69

Biochimica etBiophysica 0 Elsevier/North-Holland

Acta, 450 (1976) 69-77 Biomedical Press, Amsterdam - Printed in The Netherlands

BBA 56860

SECRETION OF LECITHIN:CHOLESTEROL FROM ISOLATED RAT HEPATOCYTES

ACYLTRANSFERASE

G. NORDBY, T. BERG, M. NILSSON and K.R. NORUM Institute for Nutrition Research, School of Medicine,

University of Oslo, Oslo (Norway)

(Received March 19th, 1976)

Summary 1. Lecithin:cholesterol acyltransferase is secreted from isolated rat hepatocytes. 2. The secretion is stimulated when serum is added to the incubation medium. 3. Optimal conditions for secretion are: 5 - lo6 hepatocytes per ml, 5 h incubation, pH 7.3-7.4 and 25% serum in the incubation medium. 4. Concomitantly with the secretion of 1ecithin:cholesterol acyltransferase there is a secretion of unesterified cholesterol and triacylglycerol. 5. Colchicine or cycloheximide in the incubation medium inhibits secretion of 1ecithin:cholesterol acyltransferase.

Introduction catalyzes the Lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) formation of cholesterol esters by promoting acyl group transfer from the Cholesterol phosphatidylcholine to the cholesterol of plasma lipoproteins. ester formed by this reaction is transferred from high density lipoproteins to other plasma lipoproteins, and in this way the acyl transfer is responsible for a major part of the cholesterol ester found in plasma [ 11. Despite increasing knowledge of 1ecithin:cholesterol acyltransferase activity in different normal physiological and pathological conditions, there exist few data which could elucidate how the enzyme production is regulated. It is known that human acyltransferase activity is positively correlated with triacylglycerol turnover [2], with concentration of unesterified cholesterol in serum [ 31 and with body weight [ 41. Dietary studies in guinea pigs have shown positive correlation between serum unesterified cholesterol, triacylglycerol and 1ecithin:cholesterol acyltransferase activity [ 51. In vitro studies have shown that apoprotein Ai [6] and apoprotein Ci [ 71 stimulate the activity of the

70

acyltransferase. The in vitro activity may also be stimulated by lipoproteins rich in triacylglycerol [8], and by phospholipids [9]. Liver perfusion studies have given direct evidence that lecithin : cholesterol acyltransferase is produced in the liver [lO,ll]. However, the interpretation of these studies may be difficult since the liver also removes the enzyme from the perfusate [ 121. To study the synthesis and secretion of the acyltransferase we have used isolated, parenchymal rat liver cells, which would seem to offer advantages over the perfused liver: many cell samples from the same liver may be incubated simultaneously in different media, and interference from nonparenchymal liver cells may be avoided. Materials and Methods [~cY-~H] Cholesterol, specific activity 12.6 Ci/mmol, was purchased from Radiochemical Center, Amersham, England, The cholesterol was purified before use by thin-layer chromatography on silica gel H (Merck, Darmstadt, Germany) in the developing system light petroleum (b.p. 60--70”(Z)/ diethyl ether/acetic acid (85 : 15 : 3, v/v). Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer, using Permablend III (Packard Instrument Co., Ill., U.S.A.) as liquid scintillator. Counting efficiency was determined by the external standard method. Essentially fatty acid-free bovine serum albumin prepared from fraction V, collagenase Type 1, colchitine, cycloheximide, HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) and kits for determination of lactate dehydrogenase (EC 1.1.1.27) were purchased from Sigma Chemicals Co., St. Louis, MO., U.S.A. Cholesterol biochemical standard and mercaptoethanol were purchased from British Drugstore Chemicals, Poole, England. Stigmasterol was bought from Koch-Light Lab., Colnbrooke, England, and nutrition medium (RPM1 1640) was obtained from Flow Lab. Limit., Irvine, U.S.A. Boehringer, Mannheim, Germany, supplied enzymes and substrates for the determination of triacylglycerol. Animals. Blood samples from adult female rats weighing 150-180 g were taken by aorta punction under ether anesthesia. The rats were kept on a low fat powdered diet containing cereals, fishmeal, groundnut oil, minerals and vitamins. On the basis of calories it contained about 8% lipids and 20% proteins. The rats had free access to food and water until they were sacrificed. The blood was allowed to clot at room temperature before centrifugation and then the serum was heated for 1 h at 59°C which almost completely inactivated the acyltransferase present in plasma. Hepatocytes were prepared from male rats weighing 200--250 g. These rats were given ordinary laboratory chow and water ad libitum. Preparation of hepatocy tes. The procedure for cell preparation was essentially as described earlier [ 131: The liver was perfused for 10 min with Ca2+-free Hanks’ solution [14] and then treated for 10 min with 0.05% (w/v) collagenase in presence of 4 mM CaC12 [ 151. The perfusion fluid was continuously equilibrated with 95% O2 and 5% CO,. The temperature was 37°C and the PI-I was kept at 7.45 by the addition of NaHCO,. Hepatocytes were separated from non-parenchymal cells by low-speed centrifugation of the total cell suspension which had been incubated for 30 min and thereafter filtered [ 161. About 300 . Chemicals.

