Unilocular And Multilocular Lipids Drops Synonym

Acids see Electrolytes: Acid-Base Balance

ADIPOSE TISSUE

G Frühbeck and J Gomez-Ambrosi, Universidad de

Navarra, Pamplona, Spain

© 2005 Elsevier Ltd. All rights reserved.

Introduction

The role of white adipose tissue (WAT) in storing and releasing lipids for oxidation by skeletal muscle and other tissues became so firmly established decades ago that a persistent lack of interest hindered the study of the extraordinarily dynamic behavior of adipocytes. However, disentangling the neuroendocrine systems that regulate energy homeostasis and adiposity has jumped to a first-priority challenge, with the recognition of obesity as one of the major public health problems. Strictly speaking, obesity is not defined as an excess of body weight but as an increased adipose tissue accretion, to the extent that health may be adversely affected. Therefore, in the last decades, adipose tissue has become the research focus of biomedical scientists for epide-miological, pathophysiological, and molecular reasons. Although the primary role of adipocytes is to store triglycerides during periods of caloric excess and to mobilize this reserve when expenditure exceeds intake, it is now widely recognized that adipose tissue lies at the heart of a complex network that participates in the regulation of a variety of quite diverse biological functions (Figure 1).

Development

Adipose tissue develops extensively in home-otherms with the proportion to body weight varying greatly among species. Adipocytes differentiate from stellate or fusiform precursor cells of mesenchymal origin. There are two processes of adipose tissue formation. In the primary fat formation, which takes place relatively early (in human fetuses the first traces of a fat organ are detectable between the 14th and 16th weeks of prenatal life), gland-like aggregations of epithe-loid precursor cells, called lipoblasts or preadipocytes, are laid down in specific locations and accumulate multiple lipid droplets becoming brown adipocytes. The secondary fat formation takes place later in fetal life (after the 23rd week of gestation) as well as in the early postnatal period, whereby the differentiation of other fusiform precursor cells that accumulate lipid to ultimately coalesce into a single large drop per cell leads to the dissemination of fat depots formed by unilocular white adipocytes in many areas of connective tissue. Adipose tissue may be partitioned by connective tissue septa into lobules. The number of fat lobules remains constant, while in the subsequent developmental phases the lobules continuously increase in size. At the sites of early fat development, a multilo-cular morphology of adipocytes predominates, reflecting the early developmental stage. Microscopic studies have shown that the second trimester may be a critical period for the development of obesity in later life. At the beginning of the third trimester, adipocytes are present in the main fat depots but are still relatively small. During embryonic development it is important to emphasize the temporospacial tight coordination of angiogenesis with the formation of fat cell clusters. At birth, body fat has been reported to

Appetite regulation

Appetite regulation

metabolism

Figure 1 Dynamic view of white adipose tissue based on the pleiotropic effects on quite diverse physiological functions.

metabolism

Figure 1 Dynamic view of white adipose tissue based on the pleiotropic effects on quite diverse physiological functions.

account for approximately 16% of total body weight (with brown fat constituting 2-5%) with an increase in body fat of around 0.7-2.8 kg during the first year of life.

Adipogenesis, i.e., the development of adipose tissue, varies according to sex and age. Furthermore, the existence of sensitive periods for changes in adipose tissue cellularity throughout life has been postulated. In this regard, two peaks of accelerated adipose mass enlargement have been established, namely after birth and between 9 and 13 years of age. The capacity for cell proliferation and differentiation is highest during the first year of life, while it is less pronounced in the years before puberty. Thereafter, the rate of cell proliferation slows down during adolescence and, in weight stable individuals, remains fairly constant throughout adulthood. In case of a maintained positive energy balance adipose mass expansion takes places initially by an enlargement of the existing fat cells. The perpetuation of this situation ends up in severe obesity where the total fat cell number can be easily trebled. Childhood-onset obesity is characterized by a combination of fat cell hyperplasia and hypertrophy, whereas in adult-onset obesity a hypertrophic growth predominates. However, it has been recently shown that adult humans are capable of new adipocyte formation, with fat tissue containing a significant proportion of cells with the ability to undergo differentiation. Interestingly, the hyper-plasic growth of fat cells in adults does not take place until the existing adipocytes reach a critical cell size.

Initially, excess energy storage starts as hyper-trophic obesity resulting from the accumulation of excess lipid in a normal number of unilocular adipose cells. In this case, adipocytes may be four times their normal size. If the positive energy balance is maintained, a hyperplasic or hypercellular obesity characterized by a greater than normal number of cells is developed. Recent observations regarding the occurrence of apoptosis in WAT have changed the traditional belief that acquisition of fat cells is irreversible. The adipose lineage originates from multipotent mesenchymal stem cells that develop into adipoblasts (Figure 2). Commitment of these adipoblasts gives rise to preadipose cells (preadipocytes), which are cells that have expressed early but not late markers and have yet to accumulate triacylglycerol stores (Figure 3). Multipotent stem cells and adipoblasts, which are found during embryonic development, are still present postnatally. The relationship between brown and white fat during development has not been completely solved. Brown adipocytes can be detected among all white fat depots in variable amounts depending on species, localization, and environmental temperature. The transformation of characteristic brown adipocytes into white fat cells can take place rapidly in numerous species and depots during postnatal development.

The morphological and functional changes that take place in the course of adipogenesis represent a shift in transcription factor expression and activity leading from a primitive, multipotent state to a final phenotype characterized by alterations in cell shape and lipid accumulation. Various redundant signaling pathways and transcription factors directly influence fat cell development by converging in the upregula-tion of PPAR7, which embodies a common and essential regulator of adipogenesis as well as of adi-pocyte hypertrophy. Among the broad panoply of transcription factors, C/EBPs and the basic helix-loop-helix family (ADD1/SREBP-1c) also stand out together with their link with the existing nutritional status. The transcriptional repression of adipogen-esis includes both active and passive mechanisms. The former directly interferes with the transcrip-tional machinery, while the latter is based on the binding of negative regulators to yield inactive forms of known activators.

Hormones, cytokines, growth factors, and nutrients influence the dynamic changes related to adipose tissue mass as well as its pattern of distribution (Figure 4). The responsiveness of fat cells to neurohumoral signals may vary according to peculiarities in the adipose lineage stage at the moment of exposure. Moreover, the simultaneous presence of some adipogenic factors at specific threshold concentrations may be a necessary requirement to trigger terminal differentiation.

