Endocr Pathol DOI 10.1007/s12022-013-9288-1

Morphological and Inflammatory Changes in Visceral Adipose Tissue During Obesity Xavier S. Revelo & Helen Luck & Shawn Winer & Daniel A. Winer

# Springer Science+Business Media New York 2013

Abstract Obesity is a major health burden worldwide and is a major factor in the development of insulin resistance and metabolic complications such as type II diabetes. Chronic nutrient excess leads to visceral adipose tissue (VAT) expansion and dysfunction in an active process that involves the adipocytes, their supporting matrix, and immune cell infiltrates. These changes contribute to adipose tissue hypoxia, adipocyte cell stress, and ultimately cell death. Accumulation of lymphocytes, macrophages, and other immune cells around dying adipocytes forms the so-called “crown-like structure”, a histological hallmark of VAT in obesity. Cross talk between immune cells in adipose tissue dictates the overall inflammatory response, ultimately leading to the production of proinflammatory mediators which directly induce insulin resistance in VAT. In this review, we summarize recent studies X. S. Revelo : H. Luck : S. Winer : D. A. Winer (*) Division of Cellular & Molecular Biology, Diabetes Research Group, Toronto General Research Institute (TGRI), University Health Network, Toronto, ON, Canada e-mail: [email protected] S. Winer : D. A. Winer Department of Pathology, University Health Network, Toronto, ON, Canada D. A. Winer Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada H. Luck : D. A. Winer Department of Immunology, University of Toronto, Toronto, ON, Canada D. A. Winer Department of Endocrinology, University of Toronto, Toronto, ON, Canada Present Address: D. A. Winer MaRS Centre 10-352, Toronto Medical Discovery Tower, 101 College St., Toronto, ON M5G1L7, Canada

demonstrating the dramatic changes that occur in visceral adipose tissue during obesity leading to low-grade chronic inflammation and metabolic disease. Keywords Obesity . Visceral adipose tissue . Inflammation . Immunology . Diabetes Obesity has reached epidemic proportions and is now one of the fastest growing health issues worldwide. The World Health Organization estimates that over 1.4 billion people are currently overweight or obese [1] and these numbers are paralleled by a growing prevalence of obesity associated complications. Consequences of obesity include insulin resistance (IR), type 2 diabetes, non-alcoholic fatty liver disease, hypertension, coronary artery disease and cancer [2–6]. By 2030, it is estimated that the total number of people affected by type 2 diabetes will increase to 366 million, up from 171 million in the year 2000 [7]. Thus, due to the immense array of health complications related to obesity, it is critical to understand the underlying mechanisms of disease associated with the obese state. One tissue which becomes extensively altered during obesity is visceral adipose tissue (VAT). During obesity, there is a marked expansion of VAT with alterations in the adipocytes themselves, their supporting matrix, and immune cell infiltrates leading to morphological and inflammatory changes in the VAT. Together, these changes contribute to IR, which is at the core of metabolic syndrome and risk for type II diabetes. This review will discuss some of the alterations to VAT during obesity, with an emphasis on inflammatory cell changes.

Basic Components, Structure and Remodeling of VAT During Obesity White adipose tissue is distributed into two major depots, subcutaneous and visceral. VAT surrounds and connects inner

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organs in the abdominal cavity and mediastinum, while subcutaneous adipose tissue accumulates in the hypodermis under the skin [8]. VAT is composed of unilocular adipocytes tightly packed together and supported by loose connective tissue with a dense network of capillaries. The nonadipocyte component of VAT is also called the stromal vascular fraction and includes the extracellular matrix (ECM), multi-potent stem cells, pre-adipocytes, fibroblasts, endothelial cells, and immune cells. The ECM proteins, including collagens type 1, 3, 4, and 6, and proteoglycans, such as fibronectin, osteonectin, osteopontin, tenascin C, and matrix metalloproteinases help anchor the adipocytes to ensure structural and functional integrity of the tissue [9, 10]. Multi-potent stem cells in VAT have the capacity to differentiate into many lineages of cells such as adipocytes, hepatocytes, neural cells and the use of these cells for tissue regeneration is an intensive area of current research [11]. Pre-adipocytes are adipocyte precursor cells with the ability to proliferate and differentiate into adipocytes during adipogenesis [12]. During obesity, there is marked expansion of the VAT with prominent changes in the adipocytes and the stromal vascular components. Adipocytes primarily show hypertrophy or enlargement, and this increase in size is influenced by an increased rate of triglyceride storage and decreased triglyceride removal rate (lipolysis followed by oxidation) [13]. Increased adipocyte size is associated with augmented pro-inflammatory cytokine secretion, and increased risk of IR [14]. On the other hand, there is less evidence to support a role for hyperplasia in the development of obesity, though this process is influenced by proliferation from precursor pre-adipocytes and stem cells [15, 16]. Indeed, Spalding et al. [17] measured the incorporation of atmospheric 14C into adipocyte DNA and reported that, even after weight loss, the number of adipocytes is roughly constant in adulthood in lean and obese individuals. Despite this lack of change in cell number within an individual, total adipocyte number between people was a major determinant of fat mass in adults. Furthermore, the turnover rate of human adipocytes was relatively high in both lean and obese people at approximately 10 % renewal per year at all adult ages and levels of body mass index [17]. Compared with lean VAT, obese adipose tissue shows increased fibrosis [10] and expression of extracellular matrix components including collagen VI [18], thrombospondins, connective tissue growth factor, matrix metalloproteinases, and osteopontin [19]. Changes in these molecules exert important effects on adiposity and glucose homeostasis. For instance, disruption of collagen VI in mice decreases weight gain on high fat diet (HFD) and improves glucose homeostasis [9] while mice lacking collagen Va3 have altered fat deposition and are insulin resistant [20]. Collagen VI expression in adipose tissue is also associated with greater metabolic risk in humans [19]. Progressive fibrosis in VAT may also function to limit the ability of adipocytes to hypertrophy in obesity, promoting the

