Lipolysis and lamellar structures in white adipose tissue of young rats: lipid movement in membranes

JOURNAL OF ULTRASTRUCTURE RESEARCH 77, 295--318 (1981)

Lipolysis and Lamellar Structures in White Adipose Tissue of Young Rats: Lipid Movement in Membranes E. JOAN B L A N C H E T T E - M A C K I E AND ROBERT 0 . S c o w Section on Endocrinology, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received March 10, 1981, and in revised form August 10, 1981 Lamellar structures with a periodicity of 40 A developed during incubation at 25°C in glutaraldehyde-fixed adipose tissue from young rats. The lameUae were found in transendothelial channels, at intercellular contacts, and in channels that extended from extracellular space to surfaces of lipid droplets in fat cells. They were also found in capillaries, associated with chylomicrons, in tissue from fed rats, and near lipid droplets of fat cells in tissue from fasted rats. The amount and distribution of lamellae varied with incubation time and metabolic state of the tissue donor. Development of lamellae under conditions causing lipolysis and accumulaton of fatty acids in fixed tissue indicates the lamellae were composed primarily of fatty acids. We conclude that fatty acids resulting from lipolysis accumulated in an interfacial continuum of external leaflets of cell membranes, and that when the continuum became crowded, fatty acids formed lamellar extensions of the continuum at different sites along its course through the tissue.

Uptake of fatty acids from chylomicrons by adipose tissue requires hydrolysis of triacylglycerol in chylomicrons by lipoprorein lipase acting at the luminal surface of capillaries (Robinson, 1970; Scow et al., 1972). The fatty acids formed are then transferred across capillary endothelium and extracellular space to fat cells where t]hey are reesterified to triacylglycerol and stored (Robinson, 1970; Blanchette-Mackie and Scow, 1971; Scow et al., 1976). Mobilization of fatty acids from adipose tissue, as during fasting, requires hydrolysis of triacylglycerol in fat cells to fatty acids by tissue lipase and transfer of fatty acids to the luminal surface of capillaries where they bind to albumin in blood for transport to other tissues (Scow and Chernick, 1970; Spector and Fletcher, 1978). We have proposed that lipolytic products may be transferred from chylomicrons to fat cells by lateral movement in a continuous interface composed of the chylomicron surface film and the external leaflets of plasma and intracellular membranes of endothelial and parenchymal cells (Scow et al., 1976,

1977a, 1980; Smith and Scow, 1979). Transfer of fatty acids from fat cells to capillary lumen could also occur by the same mechanism (Scow et al., 1980). In a cytochemical study of lipoprotein lipase activity in adult rat adipose tissue, we localized lipolytic products by forming fatty acid soaps with Pb 2+ (Blanchette-Mackie and Scow, 1971). We found that chylomicron triacylglycerol was hydrolyzed by lipoprotein lipase in tissue fixed with glutaraldehyde, and that lamellar and granular structures developed in vesicles and vacuoles of capillary endothelium, and in subendothelial space. At that time it was presumed that lipolytic products accumulated at sites of enzyme activity (Gomori, 1952; Schoefl and French, 1968; BlanchetteMackie and Scow, 1971). Subsequent studies of chylomicrons incubated with purified lipoprotein lipase showed that lipids resulting from hydrolysis of triacylglycerol accumulated in the interface between triacylglycerol and water, and extended the interface as a monolayer that lined and bilayers that spiraled within aqueous spaces 295 0022-5320/81/120295-24502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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in the chylomicron core (Blanchette-Mackie and Scow, 1976a,b). These findings suggested that lipolytic products could transfer from sites of lipolysis by lateral movement in the interracial plane, and that lipolysis would continue, in the absence of fatty acid acceptor, if the interface could expand. We then concluded that lamellar and granular structures found in incubated, fixed adipose tissue did not necessarily indicate sites of lipolysis (Scow et al., 1977a). Electron microscope studies of the mammary gland of lactating rats injected intravenously with chylomicrons also showed development of lamellar structures in capillary endothelium and extracellular spaces even though the tissues were not treated with Pb 2+ (Scow et al., 1980). Also, lamellar structures were found closely associated with cell membranes. This suggested that lamellae might form as extensions of cell membranes and, thereby, could mark in fixed tissues the route we proposed for transfer of lipolytic products from chylomicrons to cells (Scow et al., 1977a, 1980). This paper describes studies in white adipose tissue of young rats designed to determine conditions affecting formation and movement of lipolytic product in fixed tissues. Lipolytic product formed from chylomicrons was studied in tissue of fed rats and product formed from lipid in fat cells was studied in tissues of rats fasted overnight. Visualization of lipolytic product as lamellar structures was enhanced by adding tannic acid to the incubation medium. The findings provide morphological evidence for lateral movement of lipolytic product in a continuum of cell membranes. MATERIALS AND METHODS

Animals Female CD rats pregnant for 15 days were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.) and allowed free access to water and Purina NIH Open Formula Rat and Mouse Ration No. 5018 (Ralston Purina Co., St. Louis, Mo.). Two days after delivery, the size of each litter was adjusted to eight pups.

Preparation of Chylomicrons Chylomicrons were isolated from thoracic duct lymph collected for 6 hr from adult rats tube fed, after an overnight fast, 0.5 ml of corn oil (Scow et al., 1967). Chylomicrons containing triacylglycerol labeled with ~H]oleic acid were collected from rats tube fed corn oil containing tri[9,10-3H]oleoylglycerol (545 mCi/ mmole, TRA 191, Batch 45, Amersham Corp., Arlington, Ill.). The chyle was centrifuged in a swinging head rotor SW 50.1 at 24 000 rpm for 60 min at 3°C with a Spinco Model L-2-65B ultracentrifuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.). The compact floating layer of chylomicrons formed during centrifugation was collected and suspended, as described elsewhere (Scow et al., 1967), in 4% albumin in Tyrode's solution at a triacylglycerol concentration of 80-120 raM. The albumin-Tyrode's solution contained 40 mg bovine serum albumin powder (Fraction V, Lot J-40409, Armour Pharmaceutical Co., Kankakee, Ill.)/ml of glucose-free Tyrode' s solution.

Morphological Analyses Epididymal fat pads were immediately removed from decapitated rats, cut into small pieces, and processed by one of the following procedures: 1. Glutaraldehyde, tannic acid, and osmium (Glut/ TA/Os). Fixed with 3% glutaraldehyde in 0.2 M sodium cacodylate solution (pH 7.4) for 30-60 rain at 4°C, rinsed with cold sodium cacodylate solution, incubated with 1% tannic acid (Mallinckrodt, Inc., St. Louis, Mo.) in 0.2 M sodium cacodylate solution (pH 7.4) for 30-60 min at 25°C, and postfixed with 2% OsO4 in sodium cacodylate solution for 2 hr at 4°C. H. Glutaraldehyde-tannic acid and osmium (GlutTA/Os). Fixed with 3% glutaraldehyde and 1% tannic acid in sodium cacodylate solution (pH 7.4) for 30-60 min at 4°C, rinsed with cold sodium cacodylate solution, and postfixed with OsO4 in sodium cacodylate solution for 2 hr at 4°C. IlL Glutaraldehyde and osmium (Glut/Os). Fixed with 3% glutaraldehyde in sodium cacodylate solution for 30-60 min at 4°C, rinsed with cold sodium cacodylate solution, and postfixed with OsO4 in sodium cacodylate solution for 2 hr at 4°C. IV. Osmium (Os). Fixed with OsO4 in sodium cacodylate solution for 2--4 hr at 4°C. V. Glutaraldehyde, osmium, and tannic acid (Glut/ Os/TA). Fixed with 3% glutaraldehyde in sodium cacodylate solution for 30 min at 4°C, rinsed with cold sodium cacodylate solution, postfixed with OsO4 in sodium cacodylate solution for 2 hr at 4°C, rinsed with cold sodium cacodylate solution, and treated with 1% tannic acid in sodium cacodylate solution at 4°C for 30 rain. The tissues were then dehydrated rapidly with acetone (Blanchette-Mackie and Scow, 1971) and embedded in Epon 812. Sections from tissues processed by procedures I and II were stained with Pb(OH)2. All

LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE sections were examined in a Philips EM-300 electron microscope.

