Development of Mesenchymal/ Mesodermal derivatives

Preadipocytes, fibroblast-like cells present in the stromalvascular fraction of adipose tissue, can differentiate to form mature adipocytes and this capacity is present throughout life. The signal for differentiation of new adipocytes is related to nutritional state. Important stimuli for differentiation include insulin and fatty acids. Fatty acids act through members of the peroxisome proliferator-activated receptor (PPAR) family, PPARδ (also known as fatty acid-activated receptor, FAAR38) and particularly PPARγ. The natural ligand for PPARγ is probably a fatty acid derivative (Rosen and Spiegelman, 2000).

The only widely accepted marker of preadipocytes is preadipocyte factor 1 (Pref-1; also known as DLK-1 or Drosophila Homolog-like 1). Pref-1 is expressed at high levels in both white and brown preadipocytes, and expression markedly decreases upon differentiation.Pref-1 is synthesized as a transmembrane protein and is cleaved to generate a soluble 50 kDa form that acts to inhibit adipocyte differentiation .Pref-1, however, is not unique to the preadipocyte and is also expressed in placenta, pituitary, adrenal cortex, fetal liver, and pancreatic islet cells (Villena et al., 2002).

Other putative preadipocyte markers are the type VI collagen alpha 2 chain (COL6A2) and a secretory protein related to the Wnt antagonist Frzb, namely FRP2/SFRP2 The latter is particularly abundant in subcutaneous fat .Like Pref-1, both are more highly expressed in undifferentiated preadipocytes and reduced in mature adipocytes. However, neither is adipose tissue specific (Gesta et al., 2006).

Mature adipocytes in WAT and BAT are marked by the presence of PPARγ2, markers of terminal differentiation (such as Glut4 and fatty-acid synthase), and insulin-regulated glucose uptake and metabolism. In addition, WAT is characterized by the presence of leptin, whereas BAT is distinguished by the existence of UCP-1. However, white or brown preadipocytes are indistinguishable from any cell type with a fibroblast-like morphology, making them difficult to identify and study (Rosen and MacDougald, 2006).
Site-specific properties of adipose tissue depots

Upper-body obesity was associated with a much greater incidence of adverse consequences (largely related to insulin resistance and dyslipidaemia) than was lower-body obesity. Intra-abdominal ‘visceral’ adipocytes have the highest metabolic activity followed by upper-body subcutaneous adipocytes, and lowest response in lower-body adipocytes. Metabolic activity refers mainly to lipolysis: intra-abdominal adipocytes have the highest rates of lipolysis when stimulated with a β-adrenoceptor agonist, and lipolysis is least susceptible to suppression by insulin. It could be simply argued that upper-body adipocytes discharge fatty acids at a high rate, and interfere with insulin-sensitive glucose metabolism. Lower-body fat depots may be efficient at removing fat from the circulation and they are relatively resistant to loss during weight loss. Their predominance in women may imply that these are long-term fat reserves to cover eventualities such as child-bearing and nurturing if food supply runs short (Jones and Edwards,1999).

Functional differences between visceral and the subcutaneous adipocytes are related to their anatomical location. For example, implantation of adipose cells into visceral area of nude mice increased serum TNF-α and insulin resistance, while it did not increase them when it was implanted in the subcutaneous regions (Shibasaki et al., 2002).

The metabolic characteristics of the adipocytes from the subcutaneous abdominal region tend to be intermediate. In vivo data also supports these findings. Abdominal fat may directly impact hepatic free fatty acid flux due to its proximity to the portal circulation and consequently increases triglycerides synthesis and decrease hepatic insulin clearance. It was noticed that there is a marked heterogeneity in handling FFA by various fat depots. Other contributing mechanisms include abnormal expression and secretion of fat derived cytokines such as resistin, leptin, adiponectin, TNF-α and IL-6 (Shibasaki et al., 2002).

