Biochem. J. (1977) 168, 483-494
Printed in Great Britain
483
Binding, Interiorization and Degradation of Cholesteryl Ester-Labelled
Chylomicron-Remnant Particles by Rat Hepatocyte Monolayers
By CLAES-HENRIK FLOREN* and AKE NILSSON
Department ofPhysiological Chemistry, University ofLund, Lund, Sweden
(Received 16 May 1977)
1. The cholesteryl ester ofisolated chylomicron-remnant particles was efficiently degraded
by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors.
2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached
saturation and conformed to a linear double-reciprocal plot. The Vma.. was determined as
80ng of cholesteryl ester/h per mg of protein and the apparent Km as 1.4,ug of cholesteryl
ester per mg of protein. The time course for the uptake and hydrolysis suggested that
binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of
native chylomicrons were degraded to a much smaller extent and their presence had only
a small inhibitory effect on the degradation of chylomicron remnants. Intestinal
very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and
caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited
the hydrolysis of remnant cholesteryl ester by up to 50 %, but had less influence on the
amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake
and hydrolysis, whereas d= 1.21 infranatant had no effect. 4. The cholesteryl ester
hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine.
Only 28-36% of the unhydrolysed cholesteryl ester could be released from these
cells by trypsin treatment, indicating that the major portion was truly intracellular. The
particles that could be released from the cell surface by trypsin and those remaining in the
medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant
particles. Significant amounts of denser particles were thus not formed during contact
with the cell surface. 5. The presence of heparin, as well as preincubation of the
cells with heparin, increased the uptake of chylomicron remnants. This effect was most
marked in the presence of serum. A much smaller proportion of the other serum
lipoproteins was taken up, and this proportion was not increased by heparin.
Earlier studies (Stein et al., 1969; Nilsson &
Zilversmit, 1971) indicate that the degradation of the
chylomicron remnants that are formed during the
extrahepatic metabolism of chylomicrons (Redgrave,
1970; Mj0s et al., 1975) occurs mainly in the hepatocytes. During the conversion of chylomicrons into
remnant particles a loss of triacylglycerol, phospholipid and small peptides, and an enrichment in cholesteryl ester, B-peptide and arginine-rich peptide,
occurs (Mj0s et al., 1975). These or other changes,
such as the attachment of lipoprotein lipase (EC
3.1.1.34) to the lipoproteins (Felts et al., 1975),
apparently increase the affinity of the particles for
the hepatocyte surface, since remnants are taken up
more efficiently than native chylomicrons both in the
perfused liver (Noel et al., 1975; Felts et al., 1975;
Gardner & Mayes, 1976) and in suspended
(Nilsson & Akesson, 1975; Nilsson, 1977) and cultured (Floren & Nilsson, 1977) hepatocytes. In
hepatocyte cultures the hydrolysis of cholesteryl
* Address for
correspondence: Institutionen for Medicinsk Kemi 4, P.O. Box 750, S-220 07 Lund 7, Sweden.
Vol. 168
ester after- the uptake by the cells was inhibited by
colchicine and by chloroquine, suggesting a role of
the microtubules and of lysosomal enzymes in the
cholesteryl ester degradation (Floren & Nilsson,
1977).
In the present study the kinetics of degradation of
remnant cholesteryl esters in hepatocyte monolayers
was studied. In particular we examined whether the
uptake exhibited saturation kinetics and whether
native intestinal lipoproteins and HD lipoproteinst
compete for the same transport mechanism. Trypsin
treatment was used to examine whether cellular
cholesteryl ester remained in particles bound to the
cell surface or was truly intracellular. The purpose
was to examine whether binding of the remnants
to the cell surface preceded the interiorization of the
cholesteryl ester and to elucidate at what stage
t Abbreviations: HD lipoprotein, high-density lipoprotein (1.063 <d< 1.21, 6-lOnm);VLD lipoprotein, verylow-density lipoprotein (d< 1.006, Sf 20-400, 25-75nrm);
Hepes,4-(2-hydroxymethyl)-1-piperazine-ethanesulphonic
acid.
484
colchicine and chloroquine interfere with the
degradation of chylomicron-remnant cholesteryl
ester.
For instance, fusion between the remnant surface
coat and the hepatocyte plasma membrane has been
suggested as a possible mechanism for chylomicronremnant degradation (Felts et al., 1975). If such a
process is inhibited by colchicine the remnant
particles would be expected to remain bound to the
cell surface and to be released by trypsin. On the other
hand the undegraded cholesteryl ester would probably
be intracellular, if the degradation occurs by endocytosis.
A trypsin-sensitive binding of remnant particles
to the cell surface does not necessarily mean that
remnants are directly bound to cell-surface protein,
since the binding of glycosaminoglycans to hepatocytes is also decreased by mild trypsin treatment
(Kjellen et al., 1977). The possibility that glycosaminoglycans may bind to the hepatocytes and participate in remnant binding to the cell surface was
therefore investigated.
Materials and Methods
Materials
['4C]Linoleic acid, [4-14C]cholesterol and [G-3H]cholesterol were from The Radiochemical Centre,
Amersham, Bucks., U.K. Leibovitz L-15 medium,
foetal calf serum and Hepes were from Flow
Laboratories, Irvine, Ayrshire, Scotland, U.K.
Acid-soluble calf skin collagen and chloroquine
were from Sigma Chemical Co., St. Louis, MO,
U.S.A., colchicine was from BDH Chemicals,
Poole, Dorset, U.K., pig insulin was from Novo
Industri A/S, Bagsvaerd, Denmark, collagenase
[CLS, 143-180 units/mg (1 unit liberates 1,umol of
amino acid from collagen in 5h at 37°C, pH7.5)]
was from Worthington Biochemical Corp., Freehold,
NJ, U.S.A., trypsin (1 %) and EDTA (0.2%) in
phosphate-buffered saline (NaCl, 8 g/l; KCI,
0.2g/l; Na2HPO4, 1.44g/l; KH2PO4, 0.2g/l; pH7.4)
were from Statens Bakteriologiska Laboratorium,
Stockholm, Sweden, and cholesterol was from Merck,
Darmstadt, West Germany. Heparin [5000 units/ml
(2.4,ug of uronic acid/unit)] was from Vitrum AB,
Stockholm, Sweden.
Methods
Preparation of lipoproteins. Male white SpragueDawley rats, weighing 250-300g, were obtained
from Anticimex AB, Stockholm, Sweden. Thoracicduct cannulations (Bollman et al., 1948) were performed under diethyl ether anesthesia as described by
Nilsson & Akesson (1975). The rats were given 0.75 ml
of corn oil containing 1 mCi of [3H]cholesterol and
50,Ci of ['4C]linoleic acid, and, to increase the
percentage of cholesteryl ester in the chyle, in some
C.-H. FLOREN AND A. NILSSON
instances together with 50pmol of cholesterol. The
corn oil was given in three doses over 4h. Lymph was
collected and stored at 4°C with 2.Omm-disodium
EDTA present (Ontko, 1970). Chylomicrons (Sf>
400) were prepared as described by Minari &
Zilversmit (1963). The chyle was centrifuged in an
MSE 65 TC centrifuge with a 6 x 16.5 ml swing-out
rotor at 25000rev./min (70000gav.) for 100min at
10°C. The top layer was collected, and the infranatant layered under 1.1 % NaCl (d = 1.006) and
centrifuged in an MSE 6 x 14ml titanium swing-out
rotor at 33000rev./min (I33000gav.) for 18h at 10°C.
