Epididymal white adipose tissue after cold stress in rats I. Nonmitochondrial changes

JOURNAL

OF ULTRASTRUCTURE

AND

MOLECULAR

STRUCTURE

RESEARCH

101,

109-122 (1988)

Epididymal White Adipose Tissue after Cold Stress in Rats I. Nonmitochondrial

Changes

DRAGUTIN LON~AR, BJ~RN A. AFZELIUS, AND BARBARA CANNON University of Stockholm, The Wenner-Gren Institute, Biologihus

F3, S-10691 Stockholm, Sweden

Received November 29, 1988 Epididymal adipose tissue in the rat is generally considered to be “pure” white adipose tissue (WAT) with a characteristic structure and function. Previous studies in cats have, however, indicated that adipose tissue with the morphological appearance of WAT could be converted into a tissue with the morphological appearance of brown adipose tissue (BAT) by intermittent cold stress. The present electron microscopic and morphometric study describes the effect of intermittent cold stress on the epididymal WAT of young rats. The tissue volume decreased markedly as did the lipid content. The mitochondrial volume increased dramatically. The extracellular matrix was vastly reduced as was the thickness of the plasma membrane, and the number of gap junctions between adipocytes increased markedly. Indications of neoinnervation and neovascularization were observed. A great abundance of preadipocytes indicated proliferative activity of the endothelium. The low amount of lipid droplets and a relative abundance of smooth and rough endoplasmic reticulum, Golgi apparatus, and lysosomes in the epididymal WAT of cold-stressed rats gave the cells the morphological appearance of young adipocytes or preadipocytes, whereas the hypertrophic and hyperplastic mitochondria, the relative paucity of ribosomes on lipid droplet membranes, and the increased innervation and vascularization gave the cells the morphological characteristics of brown adipose tissue. o 1% Academic PRSS, hc.

Textbooks of histology generally distinguish between two types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT) (Greenwood and Johnson, 1983; Fawcett, 1986; Cormack, 1987). These two types differ in their anatomical location (Merklin, 1974; Afzelius, 1970; Hausman, 1987), function (Vernon and Clegg, 1985; Cannon and Nedergaard, 1982), and pathology (Bjorntorp and SjSStrom, 1985; Ricquier and Mory, 1984). Although these differences exist between the two types of tissue there are also some functional connections. For instance, during the perinatal period of large mammals such as cow, sheep, cat, dog, and man, the fat stores consist mainly of BAT, which later is replaced by WAT (Rasmussen, 1923; Afzelius, 1970; Hausman, 1987; Slavin, 1985). It has also been shown (Brtick, 1967; Gemmell and Alexander, 1978) that this apparent replacement of BAT with WAT could be retarded at low

ambient temperature. Our previous results (Loncar et al., 1986) showed that WAT in juvenile cats, which were exposed to cold stress, could take on the morphological appearance of adipose tissue from the newborn animals or the stimulated BAT in rodents. These experiments showed that severe cold stress in cats could cause changes which reversed adipose tissue morphologically to the perinatal stage. For small mammals, on the other hand, it has been considered that BAT and WAT are separate tissues and have separate pathways of development (Hausman, 1987; Barnard and Skala, 1970). The purpose of the present study was to investigate whether this phenomenon of reversion could be evoked not only for adipose tissue derived from BAT but also for WAT generally, regardless of its origin. We have thus exposed rats to cold stress and analyzed morphologically at the electron microscopic level their epididymal adipose 109 0889-1605/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.

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tissue. This fat pad is considered to be “pure” WAT (Wasserman and McDonald, 1963; Slavin, 1972) and to have a structure and function (accumulation and storage of lipids) that are always different from those of the heat-producing BAT (Smith, 1964; Smith and Hock, 1963). The results are described in two parts. The first, presented here, deals with all morphological changes except those in the mitochondria, which are presented in the second part (LonCar et al., in press). MATERIALS