storage of ectopic fat in other organs including liver and muscle [21]. Many of the changes that occur in VAT remodeling can be influenced by local inflammation, as several pro-inflammatory mediators, such as plasminogen activator inhibitor 1 (PAI-1) and monocyte chemotactic protein 1 (MCP-1), released in VAT can induce fibrosis [22]. Remodeling of growing adipose tissue in obesity also includes the formation of new vascular networks or angiogenesis, in a process regulated by activated adipocytes [23, 24]. To support VAT expansion, newly formed blood vessels supply hypertrophic adipocytes with nutrients, oxygen, precursor stem cells, hormones, and immune cells [24]. Therefore, vascular regression and growth factors involved in angiogenesis can be potential targets for the modulation of obesityrelated VAT inflammation [25, 26]. Given the complex interplay between the immune system and the major components of adipose tissue, it is crucial to understand how these interact, and how they contribute to local inflammation and IR. Below, we will focus on the changes in immune cell populations and function that occur in obese adipose tissue.

Inflammatory Cell Infiltrates in VAT During Obesity Obesity promotes an intricate inflammatory response that involves the production of pro-inflammatory molecules and the infiltration of immune cells to metabolic organs including adipose tissue. The notion that chronic, low-grade inflammation of VAT links obesity and type 2 diabetes emerged two decades ago with the discovery that the cytokine, tumor necrosis factor-α (TNF-α), is increased in obese mice, and that neutralization of TNF-α improves whole-body insulin sensitivity [27]. Subsequently, studies in 2003 reported a marked accumulation of macrophages in the adipose tissue of obese mice and humans [28, 29]. Although excessive production of pro-inflammatory cytokines by VAT macrophages is considered to be critical in obesity-associated adipose tissue inflammation [30, 31], recent studies have proposed novel effector and regulatory roles for other immune cells [32, 33]. New evidence implicates cells of the innate and adaptive immune system such as mast cells [34], neutrophils [35, 36], CD4+ Th1, Th17 [37], CD8+ T cells [38], B cells [39], T regulatory (Treg) [40], eosinophils [41], and innate lymphoid type 2 cells (ILC2s) [42] in the complex interplay of cells and molecules in VAT during obesity-related inflammation (Fig. 1). Below, we discuss the latest advances in our understanding of the roles of infiltrating adipose tissue immune cells in the development of obesity-induced VAT inflammation. Innate Immune Cells Inflammation of VAT is central in the development of systemic IR and is largely mediated by VAT macrophages [33].

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LEAN VAT

OBESE VAT

(Insulin sensitive)

(insulin resistant)

adipokines di ki

IL 10 IL-10

IL-1β, β IL-6, TNFα

IL-5 IL-13 IL-5, IL-10

FFA

IL-10 IFNγ IFNγ

IL-4 Elastase astase

Th2 CD4+ T

Th1 CD4+ T

Treg CD4+ T

CD8+ T

Eosinophil

B cell

ILC2 Neutrophil M2 M1 Adipocyte Macrophage Macrophage

Fig. 1 Immune cells mediate adipose tissue inflammation during obesity. In lean adipose tissue, regulatory T cells (Tregs), regulatory B cells (Bregs), CD4+ Th2 T cells, eosinophils, and innate lymphoid type 2 cells (ILC2s) secrete IL-4, IL-5, IL-10, and IL-13 promoting polarization of VAT macrophages to an M2 anti-inflammatory phenotype. During

obesity, expanded adipose tissue secretes adipokines whereas CD8+ T cells and CD4+ Th1 T cells predominate and secrete IFNγ, favoring M1 macrophage polarization and pro-inflammatory cytokine production, including IL-1β, IL-6, and TNF-α

Macrophages accumulate in VAT early during obesity in response to increased chemokines and cytokines released by adipocytes and potentially, other immune cells [31]. Although the majority of VAT macrophages are thought to be recruited from the blood, macrophages can also proliferate within tissues in a process independent of monocytes and regulated by IL-4 [43, 44]. However, more data is needed to determine whether local macrophage proliferation in VAT plays a dominant role in VAT inflammation. In expanded adipose tissue, there is a prominent shift in the activation state of macrophages from an anti-inflammatory or “M2-polarized” state towards a pro-inflammatory phenotype or “M1-polarized” [45, 46]. In general, excessive production of pro-inflammatory cytokines such as TNF-α by M1 macrophages is an important contributor towards IR whereas M2 macrophages promote insulin sensitivity by secreting IL-10 [45, 47]. Cytokines such as TNF-α are thought to directly induce IR in adipocytes through c-Jun NH(2)-terminal kinase (JNK) and inhibitor of nuclear factor kappa B kinase subunit β (IKK-β)-mediated induction of serine phosphorylation of the insulin receptor or its substrates, including insulin receptor

substrate-1 (IRS-1) [31]. The classification of macrophages into either the pro-inflammatory M1 or anti-inflammatory M2 phenotypes does not consider the complexity and functional diversity of mononuclear phagocytes which consist of discrete subpopulations with varying levels of activation [48]. A complex meshwork of signaling molecules, transcription factors, and posttranscriptional modifications likely controls macrophage polarization. For example, the nuclear hormone receptor PPARγ has been identified as a key regulator of macrophage alternative activation [47, 49]. Similarly, the adaptor protein Trib1 also has been proposed as a critical regulator of tissue-resident M2-polarized macrophage maintenance [50]. Although the role of macrophages in obesity-associated inflammation is well established, only recently has the direct role of TNF-α producing VAT macrophages in IR been specifically confirmed. Aouadi et al. used siRNA encapsulated by glucan shells to selectively ablate TNF-α exclusively in VAT macrophages leaving such cells intact in other tissues of obese mice, and reported improved whole-body glucose tolerance, ratifying the direct role of VAT macrophages in IR [51].

