2.4. The interaction of adipocytes under hypertrophy and hypoxia and infiltrated immune cells in development of adipose tissue inflammation and obesity complications

Adipocyte hypertrophy and hypoxia are crucially involved in adipose tissue inflammation via induction of pro-inflammatory cytokines, as well as of chemokines that attract immune cells in the early development of obesity. It has long been known that adipose tissue in obesity is in a heightened state of inflammation. Recently, it has been transformed by the knowledge that immune cells such as macrophages and T cells can infiltrate adipose tissue and are responsible for the majority of inflammatory cytokine production and adipose tissue inflammation. It has also been suggested that adipocytes could act as antigen-presenting cells to immune cells in adipose tissue inflammation [45].

#### 2.4.1. Macrophages

the frequency of adipocyte death was significantly associated with the adipose gene expressions of TNF-α, IL-6, and MCP-1 in adipose tissue and the development of whole-body insulin

Macrophages are extremely proficient in the removal of numerous molecules, ranging from small lipids to colonies of pathogens to dead cells. Necrosis of adipocytes, driven by hypertrophy and accelerated by obesity, is a prominent phagocytic stimulus that attracts macrophage infiltration into adipose tissue [18]. Using a transgenic animal model of inducible lipoatrophy, Pajvani et al. demonstrated that massive adipocyte death can indeed drive rapid accumulation of adipose tissue macrophages (ATMs) as an integral element in the remodeling of fat pads [38]. These observations implicate an important role of adipocyte hypertrophy in the development of adipocyte death and associated inflammatory changes in AT and obesity complication.

Elevation of pro-inflammatory cytokines in fat and circulation such as TNF-α, IL-1, IL-6, MCP-1, and PAI-1 has been documented in obesity [23,25,39]. The increase in adipokine production in adipocyte hypertrophy and hypoxia has been suggested to underlie the development of the inflammatory response in adipose tissue, which occurs in the obese state [11,40]. It has been clearly indicated that adipocyte size is an important determinant for the secretion of several inflammatory adipokines, such as leptin, IL-6, and MCP-1, thereby providing another link between adipocyte size and inflammation in obesity [41]. In the same study, there was a tendency to reduce the release of anti-inflammatory adipokines such as IL-10 and adiponectin

On the other hand, hypoxia has been proposed to be an inciting etiology of necrosis and macrophage infiltration into adipose tissue, which subsequently results in the dysregulation of the production of inflammation-related adipokines such as leptin, adiponectin, TNF-α, IL-6, and vascular endothelial growth factor (VEGF) [40,42]. Recently, hypoxia has also been reported to induce the production of PAI-1 and to inhibit the synthesis of adiponectin by 3T3- L1 adipocytes [39]. It is also reported to induce the expression of visfatin in these cells [43]. The expressions of other major adipokine production from murine or human adipocytes including angiopoietin-like protein 4 (Angptl4), interleukin-6 (IL-6), macrophage migration inhibitory factor (MIF), and VEGF [23,40,44] are also stimulated by hypoxia. Accordingly, Wang et al. mimicked hypoxia in human adipocytes for 24 h using cobalt chloride (CoCl2). It is shown that HIF-1α along with oxidative stress markers, inflammatory markers, and leptin was increased,

2.4. The interaction of adipocytes under hypertrophy and hypoxia and infiltrated immune

Adipocyte hypertrophy and hypoxia are crucially involved in adipose tissue inflammation via induction of pro-inflammatory cytokines, as well as of chemokines that attract immune cells in the early development of obesity. It has long been known that adipose tissue in obesity is in a heightened state of inflammation. Recently, it has been transformed by the knowledge that immune cells such as macrophages and T cells can infiltrate adipose tissue and are responsible

cells in development of adipose tissue inflammation and obesity complications

2.3.5. Effect of adipocyte hypertrophy on adipokine production

but conversely adiponectin was decreased during hypoxia [42].

with increasing adipocyte size [41].

resistance [37].

