SUBCUTANEOUS ADIPOSE TISSUE DEVELOPMENT IN YORKSHIRE (LEAN) AND OSSABAW (OBESE) PIGS

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1 SUBCUTANEOUS ADIPOSE TISSUE DEVELOPMENT IN YORKSHIRE (LEAN) AND OSSABAW (OBESE) PIGS G. J. Hausman and Roy J. Martin Summary University of Georgia 1, Athens Subcutaneous adipose tissue development was studied in Yorkshire (lean) and Ossabaw (obese) pigs. Three pigs of each breed were slaughtered at 1 to 2 (group 1), 14 to 20 (group 2), 56 to 70 (group 3) and 180 (group 4) days of age. Formalin and(or) Bouin's fixed paraffin sections were analyzed by various staining procedures. Fresh frozen sections were tested for lipid, esterase activity and glycogen. Fat cell size increased with age in the Ossabaw pigs. Fat cell size was similar for group 1 and 2 Yorkshire pigs. Transitional cells between spindle-shaped, basophilic cells and multilocular adipocytes were present in group I Yorkshire pigs. The transition was characterized by a loss of cytoplasmic basophilia and a change in nuclei size and shape. The nuclei of presumptive preadipocytes and small multilocular adipocytes contained one to three prominent nucleoli surrounded by an evenly dispersed, pale staining chromatin material. Cells with identical nuclear morphologies and little apparent cytoplasm were observed in adipose tissue of Ossabaw and Yorkshire pigs of all age groups. The frequency of multilocular adipocytes (percentage of the total adipocytes that were multilocular) decreased during the first 2 weeks of life in both breeds. In age group 3, multilocular adipocytes were more numerous in Ossabaw pigs than in Yorkshire pigs (20 vs < 1%). Capillary histological and histochemical changes were associated with adipocyte hypertrophy in the younger pigs of both breeds. (Key Words: Adipocytes, Capillaries, Histology, Histochemistry, Pig, Obesity.) Introduction growing pig has been determined (Anderson and Kauffman, 1973; Hood and Allen, 1973). Other components of adipose tissue in growing pigs have been studied (Mersmann et al., 1973, 1975). In the early stages of porcine adipose tissue development, there are large interstitial spaces between and around adipocytes (Moody et al., 1978). Substrate availability, and therefore adipocyte hypertrophy, may be affected by the nature of the blood vessels and connective tissue in these interstitial spaces. Changes in the stromal-vascular components of adipose tissue may precede and lead to changes in adipocyte size. A more complete understanding of adipocyte hypertrophy may be gained by studying the development of stromal-vascular components of adipose tissue. Cellular and metabolic differences between obese and lean strains of pigs have been reported (Tyrgstad et al., 1972; Steele et al., 1974; Powell and Aberle, 1975; Harbison et al., 1976; Steele and Frobish, 1976; Hood and Allen, 1977). When compared at either equal age or equal weight, percentage body fat was greater and muscle weight smaller in obese (Ossabaw) pigs than in lean pigs (Buhlinger et al., 1978). In an effort to clarify mechanisms that predispose the obese pig to greater body fat accumulation, histological and histochemical analyses