Bioch#nica er Biophysiea Aeta. 1083(1991) 173-178 ~ 1991 ElsevierSciencePublishersB.V. 0005-2760/91/$03.50 A DONIS 000527609~00155
173
Regulation of chylomicron remnant uptake in the human hepatoma cell-line Hep G2. Role of the low-density
lipoprotein receptor Q i C h e n I, C l a e s - H e n r i k F l o r 6 n 2, ,~ke N i l s s o n i a n d R e c a r e d o I n f a n t e 3 t Research Department 2 and Department of Internal Medicine. Uni~,ersi~v Hospital Lund fSwedenL 2 Department of Internal Medicine. Maim6 General Hospital. Maim6 (Sweden) and "¢Unit~ 55. INSER.~I, Hopital Saint-Antoine. Paris (France)
(Received 19 April 1990) (Revisedmanuscriptreceived 11 December1990) Key words: Hep G2; Chylomicronremnant; LDL; LDL receptor; Compactin;Mevalonolactone Uptake and degradation of chylomicron remnants by the human hepatoma cell line Hep G2 was studied. Mesenterie lymph was collected from rats and injected into hepatectomized rats to obtain chylomicron remnants. This remnant preparation was taken up and catabolized by Hep G2 cells. The uptake process was dependent on cell growth and was regulated by compaetin (a IEMG-CoA reductase inhibitor) which suppresses cholesterol synthesis and by mevalonolactone, which enhances cholesterol synthesis. A monoclonal anti LDL receptor antibody blocked binding of chylomieron remnants to Hep G2 cells to a degree, which was comparable to but generally lower than the suppression of low-density lipowotein binding. The results thus indicate that in Hep G2 cells, ehylomicron remnant uptake is regulated, similarly to low-density lipoprotein uptake and that a significant part of the remnant uptake is mediated through the LDL receptor.
Introduction Chylomicron remnants, which are potentially atherogenie lipoproteins Ill, are produced from chylomicrons in plasma, mainly by the activity of the enzyme lipoprotein lipas¢ [2]. Chylomicron remnants carry cholesterol, fat soluble vitamins and triacylglycerol to the liver [2], where they are largely taken up by hepatocytes [3-5]. The initial binding of chylomicron remnants to the cells is saturable and have characteristics compatible with a receptor-mediated endocytosis [3,4], Earlier studies postulated the presence of a specific apolipoprotein E or chylornicron remnant receptor in liver cells [6]. This receptor seems not to be regulated by cellular content of cholesterol, in contrast with the low-density lipoprotein (LDL) receptor [7]. However, chylomicron remnants are taken up by the LDL receptor in extrahepatic cells such as fibroblasts [8]. In primary culture of rat hepatocyte monolayers, chylomicron remnant uptake can also be modulated by the amount of cell cholesterol [9] and by insulin [10], in a way resembling the regulation of the LDL receptor [11,12]. Finally, Nagatu et at. [13] pro-
vided evidence, using an anti LDL receptor antibody, that uptake of chylomicron remnants to rat hepatocyte monolayers was dependent on the LDL receptor. In the present work we study the regulation of cbylomicron remnant uptake in a human hepatoma cell line (Hep G2) and compare it to that of the activity of the LDL receptor. Earlier studies have shown that Hep G2 cells express LDL receptors [14]. The cells also metabolize rat chylomicron remnants via specific binding sites and the char?.c!e.,Ss~ics of the uptake process closely resemble that of rat chylomicron remnant uptake in primary cultures of rat hepatocyte monolayers [3A,151. In the present study we ask the question wether the uptake of chylomicron remnants is regulated by growth and by cellular contents of cholesterol; features which are characteristic of the LDL receptor. We also use an anti LDL receptor antibody to try to block chylomicron remnant uptake in Hep G2 cells. Materials and Methods Cell culture
Correspondence: C.-H. Flor6n, Department of Internal Medicine, Malm~General Hospital, S-21401 MaimS,Sweden.
