On the other hand, it was shown that adipocytes secrete Th2 cytokines such as IL-4 and IL-13, which is important for alternative macrophage activation [32]. Adiponectin is also reported to induce M2 polarization, which was shown using adiponectin KO mice and adenoviral delivery of adiponectin [33].

In terms of intracellular signaling in adipose tissue macrophages, transcription factors and related machinery that regulate ATM polarization have been studied. Peroxisome proliferator– activated receptor gamma (PPARγ) is one of most striking regulators because it is known as a master regulator of adipogenesis. However, PPARγ was expressed at relatively high levels in monocytes and macrophages [34], and it was reported that PPARγ activation reduced proinflammatory cytokines in monocytes/macrophages [35, 36]. When PPARγ was knocked out in macrophages, bone marrow-derived macrophages showed impaired alternative macrophage activation, and these mice were obese, with an insulin resistant and glucose intolerant phenotype following HFD feeding [37]. PPARδ, another PPAR family member, is also important for ATM polarization. Myeloid-specific KO of PPARδ resulted in enhanced adipose tissue inflammation and insulin resistance, which is consistent with the M1 polariza‐ tion phenotype [32]. PGC-1β, a co-activator of the PPAR family, is also clearly involved in alternative macrophage activation by cooperating with STAT6, which is a critical signal mediated by Th2 cytokines [38]. In addition, Krüppel-like factor 4 (KLF4), which belongs to the zinc finger class of DNA-binding transcriptional regulators, was suggested to be a master regulator of macrophage polarization during obesity. KLF4 expression was markedly reduced in obese adipose tissue, and KLF4 deficiency exhibited an enhanced inflammatory response. In particular, myeloid specific KO of KLF4 led to obesity, insulin resistance, and impaired glucose tolerance [39]. Interferon regulatory factor 4 (IRF4) is also known to be involved in ATM polarization. Macrophage-specific IRF4 KO mice exhibited significant insulin resistance

of macrophages that express a marker for both M1 and M2 (i.e., CD11c+

to regulate activation of ATMs in adipose tissue along with obesity development.

ATMs in adipose tissue, but these

, CD209a+

) was

sensitivity, mice still contained a similar level of CD11c+

*2.2.4. Regulation of ATM polarization*

138 Glucose Homeostasis

and adipose tissue inflammation with a HFD [40].

MCP-1 (CCL2) is produced mostly by macrophages and endothelial cells and is a potent chemotactic factor for monocytes [42-44]. The level of MCP-1 in both WAT and plasma was increased in obese mice [45], suggesting that MCP-1 might be an adipokine whose expression is increased in obesity [46].

Binding of MCP-1 to its receptor CCR2 is considered crucial in obesity-induced insulin resistance. Several groups have demonstrated that mice with targeted deletions in the genes for *Mcp-1* and its receptor *Ccr2* have decreased ATM content, decreased inflammation in WAT, and protection against obesity-induced insulin resistance [46, 47]. On the contrary to this, mice overexpressing MCP-1 in adipose tissue had increased ATM and insulin resistance [46, 48]. Therefore, the MCP-1-CCR2 axis is important to promote ATM recruitment and insulin resistance in mice. Recent studies; however, have shown conflicting results and indicate a greater complexity than suggested by previous reports. In studies done by several groups, results showed that loss of MCP-1 did not attenuate obesity-associated macrophage recruit‐ ment to WAT or improve metabolic function, suggesting that MCP-1 is not pivotal for obesityinduced macrophage recruitment and systemic insulin resistance [49, 50]. Furthermore, although *Ccr2-/-*mice fed a HFD had fewer macrophages in WAT compared with wild type (WT) mice [47], CCR2 deficiency did not normalize ATM content and insulin resistance to the levels in lean mice, indicating that ATM recruitment and insulin resistance are also regulated by MCP-1-CCR2 independent signals. The intricacy and redundancy of chemokine signaling may account for these conflicting results.

## *2.3.2. CCL3, CCL5, CCR1, and CCR5*

Macrophage inflammatory protein-1 (MIP-1/CCL3) is a CC chemokine with upregulated expression in obese WAT of humans and mice. CCL3 transcript and protein are remarkably elevated in WAT of *ob/ob*, *db/db*, and DIO mice [8, 51]. In obese humans, the expression of CCL3 and its receptors CCR1 and CCR5 were increased in omental and subcutaneous WAT com‐ pared with normal weight individuals [41]. Moreover, expression of CCL3 and CCR1 in WAT was positively correlated with fasting blood insulin levels in humans [41, 52, 53]. Although many reports have shown a functional role of CCL3 in obesity, the consequences of this have not been established [54].

