The ultrastructural differentiation of brown adipose tissue in the rat

© 1969 by Academic Press, Inc.

J. ULTRASTRUCTURE RESEARCH 29, 311-332 (1969)

311

The Ultrastructural Differentiation of Brown Adipose Tissue in the Rat TUDOR BARNARD

The Wenner-Gren Institute, Stockholm, Sweden Received May 16, 1969 The development of interscapular brown adipose tissue has been observed in rats from 6 days ante partum to some days after birth. The most obvious alterations prior to birth were increases in cell volume, triglycerides, and glycogen stores as well as considerable growth of the chondriome. There was a marked differentiation of mitochondrial structure, including the formation of large intramitochondrial dense granules. At birth there was a rapid decrease in amount of glycogen and temporarily a depletion of stored triglycerides, simultaneously with increased numbers of autophagic vacuoles. The large intramitochondrial dense granules disappeared and chondriome growth was rapid during this time. In the days after birth the chondriome volume in the cells appeared to increase still further, and the triglyceride droplet fractional volume in the tissue also continued to grow. Evidence for the hypothesis that capillary endothelial cells are a major type of stem cell for adipocytes in brown adipose tissue, is discussed. During the last few years there has been a rapid increase in research into the physiological and biochemical mechanisms involved in nonshivering thermogenesis, and it has clearly been shown that brown adipose tissue is a specialized site of this heat production (3, 6, 11, 16). On the other hand, white adipose tissue serves mainly as a store for triglycerides. While most of the work on BAT 1 has been done using coldadapted or neonatal animals, some studies have also been made on ontogenetic aspects of the phenomenon (2, 7). While adequate descriptions have been made of the structure of differentiated and cold-adapted BAT (10, 16), the structural development of this tissue is unsatisfactorily documented, especially in view of the controversy over the ontogenetic relationship of BAT to WAT. The importance of such information for the proper interpretation of biochemical results has recently been highlighted (13). While the functional responses and ultrastructural changes of adipocytes in these tissues to 1 Abbreviations used: BAT, brown adipose tissue; ISBAT, interscapular brown adipose tissue; WAT, white adipose tissue; a.p., ante partum; p.p., post partum; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; OsO4, osmium tetroxide; PO4, phospate.

312

BARNARD

lowered a m b i e n t temperature or to starvation have been clearly shown to be distinct, it is n o t k n o w n whether these differences are intrinsic to the adipocytes or are m a i n l y a result of different cellular m i c r o e n v i r o n m e n t s (4, 9). I n the rat, b r o w n fat a n l a g e n first appear a b o u t 7 days ante p a r t u m a n d consist of loose n e t w o r k of capillaries a n d stellate cells, in which the first triglyceride droplets become visible a day later (14). Such observations do n o t seem to correlate with a short ultrastructural description of B A T development by N a p o l i t a n o (8). His conclusion that B A T is a tissue ontogenetically distinct f r o m W A T , was m a i n l y based u p o n the ultrastructural response of W A T adipocytes to extreme fat depletion. However, from observations made o n h u m a n a n d rat BAT, S i m o n came to the opposite conclusion that B A T is merely a temporarily arrested stage during the develo p m e n t of W A T (15). It was therefore a p p a r e n t that a reinvestigation of B A T development was necessary. I n this investigation, observations have been made o n a series of carefully times stages during the development of I S B A T of pre- a n d n e o n a t a l rats. The results amplify a n d correct previous observations, a n d they help to define the further ex-

Key to abbreviations Ad Av Ca EER En GI Go Mi

adipocyte autophagic vacuole capillary extracapillary erythrocyte endothelial cell glycogen Golgi body mitochondrion

N Nu Pc RER SER Tr V

nucleus nucleolus pericapillary cell rough endoplasmic reticulum smooth endoplasmic reticulum triglyceride droplet vesicle

Fla. 1. Six days a.p. The majority of the ceils have a dense granular cytoplasm and mitochondria with electron lucid spaces in the matrix. At top left is a cell with more compact mitochondria, x 10,000. Fla. 2. Six days a.p. Notice the micropinocytotic vesicles at the cell surface (~), and vesicles of similar size clustered around and confluent with (~-->)larger vesicles, perhaps elements of SER Free and attached ribosomes are numerous, x 60,000. FIG. 3. Five days a.p. Small electron lucid triglyceride droplets are now present in most cells. Notice the size and number of the intramitochondrial dense granules (~-->).x 10,000. FIG. 4. Three days a.p. The cytoplasm has increased in volume relative to the nuclei. The mitochondria now have more regularly arranged cristae, and the intramitochondrial granules (F-->)are larger. The "empty" space at left represents a glycogen field, but the glycogen has not been visualized dearly on this occasion. The triglyceride droplet at left center is bounded by a clearly visible osmiophilic line (~---~).x 10,000. FIG. 5. A detailed view of the osmiophilic line bounding a triglyceride droplet. A comparison of the triple-layered structures of the enlarged osmiophilic line (~->) and the cytoplasmic membrane (-+) suggests that the triglyceride droplet is bounded by a unit membrane, x 180,000. FIG. 6. In addition to the osmiophilic line at the triglyceride droplet boundary @->), numerous elements of SER are located close to the droplet (-+). Notice also the large intramitochondrial dense granules, x 27,500. FIG. 7. One day a.p. The cytoplasm is now filled with mitochondria, triglyceride droplets, and glycogen. The intramitochondrial dense granules (~-->)are very prominent in most cells. Three capillaries are also visible, x 10,000.