132 Adiposity - Omics and Molecular Understanding

Some of the consequences of adipocyte hypertrophy include fatty acid flux, vascularization, increased adipokine secretion, hypoxia, and adipocyte cell death. These adipocyte-related consequences of adipose tissue expansion are important contributors to the initiation of macrophage recruitment in morbid obesity. Macrophage infiltration in the inflamed adipose tissue results from blood monocyte influx, mainly attracted by the chemokine MCP-1, which is mainly secreted by hypertrophic adipocytes [46]. Adipose tissue macrophages (ATMs) accumulate in both the subcutaneous and visceral expanding fat depots [46]. Apart from increasing in numbers, adipose tissue macrophages are also phenotypically changed during obesity from the anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages dominantly in that of obese mice [5]. Activated M1 ATMs are the prominent source of pro-inflammatory cytokines such as TNF-α and IL-6, which can block insulin action in adipocytes via autocrine/paracrine signaling and also cause systemic insulin resistance via endocrine signaling. Of note, adipokine production during adipocyte hypertrophy and hypoxia such as free fatty acids and TNF-α has been reported to facilitate M1 phenotype switch in the state of obesity [47].

#### 2.4.2. T cells

In addition to macrophages, recent studies have revealed a growing list of other immune cells that are involved in the regulation of adipose tissue remodeling in state of obesity. It has been demonstrated that RANTES release is dependent on adipocyte size and is higher than those of obese donors. Hypoxia could also cause an increase in RANTES release [48,49]. Human adipocytes express the chemokine RANTES and are thus identified as one of the novel cellular sources of the immune mediator. Wu et al. [50] found higher RANTES mRNA levels in visceral compared with subcutaneous adipose tissue in obese humans. Elevated RANTES expression in the adipose tissue of diet-induced obese male mice is associated with increased T-cell infiltration, suggesting paracrine chemotactic effects [50]. Recently, it has been suggested that adipocytes could act as antigen presenting cells to T cells during adipose tissue inflammation [45]. major histocompatibility complex (MHC) class II molecules on adipocytes can functionally activate CD4+ T cells in an antigen-specific and contact-dependent manner [45]. Therefore, adipocytes seem to act as key regulatory cells in the control of adipose tissue inflammation through cytokine secretion and antigen presentation. The interaction involves several T lymphocyte lineages including CD4+ and CD8+ T cells, regulatory T cells, and mast cells [6,51]. In particular, the levels of CD8+ T cells are enriched in the early stages of obesity before the accumulation of ATMs [6]. It implicates a potential role for CD8+ T cells in the initiation of the subsequent inflammatory cascade. It is also suggested that adipose tissue inflammation is the coordinated inflammatory responses involving hypertrophic, hypoxic adipocytes, recruitment of ATMs, the accumulation of pro-inflammatory T cells (CD8+ and Th1 CD4+ T cells), and the loss of anti-inflammatory regulatory T cells, Th2 CD4+ T cells as well as the appearance of B cells, natural killer (NK) cells, eosinophils, neutrophils, and mast cells [6,51–53].

Furthermore, the recently discovered T helper 17 (Th17) cells represent a novel subset of CD4+ T cells, defined by their production of interleukin 17 (IL-17) [54]. Interestingly, serum IL-17 is upregulated in obese human patients [55], and obesity is positively correlated with enhanced IL-17 expression in T cells isolated from spleen [56]. Zúñiga et al. revealed that that IL-17 secreted by T cells in adipose tissue is an important negative regulator of adipogenesis via suppressing the expression of several pro-adipogenic transcription factors, including PPAR-γ and C/EBP-α [57] and also glucose metabolism to aggravate insulin resistance [58]. Thereby, increased IL-17 secretion by Th17 cells inhibits the differentiation of adipocyte-derived stem cells (ASCs) to adipocytes and also suppresses the insulin responsiveness of the adipocytes. Eljaafari et al. [59] provide intriguing evidence by using the co-culture of human ASCs with human mononuclear cells (MNCs), ASCs from obese donors augment the differentiation of naïve CD4+ T cells toward Th17 cells and change the phenotype of MNCs via increased the secretion of IFN-γ by Th17 cells. Taken together, these observations suggest an important role of IL-17 and Th17 cells in obesity-related adipose tissue dysfunction and systemic complications.