Intestinal VLD lipoproteins were collected as the
top layer. HD lipoproteins were prepared from rat
plasma containing 1 mg of disodium EDTA/ml. The
density was adjusted to d = 1.063 by adding a stock
solution of KBr and NaCl, and the plasma was then
layered under 5 ml of a salt solution with d = 1.063
(34.7g of NaCl and 59.Og of KBr/l). Centrifugation
was carried out at 34000rev./min (140000gav.) in an
MSE 6 x 14ml swing-out rotor for 18 h at 10°C. The
top layer (2cm) was removed and discarded. The
density of the infranatant was raised to d= 1.21 by
adding solid KBr and NaBr. This was overlayered
with 5ml of a salt solution with d = 1.21 (96.2g of
NaCI and 212.4g of KBr/l), and centrifuged in an
MSE 6 x 14ml titanium swing-out rotor at 39000rev./
min (l90000gav.) for 44h at 10°C. The top layer was
collected and was washed once at d = 1.21. It was then
dialysed extensively against 0.15 M-NaCl/0.3 mmdisodium EDTA, pH 7.4, at 4°C. After dialysis
concentration was carried out in a Minocon B-15
Amicon B. V., Oosterhout (N.B.) The Netherlands.
The d = 1.21 infranatant was filtered through a
0.22,m Millipore filter (Millipore Corp., Bedford,
MA, U.S.A.) and stored at -17°C until used. For
the preparation oflabelled HD lipoproteins serum was
taken from rats that had been injected intravenously
with 25,uCi of [14C]cholesterol in ethanol/0.9 %
NaCl (Nilsson & Zilversmit, 1972) 24h earlier. HD
lipoproteins were then isolated as above except that
no salt solution of d= 1.21 was layered over the
d= 1.21 infranatant. Instead the latter was centrifuged once in a 6 x 14ml titanium swing-out rotor at
34000rev./min (I40000gav.) for 20h, and the top 1 cm
was removed and washed once at d = 1.21 as above.
Purity and ac-electrophoretic mobility of the HD
lipoproteins was checked by agarose-gel electrophoresis (Noble, 1968) as described by Johansson
(1972). A faint band migrating as albumin was present
as an impurity.
Chylomicron remnants (Sf >200) were prepared
by injecting chyle into functionally hepatectomized
rats (Redgrave, 1970). After 30min blood was drawn
from the abdominal aorta. Plasma was collected by
using disodium EDTA as an anticoagulant (approx.
2.5 mg/ml of blood). The plasma was adjusted to
d= 1.063 by adding a stock solution (d= 1.35) of
1977
METABOLISM OF CHYLOMICRON REMNANTS IN HEPATOCYTE MONOLAYERS
KBr and NaCI. It was layered under 1.1 % NaCI
(d = 1.006) and centrifuged in an MSE 3 x 5 ml
swing-out rotor at 27000rev./min (70000gav.) for
2ih at 4°C. Chylomicron remnants were collected
under sterile conditions and were used within 12h.
In some experiments remnants were prepared by
treating chyle with post-heparin plasma, obtained as
described earlier (Nilsson, 1977). Chyle triacylglycerol
(26-34mg), post-heparin plasma (7.5-lOml) and
5 % bovine serum albumin (36ml; Cohn fraction V;
Serva Feinbiochemica, Heidelberg, West Germany)
were incubated for 1 h at 28°C. The density was then
adjusted to 1.063 by adding a stock solution of KBr
and NaCI (d= 1.35) and the solution was layered
under 1.1 % NaCl (d = 1.006) and centrifuged at
25000rev./min (70000gav.) for 2ih at 10°C in a 6x
16.5ml MSE swing-out rotor. The remnants were
then collected with a Pasteur pipette under sterile
conditions. Remnants prepared with post-heparin
plasma were used only when specified. In all other
experiments remnants isolated from hepatectomized
rats were used.
Preparation of rat hepatocyte monolayers. Hepatocytes were prepared by a collagenase procedure
(Berry & Friend, 1969), by using the conditions of
Seglen (1973). The apparatus was described by
Nilsson et al. (1973a). Heat-sterilized equipment
and aseptic technique were used during operation.
The perfusate contained 100pg of penicillin (KABI
AB, Stockholm, Sweden) and 50,ug of gentamicin
(Schering Corp., Kenilworth, NJ, U.S.A.)/ml and
was buffered with 40mM-Hepes to make pH adjustments unnecessary. Hepatocytes were cultured in
primary monolayers (Bissell et al., 1973; Bonney
et al., 1974) on collagen-coated 60mm Petri dishes
(Falcon no. 1007 or 3002; Falcon Plastics, Oxnard,
CA, U.S.A.) as described by Lin & Snodgrass (1975).
The culture medium was Leibovitz L-15 medium
containing 28 mM-Hepes, pH 7.4, 1 mM-sodium succinate, 100,ug of penicillin and 50,og of gentamicin/
ml: 3 x 106-8 x 106 suspended hepatocytes were
plated in each dish in a volume of 2.5 ml. The dishes
were placed in humidified air at 37°C. During the
first 21-24h the medium contained 5% foetal calf
serum. Medium was changed after 3-5 h and after
21-24h. After that, medium was changed once every
day. The hepatocytes were incubated with lipoproteins
21-24h after plating in humidified air with shaking
at 25 cycles/min. Further details were given by Floren
& Nilsson (1977).
After incubation with lipoproteins the medium
was collected and the cells were washed twice with
0.5 ml of 0.85 % NaCl. They were scraped off with a
rubber 'policeman'duringrepeatedadditions(4 x 1 ml)
of methanol/water (2:1, v/v). As a control lipoproteins were always incubated with cell-free
collagen-coated dishes. Triplicate controls were done
in each experiment.
Vol. 168
485
Trypsin treatment of monolayers was done with
0.02% EDTA/0. 1% trypsin in phosphate-buffered
saline, after the medium had been removed and the
cells washed: 1 ml of EDTA/trypsin was added and
incubated with the monolayer for 10min at 37°C.
The detached cells were sedimented at 1000rev./min
for 10min and washed once in culture medium
containing5 %foetal calf serum. Lipids were extracted
from the pooled supernatants and from the cells.
The amount of lipids that could be removed from the
cells by trypsin is regarded as surface-bound, the rest
as interiorized (Stein & Stein, 1975).
Analytical. Lipids were extracted with chloroform/
methanol (1:2 or 1:1, v/v) (Bligh & Dyer, 1959).
