The role of JAK-STAT signaling in adipose tissue function

Abstract

Adipocytes play important roles in lipid storage, energy homeostasis and whole body insulin sensitivity. The JAK-STAT pathway mediates a variety of physiological processes including development, hematopoiesis, and inflammation. Although the JAK-STAT signaling pathway occurs in all cells, this pathway can mediate cell specific responses. Studies in the last two decades have identified hormones and cytokines that activate the JAK-STAT signaling pathway. These cytokines and hormones have profound effects on adipocytes. The content of this review will introduce the types of adipocytes and immune cells that make up adipose tissue, the impact of obesity on adipose cellular composition and function, and the general constituents of the JAK-STAT pathway and how its activators regulate adipose tissue development and physiology. A summary of the identification of STAT target genes in adipocytes reveals how these transcription factors impact various areas of adipocyte metabolism including insulin action, modulation of lipid stores, and glucose homeostasis. Lastly, we will evaluate exciting new data linking the JAK-STAT pathway and brown adipose tissue and consider the future outlook in this area of investigation.

1. Introduction

1.1 Adipocytes and Adipose Tissue

Obesity is a condition of excess adipose tissue and is the most common metabolic disorder in the industrialized world. In the US alone, it affects 154.7 million individuals over the age of 20, which is approximately 25% of the adult population. This obesity epidemic has been a prelude to increases in chronic diseases. Obese individuals, particularly those with excess abdominal adipose tissue, have an elevated risk of developing Type 2 diabetes mellitus (T2DM), cardiovascular disease, and hypertension. During obesity, the production of inflammatory cytokines and reactive oxygen species within adipose tissue increases as well as ectopic lipid deposition in liver or skeletal muscle (reviewed in [58]). These consequences reflect potential causative links between adipose tissue dysfunction and insulin resistance. However, the exact nature of this relationship is still poorly understood and the subject of intense investigation. Hence, understanding adipose tissue biology is highly relevant in elucidating the pathogenesis and treatment of metabolic diseases like T2DM.

Adipocytes are highly specialized lipid storage cells that play a key role in modulating energy balance and nutrient flux in vertebrates. They provide a storage reservoir for energy in the form of lipid, which is stored as a single or multiple droplet(s) that give adipocytes their characteristic rounded morphological appearance. Adipocytes also produce and secrete numerous enzymes, hormones, cytokines, and growth factors that modulate appetite, lipid and glucose homeostasis, insulin sensitivity, inflammation, blood vessel formation, and overall energy homeostasis [3]. Several of these secreted factors, such as leptin, prolactin, interleukin-6, and cardiotrophin-1, activate the JAK-STAT pathway and are mentioned in this review. In the context of this review, we also discuss the STAT1-mediated transcriptional regulation of lipoprotein lipase, an enzyme secreted from adipocytes.

The two classical types of fat cells that have been widely studied include white and brown adipocytes. White adipocytes are important in energy storage and have three main functions – they sequester and release lipid, take up glucose in response to insulin, and secrete paracrine and endocrine factors. Brown adipocytes are predominantly classified by their high content of mitochondria containing uncoupling protein-1 (UCP-1) and contribute to energy expenditure. UCP-1 uncouples the electron transport chain from energy production and results in the release of potential energy obtained from food as heat. As a result, brown adipocytes play an important role in adaptive thermogenesis and are essential for non-shivering thermogenesis in response to cold or β3-adrenergic stimulation [23,75]. We will review two recent high impact studies that link the JAK-STAT signaling pathway to brown adipocyte differentiation and adaptive thermogenesis and mark the infancy of our understanding of JAK-STAT signaling in brown adipose tissue (BAT).

Expansion of adipose tissue occurs through both increases in the size and number of the adipocyte population. New, mature adipocytes arise via differentiation of progenitor cells within adipose tissue. Evidence exists suggesting that white and brown adipocytes derive from different types of mesenchymal progenitor cells [78]. However, innovative studies examining the development of brown-like adipocytes within white adipose tissue (WAT) recently have challenged this concept [74]. The signaling factors regulating the transition of mesenchymal progenitor cells to committed preadipocytes are poorly defined. Nonetheless, significant advances towards an understanding of adipose tissue biology have been made by studying the function of transcription factors, which regulate differentiation of committed preadipocytes, and are involved in the modulation of adipocyte gene expression. Fat cell differentiation, known as adipogenesis, proceeds as a highly coordinated and temporally defined series of events that involves the regulated expression of numerous transcription factors (reviewed in [75,104]). Several laboratories have investigated the role of STATs (Signal Transducers and Activators of Transcription) in adipocyte development and function. Additionally, studies show that many STAT activators play a critical role in the regulation of adipocyte gene expression and exhibit differential expression in conditions of obesity and/or insulin resistance [13,75].

