Assembly and secretion of very low density lipoproteins by rat liver following inhibition of protein synthesis with cycloheximide

et Biophysicn Acttr, 306 ( I 973) Scientific Publishing Company,

Biochimictr c Elsevier

I 06-1

14

Amsterdam

Printed

in The Netherlands

BBA 56229

ASSEMBLY

AND

BY RAT LIVER WJTH

SECRETION

FOLLOWING

OF VERY LOW DENSITY INHIBITION

OF PROTEIN

LlPOPROTEINS SYNTHESIS

CYCLOHEXIMIDE

A. I. KOOK,

0. STEIN

Lipid Reserrrch Loborator); Deportment of‘ Esperimental Schooi, Jer~rstriet~~ (lsraei)

H. BAR-ON,

Department Medicine

(Received

October

I 1th,

and Y. STEIN of Medicine B, Hndmsaiz University Hospitrri, and trnd Comer Research, Hebrew Utlicersity-Hrrdrrsstr/l

Medictrl

1972)

SUMMARY

Secretion of very low density lipoproteins by the perfused rat liver continued for 50-60 min following inhibition of protein synthesis induced by cycloheximide. The lipoproteins secreted during that period were immunologically and morphologically similar to those secreted during active protein synthesis. Following administration of cycloheximide, labeled palmitic acid was channeled preferentially to phospholipids. This change in the relative distribution of label between triglycerides and phospholipids in the very low density lipoproteins, isolated from the perfusate, supported the conclusion that the lipid moiety of very low density lipoproteins secreted after interruption of protein synthesis was added to preexisting apolipoprotein rather than to preformed lipoprotein. Analysis of the labeled apolipoproteins secreted after cycloheximide indicated that the hepatic apolipoprotein pool did not contain all the components at proportions normally present in secreted very low density lipoproteins.

INTRODUCTION

During the last decade the various steps of biosynthesis, intracellular transport and secretion of serum very low density lipoproteins were studied extensively’ 3. Even though the intracellular site of synthesis of protein, lipid and carbohydrate has been localized, the exact sequence of the assembly of the lipoprotein has not been clarified. Recent studies have provided evidence that some of the carbohydrate is added to the Even though the protein and lipid components lipoprotein in the Golgi apparatus4. are formed in the endoplasmic reticulum 2.3, it is not clear whether the lipid is attached to nascent polypeptide chains or whether there are separate pools of apoliproprotein and lipid from which the lipoprotein particles are formed during their passage through the cisternae of the endoplasmic reticulum. Buckley rt al.’ have studied the appearance of label in the protein and lipid moieties of serum lipoproteins following injection of labeled fatty acid and amino acid. Since the label appeared first in the lipid portion of the molecule, they concluded that newly formed lipid had been added to preexisting

ASSEMBLY

AND SECRETION

OF LIPOPROTEINS

BY LIVER

107

protein”. However, differences in pool size of fatty acids and amino acids might have contributed toward such a result. Moreover, the experimental design did not permit to distinguish between addition of lipid to preexisting lipoprotein or apolipoprotein. In a recent study’ it was shown that cycloheximide, while not interfering with fatty acid esterification, causes a preferential channeling of injected labeled fatty acid into phospholipids rather than into triglycerides. It was hoped that such a difference in the labeling pattern could serve as a marker to distinguish between very low density lipoproteins which have acquired their lipid moiety before cycloheximide and those after administration of the drug. The use of cycloheximide was also designed to elucidate the question whether secretion of very low density lipoproteins requires concommitant synthesis of proteins, or whether it may proceed in the absence of protein synthesis, utilizing apolipoproteins from a storage pool. Another aim of the present study was to investigate whether such a pool of apolipoproteins would contain all apolipoproteins in the same proportion as normally present in very low density lipoproteins of serum. MATERIALS

