Adipose tissue anatomy
Adipocytes are organized in a multi-depot organ called adipose tissue. Only one third of adipose tissue contains mature adipocytes. Combination of small blood vessels, nerve tissue, fibroblasts, and adipocyte precursor cells, known as preadipocytes, comprises the remaining two thirds. Mature adipocytes exist as two types, white and brown adipocytes. White and brown adipocytes are distinguished by differences in their color and function. White adipose tissue (WAT), which is yellow or ivory, contains predominantly white adipocytes; brown adipose tissue (BAT), which appears brown, contains predominantly brown adipocytes (Geloen et al., 1990).
Brown and white adipocytes are histologically distinct. Lipids within white adipocytes are organized within one large, ‘‘unilocular’’ droplet, the size of which can exceed 50 mm. White adipocytes are spherical, thereby allowing for maximal volume within minimal space. Its diameter is variable, ranging between 30 and 70 mm according to depot site. The lipid droplet occupies the majority of intracellular space, compressing the cytoplasm and nucleus into a thin visible rim. Lipids within brown adipocytes are organized into multiple smaller ‘‘multilocular’’ droplets. Brown adipocytes are additionally characterized by their high content of large mitochondria packed with cristae within the cytoplasm. The cells are polygonal, have centrally placed nuclei, and are relatively smaller than white adipocytes, ranging from 20 to 40 mm (Alison et al., 2005).
Collections of white adipocytes comprise fat lobules, each of which is supplied by an arteriole and surrounded by connective tissue septae. An individual adipocyte is supplied by an adjacent capillary and is additionally associated with a glycoprotein layer as well as reticular fibrils, fibroblasts, mastocytes, and macrophages. Compared with WAT, BAT contains a richer vascular tree, dense with multiple capillaries.The extensive vascularization of the tissue in combination with densely packed mitochondria at the ultrastructural level account for its ‘‘brown’’ color (Cinti, 2005).
WAT and BAT are both innervated by the noradrenergic fibers from the sympathetic nervous system. Noradrenergic fibers in WAT are mostly confined to the capillary wall, whereas in BAT they directly interface the plasma membrane of brown adipocytes via ‘‘incidental’’ synapses (Bartness and Bamshad ,1998).
WAT can be found in several anatomically distinct and separate collections, or ‘‘depots.’’ There are two major anatomic subdivisions, each with unique anatomic, metabolic, endocrine, paracrine, and autocrine properties: intra- abdominal or visceral adipose tissue and subcutaneous adipose tissue .The subcutaneous adipose tissue is divided into two distinct layers: the superficial subcutaneous adipose tissue and deep subcutaneous adipose tissue. Visceral fat is subdivided into intraperitoneal and retroperitoneal compartments. Intraperitoneal fat, itself composed of omental and mesenteric adipose tissue, comprises the majority of visceral fat (Smith et al., 2001).
White adipose tissue, composed of adipocytes with a single large lipid inclusion and a large peripherally-located nucleus, represents the predominant type of fat in humans. It is involved in a variety of physiological roles including the storage of energy-rich triglycerides, cushioning of vital structures and organs, metabolic homeostasis, immune regulation, reproduction, and angiogenesis. The imbalance of adipose tissue results in either too much fat, such as in generalized obesity, or too little fat, such as in genetic or acquired lipodystrophies and aging (Haque and Garg, 2004).
Types of adipose tissue. Depots of white adipose tissue are found in areas all over the body, with subcutaneous and intra-abdominal depots representing the main compartments for fat storage. Brown adipose tissue is abundant at birth and still present in adulthood but to a lesser extent (Modified from Gesta et al., 2007).
Histology of the subcutaneous fat
Subcutaneous fat is organized into lobules of fat cells (lipocytes or adipocytes), and the lobules are separated by thin septa of connective tissue. The thickness of the subcutaneous fat varies from one part of the body to another, with a thinner sub cutis in areas of lax skin, such as the eyelids and scrotum, and thicker in the hips and buttocks. There are also gender differences in the deposition of fat, and an increased thickness of the sub cutis results in the rounded contours of the female body (Querleux et al., 2002).