AND METHODS

Eight male Sprague-Dawley rats 3 weeks old were caged individually. The control group (four animals) remained at room temperature (22°C). The experimental group was kept at a constant cold temperature (4°C) for I week. The following week. the animals from the experimental group were exposed twice daily to a temperature of -20°C for I hr (at 8 a.m. and 8 p.m.). On Day 14 @-week-old animals) all animals were anesthetized and transcardially perfused with physiological saline solution (37°C) and then with a fixative consisting of 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. After perfusion. small pieces of epididymal adipose tissue were dissected out and immersed in the same fixative overnight. The tissue pieces were rinsed in saline solution and then postfixed in I% osmium tetroxide in the same cacodylate buffer for 1 hr at 4°C. After dehydration in cold acetone and propylene oxide, the pieces were embedded in Epon 812. Sections (70 nm thick) were made on a Reichard Ultracut E, stained with uranyl acetate and lead citrate, and examined with a JEOL 100 S electron microscope. Morphometty

The following parameters were measured: (a) the numerical relationship between multilocular and unilocular adipocytes (b) the ratio of capillaries to adipocytes in sections (c) the ratio of pericytes to adipocytes in sections (d) the ratio of nerve fibers to adipocytes in sections (e) the number of connections between adipocytes (f) the diameter of adipose cells

---

AND CANNON (g) the thickness of the adipocyte plasma membrane (h) the volume ratio of cytoplasm to lipids per adipocyte (i) the volumetric density of nuclei tj) the volumetric density of mitochondria (k) the volumetric density of Golgi apparatus (I) the numerical density of free ribosomes, polysomes, and rough endoplasmic reticulum (m) the number of ribosomes associated with lipid membranes around lipid droplets. The diameter of adipose cells, the number of pericytes, and the number of capillaries per adipose cell were determined at the optical microscopic level using eyepieces with an inbuilt calibrator and a square net. Four pieces of epididymal adipose tissue were selected randomly from each animal. One-micrometer-thick sections were cut from each block and stained with toluidine blue. From these, sections to be studied were selected randomly. Assuming cell spherity, the diameter of the adipose cells was determined. From these data, 100 of the largest measured diameters were used for calculation of the mean maximum diameter. After cold stress, many of the cells had the nucleus situated al the center. We have also measured the mean maximal diameter of these cells. From these data we calculated the mean diameter as described above. For measurement of the data marked (d) to (m) we followed the procedures developed by Weibel and coworkers (Weibel and Bolender, 1973: Weibel, 1979) and described also in a previous paper (Loncar et al., 1986). For measurement of (d) and (e), we analyzed adipocytes and their interconnections, and their relationship to nerves, in ultrathin sections. We then inspected the circumference of 2000 adipocytes taken randomly from each group at a magnification of I*_IS 000. For these analyses, we examined only such adipocytes as were situated entirely within copper grid squares. For measurement of (h) and (i) electron micrographs were examined at a magnification of * 1000; for measurement (j) at x 8000; for (k) at * 20 000; and for (g). (I), and (m) at x 50 000. A computer program was used to collect and store primary data. as well as to make final computations and statis:ics (courtesy of Mr. S. Sundelin and Dr. A. Johansyen). Volumetric densities of nuclei, mitochondria, Golgi apparatus, and ribosomes refer to the cytoplasm of the adipose cells excluding lipid droplets. All data were subjected to statistical analysis using Student’s t test for differences between controls and cold-stressed animals.

-i-.-.--__-_-.-..-____

-II__.

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FIG. I. Epididymal white adipose tissue of a rat 5 weeks old. Adipocytes are filled with large droplets (L). A thin cytoplasmic rim is seen at the periphery of the cell. Intercellular space (I), capillaries CC). and nuclei of adipocytes (N). x 1900. FIG. 2. Epididymal white adipose tissue of a cold-stressed rat. The same magnification as Fig. 1, Adipocytes have centrally located, pale nuclei (N). Small lipid droplets (L) are scattered throughout the cytoplasm and adipocytes therefore have a multilocular appearance. The cytoplasm of adipocytes contains large. roundish mitochondria (M). The intercellular space seems largely devoid of intercellular matrix. Capillaries (C) and nerves (Ne). x 1900.