Figure 2 Schematic diagram of the histogenesis of white and brown adipocytes. C/EBPs, CCAAT/enhancer binding proteins; PGC-1a, peroxisome proliferator-activated receptor-7 coactivator-1; PPAR7, peroxisome proliferator-activated receptor-7.

Structure

Adipose tissue is a special loose connective tissue dominated by adipocytes. The name of these cells is based on the presence of a large lipid droplet with 'adipo' derived from the Latin adeps meaning 'pertaining to fat.' In adipose tissue, fat cells are individually held in place by delicate reticular fibers clustering in lobular masses bounded by fibrous septa surrounded by a rich capillary network. In adults, adipocytes may comprise around 90% of adipose mass accounting only for roughly 25% of the total cell population. Thus, adipose tissue itself is composed not only of adipocytes, but also other cell types called the stroma-vascular fraction, comprising blood cells, endothelial cells, pericytes, and adipose precursor cells among others (Figure 5);

these account for the remaining 75% of the total cell population, representing a wide range of targets for extensive autocrine-paracrine cross-talk.

Adipocytes, which are typically spherical and vary enormously in size (20-200 mm in diameter, with variable volumes ranging from a few picoliters to about 3 nanoliters), are embedded in a connective tissue matrix and are uniquely adapted to store and release energy. Surplus energy is assimilated by adipocytes and stored as lipid droplets. The stored fat is composed mainly of triacylglycerols (about 95% of the total lipid content comprised principally of oleic and palmitic acids) and to a smaller degree of diacylglycerols, phospholipids, unesteri-fied fatty acids, and cholesterol. To accommodate the lipids adipocytes are capable of changing their

Mesenchymal Immature Mature stem cell Adipoblast Preadipocyte adipocyte adipocyte

expansion k \ accumulation"- Ii |

st + expansion accumulation

Molecular/ Proliferation expansion physiological „

events , Growth arrest +

and early markers' appearance emerging regulatory genes

ECM alterations Cytoskeletal remodeling LPL CD36 SREBP-1 C/EBP/3 & S PPAR7 C/EBPa GLUT4

Lipogenic enzymes aP2

Leptin & other secreted factors

Figure 3 Multistep process of adipogenesis together with events and participating regulatory elements. aP2, adipocyte fatty acid binding protein; C/EBPa, CCAAT/enhancer binding protein a; C/EBPp & S, CCAAT/enhancer binding protein p & S; CD36, fatty acid translocase; ECM, extracellular matrix; GLUT4, glucose transporter type 4; LPL, lipoprotein lipase; PPAR7, peroxisome proliferator-activated receptor-7; Pref-1, preadipocyte factor-1; SREBP-1, sterol regulatory element binding protein-1.

diameter 20-fold and their volumes by several thousandfold. However, fat cells do not increase in size indefinitely. Once a maximum capacity is attained, which in humans averages 1000 picoliters, the formation of new adipocytes from the precursor pool takes place.

Histologically, the interior of adipocytes appears unstained since the techniques of standard tissue

ADIPOGENIC FACTORS

> angiotensin II

> diet rich in saturated fat

> estrogens

> glucocorticoids . IGF-1

> long-chain fatty acids

> lysophosphatidic acid . MCSF

> prolactin

> retinoids

> thyroid hormones

ANTIADIPOGENIC FACTORS

> catecholamines . EGF

> flavonoids

• testosterone

Figure 4 Factors exerting a direct effect on adipose mass. EGF, epidermal growth factor; GH, growth hormone; IGF-1, insulin-like growth factor-1; IL-1, interleukin-1; IL-6, interleukin-6; LIF, leukemia inhibitory factor; MCSF, macrophage colony stimulating factor; PAI-1, plasminogen activator inhibitor-1; PDGF, platelet-derived growth factor; PGF2a, prostaglandin F2a; PPARs, peroxisome proliferator-activated receptors; TGF-p, transforming growth factor-p; TNF-a, tumor necrosis factor-a.

35-70% adipocytes

Stromal cell fraction

• fibroblasts

• endothelial cells •pericytes

' preadipocytes ' poorly differentiated mesenchymal cells ' very small fat cells

Figure 5 Schematic representation of cell types present in adipose tissue. WAT, white adipose tissue.

preparation dissolve out the lipids, leaving a thin rim of eosinophilic cytoplasm that typically loses its round shape during tissue processing, thus contributing to the sponge-like appearance of WAT in routine preparations for light microscopy (Figure 6 and Figure 7). Owing to the fact that about 90% of the cell volume is a lipid droplet, the small dark nucleus becomes a flattened semilunar structure pushed against the edge of the cell and the thin cytoplasmic rim is also pushed to the periphery of the adipocytes. Mature white adipose cells contain a single large lipid droplet and are described as unilocular. However, developing white adipocytes are transiently multilocular containing multiple lipid droplets before these finally coalesce into a single large drop (Figure 8). The nucleus is round or oval in young fat cells, but is cup-shaped and peripherally displaced in mature adipocytes. The cytoplasm is stretched to form a thin sheath around the fat globule, although a relatively large volume is concentrated around the nucleus. A thin external lamina called basal lamina surrounds the cell. The smooth cell membrane shows no microvilli but has abundant smooth micropinocytotic invaginations that often fuse to form small vacuoles appearing as rosette-like configurations (Figure 9). Mitochondria are few in number with loosely arranged membranous cristae. The Golgi zone is small and the cytoplasm is filled with free ribosomes, but contains only a limited number of short profiles of the granular endoplasmic reticulum. Occasional lyso-somes can be found. The coalescent lipid droplets contain a mixture of neutral fats, triglycerides, fatty acids, phospholipids, and cholesterol. A thin interface membrane separates the lipid droplet from the cytoplasmic matrix. Peripheral to this membrane is a system of parallel meridional thin filaments. Because of the size of these cells, relative to the thickness of the section, the nucleus (accounting for only one-fortieth of the cell volume) may not always be present in the section. Unilocular adipo-cytes usually appear in clumps near blood vessels, which is reasonable since the source and dispersion of material stored in fat cells depends on transportation by the vascular system.