#### 2.4.3. Others

The development of adipocyte hypertrophy and hypoxia plays an important trigger to initialize immune cells infiltration, either directly or indirectly. As mentioned above, adipocytes act as key regulatory cells in the control of adipose tissue inflammation through cytokine secretion and antigen presentation. Immune cells such as macrophages, T cells, mast cells, and eosinophils have all been implicated to substantially participate into the process of adipose tissue inflammation [60]. Besides ATMs and T cells, increased infiltration of neutrophils into adipose tissue has also been documented in high-fat diet (HFD)-induced obese mice [61]. Neutrophils secrete various proteases, such as neutrophil elastase. The depletion of neutrophil elastase in HFD-fed mice improves adipose tissue inflammation, implicating that secreted elastase from neutrophil is the key mediator of adipose tissue inflammation [61]. In addition, it has been demonstrated that eosinophil regulated macrophage activation in adipose tissue and also plays a crucial role in metabolic homeostasis. Alternatively activated (M2) macrophages induced by Th2 cytokines IL-14, which is major secreted from eosinophils. Alternatively activation of ATMs in adipose tissue is impaired in the absence of IL-4 or eosinophils [60]. Mast cells have been shown to accumulate in the visceral adipose tissue of obese mice. Mast cell KitW-sh/W-sh-deficient mice without mature mast cells are resistant to high fat diet-induced obesity and exhibit significant reduction in pro-inflammatory cytokines and chemokines and also in macrophage number in visceral adipose tissue [62]. Therefore, it appears that mast cell arrival in adipose tissue precedes the release of pro-inflammatory mediators that attract macrophages [62].

In addition, our recent study [63] has further demonstrated that COX-2 mediated PGE2 EP3 signaling during the development of adipocyte hypertrophy and hypoxia is important to recruit and interact with adipose immune cells to amplify the inflammatory responses in adipose tissue, which is also causally linked to the development of systemic insulin resistance.

#### 2.5. The regulatory mechanisms of adipocyte hypertrophy in the development of obesity

The pathogenic change of adipocyte hypertrophy during obesity is determined by two distinct processes of adiposity: adipocyte differentiation (adipogenesis) and lipogenesis. They are dependent on both of genetic predisposition and environmental surroundings. During persistent positive caloric intake, adipocyte hypertrophy might result in adipocyte dysfunction while adipogenesis is impaired [64,65].

On the other hand, the mechanisms of adipocyte differentiation have been extensively studied in recent decades. A number of key transcription factors and adipokines in adipocyte differentiation have been identified [66]. For instance, they are included peroxisome proliferatoractivated receptor (PPAR) family proteins [67], CCAAT/enhancer-binding protein (C/EBP) [68], adipocyte differentiation determination-dependent factor 1 (ADD1) [69], and sterol response element-binding protein 1 (SREBP 1) family proteins [70]. In addition, the tyrosine phosphorylated Dok1 has also been demonstrated to promote adipocyte hypertrophy by counteracting the inhibitory effect of extracellular signal–regulated kinase (ERK) on PPAR-γ [71].

In addition, sustained energy excess could facilitate the storage of energy through lipogenesis and hypertrophy of existed adipocytes than through adipogenesis with recruitment and differentiation of new adipocytes from pre-adipocytes. Eventually, it would lead to pathologic adipocyte hypertrophy that contributes to the development of adipose tissue inflammation and obesity-associated metabolic disorders [72,73].

