New Perspectives in Adipose Tissue: Structure, Function and Development

3TU

Chapter 1

The comparative anatomy of adipose tissue

Gary J. Hausman

Publisher Summary

The cellular composition of adipose tissue is highly variable and is shown to be dependent on anatomical site, species, and a host of environmental factors. Recent studies also demonstrate that adipocytes perform essential physiological functions in addition to triglyceride storage. An eventual description of the full physiological role of adipose tissue in man and animals is equally dependent on both biochemical and anatomical research. This chapter highlights some new perspectives in adipose tissue anatomy. The relationship between adipose tissue cellularity and human obesity has been extensively reviewed, which shows that early-onset obesity is associated with increased cell numbers, whereas only cell hypertrophy is associated with adult-onset obesity. Typically the stromal-vascular cells consist of macrophages, fibroblasts, pericytes, and various types of blood cells. Electron microscopic studies on cells that develop into adipocytes describe a wide variety of cellular ultrastructures. In some species, such as dog, man, mouse, and rat, mast cells are a normal component of adipose tissue that are deployed along the blood vessels. The full range of cells common to mature red bone marrow have been located in immature and mature white adipose tissue. The capillaries and primitive blood cells are formed in a process of blood island formation.

1.1 Introduction

In the time since this subject was last reviewed (Vague and Fenasse, 1965) there has been an explosion in the appreciation and knowledge of the biology of adipose tissue. The cellular composition of adipose tissue is highly variable and has been shown to be dependent on anatomical site, species and a host of environmental factors. Interest in adipose tissue cellularity was generated originally by studies that suggested that there was a relationship between age of onset of obesity and the total number of fat cells. The relationship between obesity and fat cell number remains a controversial subject and is in need of continuing research. Recent studies have also demonstrated that adipocytes perform essential physiological functions in addition to triglyceride storage (Tischler and Goldberg, 1980). An eventual description of the full physiological role of adipose tissue in man and animals is equally dependent on both biochemical and anatomical research. This review will serve to highlight some new perspectives in adipose tissue anatomy.

1.2 Anatomy of adipose tissue in the fetus

1.2.1 Identification of adipose tissue in the fetus

The anatomical sites destined to contain large adipocytes in the growing animal have been located and studied in the fetus. A multitude of studies has indicated that the extent of tissue and cell organization (i.e. the assemblage of cells and capillaries in areas that are distinct from surrounding connective tissue) prior to lipid deposition is not only dependent on the species but also on the location within a given species. For instance, the highest levels of organization are evident when the development of the interscapular brown adipose tissue (ISBAT) of rodents is considered (Hammar, 1895; Napolitano and Fawcett, 1958). On the other hand, little or no organization is evident in the development of mesenteric and subcutaneous depots in the dog, sheep, guinea-pig, rabbit and calf (Flemming, 1871; Hammar, 1895; Bell, 1909). Between these two extremes, there are intermediate levels of organization, achieved in depot development, that depend on both species and anatomical site (Bell, 1909; Wassermann, 1965; Wensvoort, 1967; Hermans, 1973; Hausman, 1977). In the development of rodent ISBAT, the presumptive tissue is completely filled with cellular condensations (that have an extensive vascular network) prior to lipid deposition (Hammar, 1895; Napolitano and Fawcett, 1958). In contrast, the development of all depots in cattle (Hammar, 1895; Bell, 1909) and pigs (Hermans, 1973; Hausman, 1977) and the development of the subcutaneous, mesenteric and gonadal depots in rodents (Hammar, 1895; Wassermann, 1965) are characterized by a low local concentration of cellular condensations during lipid deposition. Higher local concentrations of cellular condensations are present, however, during lipid deposition in all depots in the fetal sheep (Wensvoort, 1967) and goat (Thompson and Jenkins, 1970). All adipose tissue (brown and white) is eventually ‘filled’ with adipocytes through the formation of a number of fat cell clusters and the subsequent growth of these clusters (Bell, 1909; Wassermann, 1965; Wensvoort, 1967; Thompson and Jenkins, 1970; Hermans, 1973; Hausman, 1977) (Figure 1.1). The capillaries and adipocytes of the fat cell cluster are derived from the pre-existent cellular condensations.

Figure 1.1 Complete cross-sections of dorsal subcutaneous adipose tissue (over the shoulder) from a 65-day-old (a) and a 110-day-old (b) pig fetus. Lipid accumulation with age is characterized by an increase in number and size of fat cell clusters. Lipid-containing fat cell clusters (arrowed) are present only in the inner layer (I) of tissue at 65 days of gestation (a). (O = outer layer of tissue; HF = hair follicles; M = muscle; CB = connective tissue border between outer and inner layers of adipose tissue; D = dermis.) (c)–(f) Higher magnifications of fat cell cluster (arrowed) formation in the inner layer of subcutaneous tissue. The tissue becomes ‘filled’ with lipid through the formation of a number of fat cell clusters and their subsequent growth ((c), 75-day-old fetus; (d), 90-day fetus; (e), 3-day-old pig; (f) 9-day-old pig). Frozen sections stained with oil red O and Harris hematoxylin. (a),(b) × 25; (c),(d),(e),(f) × 80. (Reduced to 60% in reproduction) From Hausman (1978) with permission from The American Meat Science Association