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‘1ABLE 1 EFFECT

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PARAMETERS OF RAT

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Control group mean t SD (A) (B) (C) (D) (E) (F) (G) (H) (I) (J) (K) (L)

(M)

Adipocytes with central nucleus” (%I Number of capillaries per adipocyte” Number of adipocytes per pericyte” Number of adipocytes per nerve fiber” Number of gap junctions per adipocyte Mean maximal adipocyte diametef’ (urn) Mean maximal diameter of adipocyte with central nucleus” (pm) Adipocyte plasma membrane (thickness) (A) Adipocyte surface area on section (pm’) (1) Cytoplasm (urn’) (II) Lipids (urn’) Mean area of adipocyte nucleus (km’) Volumetric density of adipocyte nucleus” Volumetric density of mitochondriad Volumetric density of Golgi apparatu? The numerical density of ribosomes” (N/urn”) (I) Polysomes (II) Free ribosomes (III) ER ribosomex Number of ribosomes per 1 urn of lipid membrane (N/urn)

0 0.37 i 0.04 250 + 83 20 ! 7 0.001” 54 ?I 6

-100 2 IO 21 I4 f 28 176 t 21 (8%) 1983 t- 173 (92%) 33 + 7 0. I8 + 0.04 0.14 ‘-+ 0.06 o.oo.Sh 61 i 44 48 z 31 (78%) 6 i 5 (10%) 7 t 9 (12%) 12 - 6

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Cold stress group mean 2 SD 89 0.81 ,, I.1 0.93 35

+ 6”** _+-O.l*** t 2*** z 0.3**” t o..(*** i 7*-*

I9 T 5 70 t io*** 277 i- 22*** I05 ” 30 (38%)*** 172 t 48 (62%)*** 40 I 8 0.38 t 0.07*** 0.58 i- O.Ol**” 0.6 t 0.1*** 192 _t 96 89 t 40 (46%) 15 i 12 (8%) 88 rf 21 (45%) 4 + 5’

u Measured on light microscopical section. ’ Very low value; see text for details. ’ Ribosomes are present only near mitochondria situated close to lipid membrane. ’ Refer to volume of cytoplasm of adipocytes excluding the lipid droplets ***P < 0.001

RESULTS

Our morphological examination of the epididymal adipose tissue from control rats 5 weeks old (Fig. 1) agreed with previous data (Fawcett, 1952; Sheldon, 1962; Wasserman and McDonald, 1963; Wasserman. 1965; Slavin, 1979, 1985, 1987). In the following text we describe data relevant to our experiment, but exclude data on mitochondria, which will be presented separately (LonCar et al., in press). After cold stress, the fresh epididymal adipose tissue became dark brown, mainly due to a substantial reduction in the volume of the adipocytes to 5% of their original volume (Table I) and a resulting relative increase in capillaries. After perfusion (see Materials and Methods), the tissue became paler but was still more brownish than per-

fused WAT of the control group, presumably due to an increased content of mitochondria (Table I; Fig. 2). Adipocytes

The cells in the control animals appeared roundish or polygonal and had a thin peripheral rim of cytoplasm (Fig. 1). The central part of the cell contained a single lipid droplet. Numerous smaller lipid droplets were also present in the peripheral cytoplasm; 92% of the adipocyte was occupied by lipids (Table I). The space between neighboring adipocytes contained capillaries, libroblasts, pericytes, and extracellular matrix (Fig. 1). Collagen microfibrils connected adipocytes and formed an irregular meshwork (Figs. 3 and 5). After analyzing the surface of more than 2000 adipocytes.

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we found only one direct contact between two adipocytes (Table I). The distribution of the capillary meshwork was similar to that in WAT from cats (LonEar et al., 1986). Pericytes around capillaries were very rare (Table I). Sympathetic nonmyelinated nerves were present between adipocytes as free axons or, more rarely, as bundles of axons surrounded by Schwann cells. Our results showed that about 5% of the adipocyte perimeter was in contact (directly or via a gap) with nerve fibers (Table I). After cold stress, the cells became polygonal, irregular in shape, and reduced in size. Their diameters depend on the amount of lipid remaining in the cell. Only about 10% of the adipocytes remained unilocular but they contributed markedly to the relatively large mean maximal diameter of adipocytes in the cold-stressed group (Table I). The cytoplasmic rim in the periphery of these unilocular cells showed the same morphological characteristics (Fig. 4) as the multilocular adipocytes (see below). We found the average diameter of adipocytes

