Triacylglycerol secretion in very low density lipoproteins by isolated rat liver parenchymal cells

Vol. 55, No. 3,1973

BIOCHEMICAL

TRIACYLGLYCEROL

SECRETION

IN

VERY

LIVER R. Department

of

Received

LOW DENSITY

PARENCHYMAL

Sundler,

6.

Physiological

October

AND BIOPHYSICAL

LIPOPROTEINS

BY ISOLATED

RAT

CELLS1

Akesson

Chemistry,

RESEARCH COMMUNICATIONS

and

A.

Nilsson

University

of

Lund,

Lund,

Sweden

23,1973

SUMMARY Enzymatically

isolated rat liv 5 arenchymal cejls secreted labeled triacylwhen incubated with glycerol or [ H]oleic acid. The presence of fti albumin or serum did not affect the secretion, but it was strongly inhibited by cycloheximide, colchicine, EDTA and by incubating at 4’C instead of at 37’C. Analyses of incubation media by agarose gel electrophomsis and by ultracentrifugation showed that the labeled triacylglycerols were in particles with the properties of very low density lipoproteins.

glycemls

INTRODUCTION

The liver of

contributes

blood

most

are

plasma

frequently the

intact

(2,3),

form in

was

lipids of

lipoprotein

organ

by

enzymatically

interest

to

perfusion.

was

Cell

AND

In

experimental

labeled

on

parenchymal

the

cells

retains

the

capacity

system

is

rnatabo-

the

advantages

cells were therefore of

studies

compared

incubated

lipids

into

to

with the

label-

medium

METHODS

isolation

weighing were

and

ZOO-350 prepared

used

as

the

and

dialyzed

incubation.

were

g

according

carbonate-free

to

Hanks’

basic bovine

fed

a balanced

Berry

solution

skilful

medium. albumin

by the Swedish Medical technical assistance by greatfully acknowledged.

VLD[Ll,

very

low

density

and

diet Friend

buffered

incubation serum

Sprague-Oawley rats

Male

1Supported is

have

since

rat liver

by

slices.

rat liver

especially

release

the

secretion

systems

examined.

MATERIAL

2

tissue

experimental

this preparation

secretion,

and

The

and

isolated

rat liver

acid

and

whether

slices and

Suspended

or oleic

formation

organ

know

and

(11.

their

on

perfused

formation

ed glycerol

VLO*-lipoproteins

the

superior to liver

lically

of

studies

animal, of

it

major part of the non-chylomicron triacylglycerols

the

the

used

biosynthesis

of

in

with Where

(Fraction

10 mM phosphate indicated V.

Serva

d < 1.006.

961

AB,

Sweden)

Liver parsnchymal --ad libitum. (41 as described elsewhere (pH

7.41

2 % delipidated Heidelberg),

Research Council (03X-3958 Miss Elisabeth Nilsson and

(lipoproteins),

Copyright @ 1973 by Academic &em. Inc. All rights of reproduction in any form reserved.

(Anticimex

cells (21. was (51

with

and Miss

Bi-

or

03X-39691.

Gertrud

The Olsson

BIOCHEMICAL

Vol. 55, No. 3, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

18a 16-

0

I.6

60

120

INCUBATION

TIME

12 I I

18

a v w

l4

d

lo-

E 0 w

I.4

60

18.

6-

5: a 8 s

(min)

b

20

60

Figure 1 (left). Rat liver parenchymal for the time indicated. The incubation figure shows the proportion of total (a) and the total incorporation of medium [H 1 and cells + medium of about ten separate experiments. Figure acid

(right).

2 in

tion of total triacylglycerol plus

medium

without

[M

min

at

contained 3H]glycerol sham) The less Rat tion off

in

(150

oleic

acid was

approx. or 0.5

5 x

was

parenchymal Hanks’

x g for

medium

(w

The the I,

or 10 % dialyzed in

cells

umole

ml)

in

(61,

lo6

and

-“J H

1 ml

(60-60

the

with % rat

(min)

c %]g serum. in the

lycero 1 The medium

glycerolipids shown

are

of

typical

incubated with [3H]oleic figure shows the propormedium (al and the total cells (b--d 1 and cells

in

oleic

acid

a 25

ml

incubation

was

added

and

5 mini.

An

aliquot

the

medium.

Each

mg proteinland

oscillations the

rat serum previously

incubation

(2.9-5.0

l 9,lO of

stated (0.1

were

cells

buffer. radioactivity in

included

volume shaken

cells were incubated medium contained 10 glyceylipid radioactivity added [ HIglycerol into (-1 (b). The results

TIME

(bl.

