White adipose tissues (WATs) regulate energy homeostasis through their ability to store and release lipids ^[1]. Further, these tissues provide thermal insulation and act as shock absorbers ^[2]. Another critical function is to regulate activities in other organs such as the liver, lungs, heart, and skeletal muscle through the release of bioactive factors termed adipokines, making WAT the largest endocrine organ in the body [3,4]^[3][4]. Adipokines are a highly heterogeneous population of molecules that perform many and varied functions. They include hormones, cytokines, enzymes, growth factors, and chemokines ^[5]. The endocrine function of WATs depends upon the close and extensive interrelationship between adipocytes and the microvasculature ^[6]. The development of WATs and their endocrine functions throughout life depends in major part on the presence of heparan sulfate proteoglycans on both adipocyte surfaces and vascular surfaces. This review will examine the functions of HSPGs in these roles.

2. Heparan Sulfate Proteoglycans

Each HSPG consists of a core protein to which one or more heparan sulfate (HS) chains are covalently attached [17,18]^[7][8]. Adipose tissues, like other tissues and organs, contain multiple species of HSPGs that appear on both cell surfaces and in the matrix [17,18,19]^[7][8][9]. These have been allocated into full-time and part-time entities [18]^[8], and both sets are present in adipose tissues. It is important to note that adipose depots in various anatomic sites differ physiologically [20]^[10]. Gesta and co-workers [20]^[10] examined these various sites in both mice and humans to determine whether specific sets of genes might be differentially expressed. They found that a specific set of genes was more highly expressed in mouse intraabdominal epididymal depots compared to other anatomic sites. One of these highly over-expressed genes was the HSPG glypican 4. The same gene was also highly over-expressed in human visceral fat depots, except in lean individuals where it was over-expressed in subcutaneous regions. Glypican 4, like other glypicans, is glycosyl-phosphatidylinositol–anchored (GPI-anchored) to the cell membrane [18]^[8]. This allows it to migrate within the plane of the membrane, a feature that enables it to become concentrated at specific sites in the membrane. The part-time HSPG CD44 is also present in adipose tissue. CD44 plays a role in adipose tissue physiology, but it is not clear whether the HS chain is involved [21]^[11]. Betaglycan, which functions as an alternate TGFβ receptor, has been shown to be downregulated in the visceral fat of obese individuals [22]^[12]. The BM HSPG perlecan is also a major component of adipose tissues. Perlecan is a matrix PG, and its core protein contains multiple binding domains for other matrix molecules [23]^[13]. This feature enables its insertion into BMs. It also may interact with cell surfaces via the α2β1 integrin [24]^[14].