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lo6 to 400 - 10h cells were obtained from each liver, and about 95% were viable, measured as resistance to uptake of trypan blue. Incubation conditions. In order to study the secretion of lecithin:cholesterol acyltransferase the hepatocytes were resuspended in 10 ml of the medium to be tested. The incubations were carried out in lOO-ml siliconized Ehrlenmeyer flasks at 37°C with shaking (60 rev./min) for 5 h if not otherwise stated. At zero time and at the end of the incubations, the cells and the medium were separated by centrifugation (2000 rev./min for 10 min) at 0°C. In experiments where the cells were incubated at different pH or when the medium contained compounds which might influence the acyltransferase assay (colchicine, cycloheximide) the supernatants were dialyzed at +4”C overnight against 0.2 M potassium phosphate buffer (pH 7.2) before analysis. In most experiments, the hepatocytes were incubated in a solution containing 6.7 g NaCl, 0.4 g KCl, 0.06 g Na2HP04 * 2H20, 0.06 g KH,PO, * 2Hz0, 0.2 g MgSO, - 7Hz0, 14.30 g HEPES, 0.4 g CaCl, - 2Hz0, and water to 1000 ml. The pH was adjusted to 7.5 by the addition of NaOH and 1% (w/v) bovine serum albumin was added. Osmolality was approx. 298 mosM. This basic medium was modified in various experiments by addition of the components to be tested. Lecithin : cholesterol acyltransferase assay. The acyltransferase activity of the medium was assayed by adding 30 ~1 of a [3H]cholesterol/albumin emulsion [17] and 20 ~1 0.1 M mercaptoethanol to 200 ~1 of the cell incubation medium. In experiments where the cell medium contained less than 25% heatinactivated rat serum, serum was added to ensure sufficient substrate for the acyl transfer. The volumes were adjusted with 0.2 M potassium phosphate buffer (pH 7.2). The formation of cholesterol ester was independent of the amount of serum in the incubation mixture, as long as the amount of inactivated serum was between 25 and 75%. The mixture was incubated at 37°C for 24 h. The formation of cholesterol ester was linear with time in this period. The extraction of unesterified and esterified cholesterol was carried out according to Stokke and Norum [17]. Unesterified and total cholesterol were determined by gas-liquid chromatography 151. The glycerol of the triacylglycerol was determined according to the method by Wieland [ 181 and measured fluorimetrically. Lactate dehydrogenase was determined according to the method developed by Sigma Chemical Co. [ 191. Results Secretion of 1ecithin:cholesterol acyltransferase from isolated rat hepatoTable I shows that acyltransferase was secreted into the medium when rat hepatocytes were incubated in a buffered salt medium containing 1% bovine serum albumin. Replacing the salt solution with a commercial cell culture medium gave a somewhat higher enzyme secretion. The secretion was, however, enhanced considerably when heat-inactivated rat serum was added into the medium, as shown in Table I. There was an increasing enzyme secretion with increasing concentration of serum in the medium, reaching a maximum at about 50% serum. cytes.