The most extensive morphological study of fetal adipose tissue was conducted by Bell (1909) on calf fetuses. Bell showed that immature or preadipose tissue was a peculiar open-meshed tissue that had a characteristic reticular appearance and contained fine connective tissue fibers surrounding loosely arranged cells. This preadipose tissue was abundant around the kidneys, whereas small amounts were located around blood vessels in subcutaneous and omental areas. The preadipose tissue developed from a pre-existent fibrous connective tissue (Bell, 1909). In other words, immature adipose tissue was identified as a modified form of a fibrous connective tissue. However, data from many other studies (Napolitano and Fawcett, 1958; Wassermann, 1965; Wensvoort, 1967; Barnard, 1969; Hermans, 1973) do not support this concept because they do not show that a developed or mature-appearing fibrous connective tissue is present before immature adipose tissue is obvious.

Several recent studies demonstrate that fibroblasts (a normal cellular component of fibrous tissue) may become adipocytes in vivo (Napolitano, 1963; Slavin, 1979) and in vitro (Green, 1978). The major problem with this concept (Napolitano, 1963; Green, 1978; Slavin, 1979) is that although fibroblasts are distributed throughout the body, some areas of the body never contain adipocytes. This suggests that some local, as yet unknown, factor(s) is responsible for modifying fibrous connective tissue into immature adipose tissue. In recent studies (Hausman et al., 1981; Hausman, 1982a; Hausman and Martin, 1982) we have observed that the development of specific tissue components may modify fibrous connective tissue. For example, the downward and lateral growth of hair follicles into the rat dermis and hypodermis brings about the concentration and flattening of connective tissue cells; furthermore the development of immature adipose tissue in the rat hypodermis is limited to small areas immediately around fully descended hair follicles (Hausman et al., 1981). From this it would appear that the flattened and concentrated connective tissue cells developed into immature adipose tissue in the hypodermis. Developing hair follicles and sweat glands in the fetal pig (Hausman and Martin, 1982) and mammary gland ducts in the fetal rat (Hausman, 1982a), during their invasion of the connective tissue, also physically concentrate and flatten connective tissue cells; as before, immature adipose tissue is located around these tissue components. As hair follicles, sweat glands and mammary gland ducts are downgrowths of the epidermis (Ham and Cormack, 1979), when these components grow down into connective tissue some physical interaction with connective tissue cells is inevitable. The degree of interaction will be dependent on the size of the invasive tissue component and on the concentration and organization of the underlying connective tissue. The development of all exocrine and endocrine glands is characterized by epithelial downgrowth into underlying connective tissue (Ham and Cormack, 1979). During development, nerves must also grow into connective tissue. The extensive distribution of these various tissue components throughout the body could explain the extensive distribution of fetal adipose tissue.

1.2.2 Morphological correlations between fetal adipose tissue and tissue from growing animals

In fetal pigs the dorsal subcutaneous adipose tissue, at various anatomical locations, shows the same pattern of thickness as that for the backfat in older animals (Figure 1.2). This morphological characteristic is present before adipocyte lipid deposition occurs, which indicates that the difference in thickness can only be due to differences in the number of preadipocytes. Because subcutaneous adipocytes from various dorsal locations are the same size (Wood et al., 1975; Enser et al., 1976), the different thicknesses in backfat must be due to differences in cell number. The potential therefore for differences in adiposity due to anatomical site in swine backfat is present very early in development and is expressed in terms of preadipocyte number.

Figure 1.2 Subcutaneous tissue from three different regions of a 75-day-old pig fetus. Sections are (a) directly over the last three cervical vertebrae, (b) over the first and second ribs, and (c) over the first and second lumbar vertebrae. Note that the thinnest inner layer (I) is located in (c) and the thickest in (b). This pattern of thickness for the inner layer is identical to the pattern of thickness for backfat of growing and mature pigs. (O = outer layer; D = dermis; DP = dorsal-most point of the inner layer of subcutaneous tissue; M = muscle; CB = connective tissue border.) Paraffin sections stained with Picro Ponceau. × 14. (Reduced to 75% in reproduction)

Of the three layers of backfat in pigs, the middle layer is the thickest and it is the first layer to develop in the fetus (Hausman, 1978). The very early and substantial development of subcutaneous adipose tissue in both pig and human fetuses (Wassermann, 1965; Hausman, 1977) may expedite the extensive expansion of this depot in the growing pig and in man.

1.2.3 Cellularity of fetal adipose tissue

Fat cell clusters in the fetus contain cells that possess at least several lipid droplet (multilocular adipocytes). At some point in development, all adipocytes display a multilocular morphology. Multilocular cells become unilocular through the coalescence of many lipid droplets into one larger droplet (Figure 1.3). The conversion of multilocular to unilocular cells is complete at birth in bovine (Jenkinson et al., 1968) and human adipose tissue (Shaw, 1902). The process begins after birth in the pig (Mersmann et al., 1975), rat (Napolitano, 1963) and mouse (Slavin, 1979); in sheep the conversion takes place 2–3 weeks after birth and represents what is thought to be a transition of brown to white adipocytes, as revealed by electron microscopy (Gemmell et al., 1972).