3. Sulfation of Heparan Sulfates and Its Role in Adipose Tissues

The carbohydrate component of HSs consists of repeating disaccharides, each containing an amino sugar and either a uronic or iduronic acid [25]^[15]. These are linked into straight chains that are modified during synthesis. Internal cell-mediated modifications include acetylation/deacetylation, epimerization, and sulfation [25]^[15]. Sulfation is organized by a family of enzymes termed sulfotransferases [26]^[16]. Sulfotransferases are membrane-bound enzymes that are aligned in linear arrays in the Golgi so that they can organize the attachment of sulfate groups to specific regions of the HS chains. There are a maximum of four possible sulfation sites on disaccharides that contain iduronic acid and three possible sites if uronic acid is present. This means that a given HS disaccharide can have anywhere between 0 and 4 sulfate units. As sulfation is non-random, this produces GAG chains that have distinct domains that recognize heparan binding domains on select proteins [27,28,29,30]^{[17][18][19][20]}. These domains consist of five or more highly sulfated saccharide units. There are multiple sulfation patterns that are recognized by different proteins. For example, HGF binds to domains that have a high level of 6-O-S, while FGF-2 binds to domains where the principal sulfation is by N-S and 2-O-S [30]^[20]. The number of proteins that express heparan binding domains is limited. However, many of these molecules are involved in the regulation of angiogenesis and are present in adipose tissues (Table S1). As HSPGs enter the extracellular regions, they become subject to modification by other sets of cell-derived enzymes that modify the structure and sulfation of HS chains. Sulfatases are a family of enzymes that mediate the selective removal of sulfate groups from HS chains [26]^[16]. Two heparanases mediate the cleavage of specific carbohydrate linkages, thus releasing fragments of the HS chain [31]^[21]. Individuals with type I diabetes exhibit high levels of heparanases activity resulting in poorly sulfated HS associated with β-cells [32]^[22]. The fragmentation of HS chains releases chemokines that recruit inflammatory cells that destroy the β-cells. Similar events occur in mature adipose tissues exposed to elevated levels of circulating glucose. This results in the upregulation of heparanase production by adipocytes and other cells in the tissue. Additionally, elevated glucose activates inflammatory cells resulting in inflamed adipose tissues characteristic of obese individuals. These enzymes become important in diabetic individuals as the size and sulfation of HS are modified by glucose levels [31]^[21]. The HS-modifying enzymes found in adipose tissues are the same as found elsewhere; however, it is possible that their regulation is modified. Wilsie and others, using the 3T3-L1 adipogenic model, demonstrated that cell surface HSPGs play a major role in differentiating adipocytes from pre-adipocytes. They found an increase in the presence of sulfated PGs upon induction of the 3T3-L1 cells [33]^[23]. However, when the cultures were treated with xyloside compounds, a reduction in sulfation was observed and was accompanied by reduced lipid uptake by the cells. The xylosides compete with HS and CS chains for their attachment to their core proteins, resulting in a low sulfated PG [34]^[24]. Other studies have shown that apolipoprotein E-enriched very low-density lipid (VLDL) possesses heparan-binding domains that allow this molecule to be concentrated on adipocyte surfaces [33,35]^[23][25]. Adipocytes also produce and secrete lipoprotein lipase (LPL), which also associates with HS through its heparan-binding domain [33,35]^[23][25]. The mechanism by which sulfate regulates lipid uptake is not completely clear. One proposed possibility is that VLDL and LPL are concentrated in proximity to each other on the cell surface and that LPL mediates the release of triglycerides. These are then internalized by fatty acid transporters. Alternatively, VLDL is concentrated on the surface adjacent to the VLDL receptor and/or the lipoprotein receptor-related protein, which mediate the internalization of lipids [33,36]^[23][26]. This process may also occur when mature adipocytes become hypertrophic. In addition, 3T3-L1 cells grown under high glucose conditions exhibit a reduction in cell surface sulfation and a release of bound LPL [37]^[27]. In short, HS plays a role in glucose metabolism. The heparan-binding protein insulin-like growth factor binding protein-2 has been shown to interact with preadipocyte cell surfaces to inhibit their development. This further emphasizes the importance of HSPGs in the regulation of adipogenesis [38]^[28]. One of the adipocyte cell surface HSPGs is glypican-4, a glycosylphosphatidylinositol-anchored molecule [39]^[29]. The expression of this PG is higher in visceral adipose tissues than in subcutaneous adipose tissues, and its expression increases with body fat content. One of the functions of glypican-4 is to interact with the insulin receptor to enhance adipocyte differentiation and hypertrophy [39]^[29]. The core protein can be cleaved, allowing release from the cell to circulate as an adipokine. Matsuzawa’s group inhibited HS synthesis