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Subcellular distribution of newly synthesized glycerolipids in intact and homogenized rat adipocytes
(m~p. Biochem. Physiol., Vol. 63B. pp. 521 to 523
(1305-(1491 7 9 0801-0521S02.(10 (I
'C Pergamon Press Lid 1979. Printed in Great Britain
SUBCELLULAR DISTRIBUTION OF NEWLY S Y N T H E S I Z E D G L Y C E R O L I P I D S IN I N T A C T A N D H O M O G E N I Z E D RAT A D I P O C Y T E S JEAN-PAUL GIACOBINO D6partement de Biochimie m6dicale, Universit6 de Gen6ve, 20 r. Ecole de M6decine, 1211 Gen6ve 4, Suisse (Received 19 December 1978)
Abstract--1. Homogenization of rat adipocytes was found to inhibit the transfer of newly synthesized phospholipids and glycerides from the membranes to the storage lipids. 2. In intact adipocytes, only 23 + 3.4% of the phospholipids and 0.44 ___0.12°,~, of the glycerides remained bound to the membranes, while for the homogenate, the comparable values obtained were 74 + 5.2~o and 90 + 0.3~o respectively. 3. Various factors that might be involved in the liberation of the glycerolipids from the adipocyte esterification sites are discussed.
INTRODUCTION It has been shown that the fatty acid esterification enzymes of the adipocyte are located principally in the subcellular m e m b r a n e s (Giacobino & Chmelar, 1977). The glycerolipids synthesized by these enzymes in intact adipocyte are either incorporated into the adipocyte m e m b r a n e s or transferred to the adipocyte storage lipids. The m e m b r a n e lipids have been found to be composed essentially of phospholipids and the storage lipids essentially of glycerides, although studies with labeled precursors have revealed the existence of m e m b r a n e glyceride as well as cytoplasmic phospholipid pools (Angel, 1970). The present study is a n attempt to investigate the biological events that might be responsible for the observed subcellular distribution of glycerolipids and particularly for the transfer of the glycerolipids from the m e m b r a n e esterification sites to the storage lipid pool. Subcellular distribution of newly synthesized glycerolipids in intact and homogenized rat adipocytes was measured. MATERIALS AND METHODS Sprague-Dawley male rats, about 2 months old, weighing 200-250 g and fed Nafag chow (St-Gall, Switzerland) ad libitum, were used. Isolated adipocytes of epididymal and perirenal white adipose tissue were prepared according to the method of Rodbell (1964). In one type of experiment, about 2 g of the isolated adipocytes were incubated at 37°C in 5 ml of Krebs-Ringer bicarbonate buffer at pH 7.4 gassed with oxycarbon (95~o 02, 5~o CO2), containing 3.5~o bovine serum albumin and 59.4 mg~o D-glucose and to which [1-14C] palmitate (0.05 raM) had been added. In a second type of experiment the isolated adipocytes were homogenized in a Potter-Eivej hem homogenizer (1800 rev/ min, 10 up and down strokes, clearance 0.3 mm) in 1.5 vol of the Krebs-Ringer bicarbonate buffer. The homogenate was centrifuged for 20 min at 1300 g and 1 ml of the nucleifree homogenate was incubated at 37°C in a total volume of 2 ml of the Krebs-Ringer bicarbonate buffer to which nicotinic acid (0.5 mM), CDP-choline (0.2 mM), palmitoylCoA (0.1 mM) and [U-t4C]ct-glycerophosphate (0.1 mM) had been added. The experiments were stopped after 3 min by adding N-ethylmaleimide to obtain a final concen-
tration of 2 mM, which has been found to inhibit the incorporation of palmitate into adipocyte total glycerolipids by 92~o. In the experiments with isolated adipocytes, the entire mixture was then spun for 15 sec at 3000 rev/min. The fat cells were washed once with 2 mM N-ethylmaleimide in Krebs-Ringer bicarbonate buffer at 37°C and then homogenized at 4°C with a known amount (about 5 g) of nonlabeled fat cells in 3vol of 0.25M sucrose, 10mM Tris-HC1, 1 mM EDTA-Tris (pH 7.4). After 20 min centrifugation at 1300 9, the fat cake was removed and the nuclei-free homogenate was centrifuged for 45 min at 180,000 g to yield the total membrane fraction (plasma membranes + microsome + mitochondria) and the membranefree supernatant. In the experiments with homogenized adipocytes, the total membrane fraction and the membrane-free supernatant were separated in the same way. In all experiments, phospholipids and glycerides were added to the membrane fractions as carriers. The total lipids of either the fat cake, the total membrane fraction or an aliquot of the membrane-free supernatant were extracted according to the method described by Folch et al. (1957). The phospholipids were then separated from the glyceride-fatty acid fraction in silicic acid columns (Unisil, Clarkson Chemical Co., Williamsport, Pa., U.S.A.) according to the method described by Lombardi & Ugazio (19651. Finally, the glycerides were separated from the free fatty acids according to the method described by Coleman & Bell (1976). In the