Brown fat is a specialized type of adipose tissue that plays an important role in body temperature regulation. In the newborn brown fat is well developed in the neck and interscapular region. It has a limited distribution in childhood, and occurs only to a small degree in adult humans, while it is present in significant amounts in rodents and hibernating animals. The brown color is derived from a rich vascular network and abundant mitochondria and lysosomes. The individual multilocular adipocytes are frothy appearing cells due to the fact that the lipid, which does not coalesce as readily as in white fat cells and is normally stored in multiple small droplets, has been leached out during tissue

Figure 6 (A) Human subcutaneous white adipose tissue with Masson trichrome staining (10x; bar = 100mm). (B) Same tissue at a higher magnification (40x; bar = 25 mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

Figure 6 (A) Human subcutaneous white adipose tissue with Masson trichrome staining (10x; bar = 100mm). (B) Same tissue at a higher magnification (40x; bar = 25 mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

processing (Figure 10). The spherical nuclei are centrally or eccentrically located within the cell. Compared to the unilocular white adipocytes, the cytoplasm of the multilocular brown fat cell is relatively abundant and strongly stained because of the numerous mitochondria present. The mitochondria are involved in the oxidation of the stored lipid, but because they exhibit a reduced potential to carry out oxidative phosphorylation, the energy produced is released in the form of heat due to the uncoupling activity of UCP and not captured in adenosine triphosphate (ATP). Therefore, brown adipose tissue is extremely well vascularized so that the blood is warmed when it passes through the active tissue.

Figure 7 (A) Human omental white adipose tissue with Masson trichrome staining (10x; bar = 100mm). (B) Same tissue at a higher magnification (40x; bar = 25mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

Distribution

White adipose tissue may represent the largest endocrine tissue of the whole organism, especially in overweight and obese patients. The anatomical distribution of individual fat pads dispersed throughout the whole body and not connected to each other contradicts the classic organ-specific localization. WAT exhibits clear, regional differences in its sites of predilection (Table 1). The hypodermal region invariably contains fat, except in a few places such as the eyelids and the scrotum. Adipocytes also accumulate around organs like the kidneys and adrenals, in the coronary sulcus of the heart, in bone marrow, mesentery, and omentum. Unilocular fat is

Figure 8 Paraffin section of rat abdominal white adipose tissue with a hematoxylin and eosin stain showing the simultaneous presence of uni- and multilocular adipocytes (40x; bar = 25 mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

widely distributed in the subcutaneous tissue of humans but exhibits quantitative regional differences that are influenced by age and sex. In infants and young children there is a continuous subcutaneous fat layer, the panniculus adiposus, over the whole body. This layer thins out in some areas in adults but persists and grows thicker in certain other regions. The sites differ in their distribution among sexes, being responsible for the characteristic body form of males and females, termed android and ginecoid fat distribution. In males, the main regions include the nape of the neck, the subcutaneous area over the deltoid and triceps muscles, and the lumbosacral region. In females, subcutaneous fat is most abundant in the buttocks, epitrochanteric region, anterior and lateral aspects of the thighs, as well as the breasts. Additionally, extensive fat depots are found in the omentum, mesenteries, and the retroperitoneal area of both sexes. In well-nourished, sedentary individuals, the fat distribution persists and becomes more obvious with advancing age with males tending to deposit more fat in the visceral compartment. Depot-specific differences may be related not only to the metabolism of fat cells but also to their capacity to form new adipo-cytes. Additionally, regional differences may result from variations in hormone receptor distribution as well as from specific local environmental characteristics as a consequence of differences in innervation and vascularization.

Regional distribution of body fat is known to be an important indicator for metabolic and cardiovascular alterations in some individuals.

Figure 9 (A) Transmission electron micrographs with the characteristically displaced nucleus to one side and slightly flattened by the accumulated lipid. The cytoplasm of the fat cell is reduced to a thin rim around the lipid droplet (7725x). (B) The cytoplasm contains several small lipid droplets that have not yet coalesced. A few filamentous mitochondria, occasional cisternae of endoplasmic reticulum, and a moderate number of free ribosomes are usually visible (15000x). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

Figure 9 (A) Transmission electron micrographs with the characteristically displaced nucleus to one side and slightly flattened by the accumulated lipid. The cytoplasm of the fat cell is reduced to a thin rim around the lipid droplet (7725x). (B) The cytoplasm contains several small lipid droplets that have not yet coalesced. A few filamentous mitochondria, occasional cisternae of endoplasmic reticulum, and a moderate number of free ribosomes are usually visible (15000x). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra, Spain.)

(A)

Figure 10 (A) Paraffin section of rat brown adipose tissue with a hematoxylin and eosin stain (20x; bar = 50mm). (B) Same tissue at a higher magnification (40x; bar = 25mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra.)

Figure 10 (A) Paraffin section of rat brown adipose tissue with a hematoxylin and eosin stain (20x; bar = 50mm). (B) Same tissue at a higher magnification (40x; bar = 25mm). (Courtesy of Dr. M A Burrell and M Archanco, University of Navarra.)

The observation that the topographic distribution of adipose tissue is relevant to understanding the relation of obesity to disturbances in glucose and lipid metabolism was formulated before the 1950s. Since then numerous prospective studies have revealed that android or male-type obesity correlates more often with an elevated mortality and risk for the development of diabetes mellitus type 2, dyslipidemia, hypertension, and atherosclerosis than gynoid or female-type obesity. Obesity has been reported to cause or exacerbate a large number of health problems with a known impact on both life expectancy and quality of life. In this respect, the association of increased adiposity is accompanied by important pathophysiological

Table 1 Distribution of main human adipose tissue depots

Subcutaneous (approx. 80%; deep l superficial layers)

Truncal

- Cervical

- Dorsal

- Lumbar Abdominal Gluteofemoral Mammary

Visceral (approx. 20%; thoracic-abdominal-pelvic)

Intrathoracic (extra-intrapericardial) Intra-abdominopelvic

- Intraperitoneal

Omental (greater and lesser omentum) Mesenteric (epiplon, small intestine, colon, rectum) Umbilical

- Extraperitoneal

Peripancreatic (infiltrated with brown adipocytes) Perirenal (infiltrated with brown adipocytes)

- Intrapelvic

Gonadal (parametrial, retrouterine, retropubic) Urogenital (paravesical, para-retrorectal)

Intraparenchymatous (physiologically or pathologically)

Inter-intramuscular and perimuscular (inside the muscle fascia) Perivascular

Paraosseal (interface between bone and muscle) Ectopic (steatosis, intramyocardial, lypodystrophy, etc.)