## 2.6. The therapeutic implications

Furthermore, the recently discovered T helper 17 (Th17) cells represent a novel subset of CD4+ T cells, defined by their production of interleukin 17 (IL-17) [54]. Interestingly, serum IL-17 is upregulated in obese human patients [55], and obesity is positively correlated with enhanced IL-17 expression in T cells isolated from spleen [56]. Zúñiga et al. revealed that that IL-17 secreted by T cells in adipose tissue is an important negative regulator of adipogenesis via suppressing the expression of several pro-adipogenic transcription factors, including PPAR-γ and C/EBP-α [57] and also glucose metabolism to aggravate insulin resistance [58]. Thereby, increased IL-17 secretion by Th17 cells inhibits the differentiation of adipocyte-derived stem cells (ASCs) to adipocytes and also suppresses the insulin responsiveness of the adipocytes. Eljaafari et al. [59] provide intriguing evidence by using the co-culture of human ASCs with human mononuclear cells (MNCs), ASCs from obese donors augment the differentiation of naïve CD4+ T cells toward Th17 cells and change the phenotype of MNCs via increased the secretion of IFN-γ by Th17 cells. Taken together, these observations suggest an important role of IL-17 and Th17 cells in obesity-related adipose tissue dysfunction and systemic complications.

The development of adipocyte hypertrophy and hypoxia plays an important trigger to initialize immune cells infiltration, either directly or indirectly. As mentioned above, adipocytes act as key regulatory cells in the control of adipose tissue inflammation through cytokine secretion and antigen presentation. Immune cells such as macrophages, T cells, mast cells, and eosinophils have all been implicated to substantially participate into the process of adipose tissue inflammation [60]. Besides ATMs and T cells, increased infiltration of neutrophils into adipose tissue has also been documented in high-fat diet (HFD)-induced obese mice [61]. Neutrophils secrete various proteases, such as neutrophil elastase. The depletion of neutrophil elastase in HFD-fed mice improves adipose tissue inflammation, implicating that secreted elastase from neutrophil is the key mediator of adipose tissue inflammation [61]. In addition, it has been demonstrated that eosinophil regulated macrophage activation in adipose tissue and also plays a crucial role in metabolic homeostasis. Alternatively activated (M2) macrophages induced by Th2 cytokines IL-14, which is major secreted from eosinophils. Alternatively activation of ATMs in adipose tissue is impaired in the absence of IL-4 or eosinophils [60]. Mast cells have been shown to accumulate in the visceral adipose tissue of obese mice. Mast cell KitW-sh/W-sh-deficient mice without mature mast cells are resistant to high fat diet-induced obesity and exhibit significant reduction in pro-inflammatory cytokines and chemokines and also in macrophage number in visceral adipose tissue [62]. Therefore, it appears that mast cell arrival in adipose tissue precedes the release of pro-inflammatory mediators that attract macrophages [62].

In addition, our recent study [63] has further demonstrated that COX-2 mediated PGE2 EP3 signaling during the development of adipocyte hypertrophy and hypoxia is important to recruit and interact with adipose immune cells to amplify the inflammatory responses in adipose tissue, which is also causally linked to the development of systemic insulin resistance.

2.5. The regulatory mechanisms of adipocyte hypertrophy in the development of obesity

The pathogenic change of adipocyte hypertrophy during obesity is determined by two distinct processes of adiposity: adipocyte differentiation (adipogenesis) and lipogenesis. They are

2.4.3. Others

134 Adiposity - Omics and Molecular Understanding

In this chapter, we discuss the recent advance about the role of adipocytes in the control of development, growth, and remodeling of obesity-associated adipose tissue. This review article further highlights the important role of adipocytes during hypertrophy and hypoxia in the development of adipose tissue inflammation and following insulin resistance. Furthermore, the understanding of regulatory mechanism of adipocyte hypertrophy during the development of obesity could provide better strategy for the prevention and treatment of obesityassociated type 2 diabetes and metabolic syndrome.