The established hepatoblastoma cell-line Hep G2 was obtained from American type culture collection (Rockville, Maryland, U.S.A.). The cells were subcul-
174 tured at 37 ° C in flasks with a bottom area of 75 cm2, in RPMI 1640 (Gibco Laboratories, Santa Clara, CA, U.S.A.) containing 10% fetal calf serum (heat inactivated at 56°C for 30 min, from Flow laboratories (lrvine, U.K.), in the presence of benzyl penicillin (100 U/ml) and streptomycin (100 /xg/ml). The cells were grown in humidified air with 5% CO2 and were subcultured approx, once every week with a split ratio of 1 : 6. The cells were routinely screened for and shown to be free from mycoplasma using a kit with a specific DNAprobe from Gen-Probe (San Diego, CA, U.S.A.). To initiate experiments, cells were trypsinized and then seeded into 35 or 60 mm Petri plastic dishes at varying densities depending on the experiment. The medium (RPMI 1640 with 10% fetal calf serum without antibiotics) was r~utinely changed after 2 days and, if applicable, preincubations started. During the whole experimental period no antibiotics were added.
lytic activity. Low-density lipoprotein (LDL) was isolated from plasma by sequential preparative ultracentrifugation [19] using a Beckman L 5-65 ultracentrifuge with the 60 Ti rotor (Beckman instruments. Fullerton, CA, U.S.A.). The density range 1.019 < density < 1.063 k g . l -I was usually used but in some control experiments LDL with a narrower density range (1.039 < density < 1.054 k g . I -~) was used to minimize the contamination of LDL with apolipoprotein E. The results obtained, irrespective of what density range used, were the same. LDL was iodinated with 1251 by the iodine monochloride method [20] as modified tor lipoproteins [211. Unbound 1251 was removed by chromatography on a Sephadex G-25 column (Pharmacia, Uppsala, Sweden) followed by extensive dialysis against 0.15 M NaCI with 1 mM EDTA (pH 7.4). The specific activity of LDL was between 200 and 400 cpm per ng protein.
Lipoprotein isolation and labelling Mesenteric duct cannulations were performed according to Warshaw [16], and the rats were treated postoperatively, as described earlier [3]. The rats were fed 1.0 ml corn oil containing 20 mg unlabelled cholesterol plus 500/~Ci (1.85 MBq) [1,2-3H]cholesterol (Amersham ! ~ternational, Amersham, U.K.). The fat was given div.ded in three doses over 2h. Lymph was collected, added with 2 mM Na2-EDTA and stored at 4 ° C [17]. Chylomicron remnants were prepared by injecting chyle into eviscerated rats [18]. After 30 min, blood was drawn from the abdominal aorta using Na 2EDTA as anticoagulant. The density of plasma was adjusted to 1.063 kg.l -t by adding a stock solution (d = 1.35 kg. 1- i ) of KBr and NaC1. It was then layered under saline ( d = 1.006 kg. 1- I ) and centrifuged in a Beckman LS-65 ultracentrifuge with a SW Ti 40 rotor at 32000 rpm (120000 × g) for 12 h. The top layer containing chylomicron remnants was collected under sterile conditions and was used within 24 h. The particles prepared by injecting chyle into eviscerated rats had lost 98.3% of their original triacylglycerol content, as estimated from the change in the triacylglycerol/[3H]cholesteryl ester ratio in chylomicron remnants in comparison to that of the injected chylomicrons. The proportion of [3H]cholesterol in cholesteryl ester was 75.5%. The specific activity of the radioactively labelled cholesterol ranged between 5.3-6.4 dpm 3 H / n g cholesterol. The apolipoprotein and lipid composition of remnant particles prepared under identical conditions have been given earlier [3,4]. To prepare low-density lipoproteins, blood was drawn from a normo-chc,lesterolemic subject after an overnight fast into EDTA containing vacutainer tubes. Immediately, Thimerosal (sodium ethylmercuri thiosalicylate, from Sigma Chemical Company) was added to a final concentration of 25/zM in order to inhibit proteo-
LDL receptor assay LDL receptor activity was assayed under two different conditions. First, a 4-h uptake and degradation assay was carried out at 37 o C. After the preincubation period, medium was changed to RPMI plus 0.5% human serum albumin, lodinated LDL was then added either with or without a more than 20-fold excess of unlabelled LDL. After incubation for 4 h at 37°C, medium was removed and frozen at - 1 8 ° C . The cells were washed three times with 2 ml PBS containing 0.1% bovine serum albumin and then twice with 2 ml PBS. The cells were deep-frozen until assayed. To estimate specific uptake of LDL, uptake of 125I LDL (cpm associated with cells) in the absence and presence of a 20-fold excess of unlabeiled LDL was calculated. Degradation was measured as the trichloroacetic acid-soluble non-iodine ~251 radioactivity in the aledium [22]. Specific degradation was degradation in the absence minus degradation in the presence of a 20-fold excess of unlabelled LDL. LDL receptor activity was also assayed by binding of LDL to Hep G2 cells in the ,:.old [23]. After the preincubation period, the dishes were washed with 2 mi PBS containing 0.2% bovine serum albumin twice at room temperature. Ice cold RPMI with 0.5% human serum albumin containing 10 mM Hepes (pH 7.4) was then added, and the dishes were placed in the cold room for 20 min. Lipoproteins were added and left with the cells at 4 ° C for 2 h. The dishes were washed twice with 2 ml ice-cold PBS containing 0.2% bovine serum z,lbumin and then twice with 2 ml PBS without albumin. The cells were thereafter scraped with a rubber pohceman in 0.1 M NaOH. Specific binding of LDL was defined as binding in the absence of an excess of unlabelled LDL minus the binding in the presence of unlabelled LDL. Experiments using the monoclonal anti-LDL recep-
175 for antibody were also carried out under the same experimental conditions.