Hyperlipidemia

Cardiovascular disease

Cancer Obstructive sleep apnea Hyperuricemia

Atherosclerosis/ inflammation

Hyperlipidemia sleep apnea Hyperuricemia

Atherosclerosis/ inflammation

^ Psychosocial distress

Others

Figure 11 Main comorbidities associated with increased adiposity.

Metabolic syndrome Infertility

^ Psychosocial distress

Osteoarthritis

Gastrointestinal alterations

Others

Figure 11 Main comorbidities associated with increased adiposity.

alterations, which lead to the development of a wide range of comorbidities (Figure 11).

Function

Although many cell types contain small reserves of carbohydrate and lipid, the adipose tissue is the body's most capacious energy reservoir. Because of the high energy content per unit weight of fat as well as its hydrophobicity, the storage of energy in the form of triglycerides is a highly efficient biochemical phenomenon (1 g of adipose tissue contains around 800 mg triacylglycerol and only about 100 mg of water). It represents quantitatively the most variable component of the organism, ranging from a few per cent of body weight in top athletes to more than half of the total body weight in severely obese patients. The normal range is about 10-20% body fat for males and around 20-30% for females, accounting approximately for a 2-month energy reserve. During pregnancy most species accrue additional reserves of adipose tissue to help support the development of the fetus and to further facilitate the lactation period.

Energy balance regulation is an extremely complex process composed of multiple interacting homeostatic and behavioral pathways aimed at maintaining constant energy stores. It is now evident that body weight control is achieved through highly orchestrated interactions between nutrient selection, organoleptic influences, and neuroendocrine responses to diet as well as being influenced by genetic and environmental factors. The concept that circulating signals generated in proportion to body fat stores influence appetite and energy expenditure in a coordinated manner to regulate body weight was proposed almost 50 years ago. According to this model, changes in energy balance sufficient to alter body fat stores are signaled via one or more circulating factors acting in the brain to elicit compensatory changes in order to match energy intake to energy expenditure. This was formulated as the 'lipostatic theory' assuming that as adipose tissue mass enlarges, a factor that acts as a sensing hormone or 'lipostat' in a negative feedback control from adipose tissue to hypothalamic receptors informs the brain about the abundance of body fat, thereby allowing feeding behavior, metabolism, and endocrine physiology to be coupled to the nutritional state of the organism. The existing body of evidence gathered in the last decades through targeted expression or knockout of specific genes involved in different steps of the pathways controlling food intake, body weight, adiposity, or fat distribution has clearly contributed to unraveling the underlying mechanisms of energy homeostasis. The findings have fostered the notion of a far more complex system than previously thought, involving the integration of a plethora of factors.

The identification of adipose tissue as a multifunctional organ as opposed to a passive organ for the storage of excess energy in the form of fat has been brought about by the emerging body of evidence gathered during the last few decades. This pleiotro-pic nature is based on the ability of fat cells to secrete a large number of hormones, growth factors, enzymes, cytokines, complement factors, and matrix proteins, collectively termed adipokines or adipocy-tokines (Table 2, Figure 12), at the same time as expressing receptors for most of these factors (Table 3), which warrants extensive cross-talk at a local and systemic level in response to specific external stimuli or metabolic changes. The vast majority of adipocyte-derived factors have been shown to be dysregulated in alterations accompanied by changes

Table 2 Relevant factors secreted by adipose tissue into the bloodstream

Molecule

Function/effect

Adiponectin/ACRP30/AdipoQ/

apM1/GBP28 Adipsin

Angiotensinogen

Glycerol IGF-I

IL-6 Leptin

NO PAI-1

PGI2 & PGF2„ Resistin TNF-a VEGF

Plays a protective role in the pathogenesis of type 2 diabetes and cardiovascular diseases

Possible link between the complement pathway and adipose tissue metabolism Precursor of angiotensin II; regulator of blood pressure and electrolyte homeostasis Influences the rate of triacylglycerol synthesis in adipose tissue

Oxidized in tissues to produce local energy. Serve as a substrate for triglyceride and structural molecules synthesis. Involved in the development of insulin resistance Structural component of the major classes of biological lipids and gluconeogenic precursor Stimulates proliferation of a wide variety of cells and mediates many of the effects of growth hormone

Implicated in host defense, glucose and lipid metabolism, and regulation of body weight Signals to the brain about body fat stores. Regulation of appetite and energy expenditure. Wide variety of physiological functions Important regulator of vascular tone. Pleiotropic involvement in pathophysiological conditions Potent inhibitor of the fibrinolytic system

Implicated in regulatory functions such as inflammation and blood clotting, ovulation, menstruation, and acid secretion Putative role in insulin resistance May participate in inflammation

Interferes with insulin receptor signaling and is a possible cause of the development of insulin resistance in obesity Stimulation of angiogenesis

Vasoactive factors

Lipid metabolism

Immune response

Angiotensinogen Monobutyrin Adiponectin PAI-1

Eicosanoids VEGF

Tissue factor Nitric oxide

Binding proteins

Vasoactive factors

Lipid metabolism

Angiotensinogen Monobutyrin Adiponectin PAI-1

Eicosanoids VEGF

Tissue factor Nitric oxide

Binding proteins

Retinol

Adipsin ASP

Factors B and C3 CSFs IL-17 D SAA3

Growth factors

TGF/3 IGF-1 HGF NGF

Lysophosp hatidic acid

Fibronectin

Glucose metabolism

Resistin

Proteins extracellular matrix

Osteonectin

Figure 12 Factors secreted by white adipose tissue, which underlie the multifunctional nature of this endocrine organ. Although due to their pleiotropic effects some of the elements might be included in more than one physiological role, they have been included only under one function for simplicity reasons. apoE, apolipoprotein E; ASP, acylation-stimulating protein; CRP, C-reactive protein; CSFs, colony-stimulating factors; FFA, free fatty acids; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; IL-10, interleukin-10; IL-17 D, interleukin-17 D; IL-1Ra, interleukin-1 receptor antagonist; IL-1,3, interleukin-1,3; IL-6, interleukin, 6; IL-8, interleukin-8; LIF, leukemia inhibitory factor; LPL, lipoprotein lipase; MCP-1, monocyte chemoattractant protein-1; NGF, nerve growth factor; PAI-1, plasminogen activator inhibitor -1; PGF2a, prostaglandin F2a; PGI2, prostacyclin; SAA3, serum amyloid A3; sR, soluble receptor; TGF-,3, transforming growth factor-^; TNF-a, tumor necrosis factor-a; VAP-1/SSAO, vascular adhesion protein-1/semicarbazide-sensitive amine oxidase; VEGF, vascular endothelial growth factor.