of adipose tissue from growing lean and obese pigs were conducted. Materials and Methods Animals. Twelve Ossabaw (obese) pigs and 12 Yorkshire (lean) pigs were used. Three pigs of each breed were slaughtered by electrical stunning and exsanguination at 1 to 2 (group 1), 14 to 20 (group 2), 56 to 70 (group 3), and 180 (group 4) days of age. Samples of dorsal- The contribution of adipocyte number and size to adipose tissue accumulation in the most subcutaneous adipose tissue over the shoulders were removed immediately and prepared for histological and histochemical studies. 1 Dept. of Foods and Nutr., Dawson Hall. Histochemistry and Histology. Small pieces 1442 JOURNAL OF ANIMAL SCIENCE, Vol. 52, No. 6, 1981

2 ADIPOSE TISSUE DEVELOPMENT IN LEAN AND OBESE PIGS 1443 TABLE 1. BODY WEIGHTS, MULTILOCULAR ADIPOCYTE FREQUENCY (MULTILOCULAR ADIPOCYTE/TOTAL ADIPOCYTES) AND ADIPOCYTE SIZE OF GROWING LEAN AND OBESE PIGS Body weight, ga Multilocular adipocyte frequency, % Fat cell diameter, #m a Age group Obese Lean Obese Lean Obese Lean 1 (1 to 2 days) , b bc 2 (14 to 20 days) 3, , c de 3 (56 to 70 days) A d fg 4 (180 days) e hi avalues listed are means -+ SEM. b'c'd'e'f'g'h'ivertical and horizontal values with different subscripts differ (P<.O1). of the inner and outer layers of dorsal subcutaneous tissue from the shoulder region were fixed in either Bouin's fixative or 10% neutral buffered formalin and routinely processed into paraffin blocks. Other pieces were frozen in isopentane (cooled in liquid N2) and stored in an upright ultracold freezer (-60 C) until analyzed. Paraffin sections (5 to 10/lm) were stained with Harris Hematoxylin and eosin, the Periodic Acid Schiff reagents (Humason, 1972) and Harris Hematoxylin, and Picro Ponceu reagents (Humason, 1972) and Toluidine Blue (Humason, 1972). Air-dried, fresh frozen cryostat (-20 C) sections (10 to 30 Arm) were reacted for glycogen (Humason, 1972), lipid (with Oil Red O; Barka and Anderson, 1963) and nuclei (with Harris Hematoxylin). Fat Cell Size. Fat cell diameters were determined on lipid-stained cryostat sections and(or) paraffin sections according to the method of Sjostrom et al. (1971). One hundred fat cell diameters were measured for each breed at each age or age range (33 to 34 fat cell diameters were obtained for each pig). In age groups 2, 3 and 4, diameters were determined only for unilocular adipocytes. The frequency of multilocular adipocytes was determined by counting 500 adipocytes for each breed at each age or age range and recording the number that were multilocular in morphology (166 to 167 fat cells were counted from each pig). and 4. Adipocyte hypertrophy was minimal during the first 2 to 3 weeks of life in lean pigs, while fat cell size nearly tripled in the obese Results Body weights and fat cell diameters are shown in table 1. Obese pigs had larger (P<.01) fat cells than lean pigs in age groups 2, 3 Figure 1. Adipoeyte clusters in adipose tissue from a group 1 lean pig. Fresh frozen cryostat section (24 ~m) stained for lipid (Oil Red 0) and nuclei (Harris Hematoxylin) [X 180]. Note the adipocyte clusters (indicated by arrows) separated by interstitial tissues.