Incubation with chylomicron remnants The incubations of Hep G2 cells with chylomicron remnants lasted for 4 h at 3 7 ° C or for 2 h at 4 ° C . At 3 7 ° C both the uptake and the net degradation of chylomicron remnant cholesteryl ester by the cells were measured. At 4 ° C only chylomicron remnant binding to cells was measured. After the incubation period the cells were washed using the same procedure as in incubations with L D L (see above). Subsequently, the cells were scraped off the dishes with a rubber policeman during repeated additions (2 × 1 ml) of m e t h a n o l / w a t e r ( 2 : 1 , v / v ) . Lipids were then extracted from cells and media with c h l o r o f o r m / m e t h a n o l ( 1 : 2 , v / v ) [24]. Lipids were separated by thin-layer chromatography on silica gel G plates which were developed in light petrol e u m / d i e t h y l e t h e r / a c e t i c acid ( 8 0 : 2 0 : 1 , v / v ) . Spots were identified by staining with iodine vapour, and scraped into vials. 1 ml of m e t h a n o l / w a t e r (1 : 1, v / v ) and 9 ml I n s t a g e l / t o l u e n e (1 : 1, v / v ) were added and radioactivity was determined in a Packard T C 460 C D Liquid Scintillation System using the computerized external standard for quenching correction. Net hydrolysis of chylomicron remnant cholesteryl ester was calculated as described earlier [31. Thus, the percentage net hydrolysis of radioactive cholesteryl ester was calculated from the decrease in radioactivity (dpm) of cholesteryl ester, and the increase in that of free cholesterol in both cells and m e d i u m as follows: h 1 " 100 % cholesterol as ester after incubation × 100 % net ydro ysls = - % cholesterol as ester before incubation-The percentage net hydrolysis was then multiplied with the d p m cholesteryl ester added to the dish to
obtain dpllt cbolesteryl ester hydrolysed. There was no measurable net hydrolysis of lipoprotein cholesteryl ester in the cell-free controls. Cell association ( = uptake) of chylomicron remnant cholesterol is the amount of unesterified labelled cholesterol plus labelled cholesteryl ester found in the washed cells.
LDL receptor antibody The monoclonal anti-low-density lipoprotein receptor antibody (clone C7) was obtained from Amersham International (Buckinghamshire, U.K.). This antibody blocks LDL-receptor mediated uptake of LDL in cultured h u m a n fibroblasts as described by Beisiegel et al, I251. Chemical determinations Triaeylglycerol and cholesterol were determined by the commercial enzymatic kits from Boehringer Mennhelm (Mannheim, F.R.G.). The protein contents of the cells and of LDL were determined by the Lowry method [26] using humap serum albumin as a standard. Results
Growth regulated uptake of chylomicron remnants in Hep G2 cells Chylomicron remnants were added to Hep G2 cells seeded in dishes at varying cell densities. A rapid uptake of chylomicron remnants with a subsequent degradation of remnant cholesteryl ester occurred (unpublished data) in agreement with earlier data by Lenich and Ross [15]. Several experiments, where chylomicron remnant uptake and LDL receptor activity was followed during growth of the cells, were done. In one experiment (Fig. IA) the cells reached confluency 48 h after the start of ca m
1.6] A 1.2
0
0 0
2~
t~8
72
24
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INCUBATION TIME (hra)
72 ~
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INCUBATION TIME (hrs) Fig. l, Effects of cells density on the degradation and uptake of chylomicron remnant.,,and LDL. (A) Cell density curve. (lll A comparison between specific LDL degradation and chylomieron remnant cholesteryl ester degradation. ~H-labelled chylomicron remnants (containing 3.9 /~g of cholesterol ester) and 12~l-labelledLDL Icontaining 4/tg of protein) were added to two series of dishes separately. Incubations were interrupted after various time intervals a:~indicated. The medium was removed and cells ".',erev,ashed as described in Materials and Methods. Values are means of two dishes.