Morphometry The effect of incubation at 25°C on development of lamellae in fixed tissue was studied in epididymal fat pads taken from 8-day-old rats. Each fat pad, weighing about 2 mg, was divided into six equal pieces and fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate solution (pH 7.4) at 4°C for 30 rain. The tissue pieces were rinsed twice in 1% tannic acid in 0.2 M sodium cacodylate solution (pH 7.4) at 25°C. Two pieces of tissue selected at random were taken for immediate postfixation in osmium tetroxide in 0.2 M sodium cacodylate solution at 4°C for 2 hr and the others were put in tannic acid-cacodylate solution for incubation. Two pieces were removed at 30 rain, and the other two at 60 rain, for postfixation in osmium tetroxide. All specimens were dehydrated, embedded, and sectioned in the same manner as above. Electron micrographs of sections were taken randomly (Weibel and Bolender, 1973) at x 4000 and printed at a final magnification of × 10000. The printed micrograph measured 18 x 24 cm. The point counting method was used to estimate the volume fraction of lamellae and ceils (Weibel and Bolender, 1973). The points counted were intersections of lines of square lattices. The lattices, measuring 18 × 24 cm, were drawn on transparent sheets with spaces of 1.0 or 0.25 cm between lines. The coarse lattice contained 390 points and was used for measuring cell volumes, while the fine lattice contained 6240 points and was used for measuring volumes of lamellae. The lattice overlays were applied to 30 micrographs from each time group and the number of points falling on each structure relative to the total number of points falling on the micrograph were determined. In addition, the location of lamellae was recorded: in capillary lumen or associated with luminal surface of endothelium, in endothelium or associated with basal surface of endothelium, associated with preadipocytes, associated with the surface of adipocytes, or in adipocytes. The volume fraction of structures was measured by the relative number of points falling on each structure (Weibel and Bolender, 1973).

Biochemical Analyses Lipoprotein lipase activity was assayed in aqueous extracts of genital adipose tissue (Scow et al., 1977b) with rat chylomicron triacylglycerol as substrate. The number of fat pads used per assay depended on the size of tissue and the amount of activity expected. The c.hylomicrons contained triacylglycerol labeled with [3H]oleic acid. The production of fatty acid was linear with both time and amount of sample assayed. One nailliunit of activity represents production of 1 nmole of fatty acid/rain. Hydrolysis of chylomicron triacylglycerol in glutar-

297

aldehyde-fixed adipose tissue was measured in tissue taken from 14-day-old rats injected with chylomicrons containing triacylglycerol labeled with [aH]oleic acid. The rats were anesthetized with sodium pentobarbital, 4 mg/100 g body wt, given intraperitoneally 10-12 min before being injected. A suspension of chylomicrons containing about 20/zmole of triacylglycerol in 0.15 to 0.2 ml was injected into the left saphenous vein over a period of 15-25 sec, and 1.5 rain later the rat was decapitated and adipose tissue was taken for study. Genital (epididymal or parametrial) fat pads from both sides were fixed together in 5 ml of 3% glutaraldehyde in 0.2 M sodium cacodylate solution (pH 7.4) for 30 min at 4°C, and rinsed twice in 5 ml of cold 0.2 M sodium cacodylate solution, pH 7.4. One fat pad was put immediately into cold chloroform-methanol (2:1) and the other was incubated 30 rain in 5 ml of either 0.2 M sodium cacodylate solution or 1% tannic acid in 0.2 M sodium cacodylate solution (pH 7.4) at 25°C and then put into chloroform-methanol. Sometimes, one fat pad was put immediately into cold chloroformmethanol, without being fixed, while the other was fixed in glutaraldehyde solution, and rinsed in sodium cacodylate solution before being put into chloroformmethanol. Neutral lipids and fatty acids were extracted into chloroform (Folch et al., 1957) and separated into triacylglycerol and fatty acid fractions by the method of Borgstrom (1952). Thin-layer chromatography (Stein etal., 1970) of the chloroform extract of these tissues showed that more than 99% of the [aH]oleoyl moiety was either in triacylglycerol or in fatty acids. Lipids were dissolved in Beckman GP scintillation solvent for measurement of 3H content with a Beckman LS-8000/3 spectrometer. Hydrolysis of tissue triacylglycerol in glutaraldehyde-fixed adipose tissue was measured in genital fat pads of 14-day-old rats fasted 18 hr. Fat pads were taken from eight rats for each assay. The fat pads were divided into two groups, weighed, and fixed in 5 ml of cold glutaraldehyde solution. They were then rinsed twice in 5 ml of cold cacodylate solution and incubated 0 or 60 min in 5 ml of sodium cacodylate solution (pH 7.4) at 25°C. Fatty acids in the tissues were extracted into hexane and titrated (Bieberdorf etal., 1970). Column chromatography (Borgstrom, 1954) of the hexane extract of these tissues showed that fatty acids were the primary product of lipolysis. Acylglycerol content of neutral lipid fractions was measured by the method of Rapport and Alonzo (1959), and triacylglycerol content of plasma by the method of Chernick (1969). RESULTS Preliminary findings showed lamellar structures in electron micrographs of white a d i p o s e t i s s u e o f y o u n g r a t s ( F i g s . 1 a n d 2), especially when tissue was kept at room temperature after being fixed with glutar-

LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE aldehyde. They also showed that lameUae Were more tightly packed in tissue treated with tannic acid (Figs. 4 and 5). We present here evidence that the appearance of lamellar structures in f x e d tissue was affected by the preparative procedure used, and that the amount and distribution of lamellae were affected by the length of time fixed tissue was incubated at 25°C before being postfixed with osmium and by the metabolic state o f the tissue donor.

Effect of Preparative Procedure on Structure of Lamellae E f f e c t o f different p r e p a r a t i v e p r o c e dures on structure of lamellae in unincubated tissue from fed rats is shown in Figs. 1 to 5. Lamellar structures were small and irregular in tissue fixed only with osmium tetroxide at 4°C (Fig. 1). Lamellar structures were also irregular in tissue fixed with glutaraldehyde, rinsed with sodium cacodylate solution, and postfixed with osmium, all at 4°C (Fig. 2). Lamellar structures had a very regular pattern, alternating dark and light lines with a periodicity o f 40 ~ , in tissue fixed with glutaraldehyde solution containing 1% tannic acid (Figs. 4 and 5). Lamellar structures were also tightly wound in tissues incubated with tannic acid at 25°C after being fixed with glutaraldehyde (see

299

below). Tannic acid treatment did not affect the pattern of lameUar whorls, however, when applied after tissue was postfixed with osmium (Fig. 3).