AND METHODS

The perfusion system

Rat livers were perfused in situ according to the method of Mortimore7. Male rats fed ad libitum, weighing 350 g, which received IO o/osucrose in drinking water for 4 days were used as liver donors. The perfusate consisted of 20 % rat red blood cells and 0.2 “/: glucose in Krebs-Ringer bicarbonate buffer, pH 7.4. To IOO ml of perfusate 5 mg of complete amino acid mixture’, I 20 ,umoles sodium linoleate complexed to 3 g bovine serum albumin, 5 mg streptomycin, IOOOOOunits penicillin and IOO units heparin were added. The perfusion was carried out at 37 “C under O,-CO, (95 : 5, v/v). The liver was washed in situ by one passage of 40 ml of perfusate; for recirculation perfusion 60 ml of perfusate were used. Sodium linoleate (6 mM) complexed to serum albumin (4:r, molar ratio) was infused continuously into the portal vein at a rate of 120 pmoles/h, using a constant infusion pump. L-[I-‘4C]Leucine (spec. act. 62 Ci/ mole) and, when specified, sodium [g,ro-‘H,]palmitate (spec. act. 500 Ci/mole) complexed to albumin, were injected rapidly into the portal vein at the start of the experiment. At the end of each perfusion period the entire perfusate was collected and the liver was flushed with 40 ml of perfusate, which, unless specified otherwise, contained 20 klg/rnl of cycloheximide. The flush was discarded, 80 /lg of cycloheximide were with [4,5-3H,]L-leucine (spec. infused directly into the portal vein simultaneously act. 36 Ci/mmole) or [ r-14C]palmitate (spec. act. 55.2 Ci/mole) complexed to albumin and the perfusion was resumed with 60 ml of perfusate containing 20 pg/ml of cycloheximide. This concentration of cycloheximide was chosen as it was found to inhibit, within 2 min, more than 90 “/, of incorporation of labeled leucine into proteins in rat liver slices. Sodium linoleate complexed to bovine albumin was continuously infused into the portal vein at a rate of 120 /lmoles/h. Bile flow was 1.2-1.4 ml/h in control livers. After cycloheximide the bile flow was reduced to 0.4 ml/h. Ultracentrifirgal isolation oj’ very low density lipoproteins

After initial removal of red blood cells I mg/ml of ethylenediaminetetraacetate (EDTA), pH 7.4, was added to the perfusate. The perfusate was then layered under a

IO8

H. BAR-ON

er c/l.

solution of NaCl of d I .oo6 containing I mgjml EDTA (pH 7.4) and centrifuged in a 50. I rotor at 4°C for 24 h at 48000 rev./min using an L-2 65-B Beckmal~ ultracentrifu~e. The tubes were sliced and the top fraction was collected and layered again under the solution of NaCI, d I .oo6, and respun in a SW 41 rotor at 4 “C at 40000 rev./min for 24 h. On immuno~Iectrophoresis the preparations gave one precipitation line with anti serum to rat very low density lipoproteins, and did not react with the anti serum to rat albumin. Only one precipitation line was obtained with anti whole rat serum corresponding to /&lipoproteins. In one experiment, very low density lipoproteins were isolated from the perfusate by the heparin precipitation technique“. Aliquots were removed from the perfusate at specified time intervals, made cell free, adjusted to a volume of 3 ml with fresh rat serum and the very low density lipoproteins were precipitated.

The very low density Iipoprotei~ preparations isolated by ultracentrifugation were dialyzed exhaustively at 4 “C against 0.01 ‘1 ,,c,EDTA (pH 7.4) containing unlabeled leucine. The dialyzed lipoprotein was iyophilized and delipidated according to the to procedure described by Bersot et a!.” as modified by Scany and Edelstein’“, minimize the loss of protein during delipidation. Protein recovery after dialysis and were then solubilized in 0.2 M delipidation was about 80x,. The apolipoproteins Tris-HCI and 0.1 M sodium decylsulfate, pH 8.2, and dialyzed against 0.2 M TrisWC1 and 0.02 mM sodium decylsulfate, pH 8.2 (ref. to).