Adipsin ASP

Factors B and C3 CSFs IL-17 D SAA3

Growth factors

Retinol

a1-acid glycoprot. VAP-1/SSAO

TGF/3 IGF-1 HGF NGF

Lysophosp hatidic acid

Fibronectin

Glucose metabolism

Resistin

Proteins extracellular matrix

Osteonectin

Figure 12 Factors secreted by white adipose tissue, which underlie the multifunctional nature of this endocrine organ. Although due to their pleiotropic effects some of the elements might be included in more than one physiological role, they have been included only under one function for simplicity reasons. apoE, apolipoprotein E; ASP, acylation-stimulating protein; CRP, C-reactive protein; CSFs, colony-stimulating factors; FFA, free fatty acids; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; IL-10, interleukin-10; IL-17 D, interleukin-17 D; IL-1Ra, interleukin-1 receptor antagonist; IL-1,3, interleukin-1,3; IL-6, interleukin, 6; IL-8, interleukin-8; LIF, leukemia inhibitory factor; LPL, lipoprotein lipase; MCP-1, monocyte chemoattractant protein-1; NGF, nerve growth factor; PAI-1, plasminogen activator inhibitor -1; PGF2a, prostaglandin F2a; PGI2, prostacyclin; SAA3, serum amyloid A3; sR, soluble receptor; TGF-,3, transforming growth factor-^; TNF-a, tumor necrosis factor-a; VAP-1/SSAO, vascular adhesion protein-1/semicarbazide-sensitive amine oxidase; VEGF, vascular endothelial growth factor.

in adipose tissue mass such as overfeeding and lipodystrophy, thus providing evidence for their implication in the etiopathology and comorbidities asssociated with obesity and cachexia.

WAT is actively involved in cell function regulation through a complex network of endocrine, para-crine, and autocrine signals that influence the response of many tissues, including hypothalamus, pancreas, liver, skeletal muscle, kidneys, endothe-lium, and immune system, among others. Adipose tissue serves the functions of being a store for reserve energy, insulation against heat loss through the skin, and a protective padding of certain organs. A rapid turnover of stored fat can take place, and with only a few exceptions (orbit, major joints as well as palm and foot sole), the adipose tissue can be used up almost completely during starvation. Adipo-cytes are uniquely equipped to participate in the regulation of other functions such as reproduction, immune response, blood pressure control, coagulation, fibrinolysis, and angiogenesis, among others. This multifunctional nature is based on the existence of the full complement of enzymes, regulatory proteins, hormones, cytokines, and receptors needed to carry out an extensive cross-talk at both a local and systemic level in response to specific external stimuli or neuroendocrine changes. This secretory nature has prompted the view of WAT as an extremely active endocrine tissue. Interestingly, the high number and ample spectrum of genes found to be expressed in WAT together with the changes observed in samples of obese patients substantiates the view of an extraordinarily active and plastic tissue. The complex and complementary nature of the expression profile observed in adipose tissue from obese organisms reflects a plethora of adaptive changes affecting crucial physiological functions that may need to be further explored through genomic and proteomic approaches.

The endocrine activity of WAT was postulated almost 20 years ago when the tissue's ability for steroid hormone interconversion was alluded to. In recent years, especially since the discovery of lep-tin, the list of adipocyte-derived factors has been increasing at a phenomenal pace. Another way of addressing the production of adipose-derived factors is by focusing on the function they are implicated in (Figure 12). One of the best known

Table 3 Main receptors expressed by adipose tissue

Receptor

Main effect of receptor activation on adipocyte metabolism

Hormone-cytokine receptors

Adenosine

Adiponectin (AdipoRI & AdipoR2) Angiotensin II

IL-6

Insulin

Leptin (OB-R) NPY-Y1 & Y5 Prostaglandin TGF-ß TNF-a

VEGF

Catecholamine-nervous system

Muscarinic Nicotinic a1-AR a2-AR

Nuclear receptors

Androgen Estrogen

Glucocorticoids PPAR<5

PPAR7

RAR/RXR

Lipoprotein receptors

HDL LDL VLDL

Inhibition of lipolysis

Regulation of insulin sensitivity and fatty acid oxidation Increase of lipogenesis

Stimulation of prostacyclin production by mature fat cells. Interaction with insulin in regulation of adipocyte metabolism Induction of leptin and IGF-I expression. Stimulation of lipolysis Inhibition of lipolysis. Stimulation of glucose transport and oxidation LPL activity inhibition. Induction of lipolysis

Inhibition of lipolysis and stimulation of lipogenesis. Induction of glucose uptake and oxidation.

Stimulation of leptin expression Stimulation of lipolysis. Autocrine regulation of leptin expression Inhibition of lipolysis. Induction of leptin expression

Strong antilypolitic effects (PGE2). Modulation of preadipocyte differentiation (PGF2a and PGI2) Potent inhibition of adipocyte differentiation

Stimulation of lipolysis. Regulation of leptin secretion. Potent inhibition of adipocyte differentiation. Involvement in development of insulin resistance Stimulation of angiogenesis receptors

Inhibition of lipolysis Stimulation of lipolysis

Induction of inositol phosphate production and PKC activation Inhibition of lipolysis. Regulation of preadipocyte growth

Stimulation of lipolysis. Induction of thermogenesis. Reduction of leptin mRNA levels

Control of adipose tissue development (antiadipogenic signals). Modulation of leptin expression Control of adipose tissue development (proadipogenic signals). Modulation of leptin expression Stimulation of adipocyte differentiation