1.2 Other AT cell types

In addition to adipocytes, immune cells significantly contribute to the cellular composition of adipose tissue. Their presence within adipose tissue is regulated by obesity and metabolic dysfunction. The purpose of these immune cells and their relationship to metabolic dysfunction within obese adipose tissue is the subject of intense investigation and debate. Whether their presence is a cause or consequence with regards to insulin resistance is unknown, and both hypotheses have been proposed. Some types of immune cells, such as macrophages, increase in obese adipose tissue, and are associated with inflammation and metabolic disease. Yet the levels of eosinophils, which are anti-inflammatory and associated with healthy adipose tissue, decrease during obesity and insulin resistance (reviewed in [77]). Many studies suggest that adipose tissue macrophages (ATMs) are associated with insulin resistance in a manner that is dependent upon their activation status. Yet, more recent studies suggest that ATMs may have housekeeping functions in adipose tissue and may serve physiological roles in modulating lipid flux in adipocytes [47]. Interestingly, the JAK-STAT pathway was first identified and characterized as the result of immunological studies focused on understanding the signal transduction pathway utilized by interferon gamma (IFNγ) (reviewed in [84]).

Interest in adipose tissue immune cells has prompted recent studies examining the role of JAK-STAT activators and signaling in adipose tissue immune cells. Several cytokines that are activators of the JAK-STAT pathway are produced from immune cells, preadipocytes, and adipocytes within adipose tissue and have paracrine and endocrine effects on other cells with important functions in regulating metabolism and energy balance. Little is known regarding the complex interplay of JAK-STAT signaling between adipose tissue cells, but activators of this pathway have been shown to regulate development and function of both immune cells and adipocytes.

1.3 JAK-STAT signaling pathway

The STAT family of mammalian transcription factors is comprised of seven members (STATs 1-4, 5A, 5B, and 6) that have cell and tissue-specific distribution that influences their specificity and function [76]. STATs 5A and 5B, although highly homologous, are transcribed from different genes. While the expression level of STAT5A relative to STAT5B is tissue specific, the STAT5 proteins typically share similar patterns of tissue-dependent gene expression. Intriguingly, they have been shown to exhibit both redundant and non-redundant functions [94]. STATs are predominantly activated by phosphorylation of one tyrosine residue near the C-terminus that is catalyzed by a Janus Kinase (JAK). Members of the JAK family include JAKs 1-3 and Tyk2. The JAK-STAT pathway is present in all cells, mediates the action of numerous cytokines, growth factors, and hormones, and regulates diverse biological functions, including immune responses, energy expenditure, and cellular differentiation. Under basal conditions, STATs are largely inactive and localized to the cytoplasm. Upon ligand binding to a membrane-bound receptor, the receptor-associated JAKs become activated and phosphorylate tyrosine residues within the receptor, which then direct recruitment of specific STATs. STATs bind the activated receptor via their SH2 domains and become JAK substrates. Tyrosine phosphorylation of STATs results in the formation of homo- or hetero-dimers that translocate to the nucleus where they regulate transcription of specific target genes.

This review provides in depth coverage of the literature that relates to the role of JAK-STAT signaling in adipogenesis. We also address the ability of STATs to modulate fat cell function via transcriptional regulation of adipocyte-specific gene targets in response to activator stimulation. Additionally, we explore knockout studies of JAK-STAT activators in mice. These studies suggest that JAK-STAT signaling in adipose tissue plays an important role in paracrine communication between adipocytes and AT immune cells that might influence the pathogenesis of obesity. Lastly, we highlight novel studies regarding JAK-STAT signaling in brown adipose tissue.

2. Regulation of adipogenesis by STAT proteins

The first studies on the modulation of STATs during adipocyte development were performed over fifteen years ago and demonstrated that protein levels of STATs 1, 3, 5A and 5B increased during 3T3-L1 fat cell differentiation, providing the first suggestion that these STAT proteins may play a role in the transcriptional control of adipogenesis [86]. Five years later, studies in subcutaneous human primary adipocytes confirmed the up regulation of STATs 3 and 5 during differentiation [32]. However, the pattern of STAT1 protein expression during human [32] and murine [86] adipogenesis differed, suggesting species-specific regulation. Decreased STAT1 expression during adipogenesis of human adipocytes indicates that it does not promote human fat cell differentiation. There are few studies that examine the role of STATs 1 and 3 in the transcriptional control of adipogenesis. However, substantial in vitro and in vivo evidence from over a dozen independent laboratories supports the hypothesis that STAT5 promotes fat cell differentiation in mouse and man.