The precipitates were removed and the portions of
chloroform, methanol and water were adjusted to
2:1:1 (by vol.) by adding 0.1 M-KH2PO4 and
chloroform. The lower phase was washed once with
methanol/water/chloroform (48:47:3, by vol.) and
was evaporated under N2. Lipid classes were
separated by t.l.c., and radioactivity was determined
as described earlier (Nilsson, 1977). Cholesterol
was determined as described by Zak et al. (1954)
after saponification of the lipid extract (Abell
et al., 1952). Triacylglycerols and their fatty
acid composition were determined by g.l.c. of the
fatty acid methyl esters (Akesson et al., 1970).
Protein was determined as described by Lowry
et al. (1951), with bovine serum albumin as standard.
Protein content in dishes were corrected for the
presence of collagen as described earlier (Floren &
Nilsson, 1977). Uronic acid was determined by the
carbazole reaction as modified by Fransson et al.
(1968).
The percentage net hydrolysis of radioactive
cholesteryl ester was calculated from the decrease in
radioactivity (d.p.m.) of cholesteryl ester and the
increase in that of free cholesterol as follows:
% Net hydrolysis
100 % cholesterol as ester after incubation x 100
% cholesterol as ester before incubation
There was no measurable net hydrolysis of
lipoprotein cholesteryl ester in the cell-free controls.
The percentage of cholesteryl ester that was cellassociated was calculated as follows:
100 x
d.p.m. as cholesteryl ester in cells
d.p.m. as cholesteryl ester in cells and medium
When cells were trypsin-treated the percentage of
cholesteryl ester surface bound was calculated as:
d.p.m. as cholesteryl ester released by trypsin
total d.p.m. as cholesteryl ester in cells, trypsin
digest and medium
17
lOOx-
C.-H.
486
and the percentage intracellular (interiorized) cholesteryl ester calculated as:
d.p.m. as cholesteryl ester remaining in cells
after trypsinization
d.p.m. as cholesteryl ester in cells, trypsin digest
and medium
The cholesteryl ester and triacylglycerol contents of
the chylomicron remnants were calculated by comparing their cholesteryl ester and triacylglycerol
radioactivity with the specific radioactivities of
cholesteryl ester and triacylglycerol of the chyle
from which they were prepared. The degree of
hydrolysis of chylomicron triacylglycerol during
preparation of remnant particles was calculated by
comparing the cholesteryl ester/triacylglycerol radioactivity ratios in chylomicron remnants with original
chyle.
Results
Characteristics of uptake and degradation of chylomicron-remnant cholesteryl ester
In earlier experiments (Floren & Nilsson, 1977) the
esterification of added non-esterified lipoprotein
[14C]cholesterol did not exceed 1 % during a 4h
30
20 _
0
.0\C
Q-
10
0
2
3
4
Incubation time (h)
Fig. 1. Time course for the surface binding, interiorization
and hydrolysis of chylomicron-remnnant cholesteryl ester by
hepatocyte monolayers
Chylomicron remnants (0.80,ug of cholesteryl ester,
2.84,ug of triacyiglycerol, 36300d.p.m. of 3H as
cholesteryl ester, 8640d.p.m. of 3H as cholesterol)
were added to hepatocyte monolayers (3.2mg of
protein/dish) and incubated for various time intervals.
Medium was removed by suction and the cells were
washed and treated with trypsin as described under
'Methods'. Values are means±s.E.M. of two dishes.
For preparations of chylomicron remnants, lipolysis of the original chyle triacylglycerol was 96%.
A, Percentage of chylomicron-remnant cholesteryl
ester hydrolysed; *, percentage of chylornicronremnant cholesteryl ester cell-surface bound; A,
percentage of chylomicron-remnant cholesteryl ester
interiorized.
FLOR1tN
AND A. NILSSON
incubation with hepatocyte monolayers, whether
chloroquine or colchicine was present or not. The
proportion was thus very low compared with the rate
of hydrolysis of chylomicron-remnant cholesteryl
ester. The remnant cholesteryl ester hydrolysis in
4h was therefore routinely measured in singleradioisotope experiments, in which the simultaneous
esterification of cholesterol was not determined.
The time course for the uptake and hydrolysis of
remnant cholesteryl ester is shown in Fig. 1. During
the first 30-60min of incubation the hydrolysis was
slow. After that the hydrolysis was linear with time
over the 4h period studied. In the 30-60min incubations 50 % or more of the labelled cellular cholesteryl
ester could be released by trypsin. During longer
incubation periods the cell-associated cholesteryl
ester radioactivity that was intracellular increased
more than the proportion that was lost during
treatment with trypsin.
The effect of particle concentration on the
degradation of remnant cholesteryl ester is shown in
Fig. 2(a). With increasing concentration the rate of
cell association and hydrolysis approached a
saturation value, and a linear double-reciprocal plot
was obtained (Fig. 2b). The amount of cholesteryl
ester that was associated with the cells and the amount
that had been hydrolysed were about equal, and this
relation was not changed with increasing concentration of remnants (Fig. 2a).
Double-reciprocal plots for the cholesteryl ester
hydrolysis gave Vmax. and apparent Km as 40ng of
cholesteryl ester/h per mg of protein and 1.4,g of
cholesteryl ester per mg of protein respectively. The
data in Fig. 2 are from an experiment with remnants
prepared with post-heparin plasma. Similar values
for Vmax. and apparent Km (30ng of cholesteryl ester/h
per mg of protein and 1.6,ug of cholesteryl ester per
mg of protein respectively) were, however, obtained
from a less extended substrate curve with chylomicron remnants from hepatectomized rats (Flor6n
& Nilsson, 1977). The remnants prepared by treatment with post-heparin plasma are thus metabolized
at about the same rate as remnants from hepatectomized rats. Plots of cholesteryl ester association with
cells gave Vmax. of 40ng/h per mg of protein and an
apparent Km of 1.4pg of cholesteryl ester per mg of
protein. Calculation of Vmax. for the total cholesteryl
ester uptake (cell association plus hydrolysis) gave
80ng/h per mg of protein, and the apparent Km was
1.4,ug of cholesteryl ester per mg of protein (Fig. 2b).
The uptake and degradation of chylomicron
remnants by hepatocytes were found to be energydependent, since adding 20mm-NaF or 30mMNaN3 decreased the interiorization of chylomicronremnant cholesteryl ester by 73.5 ±4.7 % and
91.5±2.4% respectively. The rate of hydrolysis was
decreased by 69.7±9.2% and 89.3±1.9% respec-
tively (means±s.E.M., two dishes).
1977
METABOLISM OF CHYLOMICRON REMNANTS IN HEPATOCYTE MONOLAYERS
400
o
8.s 300
ct;
u
o
200
40-6
10
0
2
u
4
6
8
Cholesteryl ester added (pg)
487
at a very low rate. In line with this, even high concentrations of native chylomicrons had only a slight
inhibitory effect on remnant cholesteryl ester degradation (Fig. 3). Intestinal VLD lipoproteins were
degraded faster than chylomicrons, but the rate of
both cell association and hydrolysis of cholesteryl
ester was lower than for remnants. A comparison
between the uptake of chylomicrons, VLD lipoproteins and remnants is given in Table 1.