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Furthermore, specific deletion of JNK1 and JNK2 in myeloid cells also protected mice from obesity-associated IR, suggesting that the JNK signaling pathway regulates the proinflammatory functions of VAT macrophages [52]. Eosinophils, typically associated with allergy and parasitic infections, have an important role in maintaining metabolic homeostasis in lean adipose tissue [41, 42]. Eosinophils are the major producers of IL-4 and IL-13 in adipose tissue of mice fed a normal chow diet suggesting that these cells promote the differentiation and activation of antiinflammatory M2 macrophages [41]. Indeed, eosinophildeficient mice fed HFD have impaired glucose tolerance and worsened insulin sensitivity whereas helminth-induced eosinophilia in VAT reversed these effects [41]. Importantly, the homeostatic function of adipose tissue eosinophils depends on the production of IL-5 and IL-13 by innate lymphoid type 2 cells (ILC2s) [42]. Neutrophils also accumulate in VAT during diet-induced obesity and have been shown to be among the first immune cells recruited into VAT during obesity [35, 36]. Neutrophils are usually involved in the early stages of inflammatory responses and they fight infection through phagocytosis, degranulation of antimicrobial molecules, and release of neutrophil extracellular traps (NETs) [53]. Limited research has been conducted to investigate the potential role of neutrophils in the inflammation in VAT. Recently, deletion of neutrophil elastase has been shown to protect mice from obesity-induced wholebody IR, and treatment of hepatocytes with recombinant elastase degraded insulin receptor substrate 1 and caused cellular IR [36]. Further research is needed to define additional roles of neutrophils in the adipose tissue inflammatory environment caused by obesity.

Adaptive Immunity During obesity, T lymphocytes expressing CD4 and CD8 produce high amounts of inflammatory cytokines such as interferon gamma (IFN-γ) that contribute to the proinflammatory microenvironment of adipose tissue [37]. T cells likely regulate VAT inflammation and insulin sensitivity because long-term, whole-body depletion and acute specific depletion of VAT T cells improve insulin sensitivity in obese mice [37, 54]. Yang et al. [54] reported that most of the CD4+ and CD8+ T cells in VAT of obese mice are effector-memory T cells with a restricted TCR-Vβ repertoire, suggesting that specific antigens may drive the T cell response. Early in response to HFD, IFN-γ secreting CD8+ T cells increase in VAT and CD8+ T cell deficient mice fed HFD have improved insulin sensitivity and VAT inflammatory microenvironment [38, 54]. Importantly, adoptive transfer of CD8+ T cells into CD8 deficient mice causes IR and increased the numbers of M1-polarized in VAT [38]. Activated CD8+ T cells in VAT

produce increased amounts of pro-inflammatory cytokines such as IFN-γ and G-CSF, associated with HFD feeding [54]. Mature CD4+ T cells mainly differentiate into Th1 cells that produce IFN-γ or Th2 helper T cells whose effector cytokines are IL-4, IL-5, and IL-13. IFN-γ producing Th1 cells likely participate in VAT inflammation as several studies have shown accumulation of IFN-γ producing Th1 cells in VAT during obesity [37, 40, 55]. Th1-deficient IL-12p35 null mice have improved insulin sensitivity during diet-induced obesity [37] and IFN-γ, the major effector cytokine released by these cells, has been shown to inhibit insulin signaling in human adipocytes [56] suggesting that these cells directly promote IR. Diet-induced obese (DIO) mice deficient in STAT4, a key transcription factor in IL-12 induced Th1 differentiation have improved glucose tolerance and insulin sensitivity, despite similar weight gain [57]. Interestingly, adipose tissue macrophages are capable of processing and presenting MHC class II antigens which promotes CD4+ T cell proliferation and production of IFN-γ [58]. Several studies have proposed a pathogenic role for B cells in the inflammation of VAT during obesity [39]. B cells infiltrate VAT shortly after initiation of HFD feeding [59] and B cells harvested from the blood of type 2 diabetic patients have increased expression of toll-like receptors (TLRs) and produced increased amounts of IL-8 and decreased amounts of IL-10 [60]. Furthermore, HFD-fed mice lacking B cells are protected from IR despite weight gain. Transfer of IgG antibody from obese into lean mice causes glucose intolerance and IR [39]. Treatment with a depleting anti-CD20 antibody can ameliorate abnormal glucose metabolism and reduce VAT inflammation [39]. Recently, it was confirmed that HFD-fed mice lacking B cells have decreased systemic and VAT inflammation, and splenic B cells from obese mice secrete a pro-inflammatory cytokine profile [61]. Furthermore, human B cells, but not monocytes, promote Th17 T cell inflammatory cytokine production [61]. Development of chronic inflammation in VAT during obesity is counteracted by several cell types that implement antiinflammatory functions. As pro-inflammatory immune cells accumulate in VAT during obesity, the number of Foxp3+ regulatory T cells (Tregs) decrease, exacerbating adipose tissue dysfunction [37, 39, 62]. In lean adipose tissue, Tregs support monocyte differentiation into anti-inflammatory M2 macrophages [63]. Indeed, induction of Tregs in adipose tissue improves systemic metabolic abnormalities and the inflammatory tone of VAT of DIO [37] and leptin-deficient mice [64]. Importantly, the accumulation, phenotype and effector functions of VAT Tregs are regulated by PPARγ and may mediate the well-known insulin-sensitizing effects of PPARγ agonists such as thiazolidinediones [65]. Another transcription factor, Stat3, was recently found to be a key regulator of the T cell homeostasis during obesity [66]. In this study, specific ablation of Stat3 in T cells of DIO mice