Polyacrylamide gel electrophoresis was performed in 8 M urea at pH 8.9 using ro ‘A acrylamideL2 and the gels were stained with Coomassie brilliant blue’“. Radioactivity in polyacrylamide gels was determined according to Hellung-Larsent4, the scintillation system consisting of toluene-dioxane-ethanol (5 : 3 : 2, v/v/v) and 0.4 % 2,5-diphenyloxazole and o.015’%, dimethyl-~,4-bis-~-(4-methyl-5-phenyl-oxazolyl)benzene. Correction for quenching was done with the help of the channel ratio method. To estimate the efficiency of etution of radioactivity from the gels, aliquots of labeled apoiipoprotein were solidified in the gel and counted. More than 90 “/: of the applied radioactivity was recovered. To determine protein radioactivity in very low density lipoproteins, the lipoprotein was dissolved directly in Soluene or following deiipidation and counted in toluene-dioxane-ethanol scintillation system. Heparin-precipitable protein was washed twice with I o 7,; trichloroacetic acid, delipidated, dissolved and counted as above. Double isotope counting was carried out as described before’“. The organic solvents used for the delipidation of very low density lipoproteins were evaporated to dryness, dissolved in chloroform and aliquots were chromatographed on thin-layer silica gel G plates. For separation of triglycerides and phospholipids a solvent system containing light petroleun~ (30-60 “Ctdiethyl ether-glacial acetic acid (80: 20: I, v/v/v) was used. The fractions were visualized with iodine vapors and identified with the help of reference standards. The separated lipid classes were scraped into counting vials and assayed in a /&scintillation spectrometer, using a scintillation fluid containing 0.7 “/, 2,5_diphenyloxazole, 0.03 % [,4-bis-2-(4-methyl-5-phenyloxazoIyI)benzene and IO y/, naphthalene in I I of dioxane, to which 200 ml of water were added. Rat red blood cells were labeled with 5’Cr (ref. 16) and washed twice

ASSEMBLY

AND

SECRETION

OF LIPOPROTEINS

BY LIVER

‘09

with 0.9 :’ NaCl: the ‘iCr was determined in a Packard autogamma spectrometer. All radioisotopes were obtained from the Radiochemical Centre, Amersham, England. Bovine albumin (fatty-acid poor) was obtained from Pentex, Kankakee, 111. Cycloheximide was purchased from Sigma, St. Louis, Mo. RESULTS

proteins TABLE

The data presented in Table I indicate that release of very low density lipofrom the liver into the perfusate continued in spite of a block in protein synI

EFFECT OF CYCLOH~XIMIDE ON SECRETION VERY LOW DENSITY LIPOPROTEIN

OF PROTEIN

AND

TRiGLYCERIDE

OF

50&i of [i4C]leucine were infused into the portal vein at the start of perfusion period I. Cycloheximide (20 ;lgjml) was added to the wash which preceded the second period of perfusion as well as to the perfusate of the second period. In Expts z and 3, 50 @ of [3HJleucin~ were infused at the start of perfusion period Il. ‘The duration of each perfusion period was 120 min and that of the wash about to min. n.d. =- not detectable. _. -. Lubeled E.vpi. Pryfirsion C.vr,~~Vcw IUH~densiiy &wprotein in perfimtre .-- ‘_I-. __ -.----~t,~~~)d ‘NO. Itexintide fertcine Trig&wride Pm,rrirr dptn

--.

-II..

I

1

~.

_-

1%

.__

2.1

II

I

?.

‘T

-

3H

II 1 II

3 I.

1°C 3H

_.

‘IC

---.

2.5 2.3 0.7 t .4 0.3

10-3 --

319 ‘27

JH ._. _-.-

293 43 277

n.d. -..

3o

n.d.

-_ 10.3

13.8 13.0 3.8 7.2 I.7

.-. _~._ ._.. _..-.__

.-•-•---•

0

15

30

50

70

90

110

min

Fig. I. Release of bepariIt-precip~table lipoprotein (very low and Iow density lipoproteins~ by pcrfused ritt liver following inhibition of protein synthesis by cycloheximide. [t-‘*CjLeucine was administered at the start of perfusion period I. After I zo min, the perfusate was replaced by a wash containing 20 jlg!ml cycloheximide. and was followed by a second perfusion period, the perfusate containing cycloheximide. Samples were removed at time intervals starting at 15 min after the beginning of the second perfusion period to min on the abscissa). The maximal concentration of label was taken as loo”;.