3 1444 HAUSMAN AND MARTIN Figure 3. P~.raffin sections (10 jam) of group 3 obese (A) and lean (B) pigs stained with the PAS reagents and Harris Hematoxylin IX 612]. Note that the cell membranes of the (A) obese unilocular (u) adipocytes are PAS-positive, whereas those of the lean adipocytes (B) are lightly stained and are therefore less apparent. Figure 2. Histochemical status of the interstitial tissue of adipose tissue from a group 1 obese pig. Paraffin sections (10 tim) stained for connective tissue (A) (Picro Ponceu) and with the PAS reagents (B) [X 180]. Adipocyte clusters (ac) are immediately surrounded by a PAS-positive substance that does not contain connective tissue fibers (f). group 1 pigs had lumen diameters of 4 to 10 gm, with high concentrations of nuclei within and associated with the vessel walls (figure 4a). Capillaries of this nature were associated with adipocytes less than 30 ~m in diameter. Adipocytes larger than 30 /~m in diameter were associated with capillaries that had lumen diameters of 2 to 4 /~m and had few nuclei within or associated with the vessel walls (figure 4b). Blood vessel walls in PAS-stained sections were outlined in group 1, 2 and 3 lean pigs. In most instances, blood vessel walls were not outlined in group 1, 2 and 3 obese pigs. In group 4 lean and obese pigs, blood vessel walls were not stained. The PAS reaction around blood vessels indicates the presence of the endothelial basement membrane. Basement membranes around blood vessels from obese pigs may have been very thick and continuous with adipocyte basement membranes. This pigs. Adipocyte diameters increased with age (P<.01). Interstitial Tissue Histocbemistry. The subcutaneous adipose tissue of group 1 pigs was composed of many adipocyte clusters separated by interstitial tissue (IT) (figure 1). The actual amount of IT was greatest in group 1 pigs and decreased continually with increasing animal age. In group 1 lean pigs, the IT was composed primarily of connective tissue fibers that were present as either a network of small unorganized fibers or large bundles. In the obese pigs in group 1, areas immediately around the fat cell clusters were composed of a Periodic Acid Schiff (PAS) positive substance that did not contain connective tissue fibers (figure 2). Adipocyte membranes from obese pigs in groups 2 and 3 were PAS positive, while lean pigs adipocytes were negative (figure 3). Mast cells were present in the IT of obese pigs but were never observed in the lean pigs. Blood Vessel Morphology. Most capillaries in

4 ADIPOSE TISSUE DEVELOPMENT IN LEAN AND OBESE PIGS 1445 Figure 4. Changes in capillary morphology associated with increased adipocyte size. PAS- and Harris Hematoxylin-stained paraffin sections (10 ~tm) from a group 1 lean pig (A) and a group 2 obese pig (B) [X 612]. Capillaries (c) associated with small adipocytes (a) have wider lumens and more associated nuclei than do capillaries (c) associated with larger adipocytes (u). would account for the apparent lack of blood vessel definition in sections from obese pigs. Presumptive Preadipocytes. In group 1 lean pigs, there were numerous lipid-free cells with little apparent cytoplasm. These cells possessed nuclear morphologies identical to those of nuclei of small multilocular adipocytes (figure 5a,b). These nuclei were 6.5 to 9.0 /am in diameter and contained one to three prominent nucleoli surrounded by highly dispersed chromatin. This nuclear morphology was the most distinguishing characteristic of small multilocular adipocytes and preadipocytes. Apparent fibroblasts contained a prominent basophilic cytoplasm with elongated nuclei that had one to two small nucleoli and densely stained chromatin material (figure 5c). There were areas where morphologically transitional cells between fibroblasts and preadipocytes were located (figure 5c, d). These areas contained unorganized fragments of connective tissue fiber. As adipocytes hypertrophied, nuclei contained less prominent and smaller nucleoli surrounded by a more condensed chromatin (figure 5b). The major portion of the cytoplasm was located primarily on either side of the nucleus and was basophilic. The smallest multilocular adipocytes were generally in groups of three or more and were associated with capillaries. Lipid-free presumptive preadipocytes were also present in pigs in groups 2, 3 and 4 (figure 6). At a given age, the proportion of total adipocytes that were preadipocytes