Relationship o f Lamellae to Plasma Membrane in Adipose Tissue The relationship of lamellae to plasma membrane in adipose tissue is shown in Figs. 6-12. Bilayered structures extending o u t w a r d from the surface o f adipocytes were found in tissue treated with tannic acid after being postfixed with OsO4 (Figs. 6-8). When the structures were long, they formed loosely wound spirals, sometimes encircled by cytoplasmic processes (Figs. 11 and 12). The leaflets of the bilayered structures appeared to be continuous with the external leaflet of plasma membrane (Figs. 6, 7, and 12). The bilayered structures formed multilayered lamellae when tissue was incubated with tannic acid before being postfixed with OsO4 (Figs. 9 and 10). The lamellae shown in Figs. 9 and 10, however, were not as tightly p a c k e d as those shown in Figs. 13-15 and 17-26. Consequently, the relation of lamellae to plasma m e m b r a n e in tissue incubated with tannic acid could be studied only in some tissues. This difference may reflect the way in which tissues were processed: Tissues

FIGS. 1-5. Sections of developing epididymal adipose tissue. This series of micrographs shows variation in :structure of lamellae at the periphery of adipocytes associated with fixation procedures. FIG. 1. Fed 4-day-oldrat. A small lamellar structure (LW) is present at the periphery of an adipocyte (A). ER, endoplasmic reticulum; V, vesicle. Preparation: IV. Os(4°C). x 100000. FIG. 2. Fed 4-day-oldrat. Large irregular whorls of lamellae (LW) are in contact with cytoplasmic processes (P) of the adipocyte (A). Preparation: III. Glut(4°C)/Os(4°C). x 91 000. FIG. 3. Fed 4-day-old rat. A large irregular whorl of lamellae (LW) appears continuous with plasma membrane (PM) of an adipocyte (A). Preparation: V. Glut(4°C)/Os(4°C)/TA(25°C). x 148 000. FIG. 4. Fed 8-day-oldrat. A large lamellae whorl (LW), with a regular pattern of alternating dark and light lines and a periodicity of 40 A, extends outward from plasma membrane (PM) of an adipocyte (A) into extracellular space (ECS). Lamellae are present in invaginations of plasma membrane (at arrow). Basement membrane (BM) and the outer leaflet of the adipocyte plasma membrane are stained densely due to treatment with tannic acid. L, lipid, Preparation: II. Glut-TA(4°C)/Os(4°C). × 182 000. FIG. 5. Fed 8-day-oldrat. Large whorls of lamellae (LW), with a regular repeating pattern, are present at tJhe surface of an adipocyte (A) and extend outward into extracellular space (ECS). One of the lamellar whorls extends also into a vesicle (V) which is connected with another vesicle (at arrow). Basement membrane (BM), the outer leaflet (OL) of plasma membrane, and the luminal leaflet (LL) of surface invaginations are stained densely due to treatment with tannic acid. Mitochondrial membranes (M) are not stained. Preparation: II. GlutTA(4°C)/Os(4°C). x 338 000.

300

BLANCHETTE-MACKIE AND SCOW

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FIG. 6. A section of adipose tissue from a fed 8-day-old rat showing a bilayered structure extending outward from the surface of an adipocyte. One leaflet of the structure is continuous, at arrow, with the outer leaflet (OL) of plasma membrane (PM). A, adipocyte; ECS, extracellular space. Preparation: V. Glut(4°C)/Os(4°C)/ TA(4°C). x 520 000. F i t . 7. A section of adipose tissue from a fed 8-day-old rat showing a double bilayered structure extending outward from the surface of an adipocyte. A leaflet of one of the structures is continuous in this section, at arrow, with the outer leaflet (OL) of plasma membrane (PM). A, adipocyte; ECS, extracellular space. Preparation: V. Glut(4°C)/Os(4°C)/TA(4°C). x 260 000. F i t . 8. A section of adipose tissue from a fed 8-day-old rat showing a triple bilayered structure (at arrow) extending outward from the surface of an adipocyte. A, adipocyte; ECS, extracellular space. Preparation: V. Glut(4°C)/Os(4°C)/TA(4°C). × 260 000.

LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE

301

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FIG. 10. A section of adipose tissue from a fed 8-day-old rat showing several long bilayered structures extending outward from the surface of an adipocyte. These structures formed multilayered lamellae when they came together (single arrow) or curved back on themselves (double arrows). This tissue was kept in cold sodium cacodylate solution for 1 hr before being incubated with tannic acid. A, adipocyte; ECS, extracellular space; PM, plasma membrane. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 84 000.

shown in Figs. 9 and 10 were left in sodium cacodylate solution for 1 hr after being fixed with glutaraldehyde and for 15 min after being incubated with tannic acid, whereas the other tissues were quickly rinsed in sodium cacodylate solution after being fixed and after being incubated.

Distribution of Lamellar Structures Distribution of lamellar structures in adipose tissue incubated with tannic acid at

25°C after being fixed with glutaraldehyde is shown in Figs. 13-16 and 18-26. Lamellar structures were found in capillary lumen (Figs. 13-16), in transendothelial channels (Fig. 18), in extracellular space (Figs. 1923), at sites of intercellular contact (Fig. 21), in intracellular channels in preadipocytes and adipocytes (Figs. 22-26), and near lipid droplets in adipocytes (Figs. 2426). Lamellar whorls were not found in interendothelial spaces (Figs. 17 and 18), or

FIG. 9. A section of adipose tissue from a fed 8-day-old rat showing a multilayered lamellar structure connected by an apparent single bilayered structure to the surface of an adipocyte. This tissue was kept in cold sodium cacodylate solution for 1 hr before being incubated with tannic acid. A, adipocyte; ECS, extracellular space; PM, plasma membrane. Preparation: I. Glut(4°C)/TA(25°)/Os(4°C). x 120 000.

302

BLANCHETTE-MACKIE AND SCOW TABLE I

T A B L E II

VOLUME OF CELLS IN ADIPOSE TISSUE OF SUCKLING (8 DAY) RATS

DEVELOPMENT OF LAMELLAE IN GLUTARALDEHYDEFIXED ADIPOSE TISSUE OF 8-DAY OLD RATS

Group

Incubated Percentage of tissue v o l u m e a at 25°C Capillary AdipoPreadi(min) cells cytes pocytes

Group

Incu- Volume (xl04/tissue volume) of bated lamellae associated witha at 25°C Capillary AdipoPreadi(min) cells cytes pocytes

Fasted 18hr

0 30 60

8.1 ± 1.4 20.6 ± 4.9 13.3 _+ 2.6 6.9 ± 1.1 23.5 ± 3.1 18.0 ± 3.0 6 . 6 ± 2 . 1 2 6 . 0 ± 5.0 14.3 ± 3.4