56’C

otherwise

for

modified

)

a final

flask

plasma

in

triacylglycerol radioactivity

bound

for 30

liver

Rat

2 % albumin

160

120

INCUBATION

0.1

umole

(Radiochemical

heated

incubation [l/3)-

Centre,

Amer-

Erlenmeyer flask.

siliconized

per

mini

was

terminated

at

37’C

for by

180

cooling

min, on

unice.

were sedimented by centrifuga[3/41 of the supernatant was sucked cells

analysis.

962

BIOCHEMICAL

Vol. 55, No. 3, 1973

TABLE

I.

Distribution

TG, PE, PC and phosphatidylcholines

c 3H 3 glycerol

of LPC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

among

lipid

represent triacylglycerols; and lysophosphatidylcholines,

Lipid class

Incubation Modif buffer

ied

Hanks’

Cells

Medium

50.0

TG’ PE PC

94.9

10.4 39.4

LPC

0.2

extracts intraperitoneal

‘Lipid

‘Including

per

Cells

Cells

Medium

cent

of

3.5 0.6

11.5 0.2 and

injection

1.0-2.0

93.7 0.6 2.3 3.4

s yrn

from

were extracted

V/V).

The chloroform

beled washed

glycerol) or 0.1 M acetate buffer three times with methanol-l % NaCl

form.

Lipids

Lipids

rat killed (1.5 mCi).

phase,

by

obtained

by

6 vol.

by

vol.)

(60:30:5) for polar

(pH

for neutral

to 5.01

[l:l

the

lipids

and

All

were

analyses

performed

in

of

above)

hepatic

was

lipid

mixed

droplets

to give a final sample volume layered with 1.5 ml distilled rpm) ml1

97.7

19.4

0.3

26.2 0.6

1.7 0.3

after

the

(1:l

1 % NaCl

(la-

silica gel ether-acetic

chloroform-methanol-4

an MSE Superspeed 50 TC instrument at droplets ('Sf > 3000) from incubation media,

pension

46.5

H;

M NH40H

lipids.

centrifugation.

(see

on

40-60°C1-diethyl

out rotor lipid

Serum

min

extract

chromatography

by

of

Liver

(labeled oleic acid), was v/v) saturated with chloro-

Analysis

supernatant

45

1

chloroform-methanol

adding

were separated by thin-layer solvents:light petroleum Ib.p.

(75:25:1

media.

% diacylglycemls.

analysis.

acid

Medium

a male

of IT H I glycerol

Lipid

developing

and

In vivo KpZKent

radioactivity 70.2 95.6 5.5 0.4 24.0 3.2 0.3 0.9

glycemlipid

86.2

cells

medium + 10 % serum

2.2

liver

in

+ 2 % albumin and 1 r#l oleic acid

1.0

of

classes

phosphatidylethanolamines, respectively.

of

4-6’C.

a 3 x 5 ml For

0.3

ml

the of

the

swing-

flotation low-speed

rat serum and 0.1 ml of a sus(71.NaC1,O.g % and 1 M sucrose was' added

with

0.1

2 ml,

0.2

water

for 20 min. Three fractions with a syringe equipped with

in

M in sucrose. The sample was overcentrifuged at 20,000 x g,, (15.000

and

were

ml

from above (0.5, 0.5 and 2.5 When [3H]glycerol-labeled

collected

a bent

needle.

lipid droplets or rat serum (see below) were analyzed, they were substituted for the corresponding nonlabeled addition. For

the

0.2

ml

= with

isolation rat

1.063). 3.2

of

serum

diluted

0.2

to 1.2

ml

ml

with

0.9

% NaCl

containing

Na2EOTA

963

incubation

medium

a solution

sample was underlaid with 0.5

The ml

and

VLD-lipoproteins

ml

(0.1

KBr r&ml).

of (d

KBr

was (final

mixed

with

density

= 1.211 and overlayered After centrifugation at

BIOCHEMICAL

Vol. 55, No. 3, 1973

TABLE

II.

Flotation

media

after

of

incubation

AND BIOPHYSICAL

lipid

droplets

frcm

with

[3H]glycerol.

Fraction

cell

Incubation Modified buffer

Hanks’

CHl

RESEARCH COMMUNICATIONS

homogenates

--In

M*

CH1

incubation

medium

+ 2 % albumin and 1 mM oleic acid

M2

and

vivo

experiment

Liver

Serum

Hepatic droplets

horroge3 nate

per 0.3 4.4 95.3

14.0 4.0 62.0

1 [Top)

2 3 1 Cell homogenate.