Intestinal VLD lipoproteins had a more marked
inhibitory effect on cell association and degradation
of chylomicron-remnant cholesteryl ester (Fig. 4)
than chylomicrons had (Fig. 3).
Effects of HD lipoproteins and serum
Increasing concentrations of HD lipoproteins inhibited the hydrolysis of remnant cholesteryl ester.
The inhibition was about 50% at HD lipoprotein
concentrations above 280ug of protein/ml of
medium. The amount of radioactive cholesteryl
ester that was associated with the cells remained
(b)
50
r-
bo
a
0
15.
0I
10
u
0
8o
0.5
1/[S] (jg-')
Fig. 2. Effects of substrate concentration on cell
association and degradation of chylomicron remnant
cholesteryl ester
(a) Various amounts of chylomicron remnants prepared by treatment of chyle with post-heparin plasma
(0.27-11.3/pg of cholesteryl ester, 7.8-325,ug of
triacylglycerol, 13 300-564000d.p.m. of 3H as
cholesteryl ester and 14900-630000d.p.m. of 3H as
unesterified cholesterol) were incubated for 4h with
hepatocyte monolayers containing 3.7mg of cellular
protein. The triacylglycerol hydrolysis during preparation of remnants was 63.3°%. Each point is the
mean±s.E.M. of two values. o, Cholesteryl ester cellassociated (ng); A, cholesteryl ester hydrolysis (ng).
(b) Doub'e-reciprocal plot from values obtained for
total uptake (cell association and hydrolysis) of
chylomicron remnants. The reciprocal of the amount
(inpg) of chylomicron renuant cholesteryl ester added
was plotted against the reciprocal of the velocity
(ng of cholesteryl ester cell-associated and hydrolysed during a 4h incubation period).
Effects of chylomicrons and VLD lipoproteins
In earlier experiments (Floren & Nilsson, 1977) the
cholesteryl ester of native chylomicrons was degraded
Vol. 168
oA
0
0.86
1.71
2.57
3.42
Chylomicron cholesteryl ester (pg)
Fig. 3. Effect of native chylomicrons on cell association (e)
and degradation (A) of chylomicron-remnant cholesteryl
ester
Chylomicron remnants (0.63.pg of cholesteryl ester,
15.1 pg of triacylglycerol, 38600d.p.m. of ['H]cholesteryl ester, 54000d.p.m. of ['H]cholesterol) were
incubated with increasing amounts of unlabelled
native chylomicrons (20-400,pg of triacylglycerol,
0.17-3.42,pg of cholesteryl ester). The added chylomicron remnants were prepared by treatment of
chyle with post-heparin plasma; the hydrolysis of
chyle triacylglycerols was 69.1 %. Each dish contained
3.6mg of cellular protein. The values are means±
S.E.M. of two or three dishes. A similar experiment
was carried out with chylomicron remnants from
hepatectomized rats (0.77pg of cholesteryl ester,
19.1 pg of triacylglycerol, 29400d.p.m. of 3H as
cholesteryl ester, 7430 d.p.m. of 3H as unesterified
cholesterol) and the same native chylomicrons.
When 3.42,pg of chylomicron cholesteryl ester was
added the decreases in cell association and hydrolysis
of chylomicron cholesteryl ester were 37.9±0.7%
and 19.6±0.5% respectively (means±S.E.M., two
dishes).
C.-H. FLORtN AND A. NILSSON
488
Table 1. Comparison between cell association and hydrolysis of chylomicrons, VLD lipoproteins and chylomicron remnants
Labelled chylomicrons and VLD lipoproteins were added to hepatocyte monolayers in four different experiments; in
the same experiments chylomicron remnants were added to different dishes. All values are means+s.E.M.
Cholesteryl ester
Added
Expt.
lipoprotein
no.
1
Chylomicron
Chylomicron remnant
2
Chylomicron
Chylomicron remnant
3
VLD lipoprotein
Chylomicron remnant
4
VLD lipoprotein
Chylomicron remnant
Lipoprotein
Cell protein cholesteryl ester Cell-associated
(mg/dish)
added (ug)
(ng)
1.3
54.3 + 1.7
1.3
1.3
220 +6
1.36
2.6
2.34
94.5+4.0
2.6
1.69
217 +3
5.8
32.6+2.3
1.08
5.8
125 +3
0.92
2.5
0.77
39.7 +4.7
117 +7
2.5
0.85
No. of
dishes
3
3
3
3
3
3
2
3
Hydrolysed
(ng)
3.4±2.3
150 ±3
8.8+ 5.8
186 +9
16.5+4.6
125 +3
12.6+ 3.4
117 +7
i,
'0
"-
0-
25
Cd
'0
20;
'0
0 -0
00)
15
rA-
10
o
)
0
-
Q~
1oT
_
0
U
0
0
0.5
1.0
1.5
VLD lipoprotein cholesteryl ester (jg)
Fig. 4. Effects of intestinal VLD lipoproteins on cell association (a) and degradation (A) of chylomicron-remnant
cholesteryl ester
Chylomicron remnants (0.98,ug of cholesteryl ester,
11.l,pg of triacylglycerol, 29100d.p.m. of 3H as
cholesteryl ester, 6700d.p.m. of 3H as unesterified
cholesterol) were incubated with increasing amounts
of unlabelled intestinal VLD lipoproteins (0.181.42pg of cholesteryl ester, 3.1-25pg of triacylglycerol): in the added chylomicron remnants,
hydrolysis of original chyle triacylglycerol was
77.3°.). Each dish contained 5.8mg of cellular protein. Each point represents means±s.E.M. of two or
three values. The addition of intestinal VLD lipoproteins (0.71 pg of cholesteryl ester, 12.5 pg of triacylglycerol) was made in another experiment (2.5mg of
cellular protein, 0.85,ug of chylomicron-remnant
cholesteryl ester). The decrease in cell association and
hydrolysis of chylomicron-remnant cholesteryl ester
was 30.0±1.5y. and 35.5+2.9% respectively (means
+S.E.M., two dishes).
rather constant with increasing HD lipoprotein
concentrations, suggesting that HD lipoproteins
interfered with the degradation of the cholesteryl
ester rather than with the binding of remnant particles
250
500
7
.750
20
1200
HD lipoprotein protein (jig)
Fig. 5. Effects of HD lipoproteins on the cell association (0)
and degradation (A) of chylomicron-remnant cholesteryl
ester
Chylomicron remnants (0.65pg of cholesteryl ester,
3.13 pg of triacylglycerol, 19 200d.p.m. of 3H as cholesteryl ester, 5030d.p.m. of 3H as unesterified cholesterol) were incubated in dishes containing 1.6mg of
cellular protein and increasing amounts of unlabelled
rat HD lipoproteins (59-1180,ug of protein), containing 212,g of cholesterol/mg of protein. During
preparation of chylomicron remnants, hydrolysis of
original chyle triacylglycerol was 85.25%. Each value
represents means+S.E.M. of two values. The effect of
HD lipoproteins (1 180g of protein) was examined
in three other different cultures (3.1, 0.8, 3.1mg of
cellular protein/dish). The inhibition of hydrolysis
and cell association of chylomicron cholesteryl ester
was 47.8 ±3.5°% and 22.0±5.9°. respectively (means
+S.E.M., six dishes).