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improved insulin sensitivity and suppressed VAT inflammation due to a lower Th1/Treg balance and less accumulation of M1 macrophages [66]. Because of the protective role of VAT Tregs in the pathogenesis of obesity-induced VAT inflammation, manipulation of Tregs may represent a potential novel strategy for treating obesity-associated complications [62]. Similar to adipose tissue Tregs, a subset of B cells termed natural regulatory B cells (Bregs) are found in adipose tissue, primarily functioning in the lean state [67]. These Bregs are IgM+IgD+CD22+ and produce large amounts of IL-10. During obesity, B cells in VAT become more pro-inflammatory, highlighted by decreased IL-10 production from Bregs, thus facilitating CD8+ T cell IFNγ production and M1 macrophage polarization [67]. Natural Killer T Cells Natural Killer T (NKT) cells are innate-like T cells that express the natural killer receptor 1.1 (NK1.1) and specifically recognize lipid and glycolipid antigens via MHC Class 1b molecule (CD1d) [68]. NKT cells can be classified into two distinct subsets: variant (vNKT) and invariant (iNKT) cells based on the diversity of their T cell receptor (TCR) [68]. In general, NKT numbers decrease in obese mice and humans [69–72]. Invariant NKT cells, which have a restricted repertoire of TCRs, infiltrate the VAT of mice and humans [68–74] and may be one of the first immune cell types to migrate into VAT during obesity [73]. NKT cells are capable of recognizing lipid antigens [75], however, conflicting results exist regarding their pro- or anti-inflammatory functions and thus their role in obesity-induced VAT inflammation remains inconclusive. In obese mice and humans, for example, several investigators have reported the presence of NKT cells to cause M2 macrophage polarization and improved glucose tolerance during HFD, suggesting a protective role against DIO-induced IR [69–71]. In agreement, adoptive transfer of NK1.1+ T cells improved glucose intolerance and insulin signaling in genetically and diet-induced obese mice [76]. In contrast, Vα14 transgenic mice, which produce excess iNKT cells have increased dyslipidemia, worsened insulin signaling and M1polarization in response to HFD feeding [72]. Furthermore, ablation of NKT and iNKT during HFD has resulted in opposing results. In response to HFD feeding, NKT (CD1d null) and iNKT (Jα18 null) deficient mice have been reported to have similar [50, 69, 77–79], exacerbated [70, 71, 78, 80], or improved [73, 79] glucose tolerance or insulin resistance compared with WT controls. Overall, the contribution of NKT cells to adipose inflammation and insulin resistance remains inconclusive. Further research needs to elucidate their definitive roles by implementing consistent analytic and dietary manipulation methodology in controlled environmental settings [32, 81].

Adipocyte Cell Death and Hypoxia in VAT During Obesity Adipocyte Cell Death The mechanisms underlying the infiltration of the immune cells into adipose tissue during the progression of obesity remain unresolved. Cell death of engorged adipocytes during obesity has been proposed as one of the early events that triggers the migration of macrophages into VAT [82]. During weight gain in adults, adipocyte numbers remain stable whereas enlargement of fully differentiated adipocytes (hypertrophy) accounts for most of the increase in adipose tissue mass [17]. Remarkably, the majority of these infiltrating macrophages localize around dying adipocytes forming “crown-like structures” or CLS (Fig. 2) [82]. These macrophages are thought to clear the remains of dead adipocytes, including large triglyceride droplets leading to progressive lipid accumulation within the macrophages which acquire properties of foam-like cells [83]. Adipose tissue foam cells have been recently identified as a subclass of macrophages present in subcutaneous and omental fat from obese humans [84]. In obese VAT, hypertrophy and adipocyte death increase together with a higher frequency of CLS suggesting that adipocyte death is one driver of the infiltration of macrophages [82, 85]. For example, the frequency of adipocyte death increased to 16 % by week 12 and peaked at 80 % by week 16 of HFD feeding, in coincidence with highest levels of the macrophage marker CD11c and inflammatory gene expression in VAT [85]. By week 20 of HFD feeding, however, adipocyte number was restored and cell death decreased suggesting that inflammatory macrophages limited fat expansion leading to VAT tissue remodeling [85].