176 z z .-q
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Fig. 2. Effects of compactin and mevalonolactone on chytomicron remnant degradation and uptake. Cells were preincubated with either compactin 20 #M (final conc.} or 10 mM mevalonolactone (final cone.) for 48 h. Incubations were then started by changing to medium containing O.5% human serum albumin ann ehyioulicron remnants (containing 2.4 /zg cholesteryl ester). Incubations were performed at 37°C for 4 h and dishes were treated as described in Materials and Methods.
the experiment. In this experiment there was a parallellism between L D L receptor activity as assessed by a 4 h LDL degradation and chylomicrom remnant degradation (Fig. 1B). This experiment was repeated in a situation, where the cells reached confluency after 24 h and where a decline of cell number per dish subsequently occurred. Also in this situation we observed a parallellism betweeen L D L receptor activity as assessed by a 4 h LDL degradation and chylomicron remnant eholesteryl ester degradation (unpublished data). These data indicate that chylomicron remnant uptake in Hep G2 cells is influenced by cell growth and is regulated like the L D L receptor activity.
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LDL DEGRADATION ng protetn/mg cell protein
......
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LDL UPTAKE ng proteinlmg cell protein
Fig. 3. Correlations between specific LDL degradation and chylomicron remnant cholesteryl ester degradation (A) and specific LDL uptake and chylomicron remnant uptake (B). Data from an experiment similar to those described in Fig. 2.
(P < 0.0005, r = 0.97) with chylomicron remnant cholesteryl ester degradation. Specific 4 h uptake of LDL also correlated significantly (P < 0.0025, r = 0.93)
with chylomicron remnant uptake. In a second series of experiments the corresponding r values were 0.90 and 0.94. These experiments show that chylomieron r e m n a n t uptake is regulated by changes in the cellular cholesterol content and that chylomicron remnant uptake correlates significantly with the L D L receptor activity.
Blocking of L D L receptors with a specific antibody A monoclonal anti L D L receptor antibody was used in a series of experiments to elucidate if this receptor
plays a role in the binding of chyiomicron remnants by Hep G2 cells. The antibody was from the C7 clone and
has been shown to block the LDL receptor mediated u p t a k e of L D L in cultured h u m a n fibroblasts [25]. In Hep G2 cells the a n t i b o d y blocks L D L binding to Hep G2 cells by a b o u t 50% (Fig. 4). Similarly, this a n t i b o d y decreases the b i n d i n g of chylomicron remnants to Hep
Regulation of chylomicron remnant uptake by variation in celhdar cholesterol content
O
In this set of experiments the cholesterol content was modified by adding either compactin [27] which blocks de novo cholesterol synthesis or mevalonolactone which increases cholesterol synthesis [28]. C o m p a c t i n increased LDL uptake approx. 3-times and mevalonolactone decreased the L D L uptake by half (unpublished data). Mevalonolactone also decreased chylomicron remnant uptake by 64% and cholesteryl ester degradation in cells by 51% (Fig. 2). Compactin upregulated chylomicron remnant uptake and cholesteryl ester degradation by 150% and 157%, respectively (Fig. 2). ~By regulating the LDL receptor activity and concomitantly measuring chylomicron remnant uptake and degradation, correlations between L D L receptor activity and chylomicron remnant uptake could be calculated. As shown in Fig. 3, LDL receptor activity, measured by a 4 h specific degradation assay, correlated significantly
o
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;
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ANTIBODY GONG /Jg/ml Fig, 4. Effectsof anti LDL receptorantibody on LDL binding to Hep G2 cells. Incubations were started by adding different amounts of anti LDL receptor antibody and I ,ag/ml of [levi] LDL to the dishes and the experiment was carried at 4°C for 2 h. After the incubation period cells were treated as described in Materials and Methods. Each point represents the mean of two dishes.