first type of experiment, using [1-a4C]palmitate as glycerolipid precursor, the labeled fatty acids contaminating the glyceride fraction and eliminated by the method of Coleman & Bell (1976) were found to represent 17~o and 1~o respectively of the total membrane and storage lipid glyceride fractions. In the second type of experiment using [U-a4C]ct-glycerophosphate as glycerolipid precursor, a blank was performed without palmitoyl-CoA. The value of the blank was found to be less than 1~o of the value obtained in the assays performed in the presence of palmitoyl-CoA. The radioactivity was measured by liquid scintillation (Intertechnique) and the proteins according to the method described by Lowry et al., (1951). In some experiments, the homogenate was concentrated by dialysis on polyethylene glycol 6000 and in others, the Krebs-Ringer bicarbonate buffer was replaced by a medium containing 10raM NaHCO3, 375mM PO4K3, 40mM MgCI2, 40mM MgSO4, 3.5~o bovine serum albumin and 59.4 mg ~o D-glucose at pH 7.4 gassed with oxycarbon. This medium had an electrolyte composi-
521
522
JEAN-PAUL G1ACOBINO
800.
600.
400
s / t s ~ t t P
200 J
s p
s ~
J / //
3
6 12 MINUTES Fig. 1. Incorporation of labeled precursors into the total membrane glycerolipids ( - - - - - - ) or into the storage glycerolipids ( ) of isolated adipocytes (O) or of homogenized adipocytes (e). The values represent the average values + SEM of 2-3 experiments. They are expressed as nmols of labeled precursor incorporated into the total glycerolipids of the total subcellular fractions obtained from the adipose tissue of 3 rats. In the experiments with isolated adipocytes the values given for storage glycerolipids represent the sum of those obtained for the fat cake and the membrane-free supernatant. In the experiments with homogenized adipocytes the values given for storage glycerolipids are those of the membrane-free supernatant. tion similar to that of intracellular fluid and is therefore referred to as intracellular medium. RESULTS AND DISCUSSION Figure 1 illustrates the incorporation of newly synthesized glycerolipids into total membranes or storage lipids. It can be seen that in the experiments utilizing intact adipocytes the incorporation of [1-14C]palmitate into adipocyte total membrane glycerolipids reached a plateau at 3 min (2.2, 2.2 and 2.4 nmols of palmitate for the time periods 3, 6 and 12 min respectively, incorporated into the total glycerolipids of the total subcellular fractions obtained from the adipose tissue of 3 rats). On the other hand, the incorporation of[l-14C]palmitate into adipocyte storage ~lycerolipids was very rapid up to 6 min when it reached a plateau. In the experiments utilizing homogenized adipocytes, the incorporation of labeled precursors into total membrane and storage glycerolipids had still not reached a plateau at 12 min. It can also be seen that, in the experiments utilizing intact adipocytes, more labeled glycerolipids were found in the storage lipids than in the membranes. In the experi-
ments utilizing homogenized adipocytes, however, more labeled glycerolipids were found in the membranes than in the storage lipids. This difference in distribution of the glycerolipids persists up to 12 min after the beginning of the experiment. Table 1 shows the incorporation of each of the components of the newly synthesized glycerolipids into total membrane and storage lipids after a 3 min incubation period. It can be seen that homogenization results in an increase in total phospholipid synthesis and a decrease in total glyceride synthesis. The large quantity of phospholipids found in the homogenate might be due to an inhibition of phosphotidate phosphatase (EC 3.1.3.4) activity by cell disruption. The difference in distribution of the glycerolipids caused by homogenization and shown in Fig. 1. does not seem to be a simple consequence of the difference in the composition of the newly synthesized glycerolipids. As can be seen in the table, the transfer of both newly synthesized phospholipids and glycerides from the membranes to the storage lipids was, in fact, found to be greatly inhibited in the homogenate. In intact adipocytes, only 23 + 3.4% of the phospholipids and 0.44 + 0.12% of the glycerides remained
Distribution of glycerolipids
523
Table 1. Incorporation of newly synthesized glycerolipids into total membranes or storage lipids Phospholipids Total membranes Storage lipids Isolated adipocytes Homogenized adipocytes
0.79 + 0.18(4) 89 + 19 (3)
2.7 + 0.11 (4) 32 ___7.8 (3)
Glycerides Total membranes Storage lipids 0.71 + 0.17(5) 34 + 6.4 (3)
162 + 28(5) 3.69 _+ 0.63 (3)
The above are average values + SEM expressed as nmols of labeled precursor incorporated in 3 min into the phospholipids or glycerides of the total subcellular fractions obtained from the adipose tissue of 3 rats. The number of experiments is indicated in parentheses.