Regulation of fat metabolism. Plays a central role in fatty acid-controlled differentiation of preadipose cells Induction of adipocyte differentiation and insulin sensitivity Regulation of adipocyte differentiation

Stimulation of lipolysis. Regulation of leptin secretion. Induction of adipocyte differentiation. Regulation of insulin effects

Clearance and metabolism of HDL Stimulation of cholesterol uptake

Binding and internalization of VLDL particles. Involvement in lipid accumulation

Abbreviations: ACRP30, adipocyte complement-related protein of 30kDa; apM1, adipose most abundant gene transcript 1; ASP, acylation-stimulating protein; FFA, free fatty acids; GBP28, gelatin-binding protein 28; GH, growth hormone; HDL, high density lipoprotein; IGF, insulin-like growth factor; IL-6, interleukin 6; LDL, low density lipoprotein; LPL, lipoprotein lipase; NO, nitric oxide; NPY-Y1 & -Y5, neuropeptide receptors Y-1 & -5; OB-R, leptin receptor; PAI-1, plasminogen activator inhibitor -1; PGE2, prostaglandin E2; PGF2a, prostaglandin F2a; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoid x receptor; T3, triiodothyronine; TGF-ß, transforming growth factor-ß; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor; VLDL, very low-density lipoprotein; a1- & a2-AR, a1- & a2-adrenergic receptors; ßr, ß2- & ß3-AR, ß-|-, ß2- & ß3 adrenergic receptors.

aspects of WAT physiology relates to the synthesis of products involved in lipid metabolism such as perilipin, adipocyte lipid-binding protein (ALBP, FABP4, or aP2), CETP (cholesteryl ester transfer protein), and retinol binding protein (RBP). Adipose tissue has also been identified as a source of production of factors with immunological properties participating in immunity and stress responses, as is the case for ASP (acylation-simulating protein)

and metallothionein. More recently, the pivotal role of adipocyte-derived factors in cardiovascular function control such as angiotensinogen, adipo-nectin, peroxisome proliferator-activated receptor 7 angiopoietin related protein/fasting-induced adipose factor (PGAR/FIAF), and C-reactive protein (CRP) has been established. A further subsection of proteins produced by adipose tissue concerns other factors with an autocrine-paracrine function like

PPAR-7 (peroxisome proliferator-activated receptor), IGF-1, monobutyrin, and the UCPs.

It is generally assumed that under normal physiological circumstances adult humans are practically devoid of functional brown adipose tissue. As is the case in other larger mammals the functional capacity of brown adipose tissue decreases because of the relatively higher ratio between heat production from basal metabolism and the smaller surface area encountered in adult animals. In addition, clothing and indoor life have reduced the need for adaptive nonshivering thermogenesis. However, it has been recently shown that human WAT can be infiltrated with brown adipocytes expressing UCP-1.

Regulation of Metabolism

The control of fat storage and mobilization has been marked by the identification of a number of regulatory mechanisms in the last few decades. Isotopic tracer studies have clearly shown that lipids are continuously being mobilized and renewed even in individuals in energy balance. Fatty acid esterification and triglyceride hydrolysis take place continuously. The half-life of depot lipids in rodents is about 8 days, meaning that almost 10% of the fatty acid stored in adipose tissue is replaced daily by new fatty acids. The balance between lipid loss and accretion determines the net outcome on energy homeostasis.

The synthesis of triglycerides, also termed lipogen-esis, requires a supply of fatty acids and glycerol. The main sources of fatty acids are the liver and the small intestine. Fatty acids are esterified with gly-cerol phosphate in the liver to produce triglycerides. Since triglycerides are bulky polar molecules that do not cross cell membranes well, they must be hydro-lyzed to fatty acids and glycerol before entering fat cells. Serum very low-density lipoproteins (VLDLs) are the major form in which triacylglycerols are carried from the liver to WAT. Short-chain fatty acids (16 carbons or less) can be absorbed from the gastrointestinal tract and carried in chylomicra directly to the adipocyte. Inside fat cells, glycerol is mainly synthesized from glucose. In WAT, fatty acids can be synthesized from several precursors, such as glucose, lactate, and certain amino acids, with glucose being quantitatively the most important in humans. In the case of glucose, GLUT4, the principal glucose transporter of adipocytes, controls the entry of the substrate into the adipocyte. Insulin is known to stimulate glucose transport by promoting GLUT4 recruitment as well as increasing its activity. Inside the adipocyte, glucose is initially phosphorylated and then metabolized both in the cytosol and in the mitochondria to produce cytosolic acetyl-CoA with the flux being influenced by phos-phofructokinase and pyruvate dehydrogenase. Gly-cerol does not readily enter the adipocyte, but the membrane-permeable fatty acids do. Once inside the fat cells, fatty acids are re-esterified with glycerol phosphate to yield triglycerides. Lipogenesis is favored by insulin, which activates pyruvate kinase, pyruvate dehydrogenase, acetyl-CoA carboxylase, and glycerol phosphate acyltransferase. When excess nutrients are available insulin decreases acetyl-CoA entry into the tricarboxylic acid cycle while directing it towards fat synthesis. This insulin effect is antagonized by growth hormone. The gut hormones glu-cagon-like peptide 1 and gastric inhibitory peptide also increase fatty acid synthesis, while glucagon and catecholamines inactivate acetyl-CoA carboxy-lase, thus decreasing the rate of fatty acid synthesis.

The release of glycerol and free fatty acids by lipolysis plays a critical role in the ability of the organism to provide energy from triglyceride stores. In this sense, the processes of lipolysis and lipogen-esis are crucial for the attainment of body weight control. For this purpose adipocytes are equipped with a well-developed enzymatic machinery, together with a number of nonsecreted proteins and binding factors directly involved in the regulation of lipid metabolism. The hydrolysis of triglycerides from circulating VLDL and chylomicrons is catalyzed by lipoprotein lipase (LPL). This rate-limiting step plays an important role in directing fat partitioning. Although LPL controls fatty acid entry into adipocytes, fat mass has been shown to be preserved by endogenous synthesis. From observations made in patients with total LPL deficiency it can also be concluded that fat deposition can take place in the absence of LPL. A further key enzyme catalyzing a rate-limiting step of lipolysis is HSL (hormome sensitive lipase), which cleaves tria-cylglycerol to yield glycerol and fatty acids. Some fatty acids are re-esterified, so that the fatty acid: glycerol ratio leaving the cell is usually less than the theoretical 3:1. Increased concentrations of cAMP activate HSL as well as promote its movement from the cytosol to the lipid droplet surface. Cate-cholamines and glucagon are known inducers of the lipolytic activity, while the stimulation of lipolysis is attenuated by adenosine and protaglandin E2. Interestingly, HSL deficiency leads to male sterility and adipocyte hypertrophy, but not to obesity, with an unaltered basal lipolytic activity suggesting that other lipases may also play a relevant role in fat mobilization.