to the plasma membrane (Fig. 5). Considering
chylomicron-remnant cholesteryl ester interiorized
and cell-surface-bound in the presence of 59 and
11 80,g (protein weight) of HD lipoprotein, the
amount interiorized was 46.5± 1.9 ng and 41.6 ± 3.1 ng
respectively, and the amount cell-surface-bound was
47.8+0.5ng and 57.4±0.6ng respectively (means+
S.E.M., two dishes). When ['4C]cholesteryl ester1977
METABOLISM OF CHYLOMICRON REMNANTS IN HEPATOCYTE MONOLAYERS
Cholesteryl ester_
hydrolysis (%)
489
Total remaining cholesteryl ester (N)
Surface-bound (%)
(a) Pc0.001
Interiorized (%)
P <0.001
l10
N.S.
T
5
0
b)P< 0.001
P< 0.001
Il 0
N.S.
5
A
v
Fig. 6. Intracellular accumulation ofchylomicron cholesteryl ester after addition ofchloroquine (a) and colchicine (b)
Chylomicron remnants (0.63-0.85pig of cholesteryl ester, 25000-37400d.p.m. of 3H as cholesteryl ester, 42.3-79.8% of
total 3H d.p.m. as cholesteryl ester) were incubated in four different experiments (2.5-3.7mg of cellular protein) for 4h
with or without 25pM-chloroquine (a) or 33,uM-colchicine (b). The cells were trypsin-treated (see under 'Methods') and
the percentage of total remaining cholesteryl ester internalized and cell-surface bound was determined. Hatched bars
denote controls (nine dishes, two from each of three experiments, three from one experiment), and unhatched bars
inhibition with colchicine or chloroquine (nine dishes each, two from each of three experiments, three from one
experiment). Each bar is shown as a mean±S.E.M.; statistical calculations were made with Student's unpaired t test.
N.S., not significant (P>0.02).
labelled HD lipoproteins (73.6pg of protein, 23.6,ug
of cholesteryl ester, 7320d.p.m. of 14C as cholesteryl
ester, 2750 d.p.m. of 14C as unesterified cholesterol)
were added to hepatocyte monolayers (3.2mg of
cellular protein) a small but measurable cell
association of cholesteryl ester occurred (2.13 +
0.05 %, means of six observations ±S.E.M.), which
means that about 0.50,ug of cholesteryl ester was cellassociated. The net hydrolysis of cholesteryl ester did
not exceed 2 %. Per mg of protein, HD lipoprotein
inhibited degradation of remnant cholesteryl ester
more than did whole rat serum. The d= 1.21 infranatant had little or no effect, and 1 % foetal calf
serum had less effect than 1 % rat serum. However,
15 % foetal calf serum (Floren & Nilsson, 1977) and
5-10% rat serum both had marked effects on both
cell association and degradation of remnant cholesteryl ester. The inhibitory effect was also present
when complement-inactivated rat serum (heated at
56°C for 30min) was used, and was thus not caused by
a complement-mediated cytotoxic effect. When 10 %
Vol. 168
complement-inactivated rat serum was present in
incubations, the cholesteryl ester degradation decreased by about 45% and the cell association by
about 35%.
Effects of chloroquine and colchicine
The observation (Floren & Nilsson, 1977) that
colchicine and chloroquine inhibit the hydrolysis of
chylomicron-remnant cholesteryl esters after their
uptake by the cells was confirmed. The increase in
cellular unhydrolysed cholesteryl ester affected
mainly the intracellular pool, which increased much
more than the pool that could be released by trypsin
(Fig. 6). The ratio ['4C]triacylglycerol/[3H]cholesteryl ester in medium and in trypsin digest was the
same and was not influenced by the presence of the
drugs. There was no decrease in this ratio compared
with the added particles, but rather a slight increase,
suggesting some re-secretion of labelled fatty acid as
VLD-lipoprotein triacylglycerol or a faster uptake of
smaller remnants.
490
C.-H. FLORItN AND
Effects of heparin
The inhibitory effect of serum on the uptake, and
the earlier finding that heparin stimulated remnant
uptake in the presence of serum in suspended
hepatocytes (Nilsson, 1977), led us to study the effect
of heparin in the presence of serum, which is the most
physiological situation. The question was whether
heparin, or other negatively charged glycosaminoglycans, which bind to hepatocytes in monolayers
(Kjellen et al., 1977), can participate during the binding phase of remnant degradation. Incubation with
heparin would also probably cause a loss of hepatic
lipase. Whereas 4 % rat serum normally inhibited the
cholesteryl ester uptake and degradation, the
addition of 1-10 units (2.4-24,lg of uronic acid) of
heparin/ml of culture medium restored the uptake to
about the same values as in the absence of serum
(Fig. 7). The heparin effect on degradation and cell
association of remnant cholesteryl ester was also
present, but less prornounced, in incubations without
serum (Fig. 7). The effects of heparin may be due to
effects on the hepatocyte surface, since similar
effects were found with hepatocytes preincubated
with heparin (Table 2). No such stimulation of cell
association was found when [14C]cholesteryl esterlabelled rat serum lipoproteins (6770d.p.m., 80.4%
of the 14C label in cholesteryl ester) were incubated
with hepatocyte monolayers (2.6mg of cellular
protein) for 4h in a final serum concentration of 4%
in the presence or absence of heparin. In controls
A. NILSSON
Table 2. Effects of preincubation with heparin on cell
association and hydrolysis of chylomicron-remnant cholesteryl ester
Hepatocyte monolayers (2.2mg of cellular protein)
were preincubated for 2h with 1-10 units (2.4-24,ug
of uronic acid) of heparin/ml; medium was then
removed by suction, the cells were washed once
and incubated in medium with 4% complementinactivated rat serum and chylomicron remnants
for 4h. The added chylomicron remnants contained
0.56,pg of cholesteryl ester, 0.96,pg of triacylglycerol, 14900d.p.m. of [3H]cholesteryl ester and
2970d.p.m. as [3H]cholesterol. During preparation
of remnants, hydrolysis of the original chyle triacylglycerol was 94.0°%. Each value represents means
for two dishes±s.E.M. In a second experiment (3.1 mg
of cellular protein, 0.45,ug of remnant cholesteryl
ester) preincubation with heparin increased the cell
association of cholesteryl ester from 23.4±0.6% to
31.9±0.1% and the net hydrolysis from 13.6±0.5%
to 18.3 ±0.9% (means±s.E.M., three dishes).