2.1 The role of STAT5 proteins in adipocyte development

Studies of transgenic mice containing knockouts or deletions of the STAT proteins have been critical in obtaining an understanding of the function of these proteins in vivo. Deletion of STAT5A, STAT5B, or both STAT5 proteins in genetically modified mice results in impaired adipose tissue development with the double knockout mice having fat pads only 20% of the normal size [94]. Since these are non-inducible whole-body deletions of STAT5, it is unclear if the reduced adipose tissue is related to developmental deficiencies. However, a recent study provides evidence that STAT5 proteins can promote adipocyte development in vivo in a mature animal. Fibroblasts were genetically engineered to express STAT5A and injected into athymic mice. STAT5A-expressing fibroblasts conferred the formation of ectopic fat pads and demonstrated that STAT5A is physiologically capable of regulating adipose tissue development in vivo [89]. A better understanding of the ability of STAT5 to modulate endogenous fat cell differentiation awaits the creation of transgenic mice in which STAT5 is conditionally deleted or knocked out of only adipocytes or adipose tissue. To date, a role of STAT5 in the development of brown adipose tissue has not been explored.

Although in vivo studies are limited, numerous laboratories have independently demonstrated pro-adipogenic activity of STAT5 proteins using multiple murine and human non-precursor and preadipocyte cell types in culture. These studies have led to insights into the involvement of STAT5 proteins in fat cell differentiation and the mechanisms by which they promote adipogenesis, and the findings are summarized in Figure 1. In differentiating murine preadipocytes, the protein levels of both STATs 5A and 5B are increased and tightly coupled to the development of the lipid-bearing cellular phenotype and elevated expression of well-studied adipogenic transcription factors, including C/AAAT enhancer binding protein α (C/EBPα) and peroxisome proliferator-activator receptor γ (PPARγ) [88]. Furthermore, ectopic expression of STAT5A in non-precursor cells sufficiently induces fat cell differentiation [27,87]. Interestingly, STAT5B was unable to promote adipogenesis in non-precursor cells, but it did enhance STAT5A-induced fat cell differentiation, indicating distinct roles for STATs 5A and 5B in regulating adipogenesis [27]. RNA interference studies support a supplementary role of STAT5B in fat cell differentiation [38]. Studies using antisense Stat5 oligonucleotides as well as constitutively active and dominant-negative STAT5 constructs have demonstrated that STAT5 proteins mediate the pro-adipogenic activity of growth hormone on preadipocytes [39,79,108].

Figure 1. Roles of the JAK-STAT5 signaling pathway in adipose tissue.

The JAK-STAT5 signaling pathway is activated by GH, PRL, and GM-CSF, and it regulates the development and/or function of preadipocytes, adipocytes, and macrophages in adipose tissue. Substantial evidence supports that STAT5 proteins are activated in preadipocytes and that they promote adipogenesis. In mature adipocytes, multiple STAT5 target genes have been identified, demonstrating a role for STAT5 in modulating key physiological properties of adipocytes. Studies also indicate that JAK-STAT5 signaling is important in the recruitment and development of adipose tissue macrophages.

In several preadipocyte model systems, growth hormone (GH)-activated STAT5 proteins have been shown to induce PPARγ expression suggesting that STAT5 can promote adipocyte differentiation by regulating PPARγ [39]. This is supported by data showing that STAT5 can directly bind and transactivate the PPARγ promoter [39,55,100]. Many transcription factors have profound effects on adipocyte development, but PPARγ is a critical transcriptional regulator that is absolutely required for fat cell differentiation [6,61]. While the evidence suggests that STAT5 proteins regulate PPARγ expression, it is plausible that STAT5 also regulates the expression of proteins responsible for making PPARγ ligands or other proteins important in the developing and mature adipocyte.

STAT5 proteins are specifically activated by tyrosine phosphorylation almost immediately following induction of adipogenesis in 3T3-L1 cells [6,61]. Interestingly, cooperative binding of C/EBPβ and STAT5A occurs during a very early stage of adipogenesis and suggests that STAT5A is involved in chromatin remodeling and priming of regulatory sites for subsequent binding by other transcription factors [82]. Intriguingly, in human bone marrow-derived stromal cells induced to undergo adipogenesis, PPARγ also binds to the STAT5A promoter while C/EBPα and C/EBPβ bind the STAT5B promoter region [38]. Thus, there exists a complex interplay of STAT5 proteins with other adipogenic transcriptional regulators to orchestrate the stages of differentiation leading to the biochemical and morphological changes associated with the mature lipid-laden fat cell.