72 TABLE

I

SECRETION OF LECITHIN : CHOLESTEROL ACYLTRANSFERASE FROM TOCYTES. THE EFFECT OF ADDING RAT SERUM TO THE MEDIUM

ISOLATED

RAT

HEPA-

Hepatocytes were incubated for 5 h in the basic medium (see Materials and Methods) or in this medium with 25% heat-inactivated rat serum. In one of the experiments, the hepatocytes were also incubated in a nutrient medium. RPMI 1640. Acyltransferase activity in the medium was determined at zero time and after 5 h. The enzyme secretion is given per liver per h and measured as increased acyltransferase activity (nmol cholesterol ester/h) in the medium during incubation. It is assumed that one liver contains lo9 CCll8. The results of four different experiments are given. Incubation

medium

nmol cholesterol

Salt medium Nutrient medium Salt medium with 25% rat serum

6.25 8.80 12.40

Salt medium Salt medium

with 25% rat serum

4.2 10.7

Salt medium Salt medium

with 25% rat serum

3.5 9.9

Salt medium Salt medium

with 25% rat serum

4.1 11.0

ester/h

The acyltransferase in the medium most probably reflected true secretion and not leakage out of the cells. Thus, Table II demonstrates that total homogenate or cell sap from the hepatocytes had very low acyltransferase activity. This was true both for non-inc~ba~d cells and for cells incubated for 5 h. The small activity found was not different from the residual activity of the enzyme in the heat-inactivated serum used as substrate. TABLE

II

LECITHIN : CHOLESTEROL THE CELL HOMOGENATE

ACYLTRANSF~RASE ACTIVITY IN THE HEPATOCYTE AND THE CELL SAP AT ZERO TIME AND AFTER 5 h

MEDIUM,

The hepatocytes were incubated in the basic salt medium (see Materials and Methods) with 25% heatinactivated rat serum. Samples were taken at zero time and after 5 h. The cells were separated from the medium by centrifugation at 2000 rev./min for 10 min. After washing twice in 0.9% NaCl they were homogenized in their original volume in a Dounce homogenizer. The cell sap was obtained by centrifugation of the total homogenate for 2 h at 50 000 rev&in in a Beckman ultracentrifuge Model L65, rotor Ti 65. Acyltransferase activity was measured at zero time and after 5 h incubation 89 described in Materials and Methods. Acyltransferave activity is given an nmol cholesteroi ester produced in 24 h in the medium from I . lo6 hepatocytes. in the homogenate or in the cell sap. corresponding to 1 . lo6 hepatocytes. Acyltransferase Supernatants Supernatants

Oh 5h

0.26 2.09

Homogenate Homogenate

Oh 5h

0.33 0.30

Cell sap Cell sap

Oh 5h

0.22 0.34 -~-.

activity

__.___._.-_

__-_-..__

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Effect of pH and buffers on the secretion of 1ecithin:cholesterol acyltransferase. Hepatocytes secreted acyltransferase over a relatively wide pH range. The optimal pH was found to be between 7.3 and 7.4. Different types of buffers were tested. Tris buffer was toxic for the cells. HEPES and COz/NaC03 buffers were found to be the best. Since the use of HEPES buffer made incubation simpler, we routinely used this buffer in our incubations. The effect of cell concentration on the secretion of 1ecithin:cholesterol acyltransferase. Different amounts of hepatocytes were incubated in medium containing 25% heat-inactivated serum. Fig. 1 shows how the acyltransferase secretion was related to the cell concentration. When the incubation medium contained from 1 * lo6 to 5 - lo6 cells per ml, the secretion of the enzyme was directly proportional to the cell concentration. Effect of time on the secretion of 1ecithin:cholesterol acyltransferase. The secretion of acyltransferase from hepatocytes increased with time, at least for 8 h. The viability of the cells was about 95% for 5 h, thereafter it decreased to 70-80% after 8 h. Fig. 2 shows the secretion of acyltransferase into a medium containing 25% heat-inactivated rat serum. After an initial lag period of about 1 h the secretion of the enzyme was linear for about 4 h. Then the secretion rate decreased at the same time as the leakage of lactate dehydrogenase from the cells started to increase as shown in the same figure. Secretion of cholesterol and triacylglycerol from the hepatocytes. The concentration of unesterified cholesterol in the medium increased during incuba-