Figure 1.3 Unilocular adipocyte development in a 3-day-old pig. Note the presence of multilocular cells (M) surrounding the unilocular cell (arrowed). Lipid droplets (L) increase in size until they eventually coalesce to form one large droplet. Paraffin section stained with hematoxylin and eosin. × 320. (Reduced to 50% in reproduction)

The multilocular nature of functional brown adipocytes (Cameron and Smith, 1964) has been cited as evidence that brown adipose tissue (BAT) is merely a prolonged immature state of developing white adipose tissue (WAT) (Shaw, 1902; Sheldon, 1924). Early investigators noted that despite the similar cell morphology, some adipose tissue in the fetus had a ‘glandular appearance’ (Hatai, 1902; Shaw, 1902; Bonnot, 1909; Shattock, 1909; Cramer, 1920; Sheldon, 1924). The various locations where ‘glandular adipose tissue’ was located in fetal man (Hatai, 1902; Shaw, 1902; Bonnot, 1909; Shattock, 1909; Cramer, 1920), dog (Bonnot, 1909), cat (Bonnot, 1909; Sheldon, 1924) and rodents (Cramer, 1920; Sheldon, 1924) were similar to the locations of BAT in the mouse (Cameron, 1975). In almost every instance, these locations in adult man and animals contained only unilocular adipocytes (Hatai, 1902; Shaw, 1902; Bonnot, 1909; Shattock, 1909; Cramer, 1920; Sheldon, 1924; Gemmell et al., 1972; Cameron, 1975).

Morphological, chemical and physiological evidence indicates that perirenal adipose tissue from adult sheep does not convert to BAT during lipid depletion resulting from cold-acclimatization (Cox et al., 1978). Unilocular adipocytes in BAT of young adult rats apparently do convert to brown adipocytes during cold-acclimatization (6°C) (Cameron and Smith, 1964). The situation in adult man is certainly unresolved.

A number of recent studies have evaluated quantitatively the adipocyte size and number in human (Hirsch and Knittle, 1970; Novak et al., 1971; Knittle, 1972; Dauncey and Gairdner, 1975; Hager et al., 1977; Knittle et al., 1977; Dunlop et al., 1978; Bonnet et al., 1979; Enzi et al., 1980), pig (Vodovar and Desnoyers, 1978; Desnoyers et al., 1980; Hausman, 1982b; Hausman and Martin, 1982; Hausman et al., 1983), rabbit (Klein and Jenkins, 1981) and rat (Hausman et al., 1980b) fetuses or newborns (Table 1.1). Cell size data clearly indicate that at birth human adipose tissue is remarkably more mature than adipose tissues from newborn pigs, rabbits and rats (Table 1.1). Consideration of site-to-site variation is also warranted since qualitative studies have indicated that temporal lags exist between the histogenesis of various depots within a species (Shaw, 1902; Bell, 1909; Napolitano, 1963; Hermans, 1973; Slavin, 1979). For example, cells from internal depots are generally smaller than subcutaneous adipocytes (Dauncey and Gairdner, 1975; Jung et al., 1978). Considerable regional variations in cell size are evident in human subcutaneous adipose tissue (Dauncey and Gairdner, 1975; Dunlop et al., 1978; Bonnet et al., 1979). Adipocytes grouped around hair follicles in the fetal pig are smaller (Hausman, 1982b) and develop later (Hausman, 1978) than cells in the rest of the outer layer of subcutaneous adipose tissue. ‘Hair follicle adipocyte lobules’ are anatomically distinct structures (Figure 1.4) and may be present in human subcutaneous tissues (Wassermann, 1965; Ham and Cormack, 1979). Thus, the extent of cell size heterogeneity is probably extensive and considerably underestimated.

TABLE 1.1.

Fat cell size and number in fetal or newborn animals and man

Figure 1.4 Hair follicle adipocyte lobules in the outer layer (O) of subcutaneous adipose tissue from a 110-day-old pig fetus. Note that the hair follicle lobules (arrowed) are distinct structures within the outer layer of tissue. Cells within these lobules are smaller than the rest of the subcutaneous cells. These data represent an example of heterogeneity within a depot (outer layer of subcutaneous tissue). (D = dermis; M = muscle; CF = separation of outer and inner layers of tissue.) Paraffin section stained with Picro Ponceau and hematoxylin. × 17. (Reduced to 50% in reproduction) From Hausman and Martin (1982) by permission of the American Society of Animal Science, publisher of the Journal of Animal Science

Estimates of total fat cell number (FCN) (Table 1.1) should be considered with much scepticism (Kirtland and Gurr, 1979; Bonnet, 1981a). Mean cell size and body fat content are the parameters used to estimate FCN. Body fat content can only be estimated in man and a variety of techniques are used (Gurr and Kirtland, 1978; Hager, 1981). Estimates of FCN derived from studies in which the possibility of depot cell size heterogeneity is ignored are therefore likely to be subject to considerable errors (Jung et al., 1978; Bonnet et al., 1979).

Estimated FCNs in the fetal pig are much higher than in human newborns (Table 1.1). For studies on fetal pigs, body fat content was determined chemically and mean cell size from various depots was used (Vodovar and Desnoyers, 1978; Desnoyers et al., 1980). An assumption that has to be made in such studies is that all the body lipid is contained in adipocytes. However, morphological, ultrastructural and biochemical evidence from several muscles in the fetal pig indicates that large quantities of lipid are present within the muscle and muscle cells (Campione al., 1981; Darnton et al., 1983). The FCN estimates in the fetal pig must therefore be overestimates to some, as yet unknown, extent.