Although the majority of studies suggest that STAT5 proteins promote adipogenesis, there is some confounding data. For example, STAT5 activators such as GH and oncostatin M (OSM) can repress adipocyte differentiation in cell culture models of precursor cells from mice [57], rats [70], and humans [83]. This anti-adipogenic activity of STAT5 can likely be attributed to specifics of OSM signaling [57], possibly via modulation of post-translational modifications of STAT5 and/or its interacting protein partners. For studies in which primary preadipocytes [70,83] or mouse embryonic fibroblasts [57] - versus committed preadipocyte cell lines - were used to assess adipogenic potential, the developmental stage of the cell and how far it is committed to develop into an adipocyte might be responsible for the opposing effects of STAT5 on adipogenesis. Other factors could include species-specific differences in rat primary preadipocytes in response to GH or pre-exposure of the primary preadipocytes to GH in vivo [70]. Nonetheless, STAT5 proteins, particularly STAT5A, play an important role in regulating adipogenesis, and the preponderance of evidence suggests that they are pro-adipogenic in most model systems.

2.2 The role of STAT1 in adipocyte development

STAT1 expression is induced during adipocyte development in mouse cells [86]. However, mice with a targeted disruption of the Stat1 gene do not exhibit differences in weight gain, and with the exception of IFN-dependent responses, biologic responses to other cytokines were not defective [56]. Interestingly, IFNγ inhibits adipogenesis of SGBS (Simpson-Golabi-Behmel-syndrome) human fat cells [53] and rodent preadipocytes [30,40]. Although the direct role of STAT1 in the anti-adipogenic action of IFNγ was not investigated, experiments using pharmacological inhibitors indicate that the JAK-STAT1 pathway plays a central role in the ability of IFNγ to induce insulin resistance, decrease triglyceride stores, and down-regulate expression of lipogenic genes in mature human fat cells [53]. IFNγ also activates JAK2-STAT3 [4,53,85]. However, specific inhibition of JAK2 did not block IFNγ effects on fat cell development and physiology, and leptin-induced activation of JAK2-STAT3 failed to substantially decrease adipocyte differentiation and lipid accumulation [53]. Thus, it was concluded that JAK1-STAT1 primarily mediated the substantial IFNγ-induced modulation of human adipocyte functions [53].

IFNγ-null mice fed a high fat diet have smaller adipocytes in visceral WAT than wild-type mice [65]. Interestingly, there were no differences in body weight [65] similar to the STAT1 null mice on chow diet [56]. Together the presence of smaller adipocytes and lack of change in body weight in the IFNγ knockout mice suggest an increase in fat cell differentiation and indicate that IFNγ might play an inhibitory role in adipogenesis in vivo. An increase in adipocyte cell number in the IFNγ-null mice would have better supported a claim of increased adipogenesis; however, this parameter is difficult to measure and was not assessed in the study.

Knockout of IFNγ also decreased the size of the natural killer (NK) cell population in visceral but not subcutaneous WAT, while it shifted the activation phenotype of ATMs from M1 to M2 in both depots [65]. M1-type ATMs produce inflammatory cytokines that are correlated with the development of insulin resistance in obesity. A multitude of reports demonstrate an association between immune cell infiltration in visceral but not subcutaneous adipose tissue during obesity. Thus, it is not surprising that knockout of IFNγ results in more substantial changes in the population of immune cells in visceral WAT. Accordingly, it is possible that IFNγ indirectly modulates adipogenesis via its effects on the composition of immune cells within adipose tissue. The studies of IFNγ-null mice indicate that IFNγ plays a role during obesity in the regulation of inflammation and insulin sensitivity through several probable mechanisms such as modulating adipogenesis and influencing the size and composition of the AT immune cell population. However, the role of JAK-STAT1 signaling in mediating these effects of IFNγ in obesity remains to be elucidated.

The ability of IFNγ and JAK-STAT1 signaling to regulate fat cell differentiation has also been studied in the context of crosstalk with the hedgehog signaling pathway. The hedgehog signaling pathway constitutes an ancestral developmental process important in the regulation of stem cell differentiation during embryonic development and adult tissue homeostasis [7,37,98]. Sonic hedgehog (Shh) is the most widely studied homolog of the three mammalian hedgehog proteins. Shh signaling specifically blocks adipogenesis in white, but not brown adipose tissue [69,92]. IFNγ inhibits fat cell differentiation in the absence of Shh signaling [30,40,96]. However, when the Shh pathway is activated in various preadipocyte model systems, IFNγ blocks Shh signaling and rescues adipogenesis via a JAK-STAT1-dependent mechanism [96]. IFNγ-mediated inhibition of Shh signaling did not occur in Stat1−/− mouse embryonic fibroblasts (MEFs), indicating that the crosstalk depends on Stat1. Thus, STAT1 appears to regulate adipocyte differentiation via crosstalk with the Shh signaling pathway.