There are several loci determining tendency to store fat in the abdominal region. Differences in several gene expressions in visceral fat in comparison to subcutaneous fat may account for the differences in the metabolic risks between the two fat depots. Many of these genes are involved in glucose homeostasis, insulin action, or in lipid metabolism (Gabriely and Barzilai, 2003).

Twenty genes, which are mostly related to lipid metabolism and glucose homeostasis, are markedly different between the 2 types of fat. For examples angiotensinogen gene is expressed five folds higher in visceral fat compared to subcutaneous fat and PPARγ is six folds higher in visceral fat in comparison to subcutaneous fat. Similarly, the expression of resistin and adiponectin genes is also significantly higher in visceral fat (3.8 and 12.2-fold, respectively) (Gabriely and Barzilai, 2003).
Metabolic characteristics of visceral & subcutaneous fat
The uptake of fat is regulated by the enzyme lipoprotein lipase (LPL). This enzyme hydrolyzes triacylglycerols into free fatty acids, which can then be transported into the adipocyte and reesterified for storage. Greater LPL activity is associated with greater accumulation of fat. In premenopausal women, its activity is higher in the gluteal–femoral adipose areas than in the abdominal areas. The opposite is true in men, in whom LPL activity is the same or higher in the abdominal adipose areas than in the gluteal–femoral regions. Obesity related chronic diseases are associated with the location, as well as the amount, of adipose tissue on the body. Although the relative importance of total adiposity versus type of adiposity continues to be debated, the notion that an ‘apple-shaped’ (or android) body is associated with greater obesity-related health risks than a ‘pear-shaped’ (or gynoid) body is well accepted (Stevens and Truesdale, 2005).
The breakdown of fat (lipolysis) is regulated by the enzyme hormone-sensitive lipase (HSL). This enzyme releases free fatty acids, which are then released into the bloodstream and taken up by tissues, with the exception of the brain and red blood cells, for energy use or storage. The rate of basal lipolysis is higher in gluteal–femoral fat tissue than in abdominal tissue in both men and women. This may be due to greater cell size in that region. In the abdominal area, basal lipolysis is higher in subcutaneous fat than in visceral fat. However, when stimulated hormonally, rates of lipolysis may differ between men and women. Lipolytic rates have been shown to be higher in the visceral compared to the subcutaneous region in men, whereas the opposite trend is seen in women (Stevens and Truesdale, 2005).
Effects of selective removal of visceral or subcutaneous fat

Surgical removal of visceral fat in experimental animals reversed hepatic insulin resistance. It also prevented age related deterioration in peripheral and hepatic insulin action. Meanwhile it decreased gene expression of TNF-α and leptin in subcutaneous adipose tissue. Furthermore, removal of visceral fat delayed the onset of diabetes in the Zucker fatty rats, the model of obesity and diabetes (Gabriely et al., 2002).

There is a striking sex difference in body fat distribution between men and women .The regional distribution of body fat is a characteristic of masculinity and femininity. In premenopausal women a larger proportion of fat is stored in peripheral fat depots such as the breasts, hips, and thighs. Men tend to accumulate adipose tissue in the abdomen (android fat distribution) while women tend to accumulate fat in the gluteal-femoral region (gynoid fat distribution). In men, abdominal adipose tissue tends to accumulate in the visceral area to a greater extent than in women, and for a similar fat mass, men have on average a two-fold higher visceral adipose tissue accumulation compared to women (Kuk et al., 2005).