177
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I00"~
173
Regulation of chylomicron remnant uptake in the human hepatoma cell-line Hep G2. Role of the low-density
lipoprotein receptor Q i C h e n I, C l a e s - H e n r i k F l o r 6 n 2, ,~ke N i l s s o n i a n d R e c a r e d o I n f a n t e 3 t Research Department 2 and Department of Internal Medicine. Uni~,ersi~v Hospital Lund fSwedenL 2 Department of Internal Medicine. Maim6 General Hospital. Maim6 (Sweden) and "¢Unit~ 55. INSER.~I, Hopital Saint-Antoine. Paris (France)
(Received 19 April 1990) (Revisedmanuscriptreceived 11 December1990) Key words: Hep G2; Chylomicronremnant; LDL; LDL receptor; Compactin;Mevalonolactone Uptake and degradation of chylomicron remnants by the human hepatoma cell line Hep G2 was studied. Mesenterie lymph was collected from rats and injected into hepatectomized rats to obtain chylomicron remnants. This remnant preparation was taken up and catabolized by Hep G2 cells. The uptake process was dependent on cell growth and was regulated by compaetin (a IEMG-CoA reductase inhibitor) which suppresses cholesterol synthesis and by mevalonolactone, which enhances cholesterol synthesis. A monoclonal anti LDL receptor antibody blocked binding of chylomieron remnants to Hep G2 cells to a degree, which was comparable to but generally lower than the suppression of low-density lipowotein binding. The results thus indicate that in Hep G2 cells, ehylomicron remnant uptake is regulated, similarly to low-density lipoprotein uptake and that a significant part of the remnant uptake is mediated through the LDL receptor.
Introduction Chylomicron remnants, which are potentially atherogenie lipoproteins Ill, are produced from chylomicrons in plasma, mainly by the activity of the enzyme lipoprotein lipas¢ [2]. Chylomicron remnants carry cholesterol, fat soluble vitamins and triacylglycerol to the liver [2], where they are largely taken up by hepatocytes [3-5]. The initial binding of chylomicron remnants to the cells is saturable and have characteristics compatible with a receptor-mediated endocytosis [3,4], Earlier studies postulated the presence of a specific apolipoprotein E or chylornicron remnant receptor in liver cells [6]. This receptor seems not to be regulated by cellular content of cholesterol, in contrast with the low-density lipoprotein (LDL) receptor [7]. However, chylomicron remnants are taken up by the LDL receptor in extrahepatic cells such as fibroblasts [8]. In primary culture of rat hepatocyte monolayers, chylomicron remnant uptake can also be modulated by the amount of cell cholesterol [9] and by insulin [10], in a way resembling the regulation of the LDL receptor [11,12]. Finally, Nagatu et at. [13] pro-
vided evidence, using an anti LDL receptor antibody, that uptake of chylomicron remnants to rat hepatocyte monolayers was dependent on the LDL receptor. In the present work we study the regulation of cbylomicron remnant uptake in a human hepatoma cell line (Hep G2) and compare it to that of the activity of the LDL receptor. Earlier studies have shown that Hep G2 cells express LDL receptors [14]. The cells also metabolize rat chylomicron remnants via specific binding sites and the char?.c!e.,Ss~ics of the uptake process closely resemble that of rat chylomicron remnant uptake in primary cultures of rat hepatocyte monolayers [3A,151. In the present study we ask the question wether the uptake of chylomicron remnants is regulated by growth and by cellular contents of cholesterol; features which are characteristic of the LDL receptor. We also use an anti LDL receptor antibody to try to block chylomicron remnant uptake in Hep G2 cells. Materials and Methods Cell culture
Correspondence: C.-H. Flor6n, Department of Internal Medicine, Malm~General Hospital, S-21401 MaimS,Sweden.