bound to the membranes, while for the homogenate, the comparable values obtained were 74 + 5.2~o and 90 + 0.3~o respectively. Since it was found that membrane labeled glycerolipid content could not be reduced by more than 10~o by sonication, it seems likely that the glycerolipids are bound to the membranes rather than trapped in the intravesicular space. This difference in subcellular glycerolipid distribution between intact adipocyte and homogenate cannot be attributed to the different esterification substrates used since identical results were obtained when palmitoyl-CoA was replaced in the homogenate incubation medium by palmitate + ATP + CoASH. None of the expected biochemical modifications resulting from homogenization in Krebs-Ringer solution, i.e. change in cytoplasm electrolyte composition, dilution of cytoplasm proteins and decrease in ATP generation, seems to be responsible for the inhibition of the transfer of the glycerolipids from the membranes to the storage lipids. It was found, in fact, that neither homogenization in the intracellular medium described under methods, the use of homogenate that was concentrated two times, nor the addition of up to 1 mM ATP to the incubation medium had any effect on this transfer. The results do not exclude the possibility that some cytoplasmic protein(s) could be involved in the transfer of the glycerolipids from the membranes to the storage lipids but suggest that, in this case, it would be effective only at a high concentration or combined with some other substance that is lacking in the homogenate. It seems more probable, however, that the topographical modifications caused by the homogenization of the adipocyte are responsible for the observed inhibition of the transfer of glycerolipids from the membranes to the storage lipids. This would imply that, in the intact adipocyte, there is a topographical relationship between the endoplasmic reticulum where the esterification of the storage glycerolipids would take place (Giacobino, 1978), and the fat droplet, resulting in direct delivery of neosynthesized glycerolipids from the membranes to the fat droplet without prior passage through the hydrophilic cytoplasm. Electron micrographs of the adipocyte have shown, in fact, that
the smooth endoplasm~c reticulum cisternae are applied to the surface of the fat droplet (Orci & Perrelet, 1975). The results of this study suggest that the liberation of glycerolipids from the adipocyte esterification sites on the membrane is not spontaneous and occurs only when the intracellular topography is intact. Acknowledgements--We wish to thank Professor P. Favarger and Professor Simonne Rous for stimulating discussions of this work. We acknowledge Miss C. Colomb for her excellent technical assistance and Mrs J. Noebels for her valuable help in the editing of our manuscript. This work was supported as far as equipment is concerned by the Swiss National Science Foundation, grant No. 3.8970.72. REFERENCES
ANGELA. (1970) Studies on the compartmentation of lipid in adipose cells--l: Subcellular distribution, composition and transport of newly synthesized lipid:liposomes. J. Lipid Res. 11, 420-432. COLEMANR. & BELL R. M. (1976) Triacylglycerol synthesis in isolated fat cells; studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols. J. biol. Chem. 251, 4537-4543. FOLCH J., LEES M. & SLOANE STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GIACOaINOJ. P. & CHMELARM. (1977) Fatty acid esterification in adipocyte subcellular fractions. Int. J. Biochem. 8, 413-416. GIACOmNOJ. P. (1978) Role of the plasma membranes and of the intra-cellular membranes in adipocyte glycerolipid synthesis. Experientia 34, 17. LOMBARDI B. & UGAZIOG. (1965) Serum lipoproteins in rats with carbon tetrachloride-induced fatty liver. J. Lipid Res. 6, 498-505. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. ORCI L. t~ PERRELET A. (1975) Freeze-Etch Histology, pp. 159-160, Springer, Berlin. RODBELL M. (1964) Metabolism of isolated fat cells--I. Effects of hormones on glucose metabolism and lipolysis. J. biol. Chem. 239, 375-380.