The

5 min and subjected was designated cell

2

Incubation

3

ed

by

0.3 4.0 94.6

07.4

cell pellet was vigorously to recentrifugation (150 x

shaken in g for

86.0 3.7 9.5

a Vortex

5 minl.

mixer supernatant

The

for

homogenate.

homogenate

in

0.2

sucros.

M

x gav (34,000 rpml for 16 hours, the

100,000

radioactivity 12.6

msdium.

liver

Total

of glycerolipid 10.9 0.6 2.2 0.5 86.9 90.9

cent

tube

slicing.

remainder

The

was

sucked

top

fraction

off

in

(1

ml1

was

three fractions

coilect-

(1.3

ml

each).

Gel electrophoresis.

An aliquot

the

of rat plasma containing to agarose gel electrophoresis

equal

vol.

jected

ter fixation

in 5 % trichloroacetic

corresponding

gel,

to

albumin,

B-protein and the origin, described above. Preparation A

of

of

I

rat was

injected

later.

The

min.

were

droplets 60 mini

(Ref.

7).

Of

triacylglyremls, was

collected

min

at

3000

the

methods

and

lipid

cut

out

droplets

and

homogenate

10

vol.

0.25

M

in

this

sub-

from

rat serum. and killed 45 and

lipid

(20000 x gav for

fraction

98

% was

and

and 0.6 % in polar lipids. serum was prepared by centrifugation

Sodium

EDTA

(pH

7.0,

diacylglycemls

were as previously

described (2).

964

was

added

the

B-protein,

aorta

1 mg/ml)

Af-

from the gel as

sucrose

centrifugation

by

an

(91.

c1- and

between

with 1.5 mC[3H]glycerol in

was

Johansson

Lipids were extracted

at 2-4'C. Other

by

with

the

from rpm.

mixed

1 mg/mll

region

the

radioactivity

lipid

1.4 % in

7.0;

was

bands were

hepatic

homogenized the

(pH

five

a-protein,

from

medium

181 as described

respectively.

was

isolated

Na2EDTA

acid

3H glycerol-labeled I intraperitoneally liver

incubation

and

the

serum

in Blood

for 20 storec

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 3,1973

TABLE

III.

bation

Flotation

with

of

Fraction

c

fran

medium

Incubation 10

1 (d

VLO-lipoproteins

incubation

% serum per 93.1

1.0061

2 3 4

(in

modified

2 % albumin

cent

of

Hanks’

and

triacylglyceml

IV.

Analysis

liver

parenchymal

cells

Modified buffer

radioactivity

AND

As

shown

ed

glycerol

lipids

in

were

glycerol

Hanks’

incubated

gel

electmphoresis.

'H]glycsrol

with

glycemlipid

total

triacylglycerol

for

150

--In vivo experiment

~3.6~ 5.6 66.5 6.7 6.6

total

serum

o.z2 3.9 60.6 7.8 7.5

6.7l 2.6 59.5 19.5

11.7

radioactivity.

radioactivity.

DISCliSSION Figure

1 a sizeable

portion

by

liver

appeared

the

cells

of

reached

glyceml

within that

the

(31.

A

hours

30 min.

rate of comparable

(0.1

of

ce

with

is

In

the experiments

found

--in with

the

in

lipids

the

higher

synthesis of

labeled

H I oleic

acid

released well

fmm

medium.

incorporation with

glycerolipid release

synthesized

incubation

n-PI1 maximal

Experiments

P seen after incubation with albuminbound cerol was the principal labeled glycerolipid (Table 11 as incubation with [ 3d glyceml what

agamse

+ 2 % albumin and 1 n+l oleic acid

of

showed 2-3

by

medium

of

concentration was

least

media

8.7l 5.7 50.0 11.6 15.2

ci a-B B origin 1Per cent 2 Per cent

a low

incubation

Incubation

albumin

acid

1.2 2.3 3.2

of

Zone

RESULTS

incu-

93.3

3.6

Rat min.

after

buffer1

1 ITU oleic

3.3

TABLE

media

[ 3HJglyceml.

as

13H]

into

amounts

linear triacylglycerols (Fig. into

21. the

labelusing

cellular of

is

oleic

By

labeled

over at were Triacylgly-

medium

acid,in

after accordan-

vivo. labeled

glycerol

965

the

proportion

of

total

glycemlipid

BIOCHEMICAL

Vol. 55, No. 3, 1973

TABLE

V.