In a recent study, adipose tissue cellularity was evaluated in 110-day-old fetuses from lean and obese sows (Hausman et al., 1983). Fetuses were obtained from Ossabaw (obese-feral) sows and from sows selected (for 16 generations) for high backfat (obese-domestic) and for low backfat (lean-domestic) thickness. Fat cell cluster concentrations were similar throughout the depth of the subcutaneous adipose tissue taken from both obese and lean fetuses. Adipocytes from obese fetuses were however slightly larger (domestic, 23 ± 0.22 μm, feral, 21.8 ± 0.26 μm) than cells from lean fetuses (20.7 ± 0.42 μm). Another study has demonstrated that similar levels of total body lipid are present in lean and obese fetuses (McNamara and Martin, 1982). Therefore, the development of the obese condition, in these pigs, takes place after birth and fetuses from obese sows represent excellent subjects for the study of the preobese state.

1.3 Cellularity of adipose tissue

1.3.1 Measurement of adipocyte size and number

The various methods of measuring fat cell size and FCN together with the advantages and disadvantages of each are reviewed and discussed elsewhere (Gurr and Kirtland, 1978; Kirtland and Gurr, 1979; Bonnet, 1981a). Electronic particle counting (Coulter Counter) and microscopic methods have been shown to have a similar inability to record true adipocyte hyperplasia. With both of these techniques adipocytes have to hypertrophy to the same extent before they are ‘countable’ and the length of time that the cell remains ‘uncountable’ is unknown. Experiments in which animals are given [³H]-thymidine and where label is followed into stromal cells or adipocytes provide more useful measures of true adipocyte hyperplasia (Greenwood and Hirsch, 1974; Gaven-Cogneville and Swierczewski, 1979; Klyde and Hirsch, 1979; Lemonnier, 1981).

1.3.2 Methods of expressing cell size data

Expressing cellularity data as adipocyte cell size distributions can be more informative than merely reporting a mean cell size (Allen, 1976; Allen et al., 1976; Hood, 1977, 1982). When several populations of cells are present, distribution patterns are necessary to an understanding of the cellular mechanisms underlying lipid accumulation (Allen, 1976; Allen et al., 1976; Hood, 1977, 1982). Cell volume distributions, for example, reveal the relative importance of specific cell populations in accounting for the total volume of the tissue (Mersmann et al., 1973; Allen et al., 1976; Etherton, 1980) (Figure 1.5). In several experiments, cell diameter distributions have indicated that a population of small cells (20–40 μm) represents 30% of the total cells. Volume distributions, however, indicate that the population of small cells makes an insignificant (< 1 %) contribution to the total volume occupied by the cells (Mersmann et al., 1973; Etherton, 1980). Cell diameter distributions have been reported in a multitude of studies on meat animals (Anderson and Kauffman, 1973; Hood and Allen, 1973, 1977; Mersmann et al., 1973; Allen, 1976; Allen et al., 1976; Vodovar and Desnoyers, 1978; Garbutt et al., 1979; Hood and Thornton, 1979, 1980; Desnoyers et al., 1980; Etherton, 1980; Etherton et al., 1981; Powell and Aberle, 1981; Robelin, 1981). In cattle and pigs at least, increasing adiposity is associated with a bimodal distribution of cell sizes (Figure 1.5). This has been interpreted as representing the recruitment of new cells to the adipocyte pool when the existing cells have reached a critical cell size (Allen, 1976; Allen et al., 1976; Hood, 1977, 1982).

Figure 1.5 Adipocyte volume and diameter distributions for pigs with different backfat thickness. (H × Y = 109-kg Hampshire × Yorkshire barrows; BF = average backfat thickness (cm); Market = unknown-history market-weight carcasses; Sows = unknown-history heavy sow carcasses.) Note that there are biphasic diameter distributions for the market pigs and sows. The increase in biphasic nature is associated with fatter pigs (increase in BF). Also demonstrated is the small contribution to total volume made by the population (or peak) of small cells (25–50 μm). Volume distributions are ‘monophasic’ in all groups of pigs. From Allen et al (1974) with permission from The American Meat Science Association