was similar to the proportion of total adipocytes that were multilocular. Areas where apparent fibroblasts were assuming altered morphologies were present in adipose tissue from pigs in age groups 2, 3 and 4 (figure 7). Multilocular Adipocytes. The proportion of total adipocytes in each age group that were multilocular is shown in table 1. The largest multilocular adipocytes measured were 30 to 33 ~m in diameter. Two groups of adipocytes were present in only group 2 and 3 obese pigs. In these pigs, the multilocular adipocytes were present as small groups surrounded by unilocular adipocytes (figure 8a). In areas where large variation in adipocyte size was evident, the adipocytes located closest to connective tissue were always largest (figure 8b). Discussion The cellularity of adipose tissues from lean and obese pigs was different at every age analyzed. The large size of adipocytes from Ossabaw pigs in age groups 2, 3 and 4 is a common characteristic of adipose tissue from obese pigs (Allen, 1976) and obese rodents (Johnson and Hirsch, 1972). The small size of adipocytes from the youngest Ossabaw pigs is similar to findings reported for young Large White and Pietrain pigs (Moody et al., 1978). The small size at birth of the Pietrain, Large White (Moody et al., 1978) and Ossabaw pigs (present study) in relation to the Yorkshire pigs (present study) may be associated with an initial small fat cell size followed by significant hypertrophy (present study; Moody et al., 1978). The development of fetal pig adipose tissue is characterized by a slow but steady increase in fat cell size and number (Vodovar and Desnoyers, 1978). The thickness of backfat

5 1446 HAUSMAN AND MARTIN Figure 5. Variations in nuclear and cytoplasmic morphologies of developing adipocytes in adipose tissue from a group 1 lean pig. Paraffin sections (10 tim) stained with Toluidine Blue [X 612]. Note the nuclear morphologies of lipid-free preadipocytes (p) and the similar nuclei located in small multilocular adipocytes (m). Cells transitional (t) between typical fibroblasts (f) and preadipocyte and small adipocytes are also present. Nuclei of larger adipocytes (a) are darkly stained and have less prominent nuclei.

6 ADIPOSE TISSUE DEVELOPMENT IN LEAN AND OBESE PIGS 1447 from newborn and late fetal pigs is positively correlated with body weight and number of lipid-containing adipocytes (Hausman, 1978). Therefore, a smaller birth weight would be Figure 8. (A) A PAS-stained paraffin section (10 ~m) of adipose tissue from a group 2 obese pig [X 180]. Note the clusters of multilocular adipocytes (indicated by arrows) surrounded by larger unilocular adipocytes. (B) A Hematoxylin- and Eosin-stained section (10 ~m) of adipose tissue from a group 3 lean pig [X 180]. Note that the larger adipocytes (a) are located adjacent to the connective tissue fasiculi (f). Figure 7. Cellular changes around a connective tissue fasciuli in adipose tissue from a group 3 obese pig. A paraffin section stained with Toluidine Blue, A [X 180], B [X 720]. Large, spindle-shaped, basophilic cells (arrows) are located directly in the fasiculi, whereas ceils with altered morphologies (a) are located adjacent to the fasiculi. The altered cells have less basophilia and are more irregular in shape and size. associated with an immature adipose tissue in terms of smaller size and number of adipocytes. To assimilate the large amounts of dietary lipid (maternal milk), the Ossabaw pig must call upon a greater percentage of the existing adipocytes than does the larger Yorkshire pigs. The significant number of mutilocular adipocytes in the obese pigs in age group 3 indicates two populations of adipocytes. Coulter Counting techniques have demonstated biphasic adipocyte distribution in extremely fat cattle and pigs (Allen, 1976). For technical reasons, adipocytes smaller than 25 /am are not measured by Coulter Counter techniques. Therefore, these studies do not indicate at what time the population of small adipocytes (in biphasic d i s t r i b u t i o n ) w a s formed. The higher frequency of multilocular adipocytes in obese pigs than in lean pigs indicates several possibilities. Some of the adipocytes in pigs in age group 1 may grow at a much slower rate and take as long as 70 or more days to become Figure 6. Lipid-free preadipocytes (indicated by arrows) in adipose tissue from a group 2 obese pig: A paraffin section (10 tim) stained with Toluidine Blue [X 7201.