Fasted 18hr

0 30 60

0 23 ± 4.4 2 8 ± 8.8

0 12_+ 3.1 25 ± 6.8

0 3 4 + 5.8 21 ± 4 . 8

Fed

0 60

6.2 ± 1.2 28.7 ± 5.3 11.3_+ 1.4 6.2 ± 1.4 21,3 ± 4.5 13.3 ± 1.4

Fed

0 60

1 ±0.7 8+_ 3.5

0.4±0.4 3 ± 1.I

1 ± 0.7 1 4 ± 0.4

Fed + chylo0 microns iv b 30 60

6.5 + 1.6 26.1 ± 3.6 1 1 . 6 ± 2.0 8.0 ± 1.0 26.0 ± 6.0 14.0 ± 2 . 0 7.5 ± 1.4 24.3 ± 5.9 1 3 . 8 ± 4.0

Fed + chylomicronsiv b

0 30 60

7 ± 2.9 21 ± 5.3 19 ± 5 . 1

0.2 ± 0.2 7 ± 1.9 15 ± 4.0

5 ± 1.4 23_+ 5.0 20±6.8

Values given are m e a n s ± standard error of meas u r e m e n t s m a d e on 30 electron micrographs. b Tissue was taken 1.5 min after intravenous injection o f chylomicrons.

a Values given are m e a n s ± standard error of meas u r e m e n t s m a d e on 30 electron micrographs. T i s s u e was taken 1.5 min after intravenous injection of chylomicrons.

near mitochondria in adipocytes of white adipose tissue. The effects of incubation at 25°C and metabolic state of the tissue donor on amount and distribution of lamellar structures in glutaraldehyde-fixed tissue were determined with morphometric analyses of electron micrographs of adipose tissue from fasted, fed, and chylomicron-injected fed rats. Tissue was taken from the latter group 1.5 min after intravenous injection of chylomicrons. Chylomicrons were injected to increase the amount of triacylglycerol initially present in capillaries. The tissue specimens were fixed in glutaraldehyde at 4°C for 30 min, rinsed in tannic acid-cacodylate solution, and incubated in the latter solu-

tion (pH 7.4) at 25°C for 0, 30, or 60 min. The specimens were then posffixed with OsO4 at 4°C, dehydrated, embedded, sectioned, and examined with the electron microscope. Thirty micrographs were taken for each incubation time and analyzed morphometrically as described under Materials and Methods. The relative volumes of cells in tissue specimens used in this study are given in Table I. The values for each cell type were similar in each group, indicating that the sampling procedure used was satisfactory. The similarity in cell volumes between groups suggests that fasting may not affect cell volume of adipocytes. This unexpected finding, however, may be the re-

FIG. 11. A section of adipose tissue from a fed 8-day-old rat showing a large loosely w o u n d spiraling bilayered structure attached to the surface of an adipocyte. A short bilayered connection (at arrow) between the structure and p l a s m a m e m b r a n e (PM) is shown in the inset. A, adipocyte; ECS, extracellular space; ER, endoplasmic reticulum. Preparation: V. Glut(4°C)/Os(4°C)/TA(4°C). × 96 000; inset, × 320 000. Fro. 12. A section of adipose tissue from a fed 8-day-old rat showing a loosely w o u n d lamellar structure at the surface of an adipocyte. The structure is enclosed by a long curving cytoplasmic p r o c e s s (P) of the adipocyte (A). One of the bilayered extensions appears to be continuous with p l a s m a m e m b r a n e in the enclosed area as shown in the inset (at arrow). ECS, extracellular space. Preparation: V. Glut(4°C)/Os(4°C)/TA(4°C). × 64 000; inset, × 192 000.

LIPOLYSIS A N D L A M E L L A E IN ADIPOSE TISSUE

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Fro. 16. A section of adipose tissue from a fed 7-day-old rat injected with chylomicrons showing a chylomicron in contact with the surface of the capillary endothelium. Lamellae (LW) emanate from the chylomicron (Ch) and extend into the capillary lumen (Lu). A small bilayered projection (encircled) extends from the luminal leaflet of plasma membrane (PM) of the endothelium (E) and is shown in the inset at higher magnification. AS, aqueous space; BS, basal surface. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 310 000; inset, x 620 000.

FIG. 13. A section of adipose tissue from a fed 8-day-old rat injected with chylomicrons showing a capillary. The capillary lumen (Lu) contains chylomicrons (Ch) and lamellar whorls (LW) which are in contact with the capillary endothelium (E). Co, collagen; RBC, erythrocyte. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 16 500. Fro. 14. Higher magnification of portion of lanleflar whorl encircled in Fig. 13, showing the 40-/~-wide banding pattern of the whorl, x350 000. Fro. 15. A section of adipose tissue from a fed 8-day-old rat injected with chylomicrons showing a chylomicron in capillary lumen. Numerous larnellae (LW) are in contact with the chylomicron (Ch) at the luminal surface (LS) of capillary endothelium (E). Preparation: I. Glut(4°C)/TA(4°C)/Os(4°C). x 86 000.

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LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE

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FIG. 19. A section of adipose tissue from a fed 8-day-old rat showing portions of a capillary and adipocyte. A lamellar whorl (LW) is present in extracellular space (ECS) between capillary endothelium (E) and an adipocyte (A). L, lipid droplet; Lu, capillary lumen; M, mitochondria; RBC, erythrocyte. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). × 36 000.

suit of other factors, such as differences between animals in the fed state, which was :not controlled in this study. Effect of incubation. The development and location of lamellae in glutaraldehydefixed adipose tissue incubated at 25°C for different periods of time are shown in Table

II. Lamellae were not present at 0 time in adipose tissue from fasted rats. They developed during the first 30 rain of incubation and were found associated with capillary endothelium (Figs. 17, 18, and 20), adipocytes (Figs. 24 and 26), and preadipocytes (Figs. 20 and 25). There was no

FIG. 17. A section of adipose tissue from an 8-day-old rat fasted for 18 hr showing a capillary and portions of preadipocytes. This tissue was incubated in sodium cacodylate solution at 25°C for 15 rain before being fixed only in osmium tetroxide. Two large loosely wound lamellar whorls (LW) are seen in the endothelium (E), and others are present in capillary lumen (Lu) and associated with a preadipocyte (PA). Compare these lamellae with those in tissue incubated with tannic acid at 25°C after being fixed with glutaraldehyde (Fig. 18). ECS, extracellular space; N, nucleus; RBC, erythrocyte. Preparation: IV. Incubated (25°C)/Os(4°C). × 11 500. FIG. 18. A section of adipose tissue from an 8-day-old rat fasted for 18 hr showing a capillary containing five lamellar whorls (LW) within intraendothelial channels. The large lamellar whorl (large arrow) spans the width of the endothelium (E) from the basal to the luminal surface. One of the smaller lamellar whorls is in contact with the basal endothelial surface (small arrow). A portion of the large, lamella whorl (large arrow) is shown at higher magnification in the inset to demonstrate its 40-A repeating pattern. BM, Basement membrane, Co; collagen; ECS, extracellular space; IES, interendothelial space; Lu, capillary lumen; N, nucleus. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 26 000; inset, × 500 000.