tion

Effect

fmm

of

agents

and

of

rat liver

isolated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

incubation

temperature

parenchymal

on

glycerulipid

secre-

cells. 3

liver parenchytnal cells were incubated with and the glyceroc H I glycerol secretion was determined as the proportion of total glycemlipid radioactivity in the rredium. Data from at least two separate experiments are included. The figures shown represent meanpercentage + S.D. of corresponding controls (nur&er of observations). Incubation time was 60-180 min after iso-

Rat lipid

tope

addition.

Addition

~3H~glycem1 medium lipids

in

cellular + (% of control)

Glycerolipid secretion ( % of control)

Cycloheximide’ ID.035 rdl)

102.2

i- 12.8

27.2

I!

4.2

(6)

Colchicine’ (0.025 rrPl)

101.5

f

7.8

22.2

+

6.9

(4)

145.8

f

8.7

14.3

-?

3.4

(41

109.7

? 14.3

93.9

+ 12.2

(4)

105.5

f

f

(41

Na2EOTA

(3

CaEDTA MgEDTA

(1.5 (1.5

rrEl1 rrP’l) rrp1)

Incgb?tion +4c 1 2

The

+

at

cells

were

prior

Incubation

with

lipid in

found

in

the

2 % bovine

lipids

in

sence

of

the

release

albumin. of

lipids

medium

tion.

due

lipids,

incubations

labeled

cerol

among

could

be

cytoplasm

a liver

then

at

as

the

same

in

phase

of

of

led that

these

media

[Table in

oxygen

to ascertain that lipoprotein

plasma

to

the

did

not

of 11,

pre-

the enhance

the

in

the

supported medium,

with by

the

fact

release

These

the

low-speed

of

less

than

966

supematant 5 % of

the

cellular membranes that triacylglycerols of the distribution

are larger

droplets

labeled

of

cellular lipids (Table I). Another effect of the release of triacylglycerol-rich lipid droplets, of liver cells and appear as a floating layer (7,101.

of

not to cell disintegra-

labeled

irrespective

the

and

sedimentation

contamination

is further lipids

homogenate

min.

conposition

The

lysophosphatidylcholine

a gas

150

the

% rat serum.

10

for

+4’C

measure of the glycerocells were incubated

a

whether

100,000 x g for 90 min

at

conclusion

also

min

used

same

labeled

applied

secretion

indicating

major

was

under

were

to

Centrifugation

from cell

been

or

presence or absence of the

the 30

4.6

lipids.

approaches

was

has

was the

release of

Incubation

labeled

Different

medium

in

for

37’C

serum albumin

for a certain

except

7.2

60 min addition.

at

proportion

This

buffer,

This

glycerol

[ 3 H]

release.

labeled

b ted for to [ v H glycerol

preinc

inhibitor,

radioactivity

21.7

than

cell

the

small.

were the a+'I 3Hjg1y-

disintegration which

after

labeled was

occur in

centrifugation lipoproteins

the of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 3,1973

secreted

by

procedure ter

liver

in

(71

was

where

lipid

layer

(Table

incubation

on

the

in

homogenized

II

other

rat

incubation

that

associated

accordance ty

on

with

evidence

tained

by

tein

the

use

synthesis inhibiting was

pressed

after

into

the

release

for

the

of

the

inhibition

The

formation

ly

been

of

the

such

the

labeled

bivalent

cations amounts

was and

isolated

labeled

lipids

perfusion

of

(Figures experiments

la-

to contrimedia. (Table

cells

were the

almost

enof

and

were

VLD-lipoproteins.

VLDL,

had

1111

Sf-value

fact

In

pre-B-mobili-

active

by

in

into but

with

strongly 2+

the

isolated lipids

proportion

had

a

bound

released

effect

suggestions

of

(13,141. to

hepade-

similar

inhibited

were

liver,

cellular

recent

on a

Furthermore, prior

EDTA

role the

release.

glycemlipid the

obpm-

perfused

the

secretion

was inhibits

the

Neither

Colchicine

Na2EDTA

secretion

to

of

intact

plasma

lipoproteins

--in

vitro

has

perfused liver and in liver slices (11, while occurs in less complex systems requiring cofactor dispersed rat liver cells (151, rat liver microsones

rate

those

Thus,

seem

Cycloheximide

(121.