7 1448 HAUSMAN AND MARTIN unilocular. Alternatively, all age group 1 adipocytes may grow at similar rates to a limiting size, at which point a second population of adipocytes is formed. Feeding a high fat diet to mature rats results in de novo increase in adipocyte number (Klyde and Hirsch, 1979). Adipocytes may form in young pigs in direct response to the fat content of the milk diet. Therefore, the second population of adipocytes in the obese pigs may be formed at a relatively young age. This study has demonstrated that fibroblastlike cells (spindle-shaped, basophilic cells) may represent a source of presumptive adipocytes for the hyperplastic phase of adipose tissue development in young pigs. Presumptive preadipocytes in fetal porcine adipose tissue morphologically do not resemble fibroblasts at the electron (Desnoyers and Vodovar, 1974) and light microscopic levels (Hausman, 1978). This apparent dichotomy of precursor cell types in porcine adipocytes may be due to dietary influences. The stimulus for the conversion of fibroblast-like cells to adipocytes may be dietary lipid (Klyde and Hirsch, 1979). The level of blood-borne lipids in the fetal pig may be low in relation to that in the postnatal, suckling pig. Fibroblast-like cells may not convert to adipocytes in the fetal pig because the necessary stimulus is limiting. The categorization of fibroblast-like cells was made only on morphoiogy in this study. The presence of apparent preadipocytes in pigs of all age groups is in agreement with in vitro data obtained on rats and humans (Roncari and Van, 1978). The cellular transformation evident around connective tissue fasiculi in pigs of all age groups provides tentative evidence that these fasiculi represent preadipocyte reservoirs. However, preadipocytes and multilocular and small unilocular adipocytes were always surrounded by large unilocular adipocytes. Possibly, the transformed ceils along the connective tissue fasiculi migrated into areas between adipocytes where further differentiation took place. Adipocyte hypertrophy was associated with a decrease in capillary lumen diameter (from 4-10 to 2-4 /am). The smaller capillaries may slow red blood cell flow and therefore decrease substrate availability. The platueau in fat cell size around 30/am in the young lean pigs may have been coupled with the capillary changes. Adipocytes may hypertrophy rapidly up to 30 to 40 /am, at which point they may physically decrease capillary lumen diameters, thereby slowing the rate of adipocyte hypertrophy. Mast cells contain two physiologically active substances, histamine and heparin (Ham, 1969). Heparin may activate lipoprotein lipase, stimulate its production or act as a cofactor with it (Ham, 1969). Lipoprotein lipase regulates the rate-limiting step in triglyceride assimilation by adipocytes. Heparin in the mast cells of the obese animal (Ossabaw) may enhance lipoprotein lipase production. An increased enzyme activity would result in a greater capacity to deposit lipid. Lipoprotein lipase activity in adipose tissue was reported to be higher in obese pigs (Minnesota No. 1) than in lean pigs (Allen et al., 1976). Further studies are needed to determine the exact role of mast cells in adipose tissue metabolism. Capillaries were not as obvious in PAS-stained sections from obese pigs as they were in PAS sections from lean pigs in group 1, because a PAS-positive material in the interstitial spaces of adipose tissue from group 1 obese pigs made capillaries less apparent. This material may represent an expanded and abundant capillary basal lamina. The high carbohydrate content of this material may allow greater substrate flux between capillary and adipocyte than would the more organized connective tissue fibers in the interstitial spaces of lean pigs. Glucose uptake and utilization by adipose tissue of obese pigs is twice as high as that by adipose tissue of lean pigs (Martin and Herbein, 1976). The presence of mast cells in the obese pigs may also enhance substrate flow, since mast cell components such as histamine and heparin increase vascular permeability. This report establishes the occurrence of specific cellular, vascular and interstitial cytochemical and structural modifications during the development of porcine adipose tissue. An unusual interstitial histochemistry was evident during the early stages of development of obese adipose tissue. Obese adipose tissue cellular development was characterized by several populations of cells and a large unilocular adipocyte size. The cellular characteristics of developing adipose tissue for a given breed may reflect degree of adipose tissue maturity at birth, which may depend on birth weight or maturation of the endocrine system. Literature Cited Allen, C. E Cellularity of adipose tissue in meat animals. Fed. Proc. 35:2302.

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