1.3.3 FCN and size in animals and man

Total FCN has been estimated in pigs (Anderson and Kauffman, 1973; Hood and Allen, 1977), cattle (Hood and Allen, 1973; Robelin, 1981), sheep (Hood and Thornton, 1979) and man (Hirsch and Knittle, 1970; Jung et al., 1978; Kirtland and Gurr, 1979) (Table 1.2). Cell number is clearly related to body weight since the smallest species (sheep) had the lowest number of cells and the largest species (cattle) had the highest number (Table 1.2). The inherent problems in estimating FCN have been discussed thoroughly (Gurr and Kirtland, 1978; Kirtland and Gurr, 1979; Bonnet, 1981a). In the study of meat animals (Allen et al., 1976), body fat can be determined by dissection and chemical analyses whereas only estimates can be made for human subjects. The complete and thorough approach used in the study of meat animal fat deposition (Allen et al., 1976) has resulted in a keen awareness of the extensive heterogeneity in adipose tissue cellularity that can be present within a depot and, between depots, breeds and species. The extensive site-to-site variation in cell size makes it imperative that cells from at least two or three depots are measured to assure that a given mean cell size is representative of an animal or group of animals. When this has been done, studies have shown that adiposity is a function of adipocyte hypertrophy rather than cellular hyperplasia (Anderson and Kauffman, 1973; Hood and Allen, 1973, 1977; Allen, 1976; Allen et al., 1976; Hood, 1977, 1982; Jung et al., 1978; Garbutt et al., 1979; Hood and Thornton, 1979, 1980; Kirtland and Gurr, 1979; Etherton, 1980; Hausman et al., 1980b; Etherton et al., 1981; Robelin, 1981). The positive relationship between cell size and degree of fatness was well documented in one study where muscular crossbred pigs were compared to fat pigs of another breed (Hood and Allen, 1977). A difference of 16% in the amount of extramuscular carcass fat was related to a larger volume of adipocytes in fat pigs that also had 20% fewer total adipocytes than the muscular pigs (Hood and Allen, 1977). When these animals were compared at similar fat-free carcass weights, the total numbers of fat cells were similar. These data suggest that in pig carcasses the total number of adipocytes is related to the fat-free carcass weight (Hood and Allen, 1977).

TABLE 1.2.

Fat cell number in adult pigs, cattle, sheep and man

1.3.4 Variations in cellularity between depots

Average adipocyte cell size in pigs, cattle and sheep usually decreases in the following order: omental and perirenal > subcutaneous > intermuscular > intramuscular (Allen et al., 1976; Hood, 1982). While gonadal and perirenal adipocytes are usually larger than subcutaneous cells in rodent adipose tissue (Krotkiewski and Björntorp, 1976; DiGirolamo and Mendlinger, 1971; Meade and Ashwell, 1980; Pallier et al., 1980; Johnson and Goldstein, 1981). In man, omental fat cells can be considerably smaller than subcutaneous cells (Gurr and Kirtland, 1978; Jung et al., 1978).

In cattle (Hood and Allen, 1973) and pigs (Carpenter et al., 1961; Lee and Kauffman, 1974) FCN makes an important contribution to lipid accumulation in the intramuscular depot. The amount of lipid in four bovine muscles was positively related to cell number and was not related to cell size (Hood and Allen, 1973). A comparison of the cellular growth of the epididymal pads of three species indicated hypertrophic growth in adult rats and hamsters in contrast to hyperplastic growth in adult guinea-pigs (DiGirolamo and Mendlinger, 1971). In the adult rat, the perirenal depot exhibits marked hyperplasia whereas the epididymal depot increases in mass by hypertrophy only (Bertrand et al., 1978). Transplantation studies (Meade et al., 1979; Meade and Ashwell, 1980) suggest that factors determined by location, such as blood supply (Meade and Ashwell, 1980) and nervous supply, could be responsible for site-to-site variation in cell size as opposed to any inherent differences between cells.

1.3.5 Variations in cellularity within depots

Considerable variations in cell size have been recorded within all depots of the guinea-pig, where the small cells (60 μm) were seen to be arranged in pockets that were surrounded by larger cells (173 μm) (Ashwell et al., 1975). Regional variations in cell size and local cellularity are also evident in pig (Anderson et al., 1972; Enser et al., 1976; Dauncey et al., 1981; Hausman and Martin, 1981) and human (Bonnet et al., 1979) subcutaneous adipose tissue. Further research is needed, however, to elucidate the factors that are responsible for these variations within depots.

1.3.6 Obesity

The relationship between adipose tissue cellularity and human obesity has been extensively reviewed and remains a controversial subject (Stern and Greenwood, 1974; Buffer and Allen, 1979; Kirtland and Gurr, 1979; Hausman et al., 1980a; Bonnet, 1981b). A keen interest in adipocyte cellularity and obesity was spawned by several studies which showed that early-onset obesity was associated with increased cell numbers, whereas only cell hypertrophy was associated with adult-onset obesity (Hirsch and Knittle, 1970; Salans et al., 1973). Several recent studies, involving large numbers of subjects, have demonstrated that increases in cell number can occur in both obese (Krotkiewski et al., 1982) and non-obese (Chumlea et al., 1981) adult subjects. Fat cell size increased with moderate degrees of obesity while cell number increased with more marked obesity (Krotkiewski et al., 1982) and some increase in FCN in severe obesity is a generally accepted phenomenon (Jung et al., 1978; Kirtland and Gurr, 1979; Bonnet, 1981b; Chumlea et al., 1981; Krotkiewski et al., 1982). However, one study (Jung et al., 1978) showed no relationship between the calculated number of fat cells and the age at onset of obesity, and no clear distinction can be made between types of adult obese patients on the basis of their total estimated fat cell numbers (Gurr et al., 1982). Therefore, ‘hypertrophic’ and ‘hyperplastic’ may not be two discrete forms of obesity (Kirtland and Gurr, 1979). As in animals, the predominant morphological aspect of moderate obesity in man is an increase in fat cell size (Kirtland and Gurr, 1979; Bonnet, 1981b).