The established hepatoblastoma cell-line Hep G2 was obtained from American type culture collection (Rockville, Maryland, U.S.A.). The cells were subcul-
174 tured at 37 ° C in flasks with a bottom area of 75 cm2, in RPMI 1640 (Gibco Laboratories, Santa Clara, CA, U.S.A.) containing 10% fetal calf serum (heat inactivated at 56°C for 30 min, from Flow laboratories (lrvine, U.K.), in the presence of benzyl penicillin (100 U/ml) and streptomycin (100 /xg/ml). The cells were grown in humidified air with 5% CO2 and were subcultured approx, once every week with a split ratio of 1 : 6. The cells were routinely screened for and shown to be free from mycoplasma using a kit with a specific DNAprobe from Gen-Probe (San Diego, CA, U.S.A.). To initiate experiments, cells were trypsinized and then seeded into 35 or 60 mm Petri plastic dishes at varying densities depending on the experiment. The medium (RPMI 1640 with 10% fetal calf serum without antibiotics) was r~utinely changed after 2 days and, if applicable, preincubations started. During the whole experimental period no antibiotics were added.
lytic activity. Low-density lipoprotein (LDL) was isolated from plasma by sequential preparative ultracentrifugation [19] using a Beckman L 5-65 ultracentrifuge with the 60 Ti rotor (Beckman instruments. Fullerton, CA, U.S.A.). The density range 1.019 < density < 1.063 k g . l -I was usually used but in some control experiments LDL with a narrower density range (1.039 < density < 1.054 k g . I -~) was used to minimize the contamination of LDL with apolipoprotein E. The results obtained, irrespective of what density range used, were the same. LDL was iodinated with 1251 by the iodine monochloride method [20] as modified tor lipoproteins [211. Unbound 1251 was removed by chromatography on a Sephadex G-25 column (Pharmacia, Uppsala, Sweden) followed by extensive dialysis against 0.15 M NaCI with 1 mM EDTA (pH 7.4). The specific activity of LDL was between 200 and 400 cpm per ng protein.
Lipoprotein isolation and labelling Mesenteric duct cannulations were performed according to Warshaw [16], and the rats were treated postoperatively, as described earlier [3]. The rats were fed 1.0 ml corn oil containing 20 mg unlabelled cholesterol plus 500/~Ci (1.85 MBq) [1,2-3H]cholesterol (Amersham ! ~ternational, Amersham, U.K.). The fat was given div.ded in three doses over 2h. Lymph was collected, added with 2 mM Na2-EDTA and stored at 4 ° C [17]. Chylomicron remnants were prepared by injecting chyle into eviscerated rats [18]. After 30 min, blood was drawn from the abdominal aorta using Na 2EDTA as anticoagulant. The density of plasma was adjusted to 1.063 kg.l -t by adding a stock solution (d = 1.35 kg. 1- i ) of KBr and NaC1. It was then layered under saline ( d = 1.006 kg. 1- I ) and centrifuged in a Beckman LS-65 ultracentrifuge with a SW Ti 40 rotor at 32000 rpm (120000 × g) for 12 h. The top layer containing chylomicron remnants was collected under sterile conditions and was used within 24 h. The particles prepared by injecting chyle into eviscerated rats had lost 98.3% of their original triacylglycerol content, as estimated from the change in the triacylglycerol/[3H]cholesteryl ester ratio in chylomicron remnants in comparison to that of the injected chylomicrons. The proportion of [3H]cholesterol in cholesteryl ester was 75.5%. The specific activity of the radioactively labelled cholesterol ranged between 5.3-6.4 dpm 3 H / n g cholesterol. The apolipoprotein and lipid composition of remnant particles prepared under identical conditions have been given earlier [3,4]. To prepare low-density lipoproteins, blood was drawn from a normo-chc,lesterolemic subject after an overnight fast into EDTA containing vacutainer tubes. Immediately, Thimerosal (sodium ethylmercuri thiosalicylate, from Sigma Chemical Company) was added to a final concentration of 25/zM in order to inhibit proteo-