(1305-(1491 7 9 0801-0521S02.(10 (I
'C Pergamon Press Lid 1979. Printed in Great Britain
SUBCELLULAR DISTRIBUTION OF NEWLY S Y N T H E S I Z E D G L Y C E R O L I P I D S IN I N T A C T A N D H O M O G E N I Z E D RAT A D I P O C Y T E S JEAN-PAUL GIACOBINO D6partement de Biochimie m6dicale, Universit6 de Gen6ve, 20 r. Ecole de M6decine, 1211 Gen6ve 4, Suisse (Received 19 December 1978)
Abstract--1. Homogenization of rat adipocytes was found to inhibit the transfer of newly synthesized phospholipids and glycerides from the membranes to the storage lipids. 2. In intact adipocytes, only 23 + 3.4% of the phospholipids and 0.44 ___0.12°,~, of the glycerides remained bound to the membranes, while for the homogenate, the comparable values obtained were 74 + 5.2~o and 90 + 0.3~o respectively. 3. Various factors that might be involved in the liberation of the glycerolipids from the adipocyte esterification sites are discussed.
INTRODUCTION It has been shown that the fatty acid esterification enzymes of the adipocyte are located principally in the subcellular m e m b r a n e s (Giacobino & Chmelar, 1977). The glycerolipids synthesized by these enzymes in intact adipocyte are either incorporated into the adipocyte m e m b r a n e s or transferred to the adipocyte storage lipids. The m e m b r a n e lipids have been found to be composed essentially of phospholipids and the storage lipids essentially of glycerides, although studies with labeled precursors have revealed the existence of m e m b r a n e glyceride as well as cytoplasmic phospholipid pools (Angel, 1970). The present study is a n attempt to investigate the biological events that might be responsible for the observed subcellular distribution of glycerolipids and particularly for the transfer of the glycerolipids from the m e m b r a n e esterification sites to the storage lipid pool. Subcellular distribution of newly synthesized glycerolipids in intact and homogenized rat adipocytes was measured. MATERIALS AND METHODS Sprague-Dawley male rats, about 2 months old, weighing 200-250 g and fed Nafag chow (St-Gall, Switzerland) ad libitum, were used. Isolated adipocytes of epididymal and perirenal white adipose tissue were prepared according to the method of Rodbell (1964). In one type of experiment, about 2 g of the isolated adipocytes were incubated at 37°C in 5 ml of Krebs-Ringer bicarbonate buffer at pH 7.4 gassed with oxycarbon (95~o 02, 5~o CO2), containing 3.5~o bovine serum albumin and 59.4 mg~o D-glucose and to which [1-14C] palmitate (0.05 raM) had been added. In a second type of experiment the isolated adipocytes were homogenized in a Potter-Eivej hem homogenizer (1800 rev/ min, 10 up and down strokes, clearance 0.3 mm) in 1.5 vol of the Krebs-Ringer bicarbonate buffer. The homogenate was centrifuged for 20 min at 1300 g and 1 ml of the nucleifree homogenate was incubated at 37°C in a total volume of 2 ml of the Krebs-Ringer bicarbonate buffer to which nicotinic acid (0.5 mM), CDP-choline (0.2 mM), palmitoylCoA (0.1 mM) and [U-t4C]ct-glycerophosphate (0.1 mM) had been added. The experiments were stopped after 3 min by adding N-ethylmaleimide to obtain a final concen-
tration of 2 mM, which has been found to inhibit the incorporation of palmitate into adipocyte total glycerolipids by 92~o. In the experiments with isolated adipocytes, the entire mixture was then spun for 15 sec at 3000 rev/min. The fat cells were washed once with 2 mM N-ethylmaleimide in Krebs-Ringer bicarbonate buffer at 37°C and then homogenized at 4°C with a known amount (about 5 g) of nonlabeled fat cells in 3vol of 0.25M sucrose, 10mM Tris-HC1, 1 mM EDTA-Tris (pH 7.4). After 20 min centrifugation at 1300 9, the fat cake was removed and the nuclei-free homogenate was centrifuged for 45 min at 180,000 g to yield the total membrane fraction (plasma membranes + microsome + mitochondria) and the membranefree supernatant. In the experiments with homogenized adipocytes, the total membrane fraction and the membrane-free supernatant were separated in the same way. In all experiments, phospholipids and glycerides were added to the membrane fractions as carriers. The total lipids of either the fat cake, the total membrane fraction or an aliquot of the membrane-free supernatant were extracted according to the method described by Folch et al. (1957). The phospholipids were then separated from the glyceride-fatty acid fraction in silicic acid columns (Unisil, Clarkson Chemical Co., Williamsport, Pa., U.S.A.) according to the method described by Lombardi & Ugazio (19651. Finally, the glycerides were separated from the free fatty acids according to the method described by Coleman & Bell (1976). In the first type of experiment, using [1-a4C]palmitate as glycerolipid precursor, the labeled fatty acids