mill.

cetts/ml

time (h)

Fig. 1. The effect of cell concentration on 1ecithin:cholesterol acyltransferase secretion. The cells were incubated in basic medium with 26% inactivated rat serum (see Materials and Methods) for 6 h. The acyltransferase secretion from the cells was measured as increased acyltransferase act+dty in the medium (nmol cholesterol ester/h) in 1 ml of the medium. Fig. 2. Lecithhxcholesterol acyltransferase secretion and lactate dehydrogenase~leakape with duration of incubation. Hepatocytes were incubated in a basic salt medium containing 26% inactivated rat serum (see Materials and Methods). The acyltransferase secretion is given per liver and measured as increased acyltransferase activity (run01 cholesterol ester/h) in medium. The cells were homogenized in a Dounce homogenizer and the lactate dehydrogenase activity was measured in cells and medium. Lactate dehydrogenase activity in medium is given as percent of total activity. -, acyltransferase activity, expressed as nmol cholesterol ester/h: q o, percent lactate dehydrogenase leakage.

74

tion of the hepatocytes, as shown in fig. 3. This could be explained by hydrolysis of cholesterol ester in the incubation medium containing serum, as cholesterol ester decreased during the first 3 h, also shown in Fig. 3. However, the increase in cholesterol ester of the medium after that time, and the shape of the curve showing the increase in medium unesterified cholesterol made it more probable that the hepatocytes secreted lipoproteins into the medium. The true secretion of lipoproteins is confirmed by the increased appearance of triacylglycerol in the incubation medium, as Fig. 3 shows. The amount of serum in the incubation medium had a marked effect on the secretion of lipids from the liver cells. Fig. 4 shows that both triacylglycerol and unesterified cholesterol secretion increased with increasing amounts of serum in the medium, The concentration of cholesterol ester in the medium also varied, but no conclusions could be drawn from the experiment, shown in Fig. 4, regarding secretion or uptake of cholesterol ester by the hepatocytes.

The effect of ~c~o~e~irnide and co~ch~ci~e on the secretion of lec~~h~~: cholesterol acyltransferase. To test if the increased activity of acyltransferase in the medium was due to production and secretion of the enzyme by the hepatocytes, cycloheximide, which inhibits protein synthesis, or colchicine, which interferes with protein secretion, were added to the incubation medium, containing inactivated serum. Colchicine (0.5 mg/ml) lowered the acyltransferase activity in the medium from 10.7 to 3.9 nmol cholesterol ester per h.

% serum in the medium

Fig. 3. The effect of the incubation time on tbe secretion of lipids from the hepatocytes. Hepatocytes were incubated as described in Materials and Methods with 25% inactivated serum in the medium. The concentrations of cholesterol, cholesterol ester and triacylglycerol in the medium of each sample were determined as described in Materials and Methods. The amount of lipids secreted by the whole liver at q triacy1glycero1; 1j-----=, cholesterol; A--different time intervals were then ealcuiated. l -----+, cholesterol ester. Fig. 4. The effect of serum concentration in the medium on the lipid secretion. Hepatocytes were incubated in a basic salt medium (see Materials and Methods) with 0, 12.5. 25, 50 or 75% inactivated rat serum. The concentrations of cholesterol, esterified cholesterol and triacylglycerol in the medium were measured at zero time and after 5 h. The amount of lipids secreted by the whole liver in 1 h were then cholesterol ester. cholesterol; A-, triacylglycerol; n--0, calculated. e-4.