309

LIPOLYSIS AND L A M E L L A E IN A D I P O S E TISSUE T A B L E III

EFFECT OF FASTING ON L1POPROTEIN LIPASE ACTIVITY OF ADIPOSE TISSUE AND PLASMA TRIACYLGLYCEROL CONCENTRATION IN YOUNG RATS

Lipoprotein lipase activity of adipose tissue a (munit/fat pad)

Age (days) 8 9 12 14

Fed 1.4 5.9 14.3 27.5

± ± ± +

0.4 0.3 1.0 9.2

Plasma triacylglycerol concentration b (raM)

Fasted 18 hr (4) (2) (2) (3)

0.6 2.3 4.8 6.7

--+ 0.03 ± 0.3 -+ 0.6 -+ 3.3

(4) (2) (2) (2)

Fed

Fasted 18 hr

1.4 _+ 0.2 (2) -2.5 + 0.2 (2) 4.5 ± 0.2 (2)

0.3 ± 0.01 (2) -0.3 -+ 0.05 (2) 0.4 ± 0.02 (2)

a Lipoprotein lipase activity was assayed in clear homogenates of genital (epididymal and parametrial) fat pads with rat chylomicron triacylglycerol as substrate. The number of fat pads used per assay for tissue from fed rats ranged from 4-8 for age 8 days to 1-2 for age 14 days, and for tissue from fasted rats, 8-16 for age 8 days to 2-4 for age 14 days. Each assay contained tissues from approximately equal numbers of males and females. 1 munit of activity = 1 nmole of fatty acid produced per minute. Values are means _+ standard error with number of assays given in parentheses. b Values given are means _+ standard error with number of animals given in parentheses.

significant increase in lamellae after an additional 30 rain of incubation. Lamellae were present, associated with all three cell types, at 0 time in tissues from fed rats and fed rats injected with chylomicrons, and they increased markedly in both groups during incubation (Figs. 13-16, 19, 22, and 23). There were significantly more lamellae associated with adipocytes in tissue from fed rats injected with chylomicrons than in tissue from uninjected fed rats. Effect of metabolic state of tissue donor. The effect of metabolic state of tissue donor on the amount of lamellae present in capillary lumen and inside adipocytes of glutaraldehyde-fixed tissue incubated at 25°C for various periods of time is shown in Fig. 27. The fed donors were injected with chy-

lomicrons to increase the amount of triacylglycerol initially present in capillary lumen. Lamellae were present in capillary lumen, but not in adipocytes, in tissue of injected fed rats at 0 and at 30 rain. Lamellae increased in capillary lumen and appeared inside adipocytes during the second 30 rain of incubation. In tissue of fasted rats, however, lamellae were not present at either site at 0 time. Lamellae appeared inside adipocytes during the first 30 rain and increased there during the next 30 rain, while they appeared in capillary lumen only during the second 30 rain. The above findings show that lamellae in tissue of chylomicron-injected fed rats appeared first in capillary lumen and last in adipocytes, whereas lamellae in tissue of

FIG, 20. A section of adipose tissue from an 8-day-old rat fasted for 18 hr showing area of contact between a preadipocyte and a capillary. Lamellar whorls (LW) are present in the space between the preadipocyte (PA) and capillary endothelium (E) and in contact with surfaces of both cells. A larnellar whorl is also present at the luminal surface of the endothelium (arrow). A cytoplasmic process of the preadipocyte curves to appose a similar process of capillary endothelium. Note basement membrane is absent at this site. Lu, capillary lumen; RBC, erythrocytes. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 38 000. FIG. 21. A section of adipose tissue from fed 8-day-old rat showing a junction between an adipocyte and an endothelial cell. A large club-shaped process (P) of the endothelial cell (E) indents the surface of the adipocyte (A). Note the lamellar whorl between the cells, and its contact with the lipid droplet (L). Basement membrane (BM) is not present in the area of contact between cells. ECS, extracellular space; Lu, capillary lumen. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 123 000.

~i,i~ ~

310

LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE

311

F16.24. A section of adipose tissue from a 7-day-old rat fasted for 18 hr showing portions of two adipocytes. A large lamellar whorl (LW) in the cell in the lower part of the micrograph fills an intracellular channel (1C) that extends from the central lipid droplet (L) to extracellular space (ECS). Note in'the other adipocyte (A) a small • channel (arrow) extending from extracellular space toward the central lipid droplet. ER, endoplasmic reticulum; M, mitochondria. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). × 80 000.

fasted rats appeared first in adipocytes and last in capillary lumen. These findings are compatible with the concept that lamellae in tissue of fed rats represent lipolytic prodncts formed by action of lipoprotein lipase on triacylglycerol of chylomicrons in capillaries, whereas lamellae in tissue of fasted rats represent products formed by action of

tissue lipase on triacylglycerol in lipid droplets of adipocytes. Table llI shows that lipoprotein lipase activity in adipose tissue and plasma triacylglycerol concentration are both considerably higher in fed than in fasted young rats. The experiments presented in Table IV indicate clearly that lipoprotein lipase is active in glutaraldehyde-

FIO. 22. A section of adipose tissue from a fed 7-day-old rat injected with chylomicrons showing a portion of a capillary and a preadipocyte (PA). Several chylomicrons (Ch) are in the capillary lumen (Lu). A basal process (P) of capillary endothelium (E) extends through the basement membrane material to contact the preadipocyte. Lamellar whorls (LW) are present in the extracellular space (ECS), and one extends into the lumen of endoplasmic reticulum (RER) of the preadipocyte (arrow). N, nucleus, Preparation: I. Glut(4°C)/ TA(25°C)/Os(4°C). x 44 000. FI~. 23. A section of adipose tissue from a fed 7-day-old rat injected with chylomicrons showing a large lamellar whorl at the surface of a preadipocyte. The lamellar whorl (LW) also fills an intracellular channel (IC) that extends from the cell surface to the nucleus (N). The repeating pattern of the lamellae measures 40 fi,. ECS, extracellular space. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 124 000.

t~

313

LIPOLYSIS AND LAMELLAE IN ADIPOSE TISSUE

fixed tissue of fed young rats, and that triacylglycerol of injected chylomicrons is hydrolyzed to fatty acids while tissue is being fixed and when fixed tissue is incubated at 25°C. Table IV also shows that the presence of tannic acid during incubation had no effect on the amount of fatty acids produced by lipolysis. The findings presented in Table V show that triacylglycerol in glutaraldehyde-fixed tissue of fasted young rats is hydrolyzed to fatty acids when the tissue is incubated at 25°C. Since lipoprotein lipase activity in adipose tissue and plasma triacylglycerol concentration are both low in fasted young rats (Table III), it is likely that fatty acids are produced in incubated fixed tissues of fasted rats primarily by action of tissue lipase on triacylglycerol in lipid droplets of adipocytes. Effect of glucose and insulin prior to fixation on lamellar structures. Fat pads from fed rats were incubated at 38°C for 15 rain in either glucose-free Tyrode's solution or Tyrode's solution containing 1/zg/ml of insulin and 5 mM glucose before being fixed with glutaraldehyde. Electron microscopic examination of these tissues showed that lamellae were present in fat pads incubated in Tyrode's solution alone but not in fat pads incubated with insulin and glucose. This is additional evidence that lamellar structures in these studies are composed of fatty acids, products of lipolysis. DISCUSSION