~)~ ..... ~i~!~i'~'~ ii!'~

316

BARNARD

p e r i m e n t s required to clearly d e m o n s t r a t e the origin of the stem cells of B A T a n d W A T . F u r t h e r m o r e they establish a definite structural p a t t e r n against which physiological a n d b i o c h e m i c a l results on rat B A T d e v e l o p m e n t m a y be matched.

METHODS Pregnant rats, whose time of mating was known to within a few hours, and neonatal litters were obtained from Anticimex, Viby, Sweden. In this stain parturition occurred on day 23 after fertilization. The stages observed are reckoned from the day on which parturition occurred or was calculated to occur. Observations were made on both dorsal cervical and interscapular brown adipose tissue. As no important differences were apparent, only the results on ISBAT are mentioned. Reproducible fixation of good quality was not at first obtained. This was finally achieved by closely adhering to the following procedure. Pregnant rats were anesthetized by subcutaneous injection of Nembutal (Abbott, 30 mg/kg body weight), as a 3 % solution. Interscapular pads were rapidly removed from the young rats and gently sliced into very small pieces in ice-cold 2 % OsO~ in 0.135 m PO~ buffer, p H 7.2 + 0.1. When all pads had been diced the fixative was replaced with an excess ( ~ 4 0 ml/g tissue) of fresh. During fixation the vessels were continuously rotated. After 2 hours at 4°C the tissue was washed for 15 min in PO 4 buffer made isotonic with the fixative using sucrose, dehydrated, and embedded in Epon 812 using specially controlled resins (Ladd Research, Inc.) to give maximally reproducible cutting characteristics. Sections were cut on an LKB Ultrotome 1 at 600-800 A with glass knives broken on an LKB Knifemaker. After staining with lead citrate or lead citrate and uranyl acetate, they were examined in an Elmiskop l a at various magnifications.

RESULTS A t 6 days a.p., undifferentiated cells p r e d o m i n a t e d in these p r e p a r a t i o n s (Fig. 1). T h e y were characterized b y their irregular shape, low c y t o p l a s m - t o - n u c l e u s ratio, a n d g r a n u l a r cytoplasm, which at higher magnifications was seen to be p a c k e d with n u m e r o u s free polysomes. The nuclei contained one o r two p r o m i n e n t nucleoli, which were frequently l o c a t e d at the nuclear periphery. The m o d e r a t e l y a b u n d a n t r o u g h e n d o p l a s m i c reticulum h a d dilated lacunae, a n d was one of the m o s t typical u l t r a s t r u c t u r a l features of these cells. There were occasional G o l g i bodies as well as single-membrane b o u n d electron dense inclusions, which were p r o b a b l y l y s o s o m a l

FIG. 8. A few hours after birth. The substrate stores are much depleted. The intramitochondrial dense granules (~>) are numerous but of "normal" size (~ 300 A diameter). Another type ofmitochondrial inclusion is seen at ~-->. The impression is that the chondriome has grown from 1 day a.p., but the complex volume alterations in the cytoplasm might be deceiving. Autophagic vacuoles are common only at this stage. Virtually no RER is visible here, but there is some SER, mainly as small vesicles, x 17,500.