contaminating the glyceride fraction and eliminated by the method of Coleman & Bell (1976) were found to represent 17~o and 1~o respectively of the total membrane and storage lipid glyceride fractions. In the second type of experiment using [U-a4C]ct-glycerophosphate as glycerolipid precursor, a blank was performed without palmitoyl-CoA. The value of the blank was found to be less than 1~o of the value obtained in the assays performed in the presence of palmitoyl-CoA. The radioactivity was measured by liquid scintillation (Intertechnique) and the proteins according to the method described by Lowry et al., (1951). In some experiments, the homogenate was concentrated by dialysis on polyethylene glycol 6000 and in others, the Krebs-Ringer bicarbonate buffer was replaced by a medium containing 10raM NaHCO3, 375mM PO4K3, 40mM MgCI2, 40mM MgSO4, 3.5~o bovine serum albumin and 59.4 mg ~o D-glucose at pH 7.4 gassed with oxycarbon. This medium had an electrolyte composi-
521
522
JEAN-PAUL G1ACOBINO
800.
600.
400
s / t s ~ t t P
200 J
s p
s ~
J / //
3
6 12 MINUTES Fig. 1. Incorporation of labeled precursors into the total membrane glycerolipids ( - - - - - - ) or into the storage glycerolipids ( ) of isolated adipocytes (O) or of homogenized adipocytes (e). The values represent the average values + SEM of 2-3 experiments. They are expressed as nmols of labeled precursor incorporated into the total glycerolipids of the total subcellular fractions obtained from the adipose tissue of 3 rats. In the experiments with isolated adipocytes the values given for storage glycerolipids represent the sum of those obtained for the fat cake and the membrane-free supernatant. In the experiments with homogenized adipocytes the values given for storage glycerolipids are those of the membrane-free supernatant. tion similar to that of intracellular fluid and is therefore referred to as intracellular medium. RESULTS AND DISCUSSION Figure 1 illustrates the incorporation of newly synthesized glycerolipids into total membranes or storage lipids. It can be seen that in the experiments utilizing intact adipocytes the incorporation of [1-14C]palmitate into adipocyte total membrane glycerolipids reached a plateau at 3 min (2.2, 2.2 and 2.4 nmols of palmitate for the time periods 3, 6 and 12 min respectively, incorporated into the total glycerolipids of the total subcellular fractions obtained from the adipose tissue of 3 rats). On the other hand, the incorporation of[l-14C]palmitate into adipocyte storage ~lycerolipids was very rapid up to 6 min when it reached a plateau. In the experiments utilizing homogenized adipocytes, the incorporation of labeled precursors into total membrane and storage glycerolipids had still not reached a plateau at 12 min. It can also be seen that, in the experiments utilizing intact adipocytes, more labeled glycerolipids were found in the storage lipids than in the membranes. In the experi-
ments utilizing homogenized adipocytes, however, more labeled glycerolipids were found in the membranes than in the storage lipids. This difference in distribution of the glycerolipids persists up to 12 min after the beginning of the experiment. Table 1 shows the incorporation of each of the components of the newly synthesized glycerolipids into total membrane and storage lipids after a 3 min incubation period. It can be seen that homogenization results in an increase in total phospholipid synthesis and a decrease in total glyceride synthesis. The large quantity of phospholipids found in the homogenate might be due to an inhibition of phosphotidate phosphatase (EC 3.1.3.4) activity by cell disruption. The difference in distribution of the glycerolipids caused by homogenization and shown in Fig. 1. does not seem to be a simple consequence of the difference in the composition of the newly synthesized glycerolipids. As can be seen in the table, the transfer of both newly synthesized phospholipids and glycerides from the membranes to the storage lipids was, in fact, found to be greatly inhibited in the homogenate. In intact adipocytes, only 23 + 3.4% of the phospholipids and 0.44 + 0.12% of the glycerides remained
Distribution of glycerolipids
523
Table 1. Incorporation of newly synthesized glycerolipids into total membranes or storage lipids Phospholipids Total membranes Storage lipids Isolated adipocytes Homogenized adipocytes
0.79 + 0.18(4) 89 + 19 (3)
2.7 + 0.11 (4) 32 ___7.8 (3)
Glycerides Total membranes Storage lipids 0.71 + 0.17(5) 34 + 6.4 (3)
162 + 28(5) 3.69 _+ 0.63 (3)
The above are average values + SEM expressed as nmols of labeled precursor incorporated in 3 min into the phospholipids or glycerides of the total subcellular fractions obtained from the adipose tissue of 3 rats. The number of experiments is indicated in parentheses.