The lipid droplets contained in adipocytes are coated by structural proteins, such as perilipin, that stabilize the single fat drops and prevent triglyceride hydrolysis in the basal state. The phosphorylation of perilipin following adrenergic stimulation or other hormonal inputs induces a structural change of the lipid droplet that allows the hydrolysis of triglycerides. After hormonal stimulation, HSL and perilipin are phosphorylated and HSL translocates to the lipid droplet. ALBP, also termed aP2, then binds to the N-terminal region of HSL, preventing fatty acid inhibition of the enzyme's hydrolytic activity.

The function of CETP is to promote the exchange of cholesterol esters of triglycerides between plasma lipoproteins. Fasting, high-cholesterol diets as well as insulin stimulate CETP synthesis and secretion in WAT. In plasma, CETP participates in the modulation of reverse cholesterol transport by facilitating the transfer of cholesterol esters from high-density lipoprotein (HDL) to triglyceride-rich apoB-contain-ing lipoproteins. VLDLs, in particular, are converted to low-density lipoproteins (LDLs), which are subjected to hepatic clearance by the apoB/E receptor system. Adipose tissue probably represents one of the major sources of CETP in humans. Therefore, WAT represents a cholesterol storage organ, whereby peripheral cholesterol is taken up by HDL particles, acting as cholesterol efflux acceptors, and is returned for hepatic excretion. In obesity, the activity and protein mass of circulating CETP is increased showing a negative correlation with HDL concentrations at the same time as a positive correlation with fasting glycemia and insulinemia suggesting a potential link with insulin resistance.

Synthesis and secretion of RBP by adipocytes is induced by retinoic acid and shows that WAT plays an important role in retinoid storage and metabolism. In fact, RBP mRNA is one of the most abundant transcripts present in both rodent and human adipose tissue. Hepatic and renal tissues have been regarded as the main sites of RBP production, while the quantitative and physiological significance of the WAT contribution remains to be fully elucidated.

The processes participating in the control of energy balance, as well as the intermediary lipid and carbohydrate metabolism, are intricately linked by neurohumoral mediators. The coordination of the implicated molecular and biochemical pathways underlies, at least in part, the large number of intracellular and secreted proteins produced by WAT with autocrine, paracrine, and endocrine effects. The finding that WAT secretes a plethora of pleiotropic adipokines at the same time as expressing receptors for a huge range of compounds has led to the development of new insights into the functions of adipose tissue at both the basic and clinical level. At this early juncture in the course of adipose tissue research, much has been discovered. However, a great deal more remains to be learned about its physiology and clinical relevance. Given the adipocyte's versatile and ever-expanding list of secretory proteins, additional and unexpected discoveries are sure to emerge. The growth, cellular composition, and gene expression pattern of adipose tissue is under the regulation of a large selection of central mechanisms and local effectors. The exact nature and control of this complex cross-talk has not been fully elucidated and represents an exciting research topic.

Abbreviations

ACRP30/apM1/

adipocyte complement-related

GBP28

protein of 30 kDa/adipose most

abundant gene transcript

1/gelatin-binding protein 28

ADD1/SREBP-1C

adipocyte determination and

differentiation factor-1/sterol

regulatory element binding

protein-1c

ALBP/FABP4/aP2

adipocyte fatty acid binding

protein

apoE

apolipoprotein E

ASP

acylation-stimulating protein

ATP

adenosine triphosphate

cAMP

cyclic adenosin monophosphate

CD36

fatty acid translocase

C/EBPs

CCAAT/enhancer binding

proteins

CETP

cholesteryl ester transfer protein

CRP

C-reactive protein

CSF

colony-stimulating factor

ECM

extracellular matrix

EGF

epidermal growth factor

FFA

free fatty acids

FGF

fibroblast growth factor

GH

growth hormone

GLP-1

glucagon-like peptide-1

GLUT4

glucose transporter type 4

HDL

high density lipoprotein

HGF

hepatocyte growth factor

HSL

hormone-sensitive lipase

IGF

insulin-like growth factor

IL

interleukin

IL-1Ra

interleukin-1 receptor

antagonist

LDL

low density lipoprotein

LIF

leukemia inhibitory factor

LPL

lipoprotein lipase

MCP-1

monocyte chemoattractant

protein-1

MCSF

macrophage colony stimulating

factor

MIF

macrophage migration

inhibitory factor

MIP-1a

macrophage inflammatory

protein-1a

NGF

nerve growth factor

NO

nitric oxide

NPY-Y1 & -Y5

neuropeptide receptors Y-1 & -5

OB-R

leptin receptor

PAI-1

plasminogen activator inhibitor-1

PDGF

platelet-derived growth factor

PGAR/FIAF

peroxisome proliferator-

activated receptor angiopoietin

related protein/fasting-induced

adipose factor

PGC-1a

peroxisome proliferator-

activated receptor-7

coactivator-1a

PGEi

prostaglandin E2

PGFia

prostaglandin F2q!

pgi2

prostacyclin

PPAR

peroxisome proliferator-

activated receptor

Pref-1

preadipocyte factor-1

RAR

retinoic acid receptor

RBP

retinol binding protein

RXR

retinoid x receptor

SAA3

serum amyloid A3

T3

triiodothyronine

TGF-ß

transforming growth factor-^

TNF-a

tumor necrosis factor-a

UCP

uncoupling protein

VAP-1/SSAO

vascular adhesion protein-1/

semicarbazide-sensitive amine

oxidase

VEGF

vascular endothelial growth

factor

VLDL

very low density lipoprotein

WAT

white adipose tissue

a1- & a2-AR

a1- & (^-adrenergic receptors

ß1-, ßi- & ß3-AR

^2- & P3 adrenergic

receptors

See also: Cholesterol: Sources, Absorption, Function and Metabolism; Factors Determining Blood Levels. Diabetes Mellitus: Etiology and Epidemiology; Classification and Chemical Pathology; Dietary Management. Fatty Acids: Metabolism; Monounsaturated; Omega-3 Polyunsaturated; Omega-6 Polyunsaturated; Saturated; Trans Fatty Acids. Hypertension: Etiology. Lipids: Chemistry and Classification; Composition and Role of Phospholipids. Lipoproteins. Obesity: Definition, Etiology and

Assessment; Fat Distribution; Childhood Obesity; Complications; Prevention; Treatment. Pregnancy: Safe Diet for Pregnancy.