These studies demonstrate that lamellar :structures developed in glutaraldehydefixed adipose tissue of young rats, both fed and fasted, when incubated at 25°C. The ltamellae were associated with capillaries,

i

i

FED + CHYLOS

FASTED

.16

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/

/ >o

/

• In capillary lumina

=o ,12

/ /

o In adipocytes

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Y /

3O

/

/

/

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60

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60

Minutes

Fro. 27. Development of lamellae in capillary lumen and adipocytes in glutaraldehyde-fixed adipose tissues of fed rats injected with chylomicrons and fasted uninjected rats. The tissues were incubated in 1% tannic acid-0.1 M sodium cacodylate solution, pH 7.4, at 25°C.

adipocytes, and preadipocytes. They had a regular pattern of alternating dark and light bands, with a periodicity of 40 ~k, in tissues treated with tannic acid during or after fixation with glutaraldehyde. The lamellar pattern was not regular, however, in tissues treated with tannic acid after being fixed with osmium, or in tissues not treated with tannic acid (Fig. 17). Lamellae were also prevalent in tissues incubated in buffer solution (without glucose) at 25°C and then fixed with osmium alone, indicating that glutaraldehyde fixation was not necessary for development of lamellae in adipose tissue. Development of lamellar structures under conditions that caused accumulation of fatty acids in glutaraldehyde-fixed tissues (Tables II, IV, and V) suggests that the la-

FIG. 25. A section of adipose tissue from an 8-day-old rat fasted for 18 hr showing portions of three preadipocytes (PA). Lamellar whorls (LW) are present in extracellular space (ECS) between cells, and associated with an intracellular lipid droplet (L). A portion of lamellar whorl near the arrow is shown in inset to demonstrate its banding pattern of 40 ]k. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 34 000; inset, x 420 000. Fro. 26. A section of adipose tissue from an 8-day-old rat fasted for 18 hr showing a portion of an adipocyte and extracellular space. Lamellar whorls (LW) fill intracellular channels (IC) which extend from the lipid droplet (12,) to extracellular space (ECS). Lamellae are also present at the surface of the adipocyte (A). Co, collagen, M, mitochondria. Preparation: I. Glut(4°C)/TA(25°C)/Os(4°C). x 77 000.

314

B L A N C H E T T E - M A C K I E A N D SCOW T A B L E IV

HYDROLYSIS OF CHYLOMICRON TRI[ZH]ACYLGLYCEROL IN GLUTARALDEHYDE-FIXED ADIPOSE TISSUE OF FED SUCKLING (14 DAY) RATSa [aH]Acyl lipid recovered as fatty acid in tissue (% of total) Incubation medium None Cacodylate Tannic acid-cacodylate

Not fixed

Fixed

Fixed and incubated

4.9 ---

9.2 7.7 7.3

-12.6 12.9

Increase during Fixation

Incubation

4.3 +_ 0.8 b ---

-4.9 _+ 0.4 b 5.6 _+ 0.6 ~

" Tissues were taken 1.5 rain after injection of chylomicrons, fixed 30 rain in glutaraldehyde solution at 4°C, and then incubated 30 rain in cacodylate or tannic acid-cacodylate buffer, pH 7.4, at 25°C. b p < 0.025 ( N = 3-4).

mellae could be composed of fatty acids formed in fixed tissue by action of lipase on triacylglycerol, either lipoprotein lipase acting on chylomicrons in capillaries of tissue from fed rats or tissue lipase acting on lipid droplets in fat cells of tissue from fasted rats. Glucose and insulin, which enhance reesterification and thereby decrease fatty acid content in adipose tissue (Scow and Chernick, 1970), prevented development of lamellae in tissue from fed rats incubated at 25°C before being fixed. This finding is additional evidence that lamellae in incubated tissue are probably fatty acids. Association of lamellar formation with fatty acid production was also observed in our earlier studies of effect of lipoprotein lipase on isolated chylomicrons, either suspended in aqueous medium (Blanchette-Mackie

TABLE V HYDROLYSIS OF TISSUE TRIACYLGLYCEROL IN GLUTARALDEHYDE-FIXED ADIPOSE TISSUE OF SUCKLING (14 DAY) RATS FASTED 18 HRa Fatty acid content of tissue (tzmole/g) Incubation (rain) 0

60

Increase in 60 min

2.0

2.6

0.67 -+ 0.13 b

a Tissues were fixed in glutaraldehyde solution at 4°C for 30 min, and then incubated in cacodylate buffer, pH 7.4, at 25°C. P ~ 0.005 ( N = 7).

and Scow, 1973, 1976a,b) or embedded in glutaraldehyde-fixed gelatin blocks (Blanchette-Mackie and Scow, 1971). The periodicity of lamellar structures in glutaraldehyde-fixed tissues incubated with tannic acid (Figs. 4, 5, 13, 14, 18, 21, 23, 25, and 26), 40 ]k, is the same as that of lamellar structures composed of either pure oleic acid (Blanchette-Mackie and Scow, 1976a) or fatty acids formed by lipolysis of chylomicrons in vitro (Blanchette-Mackie and Scow, 1976a,b). On the basis of these observations, we conclude that the lamellar structures formed in tissues under the conditions of our study are composed primarily of fatty acids produced by enzymatic hydrolysis of triacylglycerol, in chylomicrons or intracellular lipid droplets. Distribution of lamellae in incubated fixed tissue was affected by both duration of incubation and metabolic state of the tissue donor (Table II and Fig. 27). Lamellar structures were not present at the start of incubation in fixed tissue of fasted rats, w h e r e a s they were p r e s e n t in small amounts, associated with capillary endothelium, adipocytes, and preadipocytes, in tissues of fed and chylomicron-injected fed fats. The latter agrees with the finding that hydrolysis of injected chylomicron tri[~H]acylglycerol occurred in tissue of fed rats while the tissue was being fixed with glutaraldehyde (Table IV). Lamellae developed in tissues of all groups during incu-

LIPOLYSIS AND L A M E L L A E IN ADIPOSE TISSUE

bation and were found associated with all cell types. Lamellae developed first in capillary lumen and later in adipocytes in tissue of fed rats injected with chylomicrons, whereas they developed first in adipocytes and later in capillary lumen in tissue of fasted rats. Also, lamellar structures were closely associated with chylomicrons in capillary lumen in incubated tissue of injected fed rats, and with lipid droplets in adipocytes in incubated tissue of fasted rats. These findings suggest that lamellae in tissue from fed rats are composed of fatty acids formed by hydrolysis of chylomicron triacylglycerol and transferred from capillary lumen toward the interior of adipocytes, while those in tissue from fasted rats are composed of fatty acids formed by hydrolysis of intracellular triacylglycerol and transferred from adipocytes toward capillary lumen. Distribution of lamellar structures in fixed tissue relative to duration of incubation and metabolic state of tissue donor corresponds to the direction of transfer of fatty acids in adipose tissue in vivo, from capillary to adipocytes in fed rats (Scow et al., 1976) and from adipocytes to capillary in fasted rats (Scow and Chernick, 1970). Three mechanisms have been proposed for transport of fatty acids in tissue: transport by molecular diffusion through aqueous medium and across cell membranes (Dietschy, 1978), transport within cells by fatty acid-binding protein (Ockner and Manning, 1974), and transport by lateral movement in cell membranes (Scow et al., 1976, 1977a, 1980). The concept of molecular diffusion of fatty acids requires concentration gradients across cell membranes, created by oxidation, esterification, or release of fatty acids, and presumes no difficulty in transfer of fatty acids across cell membranes or release of fatty acids from membranes to aqueous medium (Dietschy, 1978). Transfer of fatty acids across membranes, however, involves the flip-flop mechanism which is known to be slow (McConnell et al., 1972; Edidin, 1974; Lee et al., 1974). Also, desorption of long-chain