The cellular aspects of nutritionally induced obesity in rodents have been reviewed extensively (Stern and Greenwood, 1974; Hood, 1977; Buffer and Allen, 1979; Lemonnier, 1981; Johnson and Francendese, 1984). High-fat or high-sucrose diets fed to adult rodents can increase both cell size and cell number (Hausman et al., 1980a; Lemonnier, 1981). The cellular response is depot-specific such that the mode of enlargement of a particular depot is optimized in response to the increased caloric pressure (Faust et al., 1978; Lemonnier, 1981). Feeding high-fat or high-sucrose diets for 5–6 months resulted in both hypertrophy and hyperplasia in the gonadal, perirenal and inguinal depots of three strains of rats (Faust et al., 1978).

Hormonally induced obesity is also associated with alterations in adipose cellularity. Insulin administration to rats promotes adipocyte hypertrophy in a manner which is, to a certain extent, depot-dependent (Salans et al., 1972; Krotkiewski and Björntorp, 1976). Feeding a progesterone-enriched diet (Pallier et al., 1980) or giving progesterone injections (Krotkiewski and Björntorp, 1976) caused cellular alterations that were species- and depot-dependent. These studies (Salans et al., 1972; Krotkiewski and Björntorp, 1976; Pallier et al., 1980) and others (Krotkiewski and Björntorp, 1976) suggest that changes in hormonal status can modify FCN and/or size in different adipose depots.

Morphological and Coulter Counter data indicate that adipocyte hypertrophy is the consistent and usually dominant explanation for adipose depot enlargement in inherited obesity in mice (Hellman et al., 1962, 1963a,b; Taljedal and Hellman, 1962; Johnson and Hirsch, 1972), rats (Johnson and Goldstein, 1981) and pigs (Steele et al., 1974; Hood and Allen, 1977; Etherton, 1980; Hausman and Martin, 1981). An increased FCN was also recorded in the obese Zucker rat (Johnson et al., 1971), the ob/ob mouse (Johnson and Hirsch, 1972) and obese pig (Steele et al., 1974).

Frequency distribution patterns of cell sizes have indicated ‘biphasic (bimodal) distributions’ in adipose tissue from ob/ob mice (Kaplan et al., 1976), obese Zucker rats (Johnson and Goldstein, 1981) and obese (Ossabaw) pigs (Etherton, 1980). The presence of biphasic rather than monophasic distributions has been taken to represent the recruitment of a group of new cells to the adipocyte population when existing cells reach a critical size (Allen, 1976; Allen et al., 1976; Hood, 1977, 1982). Obesity caused by overfeeding or age is also characterized by ‘biphasic distributions’ in cattle (Allen, 1976) and pigs (Etherton, 1980) (Figure 1.5). These studies do not however record a similar critical (limiting) adipocyte size. In longitudinal studies of obese (Ossabaw) pigs (Hausman and Martin, 1981) and ob/ob mice (Ashwell et al., 1975), histological data indicated two populations of cell sizes in young animals. The population of small cells was apparent in obese pigs long before a large and ‘limiting’ mean cell size was achieved (Hausman and Martin, 1981). Age-associated obesity in sheep was characterized by only a ‘monophasic’ cell size distribution even though cells up to 300 μm were measured (Hood and Thornton, 1979). Therefore, an environmental signal may still be needed for adipocyte hyperplasia despite the presence of very large cells.

1.4 Stromal–vascular aspects of adipose tissue

1.4.1 The collagen matrix

Connective tissue anatomy is distinct and easy to study in fetal adipose tissue because of the low lipid content. Connective tissue is organized into complete borders around fat cell clusters in the tissues of fetuses from pigs (Figure 1.6), sheep and cattle (Bell, 1909; Wensvoort, 1967; Hausman, 1977). Complete borders per se are not however obvious in the fetal adipose tissue of man (Wassermann, 1965), rats (Hausman, 1982a) and mice (Wassermann, 1965), but the orientation of connective tissue cells and fibers in these species permits a clear distinction to be made between adipose tissue and the surrounding connective tissue (Wassermann, 1965). In all these species the connective tissue-defined areas have space present which is not occupied by fat cell clusters (Figure 1.6). Therefore, the connective tissue lobule and the adipocyte lobule (one to several fat cell clusters) are not necessarily of the same size (Figure 1.6). The size of the connective tissue lobule is more dependent on anatomical location than is the size of the adipocyte lobule. For instance, in bovine fetuses connective tissue lobules are much larger in perirenal areas than in subcutaneous sites and, furthermore, adipocyte lobule size is not affected by location in bovine fetuses (Bell, 1909). The amount of adipocyte empty space in the connective tissue lobule is dependent on local fat cell cluster concentration and is independent of fat cell size (Wassermann, 1965; Wensvoort, 1967; Hausman, 1977). Essentially, there is no connective tissue in the interscapular BAT of rodents as revealed by electron microscopy (Napolitano and Fawcett, 1958). BAT probably represents a maximum with regard to local fat cell cluster concentration (Napolitano and Fawcett, 1958). Therefore, those depots that have the largest cells in the adult animals are characterized by a low local concentration of fat cell clusters in the fetus (e.g. perirenal in cattle (Bell, 1909)). The reverse situation is also true, being exemplified by BAT depots.