Adipocytes are large, with a diameter up to 100 µm and, with hematoxylin– eosin staining, appear as empty cells with signet-ring morphology. This appearance is attributable to the fact that the flat spindle nucleus is displaced at the periphery of the cell by a single, large intracytoplasmic vacuole, which contains fat. The septa that divide the subcutaneous fat into lobules are thin and are composed of collagen and reticulin fibers. These septa house the blood and lymphatic vessels as well as the nerves. Arteries and veins of the subcutis run along the septa (Patel et al., 2002).
Each individual lobule is supplied by an arteriole branching from the septa to form capillaries into the lobule, and a capillary network surrounds each individual adipocyte. Postcapillary venules meet in veins that also run along the septa. In each microlobule, the arteriole occupies a central position, whereas the venule runs along the periphery. In contrast with the dermal vascularization, the blood supply of each subcutaneous microlobule is terminal, meaning there are no capillary connections between adjacent microlobules or between dermis and subcutaneous fat. This peculiar structure of the blood supply in subcutaneous fat explains why large vessel vasculitis involving the septal vessels usually is accompanied by little inflammation of the fat lobules, whereas when the vasculitis involves small blood vessels, there is extensive necrosis of the adipocytes with centrilobular infarct and dense inflammatory infiltrate within the lobule. The septa of the subcutaneous fat also contain a rich lymphatic plexus, which come from the dermis and transverse the subcutis, first, parallel to the surface of the skin and then vertically penetrating the deep fascia and draining into the regional lymph nodes (Karpe et al., 2002).
Necrosis of the adipocytes
In classic histopathology, changes in nuclei, namely, pyknosis, karyorrhesis, and karyolysis, are signs of cellular necrosis. In contrast, necrotic adipocytes, regardless of cause, may appear as either anucleated cells or with complete disintegration of the cellular structure. Sometimes the absence of nuclei is the only sign of necrosis of the adipocytes, and the fat cells appear as round empty bugs with no inflammatory infiltrate among them (White et al., 1996).
The most common type of fat necrosis is the so-called lipophagic necrosis, which consists of foamy macrophages laden with the lipid products released from dead adipocytes. These lipophages often exhibit a large, pale microvacuolated or granular cytoplasm. Liquefactive fat necrosis is another type of necrosis of adipocytes that produces granular wisps of amphophilic detritus. Hyalinizing fat necrosis results in mummified adipocytes surrounded by glassy homogeneous proteinaceous material that effaces their archi- tecture .Membranous fat necrosis is a late-stage necrosis of adipocytes that appears as a leathery eosinophilic or amphophilic rim of collapsed cellular organelles (Requena and Sánchez, 2001).
When membranous fat necrosis is extensive, fat microcysts devoid of cell structures appear. Ischemic fat necrosis is mostly seen at the center of the involved lobules and at first the changes are subtle. However, in fully developed lesions, the involved adipocytes at the center of the lobule appear as anucleated cells of a smaller size than normal adipocytes. Later stages of ischemic necrosis are also characterized by lipophagic necrosis. Enzymatic fat necrosis is caused by saponification of the adipocyte lipid contents by pancreatic lipase, with secondary calcium salts deposition, resulting in ghost adipocytes, which show no nuclei and granular basophilic cytoplasm (Requena and Sánchez, 2001).
WHITE ADIPOSE TISSUE (WAT)
The physiology of WAT can be grouped into 3 main categories with potentially overlapping mechanisms: lipid metabolism, glucose metabolism, and endocrine functions.
Regulation of lipid metabolism in adipocytes is controlled by 3 basic cellular functions: fatty acid (FA) uptake, lipogenesis (TG synthesis), and lipolysis (TG hydrolysis). Each of these metabolic processes can be altered in response to extracellular stimuli such as insulin, cortisol, catecholamines, growth hormone, testosterone, FFAs, and cytokines. FAs used for storage in adipocytes are derived predominantly via uptake of plasma FAs and to a lesser extent from de novo FA synthesis within the cytosol. The circulatory FA pool consists of FAs from nutrient intake as well as FAs released from adipocytes undergoing lipolysis. Uptake of FAs is facilitated by the extracellular activity of lipoprotein lipase, which varies according to nutritional and endocrine status (Ramsay, 1996).