315

fatty acids from interfaces to aqueous medium is very slow at physiological pH (Heikkila et al., 1970; Scow et al., 1979). Transfer of fatty acids between capillary lumen and adipocytes by molecular diffusion in fixed tissue seems unlikely because fatty acids are not esterified in fixed tissue of fed rats or removed from capillary endothelium by albumin in fixed tissue of fasted rats, and consequently, the concentration gradient needed for diffusion would not develop. Transport of fatty acids by the fatty acid-binding protein presumably occurs only in cytosol (Ockner and Manning, 1974). Consequently, it too would require the slow flip-flop mechanism for transfer across cell membranes and, possibly, molecular diffusion through extracellular space. Furthermore, the crosslinking action of glutaraldehyde (Jost et al., 1973) would probably immobilize the fatty acid-binding protein in fixed tissue. Transport of fatty acids by lateral movement in a continuum of cell membranes presupposes that an interfacial continuum extends between capillary lumen and lipid droplets of fat cells, and that fatty acids will locate and move in the continuum. Earlier studies showed that transendothelial channels, connecting capillary lumen with extravascular space, are present in capillaries of adipose, muscle, and mammary tissue in adult animals (Blanchette-Mackie and Scow, 1971; Simionescu et al., 1975; Scow et al., 1976, 1977a). Similar structures were found recently in capillaries of adipose tissue in young rats (Blanchette-Mackie and Scow, 1981). Since the external (luminal) leaflet of membranes lining these channels connects the external leaflet of the luminal plasma membrane with that of the basal plasma membrane, it provides an interfacial continuum across capillary endothelium. The studies in young rats also showed that cell processes of adipocytes and endothelium extended through basement membrane to make contact with each other (Blanchette-Mackie and Scow, 1981). Apparent continuity of the external leaflet of

316

C~s~ic

BLANCHETTE-MACKIE AND SCOW

L~flet

FIG. 28. Mechanism proposed for formation of lamellar whorls. Overcrowding in the external leaflet by fatty acids, represented by dots, causes collapse and outward extension of the leaflet, forming bilayered whorls.

plasma membrane of one cell with that of another cell was observed. The same studies demonstrated that intracellular channels, possibly endoplasmic reticulum, extended from extracellular space to the surface of lipid droplets in adipocytes. Thus, the luminal leaflet of membranes lining these channels provides an interfacial continuum between the external leaflet of plasma membrane and the surface of lipid droplets. These findings, then, provide morphological evidence for an interfacial continuum from the luminal surface to the basal surface of endothelial cells and from the surface of fat cells to lipid droplets within the cells, but only tentative evidence for an interfacial continuity between cells. Studies with artificial and cellular membranes have shown that lipid molecules can move rapidly in the plane of membranes (Edidin, 1974; Lee et al., 1974; McConnell et al., 1972). Morphological studies of chylomicrons incubated with purified lipoprotein lipase in medium, pH 8.1, containing limited albumin (Blanchette-Mackie and Scow, 1976a) showed that lipolytic products (fatty acids and monoacylglycerol) accumulated in the interface between triacylglycerol and the aqueous medium, and extended the interface by forming bilayers that spiraled into aqueous spaces within the chylomicron core. The findings also demonstrated that lipolytic products can transfer from the site of enzyme action by lateral movement in the interface. Similar phenomena were observed when trioleoylglycerol was applied in excess to the surface of aqueous medium, pH 7.4, in a monolayer

trough with interconnected compartments and hydrolyzed by lipoprotein lipase to oleic acid and monooleolylglycerol (Scow et al., 1979). The amphipathic products immediately located and spread throughout the interface. As they crowded the interface, surface pressure of the interface increased, hpolysls slowed down, and lipolytic products slowly disappeared from the interface. Albumin added to the aqueous subphase, to serve as a trapping agent for oleic acid and monooleolyglycerol, removed lipolytic products quickly from the interface, and thereby lowered surface pressure and enhanced lipolysis. Movement of lipolytic product in the interface was readily observed when lipase a n d albumin were added to different chambers. Electron microscopic studies have shown that monolayers of various amphipathic lipids, including long-chain fatty acid, form bilayered folds when they collapse in response to high surface pressure (Gaines, 1966; Ries et al., 1957, 1975, 1976, 1978). The above observations indicate that long-chain fatty acids in tissue at pH 7.4 could locate in the proposed interracial continuum and spread rapidly toward any area of decreased surface pressure, resulting from either removal of fatty acids from the interface or extension of the interface. In vivo, fatty acids would be removed from the interfacial continuum by esterification, oxidation, or binding to albumin in plasma (Scow et al., 1980). When these removal processes are suppressed in glutaraldehyde-fixed tissues, fatty acids in an overcrowded interface could be expected to form, at collapse pressure, a bilayered extension of the interface and thereby create space for themselves in the interface. Our present study showed that long bilayered structures, which appeared to be continuous with the external leaflet of plasma membrane (Figs. 6, 7, and 12), extended outward from the surface of adipocytes in glutaraldehyde-fixed tissues, and when tissues were incubated with tannic acid, the extensions formed tightly wound lamellar

317

LIPOLYSIS AND LAMELLAE 1N ADIPOSE TISSUE Site of FATTY ACID REMOVAL by Albumin

\

PlasmaMembrane

, ; ; ,7) ~} ~J~, }.,} , J,,,/A~k ' ~ r . . ~ / / / / / / y / / / S 2 PARENCHYMA/. CELL//' Endoplasmie Reticul. . . . .

////,//////,/All "///////~

kca~ V) ~

Siteof ESTERIFICATIONY//////

".////////

FIG. 29. Route proposed for transport of lipolytic products between capillary lumen and adipocytes in tissue by lateral movement in an interfacial continuum of cell membranes. The interfacial continuum, represented by the broad white line, is composed of the external leaflets of plasma and intracellular membranes of endothelial and parenchymal cells, and also the chylomicron surface film in fed animals. Lipolytic products formed by action of lipoprotein lipase on chylomicron triacylglycerol, shown by dots in a, enter the continuum in capillary lumen and leave the continuum in endoplasmic reticulum when they are esterified to triacylglycerol and accumulate as lipid droplets between leaflets of reticulum. Lipolytic products formed by action of tissue lipase on intracellular triacylglycerol, shown by dots in b, enter the continuum in endoplasmic reticulum and leave the continuum in capillary lumen when they bind to albumin in the blood stream. Cytoplasmic leaflets of membranes are represented by broad finely stippled lines. (Adapted from Scow et al., 1980.)