Figure 1.6 Connective tissue-defined lobules in subcutaneous adipose tissue of a 75-day-old pig fetus. Adipocyte clusters (A) are located in the center of the lobules (arrowed). Note the large area free of adipocyte clusters. Therefore, connective tissue lobules and adipocyte lobules are not the same size. Paraffin sections (10 μm) stained with (a) Picro Ponceau reagents and (b) hematoxylin and eosin. × 875. (Reduced to 50% in reproduction) From Hausman (1978) with permission from The American Meat Science Association

In the fetus, the local fat cell concentration is also correlated with the local concentration of capillaries (Wassermann, 1965; Hausman, 1978). In this regard, the inherent differences in vascularity from site to site are already evident in the fetus.

The size of the connective tissue border that surrounds the fat cell clusters is quite variable and is dependent on species and location within a given species. Little connective tissue is evident throughout the interscapular BAT of newborn rodents (Napolitano and Fawcett, 1958), whereas there is extensive connective tissue deposition in the subcutaneous depots of fetal cattle (Bell, 1909), pigs (Hausman, 1977) and rats (Hausman, 1982a). The connective tissue content of a depot or an area within a depot (Enser et al., 1976) may contribute to the regulation of cellular expression during depot enlargement. For instance, intramuscular depots in cattle and pigs are heavily infiltrated with connective tissue and these depots expand by hyperplasia (Allen et al., 1976). On the other hand, subcutaneous and perirenal depots contain less connective tissue and expand by cell hypertrophy (Allen et al., 1976). An alternative hypothesis suggests that mechanical pressure from muscle has resulted in the small cells that are seen in intramuscular depots (Waters, 1909). This latter concept is supported by work which demonstrates a relationship between the quantity of intramuscular lipid and the looseness of muscle fasciculi organization (Kauffman and Safanie, 1967). More recently we have demonstrated a relationship between the degree of collagen fiber organization and the presence of cells of typical fibroblast morphology (Hausman and Martin, 1981, 1982; Hausman et al., 1981; Hausman, 1982a; Hausman and Campion, 1982). Thus, a well-organized grouping of collagen fibers was associated with typical fibroblasts (polar, spindle-shaped basophilic cells), whereas cells with little basophilia, irregular shapes, no particular polarity and large pale-staining nuclei were located in unorganized collagen fibers. Furthermore, immature adipocytes were only present in the unorganized collagen matrix. Thus when the maturation or organization of the connective tissue matrix is delayed or prevented, conditions may result that are suitable for adipocyte development.

1.4.2 Blood vessels

The relationship between blood vessel formation and adipocyte development is reviewed elsewhere (Hausman et al., 1980a). WAT is regarded generally as a poorly vascularized tissue, especially when compared with BAT or skeletal muscle and, by contrast with BAT, the arterioles and capillaries of WAT exhibit no particular orientation (Gersh and Still, 1945; Fawcett, 1952). The capillary density per unit volume of rat subcutaneous adipose tissue has been estimated to be one-third of that in muscle (Gersh and Still, 1945). However, Gersh and Still assumed that the metabolic activity of a fat cell was confined to the cytoplasm around the lipid droplet. This cytoplasm was estimated to comprise only 2.4% of the total cell volume. Thus, if the ratio of the capillary surface to the volume of active cytoplasm is calculated, the blood supply to adipose tissue is probably richer than that to muscle (Gersh and Still, 1945).

Techniques utilizing ⁵¹Cr-tagged erythrocytes have demonstrated that the vascularity of the interscapular BAT of rats is four to six times higher than that of WAT (Hausberger and Widelitz, 1963). Similar results have been obtained using India ink-injected preparations from rats (Fawcett, 1952).

The capillary supply of mouse gonadal fat was found to be only 5% of that of subcutaneous fat when it was analyzed using radioactive microspheres (Meade and Ashwell, 1980). Although gonadal fat cells were much larger than subcutaneous fat cells from mice, transplantation studies indicated that it was the location of the fat cells rather than any intrinsic differences between the cells, that accounted for the difference in cell size between depots (Meade and Ashwell, 1980).

Traditional methodology indicated that adipose tissue blood flow per adipocyte increased with increased cell size in dogs (DiGirolamo et al., 1971), rabbits (DiGirolamo and Esposito, 1975) and man (Nielsen and Larsen, 1973). However more recent experiments, with radioactive microspheres, have demonstrated that a constant blood flow per adipocyte exists in several depots of adult rats, regardless of age or adiposity (Johnson and Francendese, 1984). These experiments also indicated concomitant changes in vasculature and FCN, since several depots exhibited hyperplasia in spite of constant blood flow per cell.

Blood flow (measured by microspheres) through the BAT of obese mice was one-half that of lean animals when non-shivering thermogenesis was induced by norepinephrine injections (Thurlby and Trayhurn, 1980). A similar study also showed that the obese Zucker rat had little BAT blood flow when stimulated with isoproterenol (Wickler et al., 1982). These studies, together with those described earlier, emphasize the importance of the role of blood vessels in adipose tissue accretion and also in the manifestation of obesity in animals.

We have studied the histochemical, ultrastructural and structural changes that occur in the blood vessels of developing WAT depots. Thus, in the immature subcutaneous adipose tissue of rats and pigs, capillary lumen diameters range between 4 and 10 μm (Hausman et al., 1980b; Hausman and Martin, 1981). Mature subcutaneous depots of both species, by contrast, contain capillaries with lumen diameters 2–4 μm (Hausman et al., 1980b; Hausman and Martin, 1981). The smaller capillaries in the mature tissue have fewer nuclei (per unit length) and thinner walls. The small lumen may make red blood cell transport physically difficult, which could result in a limiting supply of oxygen. Although such a limitation may favor lipogenesis and therefore accentuate fat cell size, the factor(s) responsible for the structural changes in the capillaries is not known.