Functions of the adipocyte.
Adipocytes utilize glucose for TG synthesis via Glut4, a transmembrane transport protein produced by fat cells. Endocrine functions include production and secretion of peptides, including leptin, AGT, PAI-1, TNF-a, adiponectin, and adipsin. AGT, angiotensinogen; ATP, adenosine triphosphate; FFA, free fatty acid; Glut4, insulindependent glucose transporter; IL-6, interleukin 6; PAI-1, plasminogen activator inhibitor type 1; TG, triacylglycerol; TNF-a, tumor necrosis factor-a; VLDL, very-low-density lipoprotein (Modified from Morrison RF, Farmer SR. J Nutr 2000:130; 3116-21).
After a meal, adipose tissue buffers digested nutrients in the form of fatty acids (FAs) stored as triacylglycerol (TG). TG stored within adipose tissue provides a reservoir of energy stores that can be utilized during times of restrictive caloric intake. TG consists of 3 molecules of FA esterified to a molecule of glycerol. The nature of hydrophobic FAs, rich with high caloric density, makes these moieties highly efficient molecules for packaging within adipose cells. The adipocyte is able to store vast amounts of TGs within intracellular storage droplets that can exceed 50 mm in diameter. During the fasted state, TGs are broken down (hydrolyzed) into free fatty acids (FFAs) and glycerol (Morrison and Farmer, 2000).
Overview of FA uptake, lipogenesis, and lipolysis.
AC, Adenylate cyclase; ACS. acyl-coenzyme A synthetase; 59-AMP, 59-adenosine monophosphate; aP2, adipocyte fatty acid binding protein; α2-AR, α 2-adrenoreceptor; ATP, adenosine triphosphate; b-AR, b-adrenoreceptor; CO2, carbon dioxide; FATP, fatty acid transporter protein; FFA, free fatty acid; GI, inhibitory G protein; GS, stimulatory G protein; Glut4, insulin-sensitive glucose transporter; glycerol 3-P, glycerol 3-phosphate; IR, insulin receptor; HSL, hormone sensitive lipase; LPL, lipoprotein lipase; p, phosphorylation; PDE3B, cAMP phosphodiesterase 3B; PKA, protein kinase C (Modified from; Morrison RF, Farmer SR. J Nutr 2000:130;3116-21 ).
Once FAs released into the circulation, serve as fuel for metabolically active tissues via FA oxidation and the release of adenosine triphosphate (ATP), the body’s main energy source. Rates of FA oxidation are determined by the need for energy in the form of ATP. Glycerol generated from FA hydrolysis is converted via glycerol kinase in the liver to the glycerol moiety needed for TG synthesis. TGs are then packaged within very-low-density lipoproteins and released into circulation (Van Dijk, 2001).
Adipocytes contribute to glucose metabolism in several ways.First, adipose tissue, in conjunction with heart and skeletal muscle, are the only known tissues to express and regulate the insulin-dependent glucose transporter, a peptide that mediates the postprandial transport of glucose from the circulation into cells for utilization and storage. Within adipocytes, glucose provides the substrate for de novo FA and glycerol synthesis (lipogenesis) via the glycolytic pathway. Second, the metabolic activity of adipocytes greatly influences glucose metabolism within other tissues. Increased lipolysis of TGs in adipocytes generates increased levels of circulatory FFAs. In the liver, FFAs have a negative regulatory role on several functions, including hepatic clearance of insulin and insulin-mediated suppression of hepatic glucose production, while at the same time exerting a positive effect on the rate of gluconeogenesis (generation of glucose from glycerol, lactate, and amino acids). The net effect of FFAs in the liver, therefore, results in the promotion of increased plasma glucose concentrations (hyperglycemia). FFAs also have a negative regulatory role on insulin sensitivity within skeletal muscle, where they induce decreased insulin-stimulated glucose utilization when produced in excess and thereby promote insulin resistance (Wajchenberg, 2000).