'whorls. Similar lamellar whorls were found i[n intracellular channels of endothelium and adipocytes, and at sites of intercellular contact. We conclude from these observations that fatty acids formed by lipolysis in glutaraldehyde-fixed tissues accumulated in an interfacial continuum of external (luminal) leaflets of plasma and intracellular membranes of both endothelium and adipocytes, and when they overcrowded the continuum, they formed lamellar whorls (Fig. 28) at various sites along the continuum and thereby marked its course through the tissue. We propose that in vivo lipolytic products would also move in an interfacial continuum of outer leaflets of membranes extending b e t w e e n capillary lumen and adipocytes of adipose tissue (Fig. 29). In tissues of fed animals, lipolytic products formed by action of lipoprotein lipase on

chylomicrons would enter the continuum in capillary lumen and be removed from the continuum in endoplasmic reticulum where they would be reesterified to triacylglycerol and accumulate as lipid droplets between leaflets of endoplasmic reticulum (Fig. 29a). In tissues of fasted rats, lipolytic products formed by action of tissue (hormone-sensitive) lipase ,on triacyglycerol in adipocytes would enter the continuum in adipocytes and be removed from the continuum at the luminal surface of capillaries by albumin in circulating plasma (Fig. 29b). We are grateful to Patricia D. Rojko and T. Ruth Fleck for their expert assistance in this study. REFERENCES BIEBERDORF, F. A., CHERNICK, S. S. AND SCOW, R. O. (1970) J. Clin. Invest. 49, 1685-1693. BLANCHETTE-MACKIE, E. J., AND Scow, R. O. (1971) J. Cell Biol. 51, 1-25.

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BLANCHETTE-MACKIE AND SCOW

BLANCHETTE-MACKIE,E. J., AND SCOW, R. O. (1973) J. Cell Biol. 58, 689-708. BLANCHETTE-MACKIE, E. J., AND SCOW, R. O. (1976a) J. Lipid Res. 17, 57-67. BLANCHETTE-MACKIE, E. J., AND SCOW, R. O. (1976b) Anat. Rec. 184, 599-609. BLANCHETTE-MACKIE,E. J., AND Scow, R. O. (1981) J. Ultrastruct. Res. 77, 277-294. BORGSTROM, B. (1952) Acta Physiol. Scand. 25, 111119. BORGSTROM, B. (1954) Acta Physiol. Scand. 30, 230239. CHERNICK, S. S. (1969) in LOWENSTEIN, J. M. (Ed.), Methods of Enzymology, Vol. 14, pp. 2416-2419, Academic Press, New York. DIETSCHY, J. M. (1978) in DIETSCHY, J. M., GOTTO, JR., A. M., AND ONTKO, J. A. (Eds.), Disturbances in Lipid and Lipoprotein Metabolism, pp. 1-28, Amer. Physiol. Soc., Bethesda, Md. EDIDIN, M. (1974) Annu. Rev. Biophys. Bioeng. 3, 179-201. FOLCH, J., LEES, M., AND STANLEY, G. H. S. (1957) J. Biol. Chem. 226, 497-509. GAINES, G. L. (1966). Insoluble Monolayers at Liquid-Gas Interfaces, pp. 144-I51, Interscience, New York. GOMORI, G. (1952) Microscopic Histochemistry, p. 200, Univ. Press, Chicago. HASTY, D. L., AND HAY, E. D. (1978) J. Cell Biol. 78, 756-768. HEIKKILA, R. E., DEAMER, D. W., AND CORNWALL, D. G. (1970) J. Lipid Res. 11, 195-200. JOST, P., BROOKS, U. J., AND GRIFFITH, O. H. (1973) J. Mol. Biol. 76, 313-318. LEE, A. G., BIRDSALL,N. J. M., AND METCALEE, J. C. (1974) in KORN, E. D. (Ed.), Methods in Membrane Biology, Vol. 2, pp. 1-156, Plenum, New York. MCCONNELL, H. M., DEVAUX, P., AND SCANDELLA, C. (1972). in Fox, C. F. (Ed.), Membrane Research, pp. 27-37, Academic Press, New York. OCKNER, R. K., AND MANNING, J. A. (1974) J. Clin. Invest. 54, 326-338. RAPPORT, M. M., AND ALONZO, M. (1959) J. Biol. Chem. 217, 195-198. RIES, H. E., JR., AND KIMBALL, W. A. (1957) in Proceedings of the Second International Congress on Surface Activity, Vol. 1, pp. 75-84, Butterworths, London.

RIES, H. E., JR., MATSUMOTO,M., UYEDA, N., AND SU1TO, E. (1975) in GODDARD, E. D. (Ed.), Advances in Chemistry: Monolayers, Number 144, pp. 285-293, Amer. Chem. Soc., Washington, D.C. RIES, H. E., JR., MATSUMOTO,M., UYEDA, N., AND SUITO, E. (1976) J. Colloid Interface Sci. 5, 396398. RIEs, H. E., JR., AND SWIFT, H. (1978) J. Colloid Interface Sci. 64, 111-119. ROBINSON, D. S. (1970) in FLORK1N, M., AND STOTZ, E. H. (Eds.), Comprehensive Biochemistry, Vol. 18, pp. 51-116, Elsevier, Amsterdam. SCHOEFL, G. I., AND FRENCH, J, E. (1968) Proc. Roy. Soc. Ser, B 169, 153-165. Scow, R. O., AND CHERNICK, S. S. (1970) in FLORKIN, M., AND STOTZ, E. H. (Eds.), Comprehensive Biochemistry, Vol. 18, pp. 19-49, Elsevier, Amsterdam. Scow, R. O., STEIN, Y., AND STEIN, O. (1967) J. Biol. Chem. 242, 4919-4924. Scow, R. O., HAMOSH, M., BLANCHETTE-MACKIE, E. J., AND EVANS, A. J. (1972) Lipids 7, 497-505. SCOW, R. O., BLANCHETTE-MACKIE, E. J., AND SMITH, L. C. (1976) Circ. Res. 39, 149-162. Scow, R. O., BLANCHETTE-MACKIE, E. J., AND SMITH, L. C. (1977a) in POLONOVSKI,J. (Ed.), Cholesterol Metabolism and Lipolytic Enzymes, pp. 143-165, Masson, New York, Scow, R. O., CHERNICK, S. S., AND FLECK, T. R. (1977b) Biochim. Biophys. Acta 487, 297-306. Scow, R. O., DESNUELLE,P., AND VERGER, R. (1979) J. Biol. Chem. 254, 6456-6463. Scow, R. O., BLANCHETTE-MACKIE,E. J., AND SMITH, L. C. (1980) Fed. Proc. 39, 2610-2617. SIMIONESCU, N., SIMIONESCU, M., AND PALADE, G. E. (1975) J. Cell Biol. 64, 586-607. SMITH, L. C., AND SCOW, R. O. (1979) Progr. Biochem. Pharmacol. 15, 109-138. SPECTOR, A. A., AND FLETCHER, J. E. (1978) in DIETSCHY, J. M., GOTTO, A. M., JR., AND ONTKO, J. A. (Eds.), Disturbances in Lipid and Lipoprotein Metabolism, pp. 229-249, Amer. Physiol. Soc., Bethesda, Md. STEIN, O., Scow, R. O., AND STEIN, Y. (1970) Amer. J. Physiol. 219, 510-518, WEIBEL, E. R., AND BOLENDER,R. P. (1973) in HAYAT, M. A. (Ed.), Principles and Techniques for Electron Microscopy, Vol. 3, pp. 237-296, Van Nostrand-Reinhold, New York.