Further Reading

Ailhaud G and Hauner H (2004) Development of white adipose tissue. In: Bray GA and Bouchard C (eds.) Handbook of Obesity. Etiology and Pathophysiology, 2nd edn, pp. 481-514. New York: Marcel Dekker, Inc.

Frayn KN, Karpe F, Fielding BA, Macdonald IA, and Coppack SW (2003) Integrative physiology of human adipose tissue. International Journal of Obesity 27: 875-888.

Fried SK and Ross RR (2004) Biology of visceral adipose tissue. In: Bray GA and Bouchard C (eds.) Handbook of Obesity. Etiology and Pathophysiology, 2nd edn, pp. 589-614. New York: Marcel Dekker, Inc.

Fruhbeck G (2004) The adipose tissue as a source of vasoactive factors. Current Medicinal Chemistry (Cardiovascular & Hematological Agents) 2: 197-208.

Fruhbeck G and Gomez-Ambrosi J (2003) Control of body weight: a physiologic and transgenic perspective. Diabetologia 46:143-172.

Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, and Burrell MA (2001) The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. American Journal of Physiology 280: E827-E847.

Gomez-Ambrosi J, Catalan V, Diez-Caballero A, Martinez-Cruz A, Gil MJ, Garcia-Foncillas J, Cienfuegos JA, Salvador J, Mato JM, and Fruhbeck G (2004) Gene expression profile of omental adipose tissue in human obesity. The FASEB Journal 18: 215-217.

Lafontan M and Berlan M (2003) Do regional differences in adipocyte biology provide new pathophysiological insights? Trends in Pharmacological Sciences 24: 276-283.

Langin D and Lafontan M (2000) Millennium fat-cell lipolysis reveals unsuspected novel tracks. Hormone and Metabolic Research 32: 443-452.

Pond CM (1999) Physiological specialisation of adipose tissue. Progress in Lipid Research 38: 225-248.

Rosen ED, Walkey CJ, Puigserver P, and Spiegelman BM (2000) Transcriptional regulation of adipogenesis. Genes and Development 14: 1293-1307.

Shen W, Wang Z, Punyanita M, Lei J, Sinav A, Kral JG, Imielinska C, Ross R, and Heymsfield SB (2003) Adipose quantification by imaging methods: a proposed classification. Obesity Research 11: 5-16.

Trayhurn P and Beattie JH (2001) Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proceedings of the Nutrition Society 60: 329-339.

Unger RH (2003) The physiology of cellular liporegulation. Annual Review of Physiology 65: 333-347.

Wajchenberg BL (2000) Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocrine Reviews 21: 697-738.

ADOLESCENTS

Contents

Nutritional Requirements Nutritional Problems

Nutritional Requirements

CHS Ruxton, Nutrition Communications, Cupar, UK J Fiore, University of Westminster, London, UK

© 2005 Elsevier Ltd. All rights reserved.

Introduction

Adolescence is the period of transition between childhood and adulthood. This reflects not only the physical and emotional changes experienced by the adolescent, but the development of dietary behaviors. Whereas younger children are characterized by their resistance to new experiences, the adolescent may use food to assert their independence, not always in a beneficial way. This section will cover development in adolescence and highlight nutrients that are important during this time. Information on adolescent energy and nutrient intakes from a broad range of countries will be presented. The findings will be put in context with dietary recommendations.

Physical Changes During Adolescence

Adolescence is generally assumed to be the period of human development from 10 to 18 years of age, a time during which rapid growth and physical maturity take place.

Growth

During prepubescent childhood, the growth of boys and girls follows a similar trajectory, although boys may be slightly taller and heavier than girls. Around the 9th year, the pubertal growth spurt, which can last up to 3.5 years, will occur in girls with boys beginning 2 years later. Girls reach their full height approximately 2 years before boys and are, therefore, the taller of the two sexes for a period of time. Current UK standards for height and weight during adolescence are presented in Table 1.

Maximum height velocity is generally seen in the year preceding menarche for girls and at around 14 years for boys. On average, weight velocity peaks at 12.9 years for girls and 14.3 years for boys. Annual growth rates during adolescence can be as much as 9 cm/8.8 kg in girls and 10.3 cm/9.8 kg in boys. Energy and protein intakes per kilogram body weight have been observed to peak during maximal growth, suggesting increased requirements during adolescence. Undernutrition in this crucial window of development can result in a slow height increment, lower peak bone mass, and delayed puberty. On the other hand, overnutrition is not without its risks. It is believed that obesity in young girls can bring about an early menarche, which then increases the risk of breast cancer in later adulthood. Menarche is deemed precocious if it occurs before the age of eight. Rising childhood obesity levels in Western countries have resulted in a rise in the proportion of girls displaying precocious menarche.

Table 1 Percentiles for height, weight, and body mass index

Age (years) Height (cm) Weight (kg) Body mass index

Table 1 Percentiles for height, weight, and body mass index

Age (years) Height (cm) Weight (kg) Body mass index

3rd

50th

97th

3rd

50th

97th

2nd

50th

99.6th

(a) Boys

11

130.8

143.2

155.8

26.1

34.5

50.9

14

17

26

16

158.9

173.0

187.4

44.9

60.2

83.2

16

20

30

18

163.3

176.4

189.7

52.0

66.2

87.9

17

21

32

(b) Girls

11

130.9

143.8

156.9

26.0

35.9

53.6

14

17

27

16

151.6

163.0

174.6

42.8

55.3

74.1

16

20

31

18

152.3

163.6

175.0

44.7

57.2

76.3

17

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