1 IO

H. BAR-ON

et al.

thesis induced by cycloheximide. The secretion of very low density lipoproteins continued for about I h (Fig. I), and the amount of protein secreted was 40-60 “0 of that released during I h prior to the introduction of cycloheximide (Table I). The very low density lipoproteins released after cycloheximide had a triglyceride to protein ratio of 5.0-5.5 (w/w) and were similar to those of very low density lipoproteins released during the first perfusion period, as well as to very low density lipoproteins isolated from rat serum. In negatively stained preparations the lipoprotein particles released before and after cycloheximide had a similar appearance {Fig. 2). On jmmunodiffusion the very low density lipoproteins isolated from Perfusate 1 and Perfusate II produced a single precipitin line against antiserum to rat very low density lipoprotein. Use was made of chromium-labeled erythrocytes in order to determine the amount of whole perfusate carried over from the first into the second perfusion period. and as seen in Table II, the amount of contamination did not exceed 0.57<. Even though thetriglyceride/protein ratio in very low density lipoproteins of Perfusate I and II remained the same, the ratio of labeled triglyceride~labeled protein declined markedly, from 13.4 to 0.9. Hence it seems that the very low density lipoproteins recovered in Perfusate II were indeed secreted during that perfusion period. The aim of the present study was to determine whether the very low density lipoproteins of perfusate II represent particles which had been fully synthesized prior to the interruption of protein synthesis, or whether formation of very low density lipoproteins continues by addition of newly synthesized lipid to preexisting (a) lipoprotein, or (b) apolipoprotein. To that end the following experiment was designed. Rat livers were perfused for too min with a perfusate cot~tainil~g [‘4~]leucitle and ~3~]paln~itic acid. The

Fig. 2. Electron trifugation from cycloheximidc.

micrographs of negatively stained very low density lipoproteins isolated by ultracenserum (a). Perfusate I (b) and Perfusate II (c), the latter after administration of ()oooo.

ASSEMBLY TABLE

AND

SECRETION

OF LIPOPROTEINS

III

II

ESTIMATION

OF RESIDUAL

“CT-LABELED

“‘Cr-labeled erythrocytes (z 10~cpm) were added were recovered at the end of the experiment.

srrwlplc~ Perfusate Wash Perfusate Liver

BY LIVER

I II

Perfusion / min 1

Percent of totui mdiocrctivit~

I20

88.5

IO

9.3

120

0.5

RED

BLOOD

CELLS

to Perfusate

IN PERFUSATE

I and more

than

II

95 “/ of counts

r.7

Perfusate Ia was removed, a brief flush was instituted and fresh Perfusate Ib was introduced which contained labeled leucine in an amount present at the end of the first IOO min of perfusion. The second perfusion period was again divided into two (lla and Ilb), separated by a brief flush, and [i4C]palmitic acid was added to Perfusate lla. When cycloheximide was used, it was introduced into the wash which followed the last 20 min of Perfusate Ib. As can be seen in Table 111, there was a fall in the [3H]triglyceride/i4C-labeled protein ratio, when the very low density lipoproteins isolated during the first IOO min (la) were compared to those isolated during the next 20 min (Ib). This ratio did not change during the subsequent periods of perfusion (lla and IIb) of 30 and 90 min each, respectively. However, when cycloheximide was introduced into the wash at the end of the first 120 min, the [3H]triglyceride/‘4C-labeled protein ratio in Perfusates lla and Ilb decreased markedly, indicating that a newly formed TABLE

III

EFFECT OF CYCLOHEXIMIDE ON THE DISTRIBUTION OF LABELED PALMITIC ACID LIPIDS OF VERY LOW DENSITY LIPOPROTElNS SECRETED INTO THE PERFUSATE The duration of the perfusion periods were: la, IOO min; lb. 20 min; followed by a wash. 50 /cCi of [‘?Z]leucine and 50 ,Ki of [3H]palmitate [LGC]leucine added to lb was equivalent to that present in la at the end added to IIa. Cycloheximidc (20 pg/ml) \vas added to the \rash which Perfusates Ila and IIb at the same concentration.

