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 (1.063 400) 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,
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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
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 (1.063 400) 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