1.4.3 Stromal cells

Separation of adipocytes from the stromal–vascular cells by digestion with collagenase has shown that one-half of the protein and 57% of the DNA in rat WAT was stromal–vascular in origin (Rodbell, 1964). More recent estimates put the stromal–vascular content at an even higher proportion of the total cell mass (Kirtland and Gurr, 1979). Several researchers have highlighted the significance of the stromal–vascular constituents of adipose tissue in the fetus (Wassermann, 1965), the growing animal (Liebelt, 1959) and starved–refed animals (Rakow et al., 1970). For instance, the growth of the lipid-free adipose depot mass follows a growth pattern similar to that of other body organs and is independent of lipid deposition (Liebelt, 1959). Obesity induced by high-fat diets is accompanied by a large increase in stromal–vascular DNA which returns to nearly preobese levels when animals are allowed to return to normal weights (Peckham et al., 1962). The significance of these characteristics of adipose tissue stromal–vascular constituents remains elusive.

Typically the stromal–vascular cells consist of macrophages, fibroblasts, pericytes and various types of blood cells. In some species (dog, man, mouse and rat), mast cells are a normal component of adipose tissue, being deployed along the blood vessels. Mast cells, although present in adipose tissue from young Ossabaw (obese) pigs, were absent from adipose tissue of Yorkshire (lean) pigs (Hausman and Martin, 1981). Obesity in rodents is usually associated with an elevated level of mast cells (Hausman et al., 1980b). Mast cell granules contain two important substances: histamine and heparin. Heparin stimulates lipoprotein lipase activity and enhances the adipose conversion of stromal cells of rat adipose tissue in vitro (Björntorp et al., 1980). Heparin released from mast cells may have a possible role in lipid metabolism but further work is needed to elucidate the exact role of mast cells in adipose tissue physiology.

Electron microscopic studies on cells that develop into adipocytes have described a wide variety of cellular ultrastructures (Napolitano, 1963; Desnoyers and Vodovar, 1974; Vodovar and Desnoyers, 1978; Iyama et al., 1979; Néchad and Barnard, 1979; Roth et al., 1981; Richardson et al., 1982). Preadipocytes have been characterized as fibroblast-like (Napolitano, 1963; Slavin, 1979), endothelial cells (Desnoyers and Vodovar, 1974; Vodovar and Desnoyers, 1978), pericytes (Iyama et al., 1979; Néchad and Barnard, 1979; Richardson et al., 1982) and macrophage-like (Roth et al., 1981). The extreme apparent variability in preadipocyte morphology may be associated with different rates of differentiation. One measure of differentiation may be the length of time between onset of cytoplasmic lipid and the onset of the multilocular-to-unilocular adipocyte conversion. In rodents, unilocular cells are evident 7–9 days after the onset of lipid deposition in preadipocyte cells (Napolitano, 1963; Slavin, 1979). Unilocular cells are present in pigs about 65 days after the onset of lipid deposition (Hausman, 1978). Because adipocyte differentiation would appear to be much faster in rodents than in pigs, the rapid rate in rodents may make it appear that fibroblasts convert directly to adipocytes with no intervening stages. In the fetal pig, the endothelial-like preadipocyte structures (Vodovar and Desnoyers, 1978) may be an intermediate stage of differentiation between fibroblasts and adipocytes.

Some stromal cells in mature depots have been identified as preadipocytes (Askew et al, 1981; Lemonnier, 1981; DeMartinis and Francendese, 1982). Preadipocytes in adult rat fat are ultrastructurally similar (Askew et al., 1981) to monocytes or immature macrophages (Ham and Cormack, 1979). However, using the presence of cytosolic lipid droplets as the sole evidence of adipocyte differentiation can only be considered as very speculative. An equally valid assumption is that these cells (Askew et al., 1981; DeMartinis and Francendese, 1982) are macrophages that have phagocytosed lipid. Quantitative data on these cells indicate that they are present in significant numbers in several depots and species (DeMartinis and Francendese, 1982).

The full range of cells common to mature red bone marrow have been located in immature (Wassermann, 1965; Hausman, 1977) and mature WAT (Tedeschi, 1965; Hausberger, 1966). A hypothesis was presented that explains the presence of the blood cells and the codevelopment of adipocytes and capillaries (Hausman et al., 1980a). In essence, capillaries and primitive blood cells are formed in a process of ‘blood island’ formation described originally by Maximow and Bloom in 1948. This concept would be compatible with a perivascular location of preadipocytes around newly formed capillaries and would explain the presence of a variety of stromal cell morphologies (Hausman et al., 1980a). BAT is particularly devoid of a wide variety of stromal cell morphologies (Napolitano and Fawcett, 1958; Dyer, 1960). The high local fat cell cluster concentration in developing BAT (Napolitano and Fawcett, 1958) indicates the possibility that most of the pre-existing precursor cells are used either as capillaries or as brown adipocytes. Therefore, there are few excess cells and few stromal cells.

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