75

Lower concentration (0.05 mg/ml) also inhibited the secretion of acyltransferase. The addition of colchicine did not alter the viability of the hepatocytes. Cycloheximide (0.1 mg/ml) lowered the secretion of acyltransferase from 10.7 to 6.3 nmol cholesterol ester per h. The viability of the cells was not influenced by cycloheximide in the medium. Discussion Perfusion studies have demonstrated that 1ecithin:cholesterol acyltransferase is secreted from the liver [ 10,111, and that the enzyme probably also is removed from circulation by this organ [ 121. In the present report incubation of isolated rat hepatocytes confirms that during incubation of the hepatocytes the cell medium gets an ability to esterify cholesterol on plasma lipoproteins. We think that this esterification is due to a secretion of the enzyme lecithin : cholesterol acyltransferase. The reaction takes place extracellulary at neutral pH and is independent of the addition of acyl-CoA. These facts rule out an esterification catalyzed by an acid hydrolase (EC 3.1.1.13) and an acyl-CoA : cholesterol acyltransferase (EC 2.3.1.26). No other enzymes catalyzing the formation of cholesterol ester are known at present. Assuming that 1 g liver contains 125 * lo6 hepatocytes and that the liver of a 200 g rat weighs about 8 g, it may be calculated that under optimal conditions (5 - lo6 cells/ml, 25% inactivated rat serum in the medium, pH 7.3) the amount of acyltransferase produced in 1 h by hepatocytes corresponding to a whole liver can form 10-23 nmol cholesterol ester/h (results from several experiments). This production is in fairly good agreement with that obtained in liver perfusion studies by Simon and Boyer [ 111 who found that the perfused liver produced acyltransferase capable of forming 16.9 nmol cholesterol ester/h. In comparison, Osuga and Portman [lo] found that the liver secreted enough acyltransferase to replace the serum enzyme pool every 5 h. A 200 g rat seems to have enough acyltransferase activity to form about 400 nmol cholesterol ester/h, assuming a rat plasma volume of 10 ml. Thus, our data indicate that the liver can produce the whole plasma pool of the acetyltransferase in about 1 day. Albumin is secreted into the medium during incubation of hepatocytes [20]. Our results suggest that acyltransferase also appears in the medium as the result of a secretion and not by leakage. The enzyme activity in the medium increases almost linearly for about 5-6 h, after a lag period of about 1 h. During this period there- is no leakage of lactate dehydrogenase from the hepatocytes into the medium. Colchicine interfers with the microtubuli system and inhibits secretion of proteins [ 211 and lipoproteins [22] from the liver. In our studies colchicine reduced the acyltransferase activity in the medium markedly. This also indicates that the enzyme is secreted from the hepatocytes. We cannot detect acyltransferase in the hepatocyte homogenate. There may be some cholesterol ester-hydrolyzing activity in the cell homogenate, or the enzyme may be stored in an inactive form, and then activated upon secretion. In addition to this possible activation of the acyltransferase there seems to be a synthesis of acyltransferase during incubation, since addition of cycloheximide to the incubation medium, reduced the enzyme secretion from the hepatocytes.