The adipocyte, an important source for many physiologically active peptides, influences a multitude of organ systems, including adipose tissue, via endocrine, paracrine, and autocrine signals. Although most of the factors secreted from adipose tissue act in an autocrine/paracrine manner to regulate adipocyte metabolism, several are released into the bloodstream where they are carried to multiple distant sites for endocrine action. Some of these secreted factors share structural properties with cytokines and are, therefore, referred to collectively as ‘‘adipocytokines’’ (Trayhurn and Beattie, 2001).
AGT, Angiotensinogen; ALBP/aP2, adipocyte lipid-binding protein; α2-AR, α 2-adrenoreceptor; β-AR, β-adrenoreceptor; ASP, acylationstimulated protein; FA, fatty acid; FFA, free fatty acid; Glut4, insulin-dependent glucose transporter; IL-6, interleukin 6; LPL, lipoprotein lipase; PAI-1, plasminogen activator inhibitor type 1; PGE2, prostaglandin E2; PGI2, prostacyclin; TG, triacylglycerol; TNF-a, tumor necrosis factor-a; VLDL, very-low-density lipoprotein (Modified from Wajchenberg BL. Endocrine Rev 2000;21:697-738).
Leptin and adiponectin, which are increased and decreased in obesity, respectively, function by regulating appetite and energy expenditure. Tumor necrosis factor–alpha (TNF-α) and interleukins-8 (IL-8) and -6 (IL-6) are all increased in obesity and are proinflammatory cytokines that promote insulin resistance and lipolysis. In addition, type 1 plasminogen activator inhibitor (PAI-1) is increased in obesity and functions to promote thrombosis by inhibiting fibrinolysis, thus acting as the main endogenous regulator of fibrinolysis (Sethi and Vidal-Puig,2007).
Adipocyte heterogeneity among different WAT depots
The distribution of WAT not only varies considerably between species but also between individuals of the same species. There are two theories about why these different fat distributions are differentially linked to metabolic complications. The first is based on anatomy and the fact that visceral fat drains its products (free fatty acids and various adipokines) into the portal circulation where they can act preferentially on the liver to affect metabolism .The second is based on the concept that fat cells in different depots have different properties causing them to be linked to a greater or lesser extent to the development of metabolic disorders. There are significant differences in expression of hundreds of genes between different depots of adipose tissue. These differences may also suggest possible differences in developmental origin of these fat cells (Vohl et al., 2004).
Differential origin of adipocytes in different WAT depots is also suggested by striking differences in adipose tissue distribution among normal individuals and especially in individuals with various forms of lipodystrophy. For example, in congenital generalized lipodystrophy, adipose tissue is almost completely absent from subcutaneous depots, intra-abdominal depots, intrathoracic regions, and bone marrow. However, these individuals still have a relatively normal amount of adipose tissue in the retro-orbital area, buccal region, palms and soles, and other areas. By contrast, familial partial lipodystrophy of the Dunnigan type is characterized by a marked loss of subcutaneous adipose tissue in the extremities and trunk but no loss of visceral, neck, or facial adipose tissue. Some lipodystrophies appear to have a segmental or dermatomal distribution (Agarwal and Garg, 2006).
Various WAT depots may be derived from distinct precursors is supported by a number of lines of evidence. First, different WAT depots have variations in chronology of appearance. In rodents, WAT develops mainly after birth, being present first in the perigonadal and subcutaneous depots, and only later in the omental depot. In humans, WAT development begins early in the second trimester of gestation and by birth is well developed in both the visceral and subcutaneous depots. Second, variations in gene expression have been observed in preadipocyte fractions from different adipose depots. Likewise, administration of monoclonal antibodies raised against adipocyte plasma membranes to chick embryos significantly reduces the weight of abdominal adipose tissue without affecting femoral or pectoral fat depots, suggesting that these depots express different membrane protein antigens than those of other depots (Gesta et al., 2006).