Collectively, these findings suggest that STAT1 may not play a major role in adipocyte differentiation and adipose tissue development under “normal” conditions in lean subjects. However, in the context of high fat diet (HFD)-induced obesity when IFNγ levels are elevated as a result of increased numbers of activated IFNγ-producing immune cells infiltrating visceral adipose tissue, JAK-STAT1 signaling may play an inhibitory role in the control of adipocyte differentiation. Further studies using genetic manipulation of Stat1 expression in adipocytes are needed to better elucidate the role of STAT1 in the control of adipogenesis in vivo. Additionally, it would be interesting to examine the phenotype of adipose tissue in Stat1 null mice following the development of diet induced obesity. Based on the available data, it seems that cross-talk with other signaling pathways and the inflammatory state of adipose tissue are important factors modulating the ability of STAT1 to regulate adipogenesis.

2.3 Role of STAT3 in the regulation of adipogenesis

The JAK2-STAT3 pathway is activated early during adipogenesis [18,101,109] and is involved in achieving maximal adipocyte differentiation potentially through modulation of C/EBPβ transcription [109]. As demonstrated by in vitro experiments, adipogenesis is suppressed by selective inhibition of JAK2 or STAT3, siRNA-induced knock-down of STAT3, or overexpression of dominant-negative STAT3 [101]. PIAS3, protein inhibitor of activated STAT3, is constitutively expressed in 3T3-L1 cells, and its activation represses STAT3 activity and inhibits fat cell differentiation [16]. In addition, a PPARγ synthetic agonist rescues adipogenesis following RNAi-induced knock-down of STAT3, suggesting that STAT3 regulation of adipocyte differentiation occurs upstream of PPARγ activation [101]. Collectively, these studies indicate that activation of the JAK2-STAT3 signaling pathway plays a role in the modulation of adipogenesis. Additional studies have shown that activation of STAT3 may promote fat cell differentiation via modulation of mitotic clonal expansion, a proliferative phase that occurs immediately following induction of adipogenesis and is necessary for differentiation of 3T3-L1 fat cells [11,18,51].

Since germ-line deletion of STAT3 is embryonic lethal [44,49], studies are lacking to demonstrate that STAT3 modulates adipocyte differentiation in vivo. However, there is a transgenic mouse model where STAT3 expression was knocked out with use of aP2 Cre. The aP2 protein is a lipid binding protein that is highly expressed in fat cells and is likely the most abundantly expressed protein in mature adipocytes. The primary phenotype of the aP2 Cre-driven STAT3 knockout mouse was increased weight and increased adipose tissue mass, associated with adipocyte hypertrophy [10]. These studies suggest that STAT3 contributes to body weight homeostasis. However, since aP2 can also be expressed in macrophages [28,29,67], brain cells, and mouse embryonic cells [97], it is unclear if these observations are solely mediated by the lack of STAT3 in adipocytes. Furthermore, since aP2-driven deletion of STAT3 does not occur until late during adipogenesis, the observed adipocyte hypertrophy is not likely a result of direct effects of STAT3 on fat cell differentiation. Overall, evidence suggests that STAT3 is capable of modulating adipogenesis. However, further investigations probing JAK-STAT3 signaling at various stages of adipogenesis in vitro and in vivo are necessary to better understand the ability of STAT3 to regulate adipocyte and adipose tissue development.

3. Role of JAKs in adipocyte development and function

Although STATs have been fairly well studied in adipocytes, there are minimal studies focusing on JAK expression, activation, and function in fat cells and adipose tissue. JAK kinases are largely controlled by tyrosine phosphorylation, rather than by expression levels. The ubiquitously expressed JAKs 1 and 2 are present at similar levels in preadipocytes and adipocytes [88], and they are expressed in adipose tissue in vivo [33]. There is some evidence that Tyk2 and JAK3 are expressed in adipose tissue [19,33]. As indicated earlier, adipose tissue is comprised of many cell types and there is no data to suggest that these two JAK family members are expressed in white adipocytes.

Both preadipocytes and adipocytes are responsive to hormones and growth factors which activate JAKs 1 and 2, including GH, prolactin (PRL), IFNγ, leukemia inhibitory factor (LIF), OSM, cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF) [4,24,39,53,66,85,90,110–112]. Currently, there is no evidence that JAKs play a STAT-independent role in modulating adipocyte differentiation in white adipose tissue. However, several cytokines that inhibit adipogenesis, including IFNγ [30,40], OSM [57,83], and neuropoietin (NP) [104], are potent activators of JAK kinases. To date, only JAKs 1 and 2 have been detected in white adipocytes and their roles are solely attributed to their ability to be activated by cytokines and confer STAT activation. There is one exception, however, in which JAK2 was shown to physically associate with aP2 in adipocytes [95]. The unphosphorylated form of JAK2 has been shown to interact with aP2 and the results of this study suggest that ligand-bound aP2 decreases JAK2 signaling [95]. Overall, there is a paucity of data regarding the role of JAKs in adipocytes. Hence, additional studies will be required to further elucidate the STAT-dependent and/or independent functions of these kinases in fat cells.