Complement- Cholesteryl
Cholesteryl
Heparin inactivated ester degrad- ester uptake
ation (%)
(units/ml) rat serum
(%)
- 25.2+0.8
16.1+0.7
7.73 + 0.76
21.7+1.2
+
1
+
25.7+0.6
13.0+1.1
2.5
+
25.3 + 0.6
14.3 +0.7
5
24.1+0.8
+
13.0+0.6
10
26.2+ 1.2
+
15.3+0.8
the cell association was 1.3±0.0%; with 1 and
2.5 units/ml of heparin present, it was 1.6±0.1%
and 1.6±0.3% respectively (means±s.E.M., for two
dishes in each case).
"0
45
o
4
6
8
lo0
Heparin (units/mil)
Fig. 7. Effects of heparin on cell association (o, *) and
hydrolysis (A, A) of chylomicron-remnant cholesteryl ester
Chylomicron remnants (0.82pg of cholesteryl ester,
3.14,ug of triacylglycerol, 37100d.p.m. of 3H as
cholesteryl ester, 9870d.p.m. as non-esterified cholesterol) were incubated for 4h with various concentrations of heparin. During preparation of the added
chylomicron remnants, hydrolysis of original chyle
triacylglycerol was 92.8%. Each dish contained
3.1 mg of cellular protein; in some dishes (-, A) 4%o
rat serum (3 mg protein/ml) was present. Each point is
the mean±s.E.M. for two or three dishes.
2
Effects on cholesterol synthesis
In experiments where the effect of chylomicron
remnants on the cholesterol biosynthesis from 3H20
was examined, no inhibition was observed in 4-12h
incubations with up to 12,ug of chylomicron-remnant
cholesteryl ester. When chylomicron remnants (15.9
,g of cholesteryl ester) were incubated with hepatocyte monolayers (2.5mg of cellular protein) for 28h,
the last 4h with 1 .5mCi of 3H20, a small decrease in
the incorporation of 3H into non-saponified lipids
occurred (73.9+1.5 compared with 83.3±1.lngatoms in controls; means+s.E.M., two dishes, 0.05 >P
>0.025, Student's unpaired t test). Chylomicron
remnants were in these cases prepared from chyle
incubated with post-heparin plasma.
Discussion
The properties of the uptake of chyle cholesteryl
ester by hepatocyte monolayers are similar to those
in vivo (Redgrave, 1970; Bergman et al., 1971;
1977
METABOLISM OF CHYLOMICRON REMNANTS IN HEPATOCYTE MONOLAYERS
Andersen et al., 1977) and in the perfused liver
(Noel et al., 1975; Felts et al., 1975; Gardner &
Mayes, 1976) in the sense that remnant particles
formed during the action of lipoprotein lipase are
taken up more efficiently than the native chyle
lipoproteins. This indicates that an increase in the
affinity of the lipoprotein particles for the hepatocyte
surface occurs during the conversion of the chylomicrons into remnant particles, and not only a
decrease in size that could increase the rate of
uptake in the intact liver by facilitating the diffusion
into the space of Disse. The size might, however,
influence the uptake by the isolated cells to some
extent, since there was a difference between the
degradation of native large (chylomicrons, Sf >400)
and small (VLD lipoproteins, Sf <400) chyle lipoproteins (Table 1). The rate of degradation of VLDlipoprotein cholesteryl ester exceeded that of chylomicron cholesteryl ester, but still it was only about
one-tenth of that observed with remnant particles
(Sf > 200). It is noteworthy that also in vivo Andersen
et al. (1977) found a greater initial uptake by the liver
(calculated as increase in hepatic cholesteryl ester
after injection of large doses of lipoproteins) of
intestinal VLD lipoproteins (Sf 30-400) and smaller
chylomicrons (Sf 400-8000) than of larger chylomicrons (Sf > 8000).
With increasing concentrations of added chylomicron remnants the rate of uptake approached
saturation kinetics (Fig. 2). The uptake was thus
dependent on saturable binding to the cells rather
than on a random endocytosis of the liquid medium.
The approximate Vmax. for the total uptake (cell
association+net hydrolysis) was 80ng of cholesteryl
ester/h per mg of cell protein (Fig. 2b). This would
correspond to about 0.16mg of cholesteryl ester/h
per 10-12g of liver, which would allow the flow of
16-32mg of fat/h to the blood as intestinal lipoprotein containing 0.5-1 % cholesteryl ester. After
injection of milligram doses of chylomicronremnant cholesteryl ester Andersen et al. (1977)
observed an initial increase in hepatic cholesteryl
ester content ofabout 22 ng/h per mg of liver per mg of
cholesterol injected per 100g rat body wt. This
corresponds to an increase of 2204ug of cholesteryl
ester/liver after injection of 2mg of cholesteryl ester
in 200g animals with a lOg liver. The increase in
hepatic cholesteryl ester in vivo was, however, linear
with the injected amount up to the highest dose
tested, i.e. 10mg of cholesterol/100g body wt. The
maximal increment in cholesteryl ester was thus
8-10-fold higher than the Vmax. for the uptake in the
present study, but Andersen et al. (1977) used remnants from chylomicrons of rats given large doses of
egg cholesterol, which may contain more cholesteryl
ester per particle. Direct comparisons are therefore
difficult to make. The interpretation of kinetic
data obtained in hepatocyte monolayers should,
Vol. 168
491
however, consider that the structure is different from
that of the intact tissue and that the proportion of the
cell surface that exhibits the specialized functions of
the sinusoidal hepatocyte surface is not necessarily
the same as in vivo.
The presence of chylomicrons, intestinal VLD
lipoproteins and serum HD lipoproteins influenced
the uptake of remnant cholesteryl ester in different
ways. VLD lipoprotein in concentrations of 1-1.5pg
ofcholesteryl ester added per dish caused a significant
inhibition of both uptake and net hydrolysis (Fig. 4).
Chylomicrons also influenced both parameters, but
the effect was less than that of VLD lipoproteins at
comparable cholesteryl ester concentrations (Fig. 3).
The effects were thus related to the rate of uptake
of the two types of particles and may be due to
decreased binding of remnants to the cells. This is
compatible with a competition for the same receptors
between remnant particles and native chyle lipo-
proteins.
HD lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to about 50 %, but
influenced the amount of labelled chylomicron
remnant cholesteryl ester that was associated with
the cells to a much lesser extent (Fig. 5). Only 1-2 %
of the added HD lipoprotein was cell-associated after
incubation. Yet this means that about 0.5,ug of
cholesteryl ester was cell-associated at the highest
HD-lipoprotein concentrations, an amount that
would be expected to cause significant inhibition of
remnant binding if it was taken up as VLD lipoprotein
(Fig. 4). The affinity of hepatocytes in monolayers for
chylomicron remnants was thus manyfold higher than
that for HD lipoproteins. The data do not provide
evidence that the same receptors are involved in the
uptake of the two lipoprotein classes, but rather that
they might share the same pathway for cholesteryl
ester hydrolysis after the uptake. Competition
between low-density lipoproteins and HD lipoproteins has been shown with human endothelial
cells (Stein & Stein, 1976) and with pig aortic smoothmuscle cells in culture (Carew et al., 1976). The
rather low rate of uptake of HD lipoproteins is in
agreement with studies on suspended hepatocytes by
Nakai et al. (1976), -whereas Drevon et al. (1977)
reported somewhat higher rates of uptake in
hepatocyte suspensions of rat serum lipoproteins
that had been subjected to the action ofphosphatidylcholine-cholesterol acyltransferase (EC 2.3.1.43)
in vitro.