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The secretion of acyltransferase is enhanced when heat-inactivated rat serum is added to the medium. Since a nutrient medium did not have the same stimulating effect, the st~ulation might not be explained as a nutrient effect alone. Addition of serum did not alter the viability of the cells. Lipids are secreted into the medium during the incubation. In our study the secretion of cholesterol and triacylglycerol increases with increasing incubation time. For the first 4-5 h there is an almost parallel secretion of cholesterol and triacylglycerol, after this time there is a more rapid secretion of triacylglycerol as compared to cholesterol. This may suggest that during incubation the pattern of lipoprotein secretion changes. After 4-5 h the secreted lipoproteins are richer in triacylglycerol. The amount of lipids secreted by our hepatocytes corresponds well with the production rate found by Goh and Heimberg [ 231 in perfused rat liver. We observed that the secretion of cholesterol and triacylglycerol was markedly stimulated by increasing amounts of serum in the medium. A stimulation of lipid secretion from rat liver has also been reported in liver perfusion studies [24], In our study we have so far no explanation of what part of the serum is responsible for the stimulating effect both on the lipid secretion and on the secretion of 1ecithin:cholesterol acyltransferase. The fact that the secretion of acyltransferase, cholesterol and triacylglycerol showed the same relationship to the serum concentration in the medium, may suggest that the acyltransferase is associated with lipoproteins during secretion. Acknowledgements The authors are indebted to Solveig Berg for skilled technical assistance. This study was supported by grants from The Norwegian Council on Cardiovascular Diseases, from Anders Jahre’s Foundation and from the Norwegian Research Council for Science and the Humanities. References 1 Glomset, J.A. and Norum, K.R. (1973) Advances in Lipid Research (Paoletti, R. and Kritchevsky, D., eds.), Vol. 11. pp. l-65, Academic Press, New York 2 Nest& P.J. (1970) CIin. Sci. 38. 593-600 3 Lacko. A.G., Rutenberg. H.L. and Soloff, L.A. (1974) Atherosclerosis 19. 297-305 4 Akanuma, Y.. Kuzuua. T., Hayasi. T.I. and Kuzuya, N. (1972) Eur. J. CIin. Invest. 3.136-141 5 Drevon, C.A. and Norum, K.R. (1976) Nutr. Metab. 18.137-151 6 Fielding, C.J., Shore. V.G. and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 46, 14931498 7 Soutar. A.K., Gamer, C.W., Baker, I-I-N., Sparrow, J.T., Jackson, R.L.. Gotto, A.M. and Smith. L.C. (1975) Biochemistry 14.2828-2836 8 Marcel, Y.L. and Vezina, C. (1973) J. Biol. Chem. 248. 8254-8259 9 Drevon, C.A. and Norum, K.R. (1976) Nutr. Metab., in the press 10 Osuga, T. and Portman. O.W. (1971) Am. J. Physiol. 220.735-741 11 Simon. J.B. and Bayer, J.L. (1970) Biochim. Biophys. Acta 218, 549-551 12 Quarfordt, S.H. and Goodman, D.S. (1969) Biochim. Biophys. Acta 176.863-872 13 Berg, T. and M&land. J. (1975) Biochim. Biophys. Acta 392. 233-241 14 Hanks, J.H. and W&ace, R.E. (1949) Proc. Sot. Exp. Biol. Med. 71,196-200 15 SegIen, P.O. (1972) Exp. Cell Res. 74.450454 16 Berg, T. and Boman, D. (1973) Biochbn. Biophys. Acta 321, 585-896 17 Stokke. K.T. and Norum, K.R. (1971) Stand. J. CIin. Lab. Invest. 27,21-27 18 Wieland, 0. (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 3, PP. 1404-1409, Sec. ed. Chemie Weinheim, Academic Press, New York

19 20 21 22 23 24

Sigma Technic. Bull. No 500 (1967) Sigma Chemicals Co., St. Louis, MO., U.S.A. Dich, J. and Gluud, C.N. (1976) Acta Physiol. stand. 94, 236-243 Redman, C.M.. Banedee, D., Howell, K. and Palade, G.E. (1975) J. Cell Biol. 66.42-59 Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 306. 142:147 Goh. E.H. and Heimberg. M. (1973) Biochem. Biophys. Res. Commun. 55,382-388 Roheim. P.S., Miller, L. and Eder. H.A. (1965) J. Biol. Chem. 240. 2994-3001

Secretion of lecithin: cholesterol acyltransferase from isolated rat hepatocytes.

69 Biochimica etBiophysica 0 Elsevier/North-Holland Acta, 450 (1976) 69-77 Biomedical Press, Amsterdam - Printed in The Netherlands BBA 56860 SECR...
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