Regional localisation of body fat is considered to be a secondary sex characteristic; it is likely that sex steroids are involved in the male and female patterns of fat deposition. This view is strengthened by the observation that variations in sex steroid levels in different phases of (reproductive) life parallel the regional differences in fat storage and fat mobilization. Until puberty, boys and girls do not differ very much in the amount of body fat and its regional distribution. From puberty onwards , differences become manifest. The ovarian production of estrogens and progesterone induce an increase in total body fat as well as selective fat deposition in the breast and gluteo-femoral region. Pubertal boys show a strong increase in fat free mass, while the amount of total body fat does not change very much. Adolescent boys lose subcutaneous fat but accumulate fat in the abdominal region, which in most boys is not very visible at that stage of development but is clearly demonstrable using imaging techniques (Kuk et al., 2005).
The sex steroid-induced regional distribution is not an all-or-nothing mechanism; it is a preferential accumulation of excess fat. Obese men and women still show their sex-specific fat accumulation but also store their fat in the ‘fat depots of the other sex’. Not only does the fat distribution differ between the sexes from puberty onwards, but the dynamics of fat cell size and fat metabolism are also different. The amount of fat in a certain depot is dependent on the number and size of the fat cells. Fat cells in the gluteal and femoral regions are larger than those in the abdominal region (Blouin et al., 2008).
The activity of lipoprotein lipase, the enzyme responsible for the accumulation of triglycerides in the fat cell, is higher in the gluteo-femoral region than in the abdominal area. Conversely, lipolysis is regulated by hormone-sensitive lipase, which, in turn, is regulated by several hormones and by the sympathetic nervous system. Catecholamines stimulate lipolysis via the β adrenergic receptor, while α2-adrenoreceptors inhibit lipolysis. Hormones affect the catecholamine receptors of the adipocytes.Testosterone stimulates the β-adrenergic receptor while estrogens/progesterone preferentially stimulate the α2-adrenoreceptors. Insulin stimulates fat accumulation. The visceral fat depot constitutes a quickly available source of calories and energy. By its close anatomical proximity to the liver it delivers fatty acids through the portal system (Blouin et al., 2008).
Dynamics of adipocyte gain and loss

Beyond two scheduled periods of growth, the first occurring from the first quarter of intrauterine life to 18 months of life and the second during puberty, WAT is unique in its potential for enormous volume change. The adipose tissue mass in lean adult humans is 9% to 18% of body weight in men and 14% to 28% in women. The fat mass can increase up to 4-fold in massively obese persons, reaching 60% to 70% of total body weight (Hausman et al., 2001).

(Modified from Kirkland JL Tchkonia T, Pirtskhalava T, Han J, Karagiannides I. Exp

Gerontol 2002; 37:757-67)
Brown adipocytes are observed in classical white fat depots, and their number increases dramatically during cold adaptation or after hormonal and/or pharmacological treatments, such as β3 adrenoceptor agonist, retinoic acid, thiazolidinediones, and leptin administration. Although these findings may represent transformation of white adipose to brown and vice versa, it seems more likely that the UCP-1-expressing brown adipocytes observed in WAT under these conditions come from recruitment and differentiation of mesenchymal progenitor cells or brown preadipocytes within the WAT. Indeed, the stromovascular fraction is a heterogeneous cell population and appears to contain pluripotent cells capable of differentiating into multiple cell lineages, including cells of mesodermal lineage (adipocytes, osteoblasts, chondrocytes, myocytes), cells of endodermal lineage (endothelial cells and hepatocytes), and cells of neuroectodermal lineage (neuronal cells and insulin- producing cells) (Fraser et al., 2006).
Apoptosis of the adipocytes

Two mechanisms for adipocyte loss have been described, apoptosis and adipocyte dedifferentiation. Adipocyte dedifferentiation, the process by which terminally differentiated cells revert morphologically and biochemically to a less differentiated precursor cell type, has been demonstrated. Fat cells that dedifferentiate in culture demonstrate a restoration of their ability to replicate. Adipocytes in culture from morbidly obese patients compared with those from lean subjects demonstrate a relative resistance to dedifferentiation, thereby highlighting the propensity of fat cells in obese persons to preserve the differentiated lipid-overfilled state (Fernyhough et al., 2005).