Particularly at early time intervals a significant
portion of the cholesteryl ester that was associated
with the cells could be released by treatment with
trypsin (Fig. 1). Binding to the outer cell surface that
depends on trypsin-digestible material (Stein &
Stein, 1975) thus precedes the interiorization and
degradation of the cholesteryl ester. Electronmicroscopic radioautographic findings suggest that
AG2
also in vivo there is an initial phase (Stein et al., 1969),
during which labelled cholesteryl ester is enriched
at the sinusoidal surface of hepatocytes. This
might give an opportunity for surface-bound enzymes
to act on the remnant particles. The ['4C]triacylglycerol/[3H]cholesteryl ester ratio and the proportion of [14C]cholesterol as ester in the material
that was released from the cells by the trypsin
digestion were, however, the same as in the particles
remaining in the medium. The data do thus not provide evidence that, in this system, remnant triacylglycerol or cholesteryl ester is digested by plasmamembrane-bound lipase (Assman et al., 1972) or
cholesteryl esterase during the binding of the particle
to the cell surface.
As in earlier experiments (Flor6n & Nilsson, 1977)
colchicine and chloroquine inhibited the hydrolysis
of cholesteryl ester after the uptake by the cells.
The inhibitors mainly increased the amount of
cellular cholesteryl ester that could not be released by
trypsin (Fig. 7). This indicates that the drugs did not
interfere with the interiorization of the particles after
the binding to the cell surface, but rather at a later
stage. If colchicine, which inhibits many processes
that involve membrane fusion (for references see
Wilson et al., 1974), had prevented a fusion between
the surface coat of the remnant particle and the
plasma membrane (Felts et al., 1975) the particles
would be expected to remain at the outer cell surface
and to come off during trypsin treatment, which was
thus not the case. Instead, the finding that chloroquine, which accumulates in lysosomes (Allison &
Young, 1964; de Duve et al., 1974) and prevents the
lysosomal degradation of protein (Wibo & Poole,
1974) and of low-density lipoproteins in fibroblasts
(Goldstein et al., 1975), also inhibits remnant cholesteryl ester degradation suggests that the degradation may occur by endocytosis and lysosomal
hydrolysis. A role of the acid lysosomal cholesterol
esterase, which is present in hepatocytes (Nilsson
et al., 1973b; Drevon et al., 1977), could then be
postulated. Theearlier observation (Flor6n &Nilsson,
1977) that chloroquine increased the cellular triacylglycerol/phospholipid radioactivity ratio during
incubations with double-labelled remnants suggests
a role of acid lipase (Teng & Kaplan, 1974) also in
the breakdown of chylomicron-remnant triacylglycerol. The inhibitory action of colchicine is also
compatible with the idea of endocytosis, since the drug
might interfere with intracellular processes such as the
fusion between endosomes and primary lysosomes
(Malawista & Bodel, 1967). The type of drug effects
seen in the monolayer studies are in good agreement
with those seen in vivo. Both anti-microtubular agents
such as colchicine and vinblastine (Nilsson, 1975),
and chloroquine (C.-H. Flor6n & A. Nilsson, unpublished work), inhibit degradation of chylomicron
cholesteryl ester after the hepatic uptake also in the
C.-H.
FLORItN
AND A. NILSSON
intact rat. Electron-microscopic radioautographic
evidence (Stein et al., 1974) and the observation that
chloroquine strongly inhibits hepatic degradation of
the VLD-lipoprotein peptide (Stein et al., 1977)
provide further evidence that B-peptide and cholesteryl ester of remnants from triacylglycerol-rich
lipoproteins may be metabolized as a unit by endocytosis. For the cholesteryl ester portion the evidence
is, however, so far indirect and based only on the
drug-inhibition studies.
Further studies of the remnant uptake in hepatocyte monolayers should include studies of the
metabolic consequences of chylomicron-remnant
degradation. Does the uptake lead to inhibition of
cholesterol synthesis and to increased cholesterol
esterification like the receptor-mediated catabolism
of low-density lipoproteins in other cell systems
(Brown et al., 1973, 1975; Kayden et al., 1976)?
In the present studies remnants did not inhibit the
incorporation of 3H20 into cholesterol in 4h incubations, i.e. the time used in most of the uptake
experiments. After a 24h preincubation with
remnants obtained from cholesterol-fed animals
there was 10-20 % decrease inthe 3H20-incorporation
over 4h. In 48h incubations with a cholesterol-rich
plasma-lipoprotein fraction from hypothyroid rats,
which may consist of remnants, Breslow et al. (1975)
noted a more significant decrease in 3-hydroxy-3methylglutaryl-coenzyme A reductase (EC 3.1.2.5).
The inhibitory effect on cholesterol biosynthesis
may thus vary significantly with the amount of remnant cholesterol added and with the incubation time.
Further studies will be necessary to establish the
conditions under which remnants inhibit cholesterol
synthesis in the hepatocyte monolayers. The infusion
of cholesterol-rich chyle lipoproteins in vivo causes
efficient inhibition of hepatic cholesterol synthesis
(for references see Nervi et al., 1975). The increased
hepatic cholesterol biosynthesis after thoracic-duct
drainage (Weis & Dietschy, 1969) suggests that the
lymph lipoproteins may be important also for the
regulation of cholesterol biosynthesis in rats that
have not received cholesterol.
In an earlier study from this laboratory (Nilsson,
1977) heparin simulated uptake or binding of chylomicron remnants by suspended hepatocytes, particularly in the presence of serum. Since negatively
charged glycosaminoglycans can bind to hepatocytes
in monolayers (Kjellen et al., 1977), the question was
raised whether heparin or heparan sulphate may
participate in the physiological degradation of chylomicron remnants. The finding that heparin did indeed
stimul ate remnant uptake in the presence of serum also
when it was present only during a preincubation period
(Table 2) indicates that it might act by binding to the
cell surface and thereby increase the affinity of the surface for remnants. Furtherstudies, including measurement of hepatic lipase, are therefore required to
1977
METABOLISM OF CHYLOMICRON REMNANTS IN HEPATOCYTE MONOLAYERS
evaluate the role of cell-bound glycosaminoglycans
in hepatic lipoprotein degradation. Two points
should, however, be stressed at this stage. First,
the positive effect ofheparin contrasts with the finding
that it inhibits the receptor-mediated binding of
low-density lipoproteins in fibroblasts (Goldstein
et al., 1976), indicating that another type of receptor
is involved in remnant degradation. Secondly, the
heparin concentrations used would be expected to
release membrane-bound hepatic lipase from the
cells. The finding that the uptake after heparin incubation increases rather than decreases therefore
speaks against a receptor function of this enzyme in
remnant degradation.