4. STAT target genes in preadipocytes and adipocytes

The regulation of tissue-specific genes has been shown to be a physiological role of STAT proteins in a variety of cell types, including adipocytes. To date, specific target genes have been identified for STATs, 1, 5A, and 5B, but not for STATs 3 and 6, in adipocytes (Table 1). The STAT target genes elucidated in fat cells code for proteins that regulate adipocyte development, insulin action, and lipid and carbohydrate metabolism. As summarized in Figure 1, many laboratories around the world have shown a role for STAT5 proteins in adipocyte and adipose tissue development in vitro and in vivo. Hence, it is not surprising that STAT5 can directly bind the PPARγ3 promoter [55] and can transactivate the PPARγ2 and PPARγ3 promoters [39,55]. PPARγ is a STAT5 target gene during adipocyte development and its modulation by STAT5 likely plays a role in the ability of STAT5 to promote adipocyte differentiation in vitro and in vivo. Studies have shown that PPARγ is also a STAT1 target gene in adipocytes. In 3T3-L1 adipocytes, STAT1 homodimers bind to an IFNγ responsive site within the PPARγ2 promoter and suggest that IFNγ-induced repression of PPARγ2 transcription [99] is mediated by the direct action of STAT1 on the PPARγ2 promoter [34]. Of note, both a dominant negative mutation in PPARγ and IFNγ signaling have been associated with the development of insulin resistance [5,53,99]. Consequently, STAT1 likely mediates the ability of IFNγ to induce insulin resistance [46,53,80,99] and inhibit adipogenesis [30,40] via transcriptional repression of PPARγ. An IFNγ-sensitive binding site for STAT1 was also discovered in the murine lipoprotein lipase (LPL) promoter [35]. LPL is the rate-limiting enzyme that catalyzes the hydrolysis of serum triglycerides from lipoproteins into free fatty acids for uptake and storage in adipose tissue. IFNγ-activated STAT1 binds to the LPL promoter in a manner that is consistent with IFNγ-induced repression of LPL expression and inhibition of LPL activity in murine adipocytes [20,30]. While STAT3 is also induced in response to IFNγ, STAT1 is a more robust mediator of IFNγ signaling in murine and human adipocytes [4,53,85]. In the described studies, STAT3 was unable to bind to the STAT1 binding sites within the PPARγ promoter [34], and LIF, a potent STAT3 activator, did not confer binding of STAT3 to the IFNγ sensitive region of the LPL promoter [35].

Table 1.

STAT Target Genes in Preadipocytes and Adipocytes.

Cell Type	STAT	STAT Activator	Target Gene
Preadipocyte	STAT5	Unknown	PPARγ
	STAT3	Unknown	C/EBPβ
Adipocyte	STAT1	IFNγ	PPARγ
		IFNγ	LPL
	STAT5	GH	aP2
		GH	AOX
		GH, PRL	FAS
		GH, PRL	PDK4

Since STAT5 proteins are activated early during adipocyte differentiation and have been shown to play such a key role in adipocyte development, it is not surprising that most studies have focused on the functions of STAT5 proteins in mature adipocytes. The promoter for acyl CoA oxidase (AOX), the rate limiting enzyme in peroxisomal fatty acid β-oxidation, contains a STAT5 binding site that modulates its gene expression in fat cells [12]. Transfection studies have shown that the promoter activity of aP2, an abundantly expressed lipid binding protein in fat cells, can be activated by STAT5 [60]. Other studies have shown that STAT5 mediates the inhibition of aP2 expression in rat primary preadipocytes [70]. This was the first study to suggest that STAT5 proteins could act as transcriptional repressors. Since that time, our own research has revealed that STAT5A can act as a transcriptional repressor in adipocytes. A STAT5A binding site in the murine fatty acid synthase (FAS) promoter mediates the repression of FAS transcription that occurs with growth hormone (GH) or prolactin (PRL) treatment [36]. FAS catalyzes the production of long chain fatty acids and is a crucial enzyme involved in de novo lipogenesis. In addition to modulation of genes associated with lipid metabolism such as AOX and FAS, STAT5 can also increase the transcription of pyruvate dehydrogenase kinase (PDK)-4, a known regulator of glycolysis that is highly induced in adipocytes by PRL or GH in a STAT5 dependent manner [103]. Under these conditions, insulin resistance accompanies the induction of PDK4. It is well known that PRL and GH are important modulators of lipid metabolism and are also potent inducers of STAT5 in adipocytes [4,60]. Hence, many of the metabolic actions of these anterior pituitary hormones could be controlled by direct modulation of target genes by STAT5 (Table 1). We also have data to indicate that STAT5 proteins regulate the expression of adiponectin, an important adipocyte hormone that modulates insulin sensitivity. In summary, STAT5 transcriptionally regulates the expression of AOX, aP2, FAS, PDK4, and adiponectin -proteins that modulate lipid and glucose metabolism and insulin sensitivity in fully differentiated fat cells (Figure 1). Although relatively few STAT5 target genes have been identified in adipocytes, it is logical to predict that other STAT5A target genes that play a role in lipid or glucose metabolism will be identified.