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with the nucleus situated in the center to be only one-third of that of cells from the control group (Table I). The shrinkage of the adipocytes led to an increase of the intercellular spaces and to a separation of the cells from one another, except at areas of intercellular contacts (Figs. 2, 6, 11, and 12). These intercellular contacts had the appearance of a gap junction. Two types of gap junctions were found: flat gap junctions (Fig. 11) and push-button gap junctions (Fig. 12). Almost all adipocytes had intercellular contact with other adipocytes in the plane of the section and also had contact with nonmyelinated nerve fibers (Figs. 7 and 8). Capillaries were seen to be more abundant in these cold-stressed animals than in the control group (Fig. 2; Table I). The Plasma Membrane and Its Associated Structures

The plasma membrane in the control state appeared as a lOO-A-wide trilaminar structure. It was covered with a basement membrane consisting of an electron-lucid

FIG. 3. Cytoplasm of two adjacent adipocytes of a rat from the control group. Although the organelles were sparse, some adipocytes contain a well-developed rough endoplasmic reticulum (E). The plasma membrane is covered by a basement membrane (arrowheads). Intercellular space (star) contains matrix material. Mitochondria (M). x 22 000. FIG. 4. Cytoplasm of a moderately depleted adipocyte from a cold-stressed rat. Although the cell has retained its unilocular appearance and has one large central lipid droplet (L) and several small peripheral ones (I), it has an increased number of large, round, pale mitochondria (M) and a prominent Golgi apparatus (G).

x 22 ooo. FIG. 5. Intercellular space (star) between two adipocytes of a control rat. The space is filled with intercellular matrix, where collagen microtibrils are visible (arrow). Note the dark, enlarged mitochondria (M) in the adipocytes. x 27 000. FIG. 6. Intercellular space (star) between adipocytes of a cold-stressed rat. No visible intercellular matrix is present. The plasma membrane of the adipocytes is naked (open arrowhead) or covered with very diffuse basement membrane (solid arrowhead). In the intercellular space there are different chylomicron-like structures (Ch) and lamellar whorls (arrow). Note the numerous, round, pale mitochondria (M) in the cytoplasm of adipocytes. x 28 000. FIGS. 7 AND 8. Longitudinal (Fig. 7) and transverse sections (Fig. 8) through sympathetic nerve fibers between two adipocytes. Note two types of vesicles in the neuroplasm: dense core (arrowhead) and transparent types (arrow) of vesicles. x 35 000 and 42 000, respectively. FIG. 9. Tangential section through the cytoplasm of an adipocyte from the control group. Vesicles of different sizes, coated (VC) and noncoated (VN), are present. Note the cytoskeletal elements (arrows) between vesicles. X 84 000. FIG. 10. Tangential section through the cytoplasm of an adipocyte of a cold-stressed rat. Most vesicles appear noncoated (VN) and of uniform size. Mitochondria (M), extracellular space (star). X 89 000. FIGS. 11 AND 12. Flat gap junction (Fig. 11) and push-button gap junction (Fig. 12) in adipocytes from a cold-stressed rat. Intercellular space (star), lipid droplets (L), mitochondria (M). x 58 000.

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and an electron-dense part, with a total width of about 500 A (Fig. 3). Outside the basement membrane, an extracellular matrix with collagen microfibrils was visible (Figs. 3 and 5). Figure 9 shows the extensive microvesicular system connected to the plasma membrane. It consisted of vesicles of different sizes, both coated and noncoated. After cold stress, the plasma membrane became thinner, only about 70 A. The outer surface was naked or covered with a thin, irregular basement membrane (Figs. 4, 6, 11, and 12), which, in turn, was in contact with a few intercellular microfibrils. Tangential sections showed that the dominant noncoated vesicles after cold stress had a diameter of 70-100 nm (Fig. 10). Ribosomes and Endoplasmic Reticulum (ER) In control animals, the cytoplasm contained ribosomes connected to the ER and possibly also connected to the surface of the adipocyte by means of the cytoskeleton. However, membrane-bound ribosomes constituted only about 12% of those present in the cytoplasm of the adipocytes (Table I), although we found a few adipocytes with a well-developed rough ER (Fig. 3). Most ribosomes (88%) in a typical adipocyte appeared as free ribosomes or polysomes (Table I). The large central lipid droplet and smaller droplets in the peripheral cytoplasmic rim were surrounded by a special ER (Francke et al., 1987) (Fig. 13). Thin profiles of ER, about 50 nm wide, were connected to the cytoplasmic ER. Ribosomes, mainly in the form of polysomes, were scattered on the cytoplasmic side of the membrane of lipid droplets (Fig. 13, Table I).