Fig. 2 Obese visceral adipose tissue (VAT) shows increased accumulation of immune cells and the formation of “crown-like structures”. VAT was harvested from C57Bl/6 mice fed high fat diet (HFD) for 12 weeks and stained with hematoxylin and eosin. Scale bar is set at 100 μm

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During diet-induced obesity, hypertrophic adipocytes degenerate and die before CLS formation suggesting that adipocyte cell death occurs prior to the arrival of macrophages [86]. In agreement, in vivo induction of adipocyte caspase 8mediated apoptosis during acute lipoatrophy precedes the appearance of CLS suggesting that macrophages are directly recruited by dying adipocytes and that CLS formation is a direct consequence of adipocyte cell death [87]. Despite the evidence indicating that dying adipocytes participate in the accumulation of immune cells of obese VAT, the exact mechanism of adipocyte cell death is less clear. Initially, it was suggested that adipocyte death occurs by necrosis since apoptosis markers were absent, but dying adipocytes exhibited features of necrotic-like cell death such as basal membrane rupture and endoplasmatic reticulum dilatation [82]. The same group, however, recently reported that hypertrophic adipocytes of leptin-deficient and HFD-induced obese mice have increased mRNA amounts of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), NOD-like receptor family, pyrin domain containing 3 (NLRP3), and caspase-1 suggesting that the NLRP3 inflammasome is activated and thus death of hypertrophic adipocytes occurs via pyroptosis [86]. During an immune response, activation of caspase-1 facilitates the maturation and release of the cytokines IL-1β and IL-18 needed to mount the inflammatory response [88]. However, during pyroptosis, robust activation of caspase-1 can also lead to inflammationassociated cell death [89]. In the context of obesity-induced VAT inflammation, the observation that enlarged adipocytes die via pyroptosis suggests that, independent of macrophage infiltration, cell death can trigger local inflammation via inflammasome activation. Hypoxia Another consequence of adipocyte hypertrophy in obesity is the development of local hypoxic regions in expanded adipose tissue due to inadequate blood supply of oxygen [90–92]. During obesity, adipose tissue hypoxia is likely the result of limited blood supply and the restricted capacity of oxygen to diffuse in enlarged adipocytes [93–95]. Areas in obese VAT showing hypoxyprobe immunoreactivity, a marker of hypoxia, predominantly colocalize with macrophages and CD3+ T cells suggesting that these immune cells are drawn preferentially to areas of hypoxia in VAT [92]. Compared to lean subjects, for example, oxygen partial pressure in abdominal adipose tissue was approximately 15 % lower in overweight or obese patients, in association with increased mRNA levels of the macrophage marker CD68 and macrophage inflammatory protein 1α [96]. It is unclear, however, whether the recruitment of macrophages to areas of hypoxia reflects local inflammation or merely the need to remove dying adipocytes [82]. Interestingly, a recent study challenged the status quo by

proposing that in VAT of obese patients, reduced oxygen extraction and mitochondrial dysfunction in VAT leads to higher oxygen partial pressure or hyperoxia [93]. These authors, however, acknowledge the heterogeneity in the study population and agree with the notion that impaired oxygen fluxes within the VAT affect adipocyte function and inflammation [93]. Evidence demonstrates that chronic hypoxia in obese adipose tissue results in cellular stress, altered adipokine secretion, and ultimately inflammation. In 3T3-L1 adipocytes, for example, the effects of hypoxia include the inhibition of adiponectin, enhanced plasminogen activator type-1 production, and upregulation of genes involved in endoplasmic reticulum (ER) stress [90]. The hypoxia-induced decrease in adiponectin likely contributes to obesity-related inflammation and IR [97]. Similarly, ER stress has been implicated in the development of inflammation and IR through the expression of unfolded protein response (UPR) proteins [98, 99]. Inflammation-related adipokines induced by hypoxia during obesity are IL-6, macrophage inhibitory factor, matrix metalloproteinases (MMPs), plasminogen activator inhibitor-1 (PAI-1), leptin, and vascular endothelial growth factor (VEGF) [100, 101]. Remarkably, the response of adipose tissue to hypoxia is largely mediated by the activation of transcription factors that act as sensors of low oxygen, most notably the hypoxic inducible factor (HIF)-1 [102]. During obesity, increased expression of the subunit HIF-1α inhibits adiponectin production and results in augmented obesity and whole-body IR [103], suggesting that the hypoxia that occurs in VAT as a consequence of adipocyte hypertrophy promotes inflammation through the recruitment of HIF-1α. Together, these findings suggest that targeting VAT oxygen tension during obesity may restore whole-body glucose homeostasis and insulin sensitivity.

Concluding Remarks During obesity, failure of adipose tissue to cope with excess of nutrients results in a marked expansion of VAT. Enormous complexity exists in the adaptations that adipocytes, their supporting matrix, and immunological components undergo, resulting in morphological changes and ultimately inflammation. Overall, obese VAT morphologically is manifested as hypertrophic and dying adipocytes set in an increased fibrotic ECM, with crown-like structure formation and immune cell infiltration. Research in the last decades has established that this chronic low-grade inflammation of VAT and release of proinflammatory cytokines is a critical link between obesity and IR. Although the health consequences of obesity-related IR are clearly severe, it is possible that IR may also represent an adaptation to unnecessary caloric intake [104]. Key changes in

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expanding adipose tissue, pivotal for the development of lowgrade inflammation, include VAT expansion, infiltration of immune cells, adipocyte death and tissue remodeling. Remarkably, the mechanisms linking these events are not mutually exclusive. Expansion of VAT, predominantly caused by hypertrophy [17], is accompanied by hypoxia which in turn is likely one of the triggers of adipocyte death. Chronic hypoxia in VAT results in cellular stress, altered adipokine secretion, and inflammation [105]. Of upmost relevance, infiltrating macrophages localize around dying adipocytes. In addition to macrophages, the list of cells of the innate and adaptive immune system cells continues to expand and now includes mast cells, neutrophils, B and T cells, eosinophils, and more recently, ILC2 cells. The outcome of obesity-induced inflammation likely depends in the balance between the pro- and anti-inflammatory functions of these immune cells [32]. Modulating the pathways involved in these processes will provide avenues for novel diagnostics and therapies to manage obesity, IR, and type II diabetes.