STAT3 is abundantly expressed in adipocytes [32,86] and mediates the action of numerous cytokines in fat cells. However, with the exception of C/EBPβ as a potential STAT3 gene target activated early in the adipogenic program [109], to date no adipocyte-specific direct target genes have been identified for STAT3 (Table 1). Although STAT6 is equivalently expressed in preadipocytes and throughout fat cell differentiation [86], only IL-4 has been shown to activate this transcription factor in 3T3-L1 preadipocytes but not in adipocytes [17]. Thus, activators, functions, and gene targets of STAT6 in both preadipocytes and adipocytes remain to be elucidated. Overall, there is almost nothing known about the identity of STAT3 and STAT6 target genes in adipocytes.

5. JAK-STAT signaling in AT immune cells

The JAK-STAT pathway was first elucidated from immunological studies in the early 1990s that were investigating cell-specific IFNγ-responsive gene transcription [84]. In the past two decades, investigators have generated a wealth of information regarding activators and functions of the JAK-STAT pathway in immune cells, inflammation, and inflammatory disorders [68,84]. Research over the past decade has established AT as a bona fide endocrine organ comprised of a heterogeneous population of preadipocytes, adipocytes, endothelial cells, connective tissue, and immune cells. Multiple lines of evidence suggest that alterations in the phenotype and/or number of AT-resident immune cells are associated with the development of insulin resistance in obese mice and humans [77]. Currently, there are only a few studies that examine activators and functions of the JAK-STAT signaling pathway in the context of AT and immunometabolism, an emerging field of investigation linking immunology and metabolism [52,77]. In this section, we discuss the ability of two known JAK-STAT activators, GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFNγ, to modulate adipose tissue function.

STAT5 plays a role in the differentiation of myeloid cells and activation of macrophages [106]. GM-CSF is a proinflammatory cytokine that signals via the JAK2-STAT5 signaling pathway [21,59,73,102]. GM-CSF knockout mice fed a HFD have increased adiposity and adipocyte size [42]. Analysis of these mice suggests that the presence of GM-CSF positively correlates with the relative number of macrophages within the mesenteric fat and the relative expression of GM-CSF differs among the fat depots. In the absence of GM-CSF, the number of ATMs in mesenteric fat declined and was accompanied by decreased expression of pro-inflammatory cytokines in mice fed a high fat diet. Additionally, GM-CSF null mice were protected from HFD-induced insulin resistance despite increased adiposity [42]. The exact mechanisms underlying the correlation between increased adiposity, adipose tissue dysfunction, and insulin resistance are active areas of investigation. Other examples [41,43], including these findings with the GM-CSF knockout mice [42], seem to rule out increased adipocyte size as the causative factor in the relationship between AT dysfunction and insulin resistance. Currently, it is not known whether STAT5 mediates these effects of GM-CSF in AT, as both STATs 1 and 3 also can be activated by this cytokine [9]. Interestingly, STAT5 signaling in hypothalamic nuclei of the brain has been implicated in the ability of GM-CSF to regulate food intake and body adiposity [48]. Overall, these data indicate that GM-CSF plays an important role in AT to recruit and activate macrophages that contribute to AT inflammation, and these actions of GM-CSF are likely mediated by the JAK-STAT signaling pathway (Figure 1).

IFNγ is produced from both natural killer (NK) cells [64] and T cells [22,71,91,107] present in adipose tissue. IFNγ can inhibit the differentiation of preadipocytes [30,40], induce insulin resistance in mature adipocytes [53,99], and decrease PPARγ expression by targeting this nuclear receptor to the ubiquitin proteasome system for degradation in adipocytes [26]. It is highly likely that the production of IFNγ from infiltrated immune cells acts in a paracrine fashion on adjacent adipocytes to result in insulin resistance. Interestingly, deletion of IFNγ in transgenic mice shifted the activation of ATMs in visceral WAT toward an alternatively activated phenotype that was associated with decreased production of inflammatory cytokines and improved insulin sensitivity [65].

Numerous other JAK-STAT activators are produced in adipose tissue and likely act in a paracrine manner to regulate the development and function of immune cells and adipocytes. For instance, IL-1 and IL-6 are secreted from ATMs [54]. The majority of IL-4 in adipose tissue is released from eosinophils, and as discussed in the next section signals via JAK-STAT6 to modulate the function of BAT and WAT [63]. Prolactin and leptin, although they also act as endocrine hormones, have direct actions on immune and fat cells in adipose tissue [2,24,25,81].