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A threefold increase in the number of ribosomes per cytoplasmic surface (Table I) and a quantitative transfer of the ribosomes from the cytoplasmic side of the lipid membrane to the cytoplasmic ER (Fig. 14; Table I) were characteristic of cold-stressed WAC. The lipid membrane lost practically all its ribosomes, whereas short polysomes were present on lipid membranes near mitochondria situated close to lipid droplets. The cytoplasm of WAC contained cisternae of smooth ER (Fig. 14) which were markedly enlarged near a well-developed Golgi apparatus (Fig. 1.5). Other Elements A Golgi apparatus (GA) was also present in adipocytes from control animals but occupied only a small fraction of the cell (Table I). With its small size and with the relatively large cytoplasmic volume, the statistical probability of finding a Golgi apparatus was small. Cytoskeletal fibers (Fig. 9) were present below the plasma membrane, but we could not observe any filaments connected to the lipid surface, as described for avian adipocytes (Luckenbill and Cohen, 1966; Wood, 1967). The Golgi apparatus occupied 0.6% of the cytoplasmic volume after cold stress (Fig. 15; Table I) and was mainly situated in the peripheral cytoplasm. Various vesicles with a diameter of 50-300 nm, as well as lysosome-like structures, were present around the Golgi apparatus (Fig. 15). Chylomicron-like structures were seen as transparent vesicles, isolated or clustered, and with diameters of 0.25-l km (Fig. 6). Lamellar whorls of a similar size were present in the intercellular space (Fig. 6), as well as in the capillary lumen near the en-

FIG. 13. Specialized portion of endoplasmic reticulum (between arrowheads) which surrounds the lipid droplets (L) in adipocytes of a control rat. On the cytoplasmic side of this lipid droplet membrane, there are numerous ribosomes (arrow). x 124 000. FIG. 14. Endoplasmic reticulum (between arrowheads) around lipid droplets (L) in an adipocyte of a coldstressed rat. Membranes are devoid of ribosomes. x 130 000. FIG. 15. Well-developed Golgi apparatuses (G) in an adipocyte of a cold-stressed rat. Lipid droplets (L), mitochondria (M), lamellar whorls (arrow), intercellular space (star). x 38 000.

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dothelial membrane, or in the mitochondrial matrix (Fig. 15). DISCUSSION

The role of WAT as a lipid reserve is well known (Vernon and Clegg, 1985). Different types of experiments that demonstrate structural changes in WAT during lipid mobilization, fasting, or diabetes have been performed (Wasserman and McDonald, 1960; Sheldon, 1962; Williamson, 1964; Williamson and Lacy, 1965; Slavin, 1972; Carpentier et al., 1977). A light microscopy study by Cameron and Smith (1964) showed a slow decrease in the size of epididymal WAT upon cold treatment and Nedergaard and Cannon (1984) showed decreases in the lipid content of epididymal adipose tissue of hamsters after cold acclimation which decreased further during hibernation. Therriault and Mellin (1971) and Faust and Miller (1981), using tritiated thymidine incorporation, reported hyperplasia of epididymal WAT upon chronic cold exposure of rats. In our results, the epididymal adipose tissue after cold stress had lost much of its lipid. This is the main reason why the entire epididymal fat pad was reduced in size and showed a relative increase in nerve and capillary connections compared to the nonstressed tissue. Our data showing approxi-

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mately 5% of the adipocytes to be innervated in the control group are in agreement with data by Slavin and Ballard (1978) obtained by examination of serial sections with high-voltage electron microscopy. Results from the cold-treated group showed that practically every adipocyte in a section was in contact with a nerve fiber. This would mean that each adipocyte after cold stress was as richly innervated as is the brown adipocyte (Bargmann et al., 1968; Suter, 1969), probably resulting from neoinnervation. The dark brown appearance of fresh epididymal WAT after cold stress was an indication of an increased vascular network. As mentioned, the diminished volume of adipocytes was attributable mainly to an increased capillary/adipocyte ratio. It was reported earlier for brown and white adipose tissue that hyperplasia of the endothelial cells can be caused by cold stress (Bukowiecki et al., 1982, 1986), as well as by endothelial growth factors produced by the preadipose cells (Castellot et al., 1980). In the experiments reported here, all these factors were present: cold stress, and a significant increase of pericytes, which could produce these endothelial growth factors and which are the potential adipocyte precursors (Iyama et al., 1979; Cinti et al., 1984). We conclude that the more than two-