by 2+

Mg

II). not

in

ribosomss (16). and secreted by the

Vl.

accordance

and

to

addition

reversed.

secretion

moiety

Furthermore,

Ca

to

glycerol

lipoprotein

completely

mechanically

thesized

of

incubation,

incubation

the

VLD-lipoproteins

in

system

fmm

plasma

cycloheximide,

in

(Table

lipids

similar

do

due

(Table

lowered.

lipids,

vivo

This

was

labeled

with

armunts

frwn

like

synthesis of

in

particles

VLDL

of

was drastically

demonstrated

as

secretion

after

water

the

IV).

agents

preincubation

protein

them,

of

glycemlipid

equimolar

of

release

incorporation

of

the

of

centrifugation

d < 1.006.

wa-

conditions

part

cells

equilibrium

(Table

inhibitory

microtubular

binding When

of

the

medium

the

the

--in

A

distilled

of the labeled

the

recovered

that

majority

the

and

tocytes

indicate

upper

disintegration

released of

electmphoresis that

without

ver

this gel

Direct

of

strongly

agamse

cell

density

particles

the

of Under

fraction

droplets

lipids

by

into

glycerol

to

triacylglycerols with

3000

with

centrifugation.

media.

Disrupting

lipid

labeled

media

labeled

than

the

a layer

move

so.

labeled

due

during

negligible

did

labeled

rapidly through

incubation

would a

serum

more flotated of

only

formed to

tirely less

in

liver

significantly

showed

> 3000

released

hand,

of

Sf

111

or

components

Analysis

analysis

Ref.

media

are

the of

and

flotate

droplets

for

adopted

cell

bute

therefore

the

particles

in

beled

and

which

The

present

results

enzymatically VLDL

secretion,

1 and

indicate

rat

isolated

21,

as is

of

judged the

same

that

plasma

liver from order

previoussynthesis additions, and

VLOL

parenchymal

are

fractional

the as

that

syn-

cells.

release

found

in

li-

M.

Stotz,

( 17- 19 1.

REFERENCES 1.

Robinson, E.M., edsl,

O.S. (19701 in Comprehensive Vo1.18, pp 51-116, Elsevier,

967

Biochemistry Amsterdam.

(Florkin,

and

Vol. 55, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL

2. Nilsson,

RESEARCH COMMUNICATIONS

A., Sundler, R. and Akesson, E. i 1973 1 European J. Biuchm., iyi press. 3. Sundler, R., Akesson, B. and Nilsson, A., to be published. 4. Berry, M.N. and Friend, O.S. (1969) 3. Cell. Riol. 43, 506-520. 5. Michael, S.E. (19611 Biochem. .I. 82, 212-218. h. Spector, A.A. and H0ak.J.C. (1969TAnal. Biochem. 32, 297-302. 7. Bar-on, H., Roheim, P.S., Stein, Cl. and Stein, Y. n971) Biochim. Eiophys. Acta l-11. -248, 8. Noble, R.P. (19681 J. Lipid Res. 9, 693-700. 13 Johansson, E.G. (19721 Stand. J. Clin. Lab. Invest. 29, suppl. 124, 7-l’;. 1;: Stein, !I. and Stein, Y. (19671 J. Cell. Hiol. 33, :lF339. 11. Bole, V.P. and Hamlin, J.T. (1962) Physiol. Rev. 42, 874-701. 12. Bar-on, H., Kook, A.I., Stein, 0. and Stein, Y. (19731 Biochim. Eiophys. Acta 306, 106-114. __ 13. Stein, Cl. and Stein, Y. (19731 Biochim. Biophys. Acta 306, 147-147. __ 14. Ilrci, L., LeMarchand, Y., Singh, A,, Assimacopoulos-Jaennet, F., Rouiller, C. and Jeanrenaud, B. (1973) Nature 244, 30-37. 15. Capuzzi, D.M. and Margolis, 5. (1971)ipids 6, 509-618. 16. Marsh, J.B. (1969) in Structural and Function51 aspects of Lipoproteins in Living Systems (Tria, E. and Scanu, A.M., E&!, pp 447-464. 17. Weinstein, I., Dishmon, G. and Heimberg, M. (1958) Biochem. Pharmacol. 15, 851-871. 18. Kimberg, M., van Harken, D.R. and Brown, T.O. (13671 Biachim. Biophys. Acta 137, 435-445. 19. Toppi= E.L. and Maves. P.A. (19721 Biochem. J. 126. 1195-311.

968