IN THE

Ila. 30 min: Ilb. 90 min. Each period was were added to perfusate la: the amount of of IOO min. 50 /Ki of [“Clpalmitate were followed Perfusion lb and \+as present in

Lipid “H

la

[’ Qleucine + [3H]palmitate [LsC]leucine [L4C]palmitate

lb IIa IIb 2

la

Ib IIa IIb

‘. i-

[L4C]leucine+ [3H]palmitate [“Qleucine [‘“Clpalmitate

“C

Trig!,‘crride

Phospho/ipid

5160

192

117 18

950 815 104

96 60

360

3534

‘4’

60

374 58 II

‘59

25 5

I2

48 32 9

Trig/J,ceride 12.3 6.0 315 ‘23

15 16

6.9 5.8 9.8

7.5 I.5

6.3 4.2

6.3 2.4 2.2

H. BAR-ON

II2

<‘f rrl.

low density lipoprotein particle was reaching the perfusate. Another indication that the very low density lipoprotein secreted after cycloheximide had been assembled in spite of the inhibition of protein synthesis could be derived from the determination of the distribution of label between triglyceride and phospholipid. As seen in Table very

ill, this ratio decreased from 26 in the very low density lipoproteins of the first IOO min (la) to 9.9 during the next 20 min (lb), but did not change appreciably during the subsequent two perfusion periods I la and I I b. A similar ratio and a comparable change in the ratio from z I .o to 7.7 was observed also in [14C]triglycer~de~‘4C-labele~~ phospholipid which became labeled following intr(~duction of [‘4C]palmitio acid during the last I 20 min of perfusion. After cycloheximide a marked decrease in the ratio of labeled triglyceride to labeled phospholipid in the very low density lipoproteins was encountered. As seen in Table Ill, the ratio of [“H]triglyceride/“H-labeled phospholipid continued to fall during the last two perfusion periods. The ratio of [14C]tr~glyceride~ ‘“C-labeled phospholipid in Perfusate IIa was much lower than that of [“Hltriglyceridej”H-labeled phospholipid found during the first IOO min of perfusion. This experiment was repeated three times and similar resufts were obtained. The very low density lipoproteins isolated from perfusates were delipid~ted and the distribution of the radioactivity among the different bands was determined following electrophoresis of the labeled apolipoproteins on polyacrylamide gel. In all control perfusates about 40’!;, of the label was recovered in a band just below the stacking gel and JO--40 ‘:i; was found in the three fast moving bands. However, following introduction of cycloheximide, there was an increase in the percent of label recovered at the beginning of the running gel and a reciprocal decrease in that found in the fast moving components. This finding was further amplified when very low density lipoproteins isolated from four perfusion periods {two prior to and two after cycloheximide) were analyzed (Fig. 3). While there was no change in the distribution of label among the various bands during the two control periods, following cycloheximide administration the fast moving bands were stained much more faintly and contained relatively less radioactivity.

1.3

lb

IIa

II b

Fig. 3. Distribution of label among the various hands following separation ol‘apolipoproteins of very low density lipoproteins by polyacrylamide gel clectrophoresis. [I-“C]Leucine was present in the pcrfusate during perfusion period la and lb. Cycloheximide, 20 l/g/ml, was added to the wash which replaced Pcrfusate lb and was present during perfusion period Ila and Ilb. too irg of delipidated ~polipoprot~iii were applied to each gel. More rccoxwed in the region of ihe bands.