in 3T3-L1 cells using CRISPR-Cas9 technology to delete the Ext1 gene, an enzyme involved in HS synthesis and found that this loss resulted in reduced glucose uptake and insulin-dependent intracellular signaling via the BMP4-FGF1 pathways [40]^[30]. They further developed mutant mice in which HS chain synthesis was partially inhibited and found a reduction in visceral adipose tissues. This group also demonstrated the role of HS in regulating insulin secretion by pancreatic β-cells [32,41]^[22][31]. Thus, HS plays a dynamic role in multiple organs to regulate glucose levels. Cell surface HSPGs also act as co-receptors for some growth factors. Fibroblast growth factor-2 and hepatocyte growth factor, both considered adipokines, bind to HS, which then presents these molecules in an active manner to the appropriate cell surface receptors to initiate signal cascades [42]^[32]. These factors combine with other HB-binding factors to regulate angiogenesis in adipose tissues: these include vascular endothelial growth factors (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), transforming growth factor- β (TGF- β), and angiopoitin like protein 4 [43]^[33]. HB-EGF is produced by multiple cells in adipose tissues: macrophages, vascular endothelial cells, and adipocytes. Its expression is upregulated by oxidative stress and obesity. One of its functions is to increase lipoprotein production by the liver [44]^[34]. Heparan-binding EGF is produced in a variety of cells and organs as a transmembrane-bound molecule that can be released by protease cleavage. In adipose tissues, it is produced by adipocytes and pro-inflammatory macrophages [41,45]^[31][35]. It circulates and concentrates in the vasculature, particularly in the liver, where it increases lipoprotein production. It also activates the EGFR and ERB4 and induces the production of oxidants. Importantly, it has been shown to be upregulated in obesity [44]^[34]. Adiponectin is produced preferentially by subcutaneous adipocytes and is released into the circulation of lean versus obese individuals, where it promotes insulin sensitivity [46,47]^[36][37]. Both pro- and anti-angiogenic properties have been ascribed to this factor ^[6]. This adipokine does not possess an HB-binding domain, but it aggregates with PDGF-BB that does associate with HS [48]^[38]. This association indicates that it tends to concentrate in subendothelial regions where it may interfere with the activities of PDGF. Leptin, a product of adipocytes, is released into the circulation, where it travels to the hypothalamus to regulate satiety ^[3]. It is more highly produced in obese individuals than in lean individuals. Leptin also plays a role in adipose tissues to promote the formation of fenestrated microvessels. In this regard, it acts in a similar manner to VEGF-A, which at high concentrations also promotes leaky vessels ^[5]. Adipose tissues are highly vascularized, and this feature is modulated as these tissues expand or contract. Adipocytes produce angiogenic factors such as VEGF-A₁₆₅, HGF, PDGF-BB, and ANGPL-4 play roles in vascular expansion or contraction [6,42]^[6][32]. All these molecules contain HB-binding domains. As such, these and other HB-binding molecules become concentrated and protected by their presence in BMs. BMs release these molecules gradually, creating gradients [49]^[39] that are necessary for development events such as angiogenesis. Perlecan has been shown to regulate neo-angiogenesis by modifying the concentration of VEGF and its interaction with the VEGF receptor-2 on vascular endothelial cells [50]^[40]. The controlled release of angiogenic factors such as FGF-2 plays a role in adipose tissue engineering [51,52]^[41][42]. These molecules are also released from adipose tissues and can also be considered adipokines ^[4]. HSPGs in BMs play critical roles in the sequestration, concentration, and protection of heparan-binding factors [25]^[15]. Furthermore, these structures regulate the diffusion of these molecules, thus creating gradients important for cellular communication and guidance in migration. One area where diffusion gradients are critical is angiogenesis. Molecules like VEGF-a, PDGF, and HGF play critical roles [50]^[40]. Adipose tissues undergo constant modification. Both pro- and anti-angiogenic factors play roles in these processes ^[6].

4. In Vitro Adipogenesis

Cell culture has been extensively employed to study adipocyte development. Early studies concentrated on the use of murine 3T3-L1 cells [54]^[43]. However, human adipose stem cells (ASCs) and mesenchymal stem cells (MSCs) have more recently been shown to develop as adipocytes following induction [16]^[44]. With the identification and isolation of human adipose stem cells, it has become possible to generate more complex organ-like cultures. All of these populations, when cultured in an adipocyte induction medium, undergo a differentiation profile very similar to that seen in vivo. Laminin is an early secreted product and is followed by other BM molecules [15,16,55]^[44][45][46]. Each adipocyte in the culture is surrounded by a BM upon completion of the induction process. Induced cells acquire the CD146 antigen early upon induction. This provides a marker to separate responsive versus non-responsive cells.