In addition to macrophages, T cells, and NK cells that have already been mentioned, recent prominent studies have also examined the roles of B cells [15,105], mast cells [1,50], and neutrophils [93] in mediating insulin resistance associated with obesity. B cells, mast cells and neutrophils have all been shown to increase in number and/or shift activation status within adipose tissue upon feeding mice HFD [1,93,105]. Using genetic knockout, immuno-neutralization, or pharmacological inactivation techniques, loss of any of these immune cells or their direct mediators, such as elastase for neutrophils, was associated with improved insulin sensitivity. Similarly, attempts to increase B cells, neutrophils, or mast cells correlate with increased insulin resistance [50,93,105], thus establishing these immune cells as modulators of insulin resistance. To date, no studies have examined the role of the JAK-STAT pathway in mediating the action of B cells or neutrophils in adipose tissue. However, it was shown that mast cells promote angiogenesis in AT in an IL-6 and IFNγ-dependent manner [50]. Activation of the JAK-STAT pathways was not assessed in these studies; therefore, future studies are needed to evaluate the role of this pathway in mast cell action. As the immunometabolism field continues to develop, we propose that future studies of JAK-STAT signaling within adipose tissue will be an exciting new area of research that will lead to additional insights into the mechanism(s) by which AT immune cells communicate with adipocytes and/or other AT immune cells to modulate insulin sensitivity.

6. Emerging roles of JAK-STAT pathway in brown adipose tissue

The last few years have been accompanied by a large increase in studies on BAT. Although originally thought to be absent in adult humans, there is now evidence to support the presence of BAT and its association with metabolic health [14,62]. In addition, many transgenic mouse models have been generated that strongly suggest that increased BAT mass is associated with increases in energy expenditure that are associated with improvements in metabolic health [8]. The opposite is also true, and mice lacking BAT are obese and have metabolic impairments [31,45]. In the last few months, two high impact studies have linked the JAK-STAT signaling pathway to BAT, and these findings are summarized in Figure 2. It is largely accepted that macrophages in WAT play a role in the pathogenesis of insulin resistance and T2DM (reviewed in [72]). It is not clear whether macrophages in BAT are also important, and this topic is controversial. However, a recent study clearly demonstrates the presence of AAMs (alternatively activated macrophages) in BAT [63]. These macrophages are sensitive to IL-4, which acts via the JAK-STAT6 signaling pathway. The activation of this pathway results in the production of norepinephrine (NE). Among other physiological effects, NE can activate lipolysis in WAT. The hypothesis developed from these recent studies suggests that NE produced from alternatively activated macrophages in BAT acts on WAT to modulate free fatty acid levels (Figure 2). The use of IL-4 receptor null mice and STAT6 null mice reveals that these proteins are necessary for adaptive thermogenesis [63].

Figure 2. JAK-STAT signaling in white and brown adipose tissue.

Different transcriptional programs control the adipogenesis of white and brown adipocytes, and different JAK and STAT members have been implicated in these programs. Cytokine and hormone activators of the JAK-STAT pathway are produced and utilized by various cell types present within adipose tissue to modulate the physiological function of WAT and BAT.

Another study linking BAT with the JAK-STAT pathway provides evidence that the JAK kinase Tyk2 plays an important role in BAT development. In vitro studies strongly provide evidence that Tyk2 promotes adipogenesis. Moreover, Tyk2 null mice are obese and have impaired glucose tolerance. The phenotype of these mice can be rescued by aP2 driven expression of a constitutively active STAT3 protein [19]. In summary, this study suggests that the Tyk2-STAT3 signaling pathway plays a role in regulating BAT development (Figure 2). These are the first two studies to indicate the potential importance of the JAK-STAT signaling pathway in regulating the production and function of BAT.

7. Conclusions and Outlook

In conclusion, modulation of the JAK-STAT pathway can regulate adipocyte development and function. An emerging area of investigation linking JAK-STAT signaling and adipocyte function will clearly be studies to examine the interplay of immune cells and adipocytes in both brown and white adipose tissue. Additional studies in both cultured adipocytes and in adipose tissue will be needed to reveal comprehensive roles of the JAK-STAT family members in adipocytes, obesity, and insulin resistance. Although tyrosine phosphorylation is critical for canonical STAT activation, other covalent modifications such as serine phosphorylation, acetylation, methylation and sumoylation can also occur (reviewed in [84]), and studies of these STAT modifications are in their infancy. Moreover, the non-canonical mechanisms and functions of JAKs and STATs in adipocytes have not been well studied. Recent investigations indicate that STATs can participate in chromatin organization and mitochondrial respiration in ways that are independent of transcriptional regulation. All of these topics will likely be intense areas of investigation in adipocyte biology in the near future and will hopefully lead to the identification of new therapeutic targets for metabolic diseases.