bound to the membranes, while for the homogenate, the comparable values obtained were 74 + 5.2~o and 90 + 0.3~o respectively. Since it was found that membrane labeled glycerolipid content could not be reduced by more than 10~o by sonication, it seems likely that the glycerolipids are bound to the membranes rather than trapped in the intravesicular space. This difference in subcellular glycerolipid distribution between intact adipocyte and homogenate cannot be attributed to the different esterification substrates used since identical results were obtained when palmitoyl-CoA was replaced in the homogenate incubation medium by palmitate + ATP + CoASH. None of the expected biochemical modifications resulting from homogenization in Krebs-Ringer solution, i.e. change in cytoplasm electrolyte composition, dilution of cytoplasm proteins and decrease in ATP generation, seems to be responsible for the inhibition of the transfer of the glycerolipids from the membranes to the storage lipids. It was found, in fact, that neither homogenization in the intracellular medium described under methods, the use of homogenate that was concentrated two times, nor the addition of up to 1 mM ATP to the incubation medium had any effect on this transfer. The results do not exclude the possibility that some cytoplasmic protein(s) could be involved in the transfer of the glycerolipids from the membranes to the storage lipids but suggest that, in this case, it would be effective only at a high concentration or combined with some other substance that is lacking in the homogenate. It seems more probable, however, that the topographical modifications caused by the homogenization of the adipocyte are responsible for the observed inhibition of the transfer of glycerolipids from the membranes to the storage lipids. This would imply that, in the intact adipocyte, there is a topographical relationship between the endoplasmic reticulum where the esterification of the storage glycerolipids would take place (Giacobino, 1978), and the fat droplet, resulting in direct delivery of neosynthesized glycerolipids from the membranes to the fat droplet without prior passage through the hydrophilic cytoplasm. Electron micrographs of the adipocyte have shown, in fact, that
the smooth endoplasm~c reticulum cisternae are applied to the surface of the fat droplet (Orci & Perrelet, 1975). The results of this study suggest that the liberation of glycerolipids from the adipocyte esterification sites on the membrane is not spontaneous and occurs only when the intracellular topography is intact. Acknowledgements--We wish to thank Professor P. Favarger and Professor Simonne Rous for stimulating discussions of this work. We acknowledge Miss C. Colomb for her excellent technical assistance and Mrs J. Noebels for her valuable help in the editing of our manuscript. This work was supported as far as equipment is concerned by the Swiss National Science Foundation, grant No. 3.8970.72. REFERENCES
ANGELA. (1970) Studies on the compartmentation of lipid in adipose cells--l: Subcellular distribution, composition and transport of newly synthesized lipid:liposomes. J. Lipid Res. 11, 420-432. COLEMANR. & BELL R. M. (1976) Triacylglycerol synthesis in isolated fat cells; studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols. J. biol. Chem. 251, 4537-4543. FOLCH J., LEES M. & SLOANE STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GIACOaINOJ. P. & CHMELARM. (1977) Fatty acid esterification in adipocyte subcellular fractions. Int. J. Biochem. 8, 413-416. GIACOmNOJ. P. (1978) Role of the plasma membranes and of the intra-cellular membranes in adipocyte glycerolipid synthesis. Experientia 34, 17. LOMBARDI B. & UGAZIOG. (1965) Serum lipoproteins in rats with carbon tetrachloride-induced fatty liver. J. Lipid Res. 6, 498-505. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. ORCI L. t~ PERRELET A. (1975) Freeze-Etch Histology, pp. 159-160, Springer, Berlin. RODBELL M. (1964) Metabolism of isolated fat cells--I. Effects of hormones on glucose metabolism and lipolysis. J. biol. Chem. 239, 375-380.