318

BARNARD

precursors. The number of cytoplasmic vesicles was variable; when present in relatively large numbers, rosette-like structures were sometimes also seen, as if vesicles were fusing together (Fig. 2). Similar structures have been reported in adipocytes from W A T of fasted rats (18). Triglyceride droplets were almost entirely absent, those which were present being only a few microns across and electron lucid. A few cells contained mitochondria which were small and compact, most cells had mitochondria which were larger and which frequently contained electron-lucid spaces in the matrix. Such spaces might be fixation artifacts but have been reported as associated with mitochondrial D N A in many developing tissues (11): in rat ISBAT, in contrast to hamster (unpublished observations), mitochondrial D N A was never certainly identified. In both types of mitochondria there were rather few cristae. In cells containing triglyceride droplets the mitochondria had dense granules in their matrices. By 5 days a.p., the cellular organization of the tissue had become fairly compact (Fig. 3). The cells themselves were, however, little more differentiated and the cytoplasm-to-nucleus ratio was still low. Most of them contained several triglyceride droplets. The R E R was less noticeable, while especially at the cell periphery one or two Golgi bodies, sometimes of large size, might be seen. Mitochondrial ultrastructure was not obviously altered. At higher magnifications, glycogen particles as well as polysomes were identified in the cytoplasm. After 2 more days of development (3 a.p.) more obvious changes had occurred in the cells (Fig. 4). They had become packed together so that their profiles were polygonal, and the cytoplasm-to-nucleus ratio was markedly increased. A layer of ribosomelike particles was clustered along the inner margin of the nuclear periphery, while most of the nucleoli had returned to a central location. Larger stores of both glycogen and triglycerides had accumulated. Frequently, the electron-lucid triglyceride droplet profiles were bounded by an electron-dense line, but it has not yet definitely been resolved whether this line represented a unit membrane or merely a layer of osmiophilic groups oriented at the lipid/cytoplasm interface. The former interpretation is suggested by images such as the one shown in Fig. 5. Smallish lacunae of SER became more prominent in the cytoplasm, and some of these were frequently located FIG. 9. Four days p.p. The triglyceride droplets have increased enormously and occupy about half the cytoplasm volume. "Normal" intramitochondrial dense granules can be seen within the mitochondria (v--~).x 10,000. FIG. 10. Three days a.p. Notice the more-or-less continuous gradient of ultrastructural organization from the endothelial cell with its expanded RER, through the small pericapillary cell with prominent dilated RER and largish mitochondria, to the differentiating adipocytes, having less hypertrophied RER and large mitochondria with moderately organized arrays of cristae and large intramitochondrial dense granules (~-->).The extra capillary erythrocyte, present in a field with apparently undamaged structural relationships, indicates that capillary endothelial cells may develop into adipocytes. A "giant mitochondrion" is seen at H--~.x 10,000.

320

BARNARD

close to the boundaries of triglyceride droplets (Fig. 6). The mitochondria might have become more numerous, and their internal structure was more regularly ordered. The intramitochondrial dense granules had increased in size: these structures as well as morphometric observations on development of the chondriome are more fully reported in another paper (1). One day before birth the adipocyte cytoplasm had further increased in volume compared to the nucleus (Fig. 7). SER was more abundant then RER, and scattered through the cytoplasm and at the cell periphery were numerous small vesicles, about 500 A in diameter, resembling micropinocytotic vesicles; (the relatively high OsO~ concentration may be expected to have caused some vesiculation of cytomembranes). Golgi bodies appeared to be somewhat smaller than at earlier stages. By this time the mitochondria were particularly obvious, partly because of their abundance and numerous cristae, but also because of the large size and numbers of the intramitochondrial dense granules. Both triglyceride droplets and glycogen deposits had further increased. Marked alterations occurred in the tissue over birth, most of them after this event. The most obvious changes were an extremely rapid depletion of substrate deposits, which was only temporary for triglycerides, and a considerable increase in the chondriome (Fig. 8). Furthermore the size of the intramitochondrial dense granules so prominent at 1 day a.p. was now very much smaller, though usually these were still numerous. Autophagic vacuoles were temporarily common: their presence during rapid mobilization of lipids in BAT has previously been reported (8). The other membranous systems of the cytoplasm did not change noticeably from the antenatal state, except that micropinocytotic vesicles and SER lacunae were more abundant at the peripheries of some cells. Further differentiation in the cells was most marked as a restoration and continued increase of the volume of triglyceride droplets, a reduction of both SER and RER to low levels and perhaps an additional growth of the mitochondria (Fig. 9). While eventually there might be only a few large triglyceride droplets within one cell, in rat ISBAT this tendency was seldom so extreme as in adipocytes of WAT. Around birth and for some days thereafter, the cellular homogeneity of the tissue at any stage was high, approximately 70 % of the total tissue volume being filled by the developing adipocytes, the only other at all common cell type being cells with small, roundish profiles usually located near a capillary. This cell type is ultrastructurally similar, not only to the cells predominant at 6 days a.p., but also to certain endothelial cells having abundant dilated RER (Fig. 10). The remainder of the tissue is filled with capillaries and extracellular space.