Keophiphath et al. identified CCL5 as the most upregulated gene in human pre-adipocytes provided with macrophage-secreted factors [55]. Although its role and its target receptors in human WAT are unknown, this chemokine is involved in blood monocyte recruitment to inflammatory sites by binding to the G-protein-coupled receptors CCR1, CCR3, and CCR5. CCL5 production in fibroblasts, platelets, and monocytes/macrophages is a known feature of inflammatory disorders [56]. In atherosclerosis, CCL5, via CCR1 and CCR5, contributes to transmigration of monocytes and T cells in atherogenic lesions [57].

required for activated tissue macrophages and dendritic cells [81-87]. Nara et al. generat‐ ed *Cxcl14* deficient mice and described that CXCL14 is involved in the obesity-induced infiltration of macrophages into WAT, serum adipokine levels, hepatic steatosis, and attenuation of insulin signaling in skeletal muscle; thereby, contributing to systemic insulin

Adipokines Involved in Macrophage Recruitment

http://dx.doi.org/10.5772/57558

141

Osteopontin (OPN) is a secreted matrix glycoprotein and proinflammatory cytokine that has previously been reported as a major element of cell-mediated immunity [89]. Many studies have provided evidence that OPN is secreted by macrophages at sites of inflammation where it mediates monocyte adhesion [90], migration [91], differentiation [92], and phagocytosis [93]. OPN play a role in the development of atherosclerosis. OPN induces chemotaxis of monocytes and elevates cellular migration through a direct interaction with its receptors [94, 95]. No‐ miyama et al. demonstrated that OPN secretion is upregulated during obesity and greatly expressed in ATMs of DIO mice, characterizing OPN as an adipokine. OPN deficiency attenuated ATM accumulation, adipose tissue inflammation and improved whole body

AIM [97] is incorporated into adipocytes via CD36-associated endocytosis, and it mediated lipolysis by suppressing the activity of fatty acid synthase (FAS) [98]. AIM is a member of the scavenger receptor cysteine-rich superfamily and was initially characterized as an apoptosis inhibitor that supports the survival of macrophages against apoptosis-inducing stimuli [97]. AIM is a direct target for regulation by nuclear receptor liver X receptor/retinoid X receptor (LXR/RXR) heterodimers [99, 100], and it is exclusively produced by tissue macrophages. As a secreted molecule, AIM is found in both human and mouse blood [97, 100-103] and increases in blood with the progression of obesity in DIO mice [98]. AIM-associated lipolysis is respon‐ sible for the obesity-induced recruitment of ATMs. Kurokawa et al. demonstrated the role of AIM in the initiation of adipose tissue inflammation that links obesity and insulin resistance [104]. Firstly, AIM-induced lipolysis is required for macrophage recruitment into obese adipose tissues. Increased blood AIM levels induce dynamic lipolysis in obese adipose tissues, augmenting local extracellular fatty acid concentrations to a level sufficient for the stimulation of TLR4, which promotes chemokine production by adipocytes and macrophage infiltration. Secondly, an increase in blood AIM is required as well as adipocyte hypertrophy for the initiation of macrophage recruitment. In *AIM* deficient mice, although the level of AIMindependent lipolysis escalated in line with adipocyte hypertrophy [98], it may not reach a level sufficient for macrophage infiltration. Thirdly, crosstalk between adipocytes and macrophages within adipose tissue establishes a vicious circle that accelerates inflammation; saturated fatty acids brought about by lipolysis activated TLR4 to induce TNFα, which in turn activated the TNFα receptor to produce inflammatory cytokines [105]. This response induces

a further progression of inflammation, lipolysis, and macrophage recruitment.

resistance in DIO mice [88].

*2.3.6. Osteopontin*

insulin resistance [96].

*2.3.7. Apoptosis inhibitor of macrophage (AIM/CD5L)*

Kitade et al. revealed that CCR5 plays a crucial role in the regulation of adipose tissue inflammation in obesity and the development of insulin resistance [58]. Expression of CCR5 and its ligands is highly increased in WAT of both *ob/ob* and DIO mice. FACS analysis clearly demonstrated that CCR5+ macrophages accumulate in WAT of obese mice. The loss of CCR5 improved obesity-induced insulin resistance in mice. Both *Ccr5-/-*mice fed a HFD and mice deficient in *Ccr5* bone marrow-derived cells showed ameliorated insulin sensitivity and protection from obesity-induced insulin resistance via reduction of ATM accumulation.