BROWN ADIPOSE TISSUE (BAT)
Development of brown adipocytes begins at the 20th week of pregnancy and continues until shortly after birth, at which time BAT comprises 1% of body weight. In neonates and newborn children, BAT can be found in several areas, including the interscapular region, surrounding blood vessels, muscles in the neck, in the axillae, along the great vessels, trachea, esophagus at the thoracic inlet, and around the abdominal aorta, pancreas, adrenal glands, and kidneys (Cannon and Nedergaard, 2004).
White adipocytes, which are the primary site of triglyceride/energy storage, but also brown adipocytes, which are important in both basal and inducible energy expenditure in the form of thermogenesis . This occurs through expression of uncoupling protein-1 (UCP-1), a 32 kDa protein found in the inner mitochondrial membrane, that allows dissipation of the proton electrochemical gradient generated by respiration in the form of heat (Cannon and Nedergaard, 2004).
In small mammals, such as rodents, BAT persists throughout its lifespan. In large mammals and humans, however, brown adipocytes from BAT depots undergo a morphologic transformation in which they rapidly accumulate lipids, become unilocular, and lose the ultrastructural and molecular properties that define them, including mitochondria. As a consequence, there are no discrete collections of BAT that can be found in the adult (Himms-Hagen, 2001).
BAT and WAT both express many of the same adipocyte-specific genes needed for lipid synthesis and hydrolysis.BAT has emerged as an independent organ with specific protein expression patterns and unique purpose .The mitochondrial protein UCP-1 (or thermogenin), which is expressed exclusively in BAT, is responsible for mediating the basic function of brown fat cells, namely the transfer of energy from food into heat. UCP-1 activity, triggered in response to adrenergic signaling via the sympathetic nervous system, is up-regulated human neonates whenever extra heat is needed, such as during episodes of cold exposure, during the postnatal period, entry into a febrile state (Commins et al.,1999).
Physiologically, the heat produced by BAT and the resulting decrease in metabolic efficiency has significant impact. BAT not only provides an important adaptive mechanism for acute body temperature regulation, but also functions as protection against obesity (Sell et al., 2004).
Although it is unclear to what extent BAT might play a role in energy balance in adult humans, it was estimated that as little as 50 g of BAT could account for 20% of daily energy expenditure, if maximally stimulated. Furthermore, up to 24% of the increase in metabolism in lean men produced by ephedrine has been attributed to BAT. It has been suggested that the age-related decline in thermogenesis and regulatory energy expenditure in humans and rodents is associated with a reduction in the amount of functional BAT (Almind et al., 2007).
BAT and obesity
In human fetuses and newborns, BAT is found in axillary, cervical, perirenal, and periadrenal regions but decreases shortly after birth and has traditionally been considered insignificant in adults, except perhaps in patients with pheochromocytoma, where adrenergic activity is extremely high , or in outdoor workers in northern climes subject to prolonged cold exposure . However, morphological and scanning studies have shown that brown fat in humans may not be as rare as once believed. Areas of metabolically active brown fat can be detected in the cervical, supraclavicular, axillary, and paravertebral regions of normal individuals. UCP-1 mRNA can be detected in human WAT and is further induced by the antidiabetic drugs thiazolidinediones , suggesting some admixture of BAT in WAT depots (Nedergaard et al.,2007).
There is good evidence for functional BAT in human newborns, who are able to double their resting energy expenditure without shivering when transferred from a thermoneutral temperature (34°C) to a mildly cold environment (28°C). The rapid initiation of adaptive nonshivering thermogenesis in infants born with a mature hypothalamic-pituitary axis at birth is vital for enabling the newborn to maintain body temperature following exposure to cold in the extrauterine environment. Newborns possess large depots of BAT and use approximately 50% of their intake for cold-induced nonshivering thermogenesis under these conditions (Himms-Hagen and Ricquier, 1998).
The relative functional capacity of BAT, however, appears to decrease with age and increasing size, as evidenced by the rapid loss of UCP-1 mRNA soon after birth, followed by a gradual loss of UCP-1 protein. This phenomenon has been ascribed to several attributes of adult humans: a relatively higher ratio between heat production from basal metabolism and smaller surface area in adults compared with newborns as well as the utilization of clothing and indoor life for protection from cold (Heasman et al.,2000).