Acknowledgments This work was supported in part by CIHR grant 119414 (DW), and CDA grants OG-3-12-3844 (DW) and CS-5-12-3886 (DW). XR is the recipient of a Banting & Best Diabetes Centre Fellowship (Funded by Eli Lilly Canada). The authors have declared that no conflict of interest exists.

References 1. Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser, 2000. 894: p. i–xii, 1–253. 2. Gualillo, O., Mediators of inflammation in obesity and its comorbidities. Mediators Inflamm, 2010. 2010. 3. Kahn, S.E., R.L. Hull, and K.M. Utzschneider, Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature, 2006. 444(7121): p. 840–6. 4. Khandekar, M.J., P. Cohen, and B.M. Spiegelman, Molecular mechanisms of cancer development in obesity. Nat Rev Cancer, 2011. 11(12): p. 886–95. 5. Masuoka, H.C. and N. Chalasani, Nonalcoholic fatty liver disease: an emerging threat to obese and diabetic individuals. Ann N Y Acad Sci, 2013. 1281: p. 106–22. 6. Rask-Madsen, C. and C.R. Kahn, Tissue-specific insulin signaling, metabolic syndrome, and cardiovascular disease. Arterioscler Thromb Vasc Biol, 2012. 32(9): p. 2052–9. 7. Wild, S., et al., Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care, 2004. 27(5): p. 1047–53. 8. Wronska, A. and Z. Kmiec, Structural and biochemical characteristics of various white adipose tissue depots. Acta Physiol (Oxf), 2012. 205(2): p. 194–208. 9. Khan, T., et al., Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI. Mol Cell Biol, 2009. 29(6): p. 1575–91. 10. Divoux, A. and K. Clement, Architecture and the extracellular matrix: the still unappreciated components of the adipose tissue. Obes Rev, 2011. 12(5): p. e494–503.

11. Yarak, S. and O.K. Okamoto, Human adipose-derived stem cells: current challenges and clinical perspectives. An Bras Dermatol, 2010. 85(5): p. 647–56. 12. Mortensen, S.B., et al., Membrane-tethered delta-like 1 homolog (DLK1) restricts adipose tissue size by inhibiting preadipocyte proliferation. Diabetes, 2012. 61(11): p. 2814–22. 13. Arner, P., Human fat cell lipolysis: biochemistry, regulation and clinical role. Best Pract Res Clin Endocrinol Metab, 2005. 19(4): p. 471–82. 14. Skurk, T., et al., Relationship between adipocyte size and adipokine expression and secretion. J Clin Endocrinol Metab, 2007. 92(3): p. 1023–33. 15. Meyer, L.K., et al., Adipose tissue depot and cell size dependency of adiponectin synthesis and secretion in human obesity. Adipocyte, 2013. 2(4): p. 217–26. 16. Traustadottir, G.A., et al., Preadipocytes proliferate and differentiate under the guidance of Delta-like 1 homolog (DLK1). Adipocyte, 2013. 2(4): p. 272–5. 17. Spalding, K.L., et al., Dynamics of fat cell turnover in humans. Nature, 2008. 453(7196): p. 783–7. 18. Pasarica, M., et al., Adipose tissue collagen VI in obesity. J Clin Endocrinol Metab, 2009. 94(12): p. 5155–62. 19. Mariman, E.C. and P. Wang, Adipocyte extracellular matrix composition, dynamics and role in obesity. Cell Mol Life Sci, 2010. 67(8): p. 1277–92. 20. Huang, G., et al., alpha3(V) collagen is critical for glucose homeostasis in mice due to effects in pancreatic islets and peripheral tissues. J Clin Invest, 2011. 121(2): p. 769–83. 21. Szendroedi, J. and M. Roden, Ectopic lipids and organ function. Curr Opin Lipidol, 2009. 20(1): p. 50–6. 22. Sell, H. and J. Eckel, Adipose tissue inflammation: novel insight into the role of macrophages and lymphocytes. Curr Opin Clin Nutr Metab Care, 2010. 13(4): p. 366–70. 23. Cao, Y., Angiogenesis modulates adipogenesis and obesity. J Clin Invest, 2007. 117(9): p. 2362–8. 24. Cao, Y., Adipose tissue angiogenesis as a therapeutic target for obesity and metabolic diseases. Nat Rev Drug Discov, 2010. 9(2): p. 107–15. 25. Brakenhielm, E., et al., Angiogenesis inhibitor, TNP-470, prevents diet-induced and genetic obesity in mice. Circ Res, 2004. 94(12): p. 1579–88. 26. Barnhart, K.F., et al., A peptidomimetic targeting white fat causes weight loss and improved insulin resistance in obese monkeys. Sci Transl Med, 2011. 3(108): p. 108ra112. 27. Hotamisligil, G.S., N.S. Shargill, and B.M. Spiegelman, Adipose expression of tumor necrosis factor-alpha: direct role in obesitylinked insulin resistance. Science, 1993. 259(5091): p. 87–91. 28. Weisberg, S.P., et al., Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest, 2003. 112(12): p. 1796– 808. 29. Xu, H., et al., Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest, 2003. 112(12): p. 1821–30. 30. Chawla, A., K.D. Nguyen, and Y.P. Goh, Macrophage-mediated inflammation in metabolic disease. Nat Rev Immunol, 2011. 11(11): p. 738–49. 31. Olefsky, J.M. and C.K. Glass, Macrophages, inflammation, and insulin resistance. Annu Rev Physiol, 2010. 72: p. 219–46. 32. Mathis, D., Immunological goings-on in visceral adipose tissue. Cell Metab, 2013. 17(6): p. 851–9. 33. Osborn, O. and J.M. Olefsky, The cellular and signaling networks linking the immune system and metabolism in disease. Nat Med, 2012. 18(3): p. 363–74. 34. Liu, J., et al., Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice. Nat Med, 2009. 15(8): p. 940–5.