FIGS. 16 AND 17. Schematic representation of the morphological characteristics observed in the epididymal adipose tissue of control (Fig. 16) and cold-stressed (Fig. 17) rats. FIG. 16. Five-week-old rats have typical white adipose tissue in the epididymal region. These unilocular white adipocytes each have a centrally located lipid droplet (5). Smaller droplets (5) are present also in the peripheral cytoplasmic rim. Around the droplets are cistemae of endoplasmic reticulum (4), whose membranes bear numerous ribosomes on the cytoplasmic side. In the cytoplasm there are also dark, elongated nuclei (7) and small, cylindrical mitochondria with a dark matrix (6). The plasma membrane (1) is covered by a basement membrane (2). The intercellular space (3) contains an extracellular matrix with collagen microtibrils. FIG. 17. Epididymal adipose tissue in a cold-stressed rat. Adipocytes are markedly smaller, with centrally located nuclei (7). Small lipid droplets (5) are scattered throughout the cytoplasm, giving the cells a multilocular appearance. The surface of the lipid droplets is still covered by special endoplasmic reticulum (4) but is devoid of ribosomes. Very developed Golgi apparatus (9) and numerous, round mitochondria (6) with long straight cristae and pale matrix are present in the cytoplasm. The plasma membrane (1) is largely devoid of a basement membrane, and adjacent adipocytes make numerous intercellular contacts-gap junctions (8). The enlarged intercellular space (caused by a shrinkage of the adipocytes) (3) is devoid of matrix but consists of numerous chylornicron-like structures and lamellar whorls (3). Between the adipocytes, there are numerous nerves (10) and capillaries (11). The morphological appearance of the epididymal adipose tissue after cold stress resembles brown adipose tissue more than white.

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fold increase in vascularization per tissue unit was caused not only by a shrinkage of adipocytes but also by neovascularization. Numerous chylomicron-like structures and lamellar whorls, present in the intercellular space and in the luminal surface of the endothelial cells, were interpreted as signs of an intensive lipid traffic, similar to the situation during starvation (Wasserman and McDonald, 1960, 1963; Blanchette-Mackie and Scow, 1981b). Plasma membrane and structures related to it in adipocytes have been described under normal and experimental conditions with various techniques (Sheldon, 1962; Slavin, 1972, 1985; Carpentier et al., 1976, 1977; Chaplowski et al., 1983; Blanchette-Mackie and Scow, 1981a, b, 1982). Using freeze-fracture techniques, Carpentier et al. (1977) found an increased local concentration of intramembranous particles per unit area during lipolysis, i.e., that lipolysis caused a change in the component of the membrane. Our finding of a decreased thickness of the plasma membrane in cold-stressed animals is probably similar to the observation by Carpentier’s group. The basement membrane in coldexposed epididymal adipocytes was absent, or only poorly developed. This is interpreted as an inability of the extracellular matrix and collagen fibers to bind to adipocytes after exposure to cold, and the subsequent shrinkage of the adipocytes during delipidation. The adipocytes had probably lost much of their extracellular matrix, including the basement membrane, which is not tightly connected to the plasma membrane during delipidation (Slavin, 1972, 1985). Perhaps as a result of this loss of extracellular matrix, the plasma membrane could make new structural connections, which may be the reason for the increased number of gap junctions observed. With an increased amount of intercellular contacts, with a relatively low lipid content, and with a poorly developed basement membrane, the cold-stressed epididymal adipocytes resembled young adipocytes or preadipo-