LDL receptor assay LDL receptor activity was assayed under two different conditions. First, a 4-h uptake and degradation assay was carried out at 37 o C. After the preincubation period, medium was changed to RPMI plus 0.5% human serum albumin, lodinated LDL was then added either with or without a more than 20-fold excess of unlabelled LDL. After incubation for 4 h at 37°C, medium was removed and frozen at - 1 8 ° C . The cells were washed three times with 2 ml PBS containing 0.1% bovine serum albumin and then twice with 2 ml PBS. The cells were deep-frozen until assayed. To estimate specific uptake of LDL, uptake of 125I LDL (cpm associated with cells) in the absence and presence of a 20-fold excess of unlabeiled LDL was calculated. Degradation was measured as the trichloroacetic acid-soluble non-iodine ~251 radioactivity in the aledium [22]. Specific degradation was degradation in the absence minus degradation in the presence of a 20-fold excess of unlabelled LDL. LDL receptor activity was also assayed by binding of LDL to Hep G2 cells in the ,:.old [23]. After the preincubation period, the dishes were washed with 2 mi PBS containing 0.2% bovine serum albumin twice at room temperature. Ice cold RPMI with 0.5% human serum albumin containing 10 mM Hepes (pH 7.4) was then added, and the dishes were placed in the cold room for 20 min. Lipoproteins were added and left with the cells at 4 ° C for 2 h. The dishes were washed twice with 2 ml ice-cold PBS containing 0.2% bovine serum z,lbumin and then twice with 2 ml PBS without albumin. The cells were thereafter scraped with a rubber pohceman in 0.1 M NaOH. Specific binding of LDL was defined as binding in the absence of an excess of unlabelled LDL minus the binding in the presence of unlabelled LDL. Experiments using the monoclonal anti-LDL recep-
175 for antibody were also carried out under the same experimental conditions.
Incubation with chylomicron remnants The incubations of Hep G2 cells with chylomicron remnants lasted for 4 h at 3 7 ° C or for 2 h at 4 ° C . At 3 7 ° C both the uptake and the net degradation of chylomicron remnant cholesteryl ester by the cells were measured. At 4 ° C only chylomicron remnant binding to cells was measured. After the incubation period the cells were washed using the same procedure as in incubations with L D L (see above). Subsequently, the cells were scraped off the dishes with a rubber policeman during repeated additions (2 × 1 ml) of m e t h a n o l / w a t e r ( 2 : 1 , v / v ) . Lipids were then extracted from cells and media with c h l o r o f o r m / m e t h a n o l ( 1 : 2 , v / v ) [24]. Lipids were separated by thin-layer chromatography on silica gel G plates which were developed in light petrol e u m / d i e t h y l e t h e r / a c e t i c acid ( 8 0 : 2 0 : 1 , v / v ) . Spots were identified by staining with iodine vapour, and scraped into vials. 1 ml of m e t h a n o l / w a t e r (1 : 1, v / v ) and 9 ml I n s t a g e l / t o l u e n e (1 : 1, v / v ) were added and radioactivity was determined in a Packard T C 460 C D Liquid Scintillation System using the computerized external standard for quenching correction. Net hydrolysis of chylomicron remnant cholesteryl ester was calculated as described earlier [31. Thus, the percentage net hydrolysis of radioactive cholesteryl ester was calculated from the decrease in radioactivity (dpm) of cholesteryl ester, and the increase in that of free cholesterol in both cells and m e d i u m as follows: h 1 " 100 % cholesterol as ester after incubation × 100 % net ydro ysls = - % cholesterol as ester before incubation-The percentage net hydrolysis was then multiplied with the d p m cholesteryl ester added to the dish to
obtain dpllt cbolesteryl ester hydrolysed. There was no measurable net hydrolysis of lipoprotein cholesteryl ester in the cell-free controls. Cell association ( = uptake) of chylomicron remnant cholesterol is the amount of unesterified labelled cholesterol plus labelled cholesteryl ester found in the washed cells.