It has been assumed that healthy young adults are devoid of functional BAT, a theory that is currently being revised. Small islands of cells expressing UCP-1 mRNA from adult humans have been detected within adipose tissue depots previously thought to contain white adipocytes only. Calculations based on DNA measurements from intraperitoneal adipose tissue samples approximate that 1 in 100 to 200 adipocytes from WAT are, in fact, brown. These brown fat cells are evenly interspersed among their white counterparts within white adipose depots. Humans with a polymorphism in the promoter region of the UCP-1 gene demonstrate greater weight gain, lower weight loss under a low-calorie diet, and other metabolic features of the obese phenotype, including hypercholesterolemia, hypertriglyceridemia, and hyperglycemia Likewise, β3-AR polymorphism has been associated with impaired lipolytic function in WAT, a lower resting metabolic rate, as well as a tendency to gain weight in those with obesity. A synergistic effect in individuals with combined polymorphisms of UCP-1 and β3-AR, as evidenced by a higher risk for weight gain and a significantly lower basal metabolic rate, suggests a link between energy balance, obesity, and BAT in humans (Ramis et al., 2004).
In rodents, targeted ablation of BAT results in diet induced obesity, diabetes, and hyperlipidemia. UCP-1-deficient mice also exhibit increased susceptibility to age- and diet-related obesity. Depots of UCP-1-positive brown adipocytes have been identified, interspersed between skeletal muscle bundles in the legs of an obesity-resistant strain of mice, suggesting that “ectopic brown adipocytes” may play an important role in the regulation of whole-body energy homeostasis .The potential of inducing even small amounts of brown fat in adult humans could provide a new approach to the treatment and/or prevention of obesity and its metabolic complications (Almind et al., 2007).
The origin of adipose tissue
Adipose tissue is regarded as having a mesodermal origin.The formation of the mesoderm begins with the migration of a layer of cells between the primitive endoderm and ectoderm. This layer spreads along the anteroposterior and dorsoventral axes of the developing embryo giving rise to the axial, intermediate, lateral plate, and paraxial mesoderm. The paraxial mesoderm, after its segmentation into somites, gives rise to the axial skeleton and muscles of the trunk. The lateral plate mesoderm generates the skeleton and muscles of the limbs. The bones and muscles of the skull and face appear to be of ectodermal origin, specifically the neural crest (Bronner-Fraser, 1994).
Neural crest stem cells have been reported to be able to differentiate into adipocytes in culture .Each of these regions are presumed to give rise to local adipose tissue. Such a developmental origin is consistent with the occurrence of various forms of partial lipodystrophy and with the differential expression of genes involved in development and patterning between different fat depots. The interscapular brown fat is derived from the paraxial mesoderm (Atit et al., 2006).
Mesenchymal stem cells (MSCs) were initially identified in postnatal human bone marrow and have been used to model differentiating mesoderm. MSCs are capable of differentiating into adipocytes, osteoblasts, chondrocytes, myoblasts, and connective tissue. MSC gives rise to a common early precursor (adipoblast), which in turn develops into committed white and brown preadipocytes that under appropriate stimulatory conditions differentiate into mature adipocytes of different types. It is not clear if separate adipoblasts and/or preadipocytes for brown and white fat exist or if there are different white preadipocytes for different white adipose depots (Billon et al., 2007).
The transition from preadipocyte to adipocyte involves four stages: growth arrest, clonal expansion, early differentiation, and terminal differentiation. These stages are orchestrated by a transcriptional cascade involving the nuclear receptor PPARγ and members of the C/EBPs family. Two isoforms of PPARγ (PPARγ1 and PPARγ2) are generated by alternative splicing and promoter usage of the PPARγ gene. Although both are expressed in adipocytes, PPARγ2 has been regarded as a specific marker of fat. PPARγ1 can compensate for loss of PPARγ2 (Farmer, 2006).
The trans-differentiation of white adipocytes into brown can be induced under certain conditions, thereby refuting the notion that stem cell commitment to and differentiation into the white or brown cell lineage is permanent (Hansen et al.,2004).