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cytes (Loewenstein, 1979; Hiragun, 1985). Nerves and capillaries could more easily enter into the intercellular space and establish contacts with the surface of the adipocytes. Changes in the character of plasmalemmal vesicles represented another reorganization of the adipocytes during lipid mobilization and so did the increase in ER, Golgi apparatus, and lysosomes. As a result, the cells resembled young adipocytes also in these aspects (Iyama et al., 1979; Hausman and Richardson, 1982; Cinti et al., 1984, 1985). An increase in the number and a change in the structure of mitochondria (Loncar et al., in press), and an increase in the number of ribosomes and their detachment from lipid membranes surrounding the ER to the ER in the cytoplasm were also signs of a changed cell activity. Our previous paper (LonEar et al., 1986) showed similar morphological changes in cold-exposed perirenal WAT of cats. The difference in innervatioin per adipocyte in cold-exposed WAT of cats was more pronounced, whereas changes in the intercellular space were less so compared with the cold-stressed WAT of rats. On the other hand, mitochondrial changes in epididymal WAT of rat (LonEar et al., in press) were more dramatic than those in cat (LonCar et al., 1986). It would seem that perirenal WAT from cats could more easily achieve the ultrastructural characteristics of BAT, i.e., attain those characteristics which the tissue had in its perinatal period (Rasmussen, 1923; Afzelius, 1970), whereas the epididymal WAT of rats which has never had a BAT histology is less easily transformed into a BAT-like tissue (Hausman, 1987). In summary, our results have shown that the ultrastructure of the epididymal WAT of young rats exposed to cold stress changed markedly. In previous studies (Cameron and Smith, 1964; Therriault and Mellin, 1971; Faust and Miller, 1981), a milder cold stress was used and the results reported were consequently less pronounced. We report that under our conditions, the ultrastructure of the adipocytes

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resembled that of young adipocytes or preadipocytes, i.e., that cold stress caused a morphological reversion of the tissue which may be followed by an increased vascularization and innervation. The markedly increased mitochondriogenesis which was noted also resulted in the tissue morphologically resembling BAT. These changes are discussed in (LonEar et al., in press). This work was supported by grants ish Natural Science Research Council.

from

the Swed-

REFERENCES AFZELIUS, B. A. (1970) in LINDBERG, O., (Ed.), Brown Adipose Tissue, pp. l-31, American Elsevier, New York. BARGMANN, W., v. HEHN, G., AND LINDNER, E. (1968) Z. Zellforsch. Mikroskop. Anat. 85, 601413. BARNARD, T., AND SKALA, J. (1970) in LINDBERG, 0. (Ed.), Brown Adipose Tissue, pp. 33-72, American Elsevier, New York. BJ~RNTORP, P., AND SJ~STR~M, L. (1985) in CRYER, A., AND VAN, R. L. R. (Eds.), New Perspectives in Adipose Tissue: Structure, Function and Development, pp. 447-458, Butterworths, London. BLANCHETTE-MACKIE, E. J., AND Scow, R. 0. (198la) J. Ultrastruct. Res. 77, 277-294. BLANCHETTE-MACKIE, E. J., AND Scow, R. 0. (198lb) J. Ultrastruct. Res. 77, 295-318. BLANCHETTE-MACKIE, E. J., AND Scow, R. 0. (1982) Anat. Rec. 203, 205-219. BR&K, K. (1967) Naturwissenschaften 54, 155-162. BUKOWIECKI, L., COLLET, A. J., FOLLEA, N., C&JAY, G., AND JAHJAH, L. (1982) Amer. J. Physiol. 242, E353-E359. BUKOWIECKI, L. J., GELOEN, A., AND COLLET, A. J. (1986) Amer. J. Physiol. 250, C88O-C887. CAMERON, I. L., AND SMITH, R. E. (1964) J. Cell Biol. 23, 89-100. CANNON, B., AND NEDERGAARD, J. (1982) in JONES, C. T. (Ed.), The Biochemical Development of the Fetus and Neonate, pp. 697-730, Elsevier Biomedical Press, Amsterdam. CARPENTIER, J. L., PERRELET, A., AND ORCI, L. (1976) J. Lipid Res. 17, 335-342. CARPENTIER, J. L., PERRELET, A., AND ORCI, L. (1977) J. Cell Biol. 72, 104-117. CASTELLOT, J. J., KARNOVSKY, M. J., AND SPIEGELMAN, B. M. (1980) Proc. Nat/. Acad. Sci. USA 77, 6007-6011. CHAPLOWSKI, F. J., BERTRAND, B. K., PESSIN, J., OKA, Y., AND CZECH, M. P. (1983) Eur. J. Cell Biol. 32, 24-30. CINTI, S., CIGOLINI, M., BOSELLO, O., AND BJ~RNTORP, P. (1984) J. Submicrosc. Cytol. 16, 243-251.

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