Miss Gertrud Olsson provided skilful technical assistance. The work was supported by grants from: the
Swedish Medical Research Council (grant no. 03X-3969);
the Medical Faculty, University of Lund; Albert Pahissons
Stiftelse; Alfred Osterlunds Stiltelse; and Svenska
Livforsakringsbolags Nmnnd for Medicinsk Forskning.
We thank Dr. Anders Malmstrom, at this Institute for the
determinations of uronic acid.
References
Abell, L. L., Levy, B. B., Brodie, B. B. & Kendall, F. E.
(1952) J. Biol. Chem. 195, 357-366
Akesson, B., Elvoson, J. & Arvidson, G. (1970) Biochim.
Biophys. Acta 210, 15-27
Allison, A. C. & Young, M. R. (1964) Life Sci. 3, 14071414
Andersen, J. M., Nervi, F. 0. & Dietschy, J. M. (1977)
Biochim. Biophys. Acta 486, 298-307
Assman, G., Krauss, R. M., Fredrickson, D. S. & Levy,
R. I. (1972) J. Biol. Chem. 248, 1992-1999
Bergman, E. N., Havel, R. J., Wolfe, B. M. & B0hmer, T.
(1971) J. Clin. Invest. 50, 1831-1839
Berry, M. N. & Friend, D. S. (1969) 1. Cell Biol. 43,
506-520
Bissell, D. M., Hammaker, L. E. & Meyer, U. A. (1973)
J. Cell Biol. 59, 722-734
Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol.
37, 911-917
Bollman, J. L., Cain, J. C. & Grindley, J. H. (1948) J. Lab.
Clin. Med. 33, 1349-1354
Bonney, R. J., Becker, J. E., Walker, P. R. & Potter, V. R.
(1974) In Vitro 9, 399413
Breslow, J. L., Spaulding, D. R., Lothrop, D. A. & Clowes,
A. W. (1975) Biochem. Biophys. Res. Comnmun. 67,
119-125
Brown, M. S., Dana, S. E. & Goldstein, M. S. (1973)
Proc. Natl. Acad. Sci. U.S.A. 70, 2162-2166
Brown, M. S., Dana, S. E. & Goldstein, J. L. (1975)
J. Biol. Chem. 250, 40254027
Carew, T. E., Koschinsky, T. & Steinberg, D. (1976)
Clin. Res. 24, 154A
de Duve, C., de Barsay, T., Poole, B., Trouet, A.,
Tulkens, P. & von Hoof, F. (1974) Biochem. Pharmacol.
23, 2495-2531
Vol. -168
493
Drevon, C. A., Berg, T. & Norum, K. R. (1977)
Biochim. Biophys. Acta 487, 122-136
Felts, J. M., Itakura, H. & Crane, R. T. (1975) Biochem.
Biophys. Res. Commun. 66, 1467-1475
Flor6n, C.-H. & Nilsson, A. (1977) Biochem. Biophys. Res.
Con*mun. 74, 520-528
Fransson, L.-A., Rhod6n, L. & Spoch, M. L. (1968)
Anal. Biochent. 21, 317-330
Gardner, R. S. & Mayes, P. A. (1976) Biochem. Soc.
Trans. 4, 715-716
Goldstein, J. L., Brunschede, G. Y. & Brown, M. S. (1975)
.T. Biol. Chem. 250, 7854-7862
Goldstein, J. L., Basu, S. K., Brunschede, G. Y. & Brown,
M. S. (1976) Cell 7, 85-95
Johansson, B. G. (1972) Scand. J. Clin. Lab. Invest.
Suppl. 124, 7-19
Kayden, H. J., Hatam, L. & Beratis, N. G. (1976) Biochemistry 15, 521-528
Kjell6n, L., Oldberg, A., Rubin, K. & Hook, M. (1977)
Biochem. Biophys. Res. Commun. 74, 126-133
Lin, R. C. & Snodgrass, P. J. (1975) Biochern. Biophys.
Res. Commun. 64,725-734
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,
R. J. (1951) J. Biol. Chem. 193, 265-275
Malawista, S. E. & Bodel, P. T. (1967) J. Clin. Invest. 46,
786-796
Minari, 0. & Zilversmit, D. B. (1963) J. Lipid Res. 4,
424-436
Mj0s, 0. D., Faergeman, O., Hamilton, R. L. & Havel,
R. J. (1975) J. Clin. Invest. 56, 603-615
Nakai, T., Otto, P. S., Kennedy, D. L. & Whayne, T. F.,
Jr. (1976) J. Biol. Chern. 251, 4914-4921
Nervi, F. O., Weis, H. J. & Dietschy, J. M. (1975) J. Biol.
Chem. 250, 4145-4151
Nilsson, A. (1975) Biochemn. Biophys. Res. Conmrun. 66,
60-66
Nilsson, A. (1977) Biochernt. J. 162, 367-377
Nilsson, A. & Akesson, B. (1975) FEBSLett. 51, 219-224
Nilsson, A. & Zilversmit, D. B. (1971) Biochim. Biophys.
Acta 248, 137-142
Nilsson, A. & Zilversmit, D. B. (1972) J. Lipid Res. 13,
32-38
Nilsson, A., Sundler, R. & Akesson, B. (1973a) Eur. J.
Biochem. 39, 613-620
Nilsson, A., Nord6n, H. & Wilhelmsson, L. (1973b)
Biochim. Biophys. Acta 296, 593-603
Noble, R. P. (1968) J. Lipid Res. 9, 693-700
Noel, S.-P., Dolphin, P. J. & Rubenstein, D. (1975)
Biochem. Biophys. Res. Comman. 63, 764-772
Ontko, J. A. (1970) J. Lipid Res. 11, 367-375
Redgrave, T. G. (1970) J. Clin. Invest. 49, 465-471
Seglen, P. 0. (1973) Exp. Cell Res. 82, 391-398
Stein, 0. & Stein, Y. (1975) Biochim. Biophys. Acta 398,
377-384
Stein, 0. & Stein, Y. (1976) Biochim. Biophys. Acta 431,
363-368
Stein, O., Stein, Y., Goodman, D. S. & Fidge, N. H.
(1969) J. Cell Biol. 43, 410-431
Stein, O., Rachmailewitz, D., Sanger, L., Eisenberg, S. &
Stein, Y. (1974) Biochim. Biophys. Acta 360, 205-216
Stein, Y., Ebin, V., Bar-On, H. & Stein, 0. (1977)
Biochim. Biophys. Acta 486, 286-297
494
Teng, M.-H. & Kaplan, A. (1974) J. Biol. Chem. 249,
1065-1070
Weis, H. J. & Dietschy, J. M. (1969) J. Clin. Invest. 48,
2398-2408
Wibo, M. & Poole, B. (1974) J. Cell Biol. 63,430-440
C.-H. FLORtN AND A. NILSSON
Wilson, L., Bamburg, J. R., Mizel, S. B., Grisham, L. M.
& Creswell, K. M. (1974) Fed. Proc. Fed. Am. Soc.
Exp. Biol. 33,158-166
Zak, B., Moss, N., Boyle, A. J. & Zlatkis, A. (1954)
Anal. Chem. 26, 776-777
1977