Endocr Pathol 35. Elgazar-Carmon, V., et al., Neutrophils transiently infiltrate intraabdominal fat early in the course of high-fat feeding. J Lipid Res, 2008. 49(9): p. 1894–903. 36. Talukdar, S., et al., Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Nat Med, 2012. 37. Winer, S., et al., Normalization of obesity-associated insulin resistance through immunotherapy. Nat Med, 2009. 15(8): p. 921–9. 38. Nishimura, S., et al., CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nat Med, 2009. 15(8): p. 914–20. 39. Winer, D.A., et al., B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nat Med, 2011. 17(5): p. 610–7. 40. Feuerer, M., et al., Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med, 2009. 15(8): p. 930–9. 41. Wu, D., et al., Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science, 2011. 332(6026): p. 243–7. 42. Molofsky, A.B.., et al., Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med, 2013. 210(3): p. 535–49. 43. Jenkins, S.J., et al., Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science, 2011. 332(6035): p. 1284–1288. 44. Jenkins, S.J., et al., IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. The Journal of Experimental Medicine, 2013. 45. Lumeng, C.N., J.L. Bodzin, and A.R. Saltiel, Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest, 2007. 117(1): p. 175–84. 46. Lumeng, C.N., et al., Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes, 2007. 56(1): p. 16–23. 47. Odegaard, J.I., et al., Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature, 2007. 447(7148): p. 1116–20. 48. Kawakami, T., et al., Resident renal mononuclear phagocytes comprise five discrete populations with distinct phenotypes and functions. J Immunol, 2013. 191 (6): p. 3358–72. 49. Stienstra, R., et al., Peroxisome proliferator-activated receptor gamma activation promotes infiltration of alternatively activated macrophages into adipose tissue. J Biol Chem, 2008. 283(33): p. 22620–7. 50. Satoh, T., et al., Critical role of Trib1 in differentiation of tissueresident M2-like macrophages. Nature, 2013. 495(7442): p. 524–8. 51. Aouadi, M., et al., Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice. Proc Natl Acad Sci U S A, 2013. 110(20): p. 8278–83. 52. Han, M.S., et al., JNK expression by macrophages promotes obesity-induced insulin resistance and inflammation. Science, 2013. 339(6116): p. 218–22. 53. Brinkmann, V., et al., Neutrophil extracellular traps kill bacteria. Science, 2004. 303(5663): p. 1532–5. 54. Yang, H., et al., Obesity increases the production of proinflammatory mediators from adipose tissue T cells and compromises TCR repertoire diversity: implications for systemic inflammation and insulin resistance. J Immunol, 2010. 185 (3): p. 1836–45. 55. Strissel, K.J., et al., T-cell recruitment and Th1 polarization in adipose tissue during diet-induced obesity in C57BL/6 mice. Obesity (Silver Spring), 2010. 18(10): p. 1918–25. 56. McGillicuddy, F.C., et al., Interferon gamma attenuates insulin signaling, lipid storage, and differentiation in human adipocytes via activation of the JAK/STAT pathway. J Biol Chem, 2009. 284(46): p. 31936–44.

57. Dobrian, A.D., et al., Signal transducer and activator of transcription (STAT) 4 deficiency reduces obesity induced insulin resistance and adipose tissue inflammation. Diabetes, 2013. 58. Morris, D.L., et al., Adipose tissue macrophages function as antigen-presenting cells and regulate adipose tissue CD4+ T cells in mice. Diabetes, 2013. 62(8): p. 2762–72. 59. Duffaut, C., et al., Unexpected trafficking of immune cells within the adipose tissue during the onset of obesity. Biochem Biophys Res Commun, 2009. 384(4): p. 482–5. 60. Jagannathan, M., et al., Toll-like receptors regulate B cell cytokine production in patients with diabetes. Diabetologia, 2010. 53(7): p. 1461–71. 61. DeFuria, J., et al., B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile. Proc Natl Acad Sci U S A, 2013. 110(13): p. 5133–8. 62. Cipolletta, D., et al., Tissular T(regs): a unique population of adipose-tissue-resident Foxp3+CD4+ T cells that impacts organismal metabolism. Semin Immunol, 2011. 23(6): p. 431–7. 63. Tiemessen, M.M., et al., CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proc Natl Acad Sci U S A, 2007. 104(49): p. 19446–51. 64. Ilan, Y., et al., Induction of regulatory T cells decreases adipose inflammation and alleviates insulin resistance in ob/ob mice. Proceedings of the National Academy of Sciences, 2010. 107(21): p. 9765–9770. 65. Cipolletta, D., et al., PPAR-gamma is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature, 2012. 486(7404): p. 549–53. 66. Priceman, S.J., et al., Regulation of adipose tissue T cell subsets by Stat3 is crucial for diet-induced obesity and insulin resistance. Proc Natl Acad Sci U S A, 2013. 110(32): p. 13079–84. 67. Nishimura, S., et al., Adipose natural regulatory B cells negatively control adipose tissue inflammation. Cell metabolism, 2013. 68. Rossjohn, J., et al., Recognition of CD1d-restricted antigens by natural killer T cells. Nat Rev Immunol, 2012. 12(12): p. 845–57. 69. Ji, Y., et al., Activation of natural killer T cells promotes M2 Macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signaling axis in obesity. J Biol Chem, 2012. 287(17): p. 13561–71. 70. Schipper, H.S., et al., Natural killer T cells in adipose tissue prevent insulin resistance. J Clin Invest, 2012. 122(9): p. 3343–54. 71. Lynch, L., et al., Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production. Immunity, 2012. 37(3): p. 574–87. 72. Subramanian, S., et al., Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice. J Lipid Res, 2013. 54(10): p. 2831–41. 73. Wu, L., et al., Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice. Proc Natl Acad Sci U S A, 2012. 109(19): p. E1143–52. 74. Lynch, L., et al., Invariant NKT cells and CD1d(+) cells amass in human omentum and are depleted in patients with cancer and obesity. Eur J Immunol, 2009. 39(7): p. 1893–901. 75. Mallevaey, T. and T. Selvanantham, Strategy of lipid recognition by invariant natural killer T cells: ‘one for all and all for one’. Immunology, 2012. 136(3): p. 273–82. 76. Elinav, E., et al., Adoptive transfer of regulatory NKT lymphocytes ameliorates non-alcoholic steatohepatitis and glucose intolerance in ob/ob mice and is associated with intrahepatic CD8 trapping. J Pathol, 2006. 209(1): p. 121–8. 77. Mantell, B.S., et al., Mice lacking NKT cells but with a complete complement of CD8+ T-cells are not protected against the metabolic abnormalities of diet-induced obesity. PLoS One, 2011. 6(6): p. e19831.