Food restriction alone, however, has not been shown to cause a decrease in adipocyte cell number. There are two documented mechanisms by which apoptosis can be triggered in cells: a mitochondria dependent pathway or via a cell surface death receptor-mediated pathway, both of which lead to the activation of a cascade of proteases, called caspases, followed by cleavage of nuclear and cytoplasmic substrates, DNA fragmentation, and ultimately, removal of the apoptotic cells by phagocytosis. In mitochondria-dependent pathways, control of cell death depends on the balance of proapoptotic and antiapoptotic Bcl-2 regulatory proteins expressed within mitochondria of a given cell population; cells with a greater number of proapoptotic proteins are sensitive to death, whereas cells with a greater number of antiapoptotic or protective proteins are usually resistant (Gupta, 2001).
Additional apoptotic signaling pathways can be initiated specifically and independently by a family of 9 documented death receptors that reside on the surface of cells. The best characterized receptors are CD95 (also called Fas or Apo1) and TNF receptor 1 (also called p55 or CD120a). The insulin-like growth factor I/IGF-I receptor system was found to protect human fat cells from apoptosis by maintaining the expression of antiapoptotic proteins. Inhibition of insulin-like growth factor I dramatically sensitizes human adipocytes to death-receptor mediated apoptosis. Adipocyte apoptosis has been detected endogenously in humans with various pathologic conditions associated with weight loss, such as malignancy and protease inhibitor- associated lipoatrophy in HIV disease (Scaffidi et al., 1998).

Regulators of adipocyte number

The proinflammatory cytokine TNF-α is an important regulator of fat mass. TNF- α limits the development of adipose tissue via multiple mechanisms, including induction of insulin resistance and leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. TNF- α has been shown to initiate apoptosis in both human preadipocytes and mature adipocytes from subcutaneous and visceral adipose tissue (Gasic et al., 1999).

Adipocyte-secreted leptin exerts complex regulatory effects on adipose tissue mass. Leptin’s dual action centrally and peripherally results in metabolic and behavioral changes that increase loss of adipose tissue. Known pathways include suppression of food intake and increased thermogenesis . Leptin acts centrally to trigger a process that results in fat cell apoptosis within the adipose tissue organ (Gullicksen et al., 2003).

The principal substrate for lipolytic enzymes, cellular TG, is stored in cytosolic LDs in essentially all tissues of the body. LDs exhibit a particle core composed of TG and cholesteryl esters and are surrounded by a phospholipid monolayer that contains numerous proteins with structural, regulatory or enzymatic functions (Ducharme and Bickel, 2008).

TG is hydrolyzed in a sequential process involving different lipases. ATGL and HSL are necessary for proper hydrolysis of tri- and diglycerides, respectively. The last step in lipolysis is performed by monoglyceride lipase (MGL), which hydrolyzes monoglycerides to form glycerol and fatty acids. The activity of ATGL and HSL is tightly regulated by hormones (Guilherme et al., 2008).

The classical pathway activates lipolysis via catecholamines and their interaction with β-adrenergic receptors. Β-adrenergic stimulation of the G protein coupled receptor activates adenylate cyclase increasing cellular cAMP levels. cAMP binding to protein kinase A induces the lipolytic breakdown of fat. Alternative signaling pathways involve the 5′-AMP activated protein kinase pathway, the extracellular-signal-regulated kinase pathway (ERK), or growth hormone and cytokine signaling. An additional pathway has been described involving natriuretic peptide, activation of guanyl cyclase, and the cGMP mediating activation of protein kinase G (Lafontan et al., 2008).

The adipocyte regulates how much fat it will store, partly through its own gene products. LPL in the adipose tissue capillaries, itself a product of adipocyte gene expression, generates excess of fatty acids, and the adipocyte takes up and esterifies a proportion of these: the remainder mix with NEFAs released from adipocyte lipolysis. Production of ASP (an adipocyte-derived stimulator of fat storage) represents one way in which the adipocyte may regulate how much fat it takes up. The opposing pathway, lipolysis of adipocyte TG, is again a function of the adipocyte (Frayn et al., 1995).

The integrative nature of adipose tissue physiology is apparent. While individual adipocytes may expand or contract, at some point in the process of fat storage there is a need for more adipocytes. The differentiation of new adipocytes is regulated by many factors in common with the pathway of fat deposition (Smith et al., 2000).