than

~5 :‘;, of the total

radioactivity

in the gel was

ASSEMBLY

AND SECRETION

OF LIPOPROTEINS

BY LIVER

113

DISCUSSION

In the present study attempts were made to analyze more closely the secretory pathway of serum very low density lipoproteins. With the help of cycloheximide, at a dose which caused a very rapid and almost complete inhibition of protein synthesis (as evidenced by the lack of appearance of 3H-labeled protein in the perfusate) it was found that secretion of lipoproteins, prelabeled in the protein moiety with r4C, is not dependent on concomitant protein synthesis. A similar conclusion was drawn also by Jamieson and Palade17, who studied the dependence of the secretory pathway in exocrine pancreas on continuous protein synthesis. Since cycloheximide does not inhibit fatty acid esterification’, it provided an opportunity to determine whether the lipoproteins released after inhibition of protein synthesis had been fully assembled, or whether lipid was added to preexisting lipoprotein or apolipoprotein. Even though the very low density lipoprotein particles released after cycloheximide had a similar triglyceride content and ultrastructural appearance as the normal very low density lipoprotein, it became evident that they have acquired most of their lipid complement after the interrruption of protein synthesis. This is supported by the finding of a pronounced fall in the ratio of [3H]triglyceride to 14C-labeled protein in very low density lipoproteins released after cycloheximide. Since both labels were present in the liver prior to cycloheximide, it is plausible that the lipoprotein particles secreted after cycloheximide were not assembled fully prior to interruption of protein synthesis. Another feature which might help to differentiate between very low density lipoproteins assembled before or after cycloheximide is the marked change in the relative distribution of labeled fatty acid between triglycerides and phospholipids. Such a preferential channeling of the label into phospholipids in liver of intact rats had been described after administration of cycloheximide’. Moreover, the much lower ratio of labeled triglyceride to phospholipid in very low density lipoproteins obtained after cycloheximide, irrespective of whether the fatty acid had been administered at the beginning of perfusion or after cycloheximide, suggests that the lipid moiety was added to preexisting apolipoprotein rather than to stored lipoprotein. The size ofthe apolipoprotein pool is most probably rather limited, as indicated by the flattening of the curve of release of very low density lipoproteins 50-60 min after inhibition of protein synthesis. The relative enrichment of label in the bands corresponding to the p component of very low density lipoproteins and the reduction in stainability and labeling of the fast moving bands indicates that the apolipoprotein pool may not contain all of the components at proportions normally present in the secreted very low density lipoproteins. The decrease in the percent of label recovered in the fast moving components of very low density lipoproteins was apparently not due to a loss of these peptides to high density lipoproteins, since a comparable decrease of label in the fast moving bands occurred also in high density lipoproteins after cycloheximide (unpublished). The present experimental design did not allow us to determine whether the different apolipoproteins do have different turnover rates or whether all the apoproteins are required for the secretion of very low density lipoproteins.

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H. BAR-ON

et rrl.

ACKNOWLEDGEMENTS

This investigation was supported in part by research grants from the U.S. Department of Agriculture, Grant No. FG-Is-285 and by a research grant from the Myra Kurland Heart Fund, Chicago, Ill. The excellent assistance of Mr G. Hollander, Mrs Y. (Galanti) Dabach and Miss E. Mamuka is gratefully acknowledged. REFERENCES I Stein, 0. and Stein, Y. (1965) Isr. J. Me& Sci. I, 378-388 Stein, 0. and Stein, Y. (1967) J. Cell Biol. 33, 319-339 3 Stein, 0.. Bar-On, H. and Stein, Y. (1972) in Progress in Lirer Risecrses (Popper, H. and Schaffner, F., eds), Vol. IV, pp. 45-62, Grune and Stratton, New York 4 Lo, C. H. and Marsh, J. B. (1970) J. Biol. C/rem. 245, 5001-5006 5 Buckley, 1. T., Delahunty. T. I. and Rubinstein, D. (1968) Curt. J. Biochc~m. 46, 341~349 6 Bar-On, H., Stein, 0. and Stein, Y. (1972) Biochirn. Biophys. Acta 270, 444-452 7 Mortimore. G. E. (1961) AN?. J. Physiol. zoo. 1315-1319 8 Jefferson. L. S. and Korner, A. (1967) Bioclrem. J. 104, 826-832 9 Burstein, M., Scholnick. H. R. and Mot%, R. (1970) J. Lipid Res. I I, 583-595 IO Bet-sot, T. P.. Brown, W. V., Levy, R. J., Windmueller, H. G.. Fredrickson, D. E. and LcQuire. V.S. (1970) Biochemistry 9, 3427-3433 I I Scanu, A. M. and Edelstein, C. (1971) Anal. Biochenl. 44. 576-588 12 Reisfeld, R. A. and Small, P. A. (1966) Science 152, 1253-1255 13 Chrambach. A., Reisfeld, R. A., Wyckoff, M. and Zaccari, J. (1967) Anal. Biochetrr. 20, 150-154 14 Hellung-Larsen, P. (1971) Anal. Biochewz. 39. 454-461 15 Stein, 0. and Stein, Y. (1963) Biockim. Biophys. Acra 70, 517-530 16 Dacie, J. V. and Lewis, S. M. (1968) Practical Haematobg_v, 4th edn, p. 376. J. A. Churchill. London 17 Jamieson, J. D. and Palade, G. E. (1968) J. Celf Biof. 39. 589-603 2