Endocr Pathol 78. Kotas, M.E., et al., Impact of CD1d deficiency on metabolism. PLoS One, 2011. 6(9): p. e25478. 79. Satoh, M., et al., Type II NKT cells stimulate diet-induced obesity by mediating adipose tissue inflammation, steatohepatitis and insulin resistance. PLoS One, 2012. 7(2): p. e30568. 80. Ji, Y., et al., Short term high fat diet challenge promotes alternative macrophage polarization in adipose tissue via natural killer T cells and interleukin-4. J Biol Chem, 2012. 287(29): p. 24378–86. 81. Wu, L. and L. Van Kaer, Contribution of lipid-reactive natural killer T cells to obesity-associated inflammation and insulin resistance. Adipocyte, 2013. 2(1): p. 12–16. 82. Cinti, S., et al., Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. Journal of Lipid Research, 2005. 46(11): p. 2347–2355. 83. Prieur, X., et al., Differential lipid partitioning between adipocytes and tissue macrophages modulates macrophage lipotoxicity and M2/ M1 polarization in obese mice. Diabetes, 2011. 60(3): p. 797–809. 84. Shapiro, H., et al., Adipose tissue foam cells are present in human obesity. J Clin Endocrinol Metab, 2013. 98(3): p. 1173–81. 85. Strissel, K.J., et al., Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes, 2007. 56(12): p. 2910–8. 86. Giordano, A., et al., Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis. J Lipid Res, 2013. 54(9): p. 2423– 36. 87. Murano, I., et al., Time course of histomorphological changes in adipose tissue upon acute lipoatrophy. Nutr Metab Cardiovasc Dis, 2013. 23(8): p. 723–31. 88. Miao, E.A., et al., Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol, 2010. 11(12): p. 1136–1142. 89. Galluzzi, L., et al., Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ, 2012. 19(1): p. 107–20. 90. Hosogai, N., et al., Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes, 2007. 56(4): p. 901–11. 91. Ye, J., et al., Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol Metab, 2007. 293(4): p. E1118–28. 92. Rausch, M.E., et al., Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration. Int J Obes (Lond), 2008. 32(3): p. 451–63.

93. Goossens, G.H., et al., Increased adipose tissue oxygen tension in obese compared with lean men is accompanied by insulin resistance, impaired adipose tissue capillarization, and inflammation. Circulation, 2011. 124(1): p. 67–76. 94. Blaak, E.E., et al., β-adrenergic stimulation and abdominal subcutaneous fat blood flow in lean, obese, and reduced-obese subjects. Metabolism, 1995. 44(2): p. 183–187. 95. Trayhurn, P., Hypoxia and adipose tissue function and dysfunction in obesity. Physiological Reviews, 2013. 93(1): p. 1–21. 96. Pasarica, M., et al., Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes, 2009. 58(3): p. 718–725. 97. Yamauchi, T., et al., The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med, 2001. 7(8): p. 941–6. 98. Boden, G., et al., Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals. Diabetes, 2008. 57(9): p. 2438–44. 99. Gregor, M.F., et al., Endoplasmic reticulum stress is reduced in tissues of obese subjects after weight loss. Diabetes, 2009. 58(3): p. 693–700. 100. Chen, B., et al., Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes. Biochem Biophys Res Commun, 2006. 341(2): p. 549–56. 101. Lolmede, K., et al., Effects of hypoxia on the expression of proangiogenic factors in differentiated 3T3-F442A adipocytes. Int J Obes Relat Metab Disord, 2003. 27(10): p. 1187–95. 102. Wang, G.L. and G.L. Semenza, General involvement of hypoxiainducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A, 1993. 90(9): p. 4304–8. 103. Jiang, C., et al., Disruption of hypoxia-inducible factor 1 in adipocytes improves insulin sensitivity and decreases adiposity in high-fat diet-fed mice. Diabetes, 2011. 60 (10): p. 2484–2495. 104. Odegaard, J.I. and A. Chawla, Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis. Science, 2013. 339(6116): p. 172–7. 105. Trayhurn, P., Hypoxia and adipose tissue function and dysfunction in obesity. Physiol Rev, 2013. 93(1): p. 1–21.