DIFFERENTIATION OF BROWN ADIPOSE TISSUE

321

DISCUSSION The results of this investigation have extended the observations of Sidman (14) and have confirmed and amplified, up to the stage of the multilocular adipocyte, the description of Simon (15). In spite of the existence of a gradient of maturation within each tissue lobule (I5), the cellular homogeneity of the images from any one stage during this investigation was rather remarkable; it is presumably an artifact of the smaller fields observed with the electron microscope than with the light microscope. Other differences between these two works are more interesting. Thus, Simon did not mention the large, numerous intramitochondrial dense granules, which are such a noticeable feature of the prenatal rat and which also occur in the ISBAT of prenatal guinea pig (D. Rafael, personal communication) and to a lesser extent in the fetal rabbit close to term (unpublished observations): hence they may be considered a normal feature of BAT differentiation. As Simon's pictures of adipogenic reticular cell mitochondria (Figs. 4 and 5 of ref. 15) and of an adipoblast (Fig. 6, ibid) could come, in my experience, only from tissue of postnatal rats, and as it was nowhere stated that prenatal rat tissue was examined by electron microscopy, it seems most probable that Simon observed prenatal rat tissue only by light microscopy. The cells seen at 6 days a.p. in ISBAT appear quite similar to the stem cells "indistinguishable from a typical fibroblast" in WAT fat pads at 9 days after birth. This is also the case when such WAT stem cells are compared to the small pericapillary cells seen in ISBAT (Fig. 10). However, most other details of ontogenesis in the two tissues differ, especially with respect to the chondriome, the disposition of the triglyceride droplets and the relative amounts of glycogen particles (8). It is interesting here to recall Napolitano's comment that the response of adipose tissue cells to experimental treatment may depend not only upon the cells themselves but also upon their microenvironment in the tissue: this could be equally apposite for developmental, as for experimental, processes (9). Curiously, Napolitano's description of differentiation in BAT, based on unpublished material, differed from the present observations both qualitatively and temporally. Thus at 6 days a.p. differentiation had scarcely begun in my material, while Napolitano claimed that, at this stage, brown fat cells indistinguishable in fine structural details from those of the newborn animal were present (8). Again, there was no mention of intramitochondrial large dense granules. Thus, ultrastructural evidence indicates that during tissue differentiation, adipocytes of both BAT and WAT develop from a fibroblast-like stem cell. In addition, the present observations have shown that there are close structural similarities in BAT between what are apparently adipocyte stem cells and endothelial cells. It is interesting that there is also autoradiographic evidence that during cold-adaptation

322

BARNARD

of 5-week-old rats, endothelial cells may be precursors of new adipocytes in BAT (6, 19).

Thus, it seems likely that, in ISBAT, a major pathway for production of adipocyte stem cells during both tissue differentiation and cold adaptation may start from capillary endothelial ceils. An outstanding problem of the relationship of white and brown adipose tissues is therefore the origin of the fibroblast-like stem cell of adipocytes in white adipose tissue. Is this a fibroblast, is it derived from a differentiated cell type such as the capillary endothelial cell, or is it a totipotent primitive mesenchymal cell? I thank Dr. Bj6rn Afzelius for his advice during this work and Miss Ursula Mann for her skillful help. REFERENCES 1. 2. 3. 4. 5. 6.

7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

BARNARD,T. and LINDBERG,O., J. Ultrastruct. Res. 29, 293 (1969). BRI~CK,K. and WUNNENBERG,B. Federation Proc. 25, 1332 (1966). HULL,D., Brit. Med. Bull. 22, 92 (1966). HULL, D. and SEGALL,M., Nature 212, 469 (1966). HUNT, T. and HLrNT,E., Anat. Record 157, 537 (1967). JOEL, C., in REYNOLD, A. E. and CAHILL, G. F., Jr. (Eds.), Handbook of Physiology, Section V. Ch. 9, p. 59. Amer. Physiol. Soc., Washington, D.C., 1965. LAGERSPETZ,K. Y. H., Helgoldnder Wiss. Meeresunters. 14, 559 (1966). NAPOLITANO,L., J. CellBiol. 18, 663 (1963). - i n REYNOLD,A. E. and CAHILL,G. F., Jr. (Eds.) Handbook of Physiology, Section V, Ch. 12, p. 109. Amer. Physiol. Soc., Washington, D.C., 1965. NAPOLITANO,L. and FAWCETT,D., J. Biophys. Biochem. Cytol. 4, 685 (1958). NASS, M. K. M., NASS, S. and AEZELIUS,B. A., Exptl. CellRes. 37, 516 (1965). PRUSINER,S. and Po~, M., Nature 220, 235 (1968). RAFAEL,J., KLASS,D. and HOHORST,H.-J., Z. Physiol. Chem. 349, 1711 (1968). SIDMAN,R., Anat. Record 124, 581 (1956). SIMON,G., in REYNOLD,A. E. and CAHILL,G. F., Jr. (Eds.), Handbook of Physiology, Section V, Ch. 10, p. 87. Amer. Physiol. Soc., Washington, D.C., 1965. STEINER,G. and CAHILL,G., Am. or. PhysioL 207, 840 (1964). SUTER,E. R., J. Ultrastruct. Res. 26, 216 (1969). WILLIAMSON,J. R.. J. Cell Biol. 20, 57 (1964). CAMERON.I. L. and SM~XH,R. E., J. Cell Biol. 23, 89 (1964).