LDL receptor antibody The monoclonal anti-low-density lipoprotein receptor antibody (clone C7) was obtained from Amersham International (Buckinghamshire, U.K.). This antibody blocks LDL-receptor mediated uptake of LDL in cultured h u m a n fibroblasts as described by Beisiegel et al, I251. Chemical determinations Triaeylglycerol and cholesterol were determined by the commercial enzymatic kits from Boehringer Mennhelm (Mannheim, F.R.G.). The protein contents of the cells and of LDL were determined by the Lowry method [26] using humap serum albumin as a standard. Results
Growth regulated uptake of chylomicron remnants in Hep G2 cells Chylomicron remnants were added to Hep G2 cells seeded in dishes at varying cell densities. A rapid uptake of chylomicron remnants with a subsequent degradation of remnant cholesteryl ester occurred (unpublished data) in agreement with earlier data by Lenich and Ross [15]. Several experiments, where chylomicron remnant uptake and LDL receptor activity was followed during growth of the cells, were done. In one experiment (Fig. IA) the cells reached confluency 48 h after the start of ca m
1.6] A 1.2
0
0 0
2~
t~8
72
24
t+B
INCUBATION TIME (hra)
72 ~
z
g
INCUBATION TIME (hrs) Fig. l, Effects of cells density on the degradation and uptake of chylomicron remnant.,,and LDL. (A) Cell density curve. (lll A comparison between specific LDL degradation and chylomieron remnant cholesteryl ester degradation. ~H-labelled chylomicron remnants (containing 3.9 /~g of cholesterol ester) and 12~l-labelledLDL Icontaining 4/tg of protein) were added to two series of dishes separately. Incubations were interrupted after various time intervals a:~indicated. The medium was removed and cells ".',erev,ashed as described in Materials and Methods. Values are means of two dishes.
176 z z .-q
ua e
300
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2°°tI 100
~
8
t#
501 o
~o
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200
¢ ~
~g
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Fig. 2. Effects of compactin and mevalonolactone on chytomicron remnant degradation and uptake. Cells were preincubated with either compactin 20 #M (final conc.} or 10 mM mevalonolactone (final cone.) for 48 h. Incubations were then started by changing to medium containing O.5% human serum albumin ann ehyioulicron remnants (containing 2.4 /zg cholesteryl ester). Incubations were performed at 37°C for 4 h and dishes were treated as described in Materials and Methods.
the experiment. In this experiment there was a parallellism between L D L receptor activity as assessed by a 4 h LDL degradation and chylomicrom remnant degradation (Fig. 1B). This experiment was repeated in a situation, where the cells reached confluency after 24 h and where a decline of cell number per dish subsequently occurred. Also in this situation we observed a parallellism betweeen L D L receptor activity as assessed by a 4 h LDL degradation and chylomicron remnant eholesteryl ester degradation (unpublished data). These data indicate that chylomicron remnant uptake in Hep G2 cells is influenced by cell growth and is regulated like the L D L receptor activity.
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Fig. 3. Correlations between specific LDL degradation and chylomicron remnant cholesteryl ester degradation (A) and specific LDL uptake and chylomicron remnant uptake (B). Data from an experiment similar to those described in Fig. 2.
(P < 0.0005, r = 0.97) with chylomicron remnant cholesteryl ester degradation. Specific 4 h uptake of LDL also correlated significantly (P < 0.0025, r = 0.93)
with chylomicron remnant uptake. In a second series of experiments the corresponding r values were 0.90 and 0.94. These experiments show that chylomieron r e m n a n t uptake is regulated by changes in the cellular cholesterol content and that chylomicron remnant uptake correlates significantly with the L D L receptor activity.
Blocking of L D L receptors with a specific antibody A monoclonal anti L D L receptor antibody was used in a series of experiments to elucidate if this receptor
plays a role in the binding of chyiomicron remnants by Hep G2 cells. The antibody was from the C7 clone and
has been shown to block the LDL receptor mediated u p t a k e of L D L in cultured h u m a n fibroblasts [25]. In Hep G2 cells the a n t i b o d y blocks L D L binding to Hep G2 cells by a b o u t 50% (Fig. 4). Similarly, this a n t i b o d y decreases the b i n d i n g of chylomicron remnants to Hep
Regulation of chylomicron remnant uptake by variation in celhdar cholesterol content
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In this set of experiments the cholesterol content was modified by adding either compactin [27] which blocks de novo cholesterol synthesis or mevalonolactone which increases cholesterol synthesis [28]. C o m p a c t i n increased LDL uptake approx. 3-times and mevalonolactone decreased the L D L uptake by half (unpublished data). Mevalonolactone also decreased chylomicron remnant uptake by 64% and cholesteryl ester degradation in cells by 51% (Fig. 2). Compactin upregulated chylomicron remnant uptake and cholesteryl ester degradation by 150% and 157%, respectively (Fig. 2). ~By regulating the LDL receptor activity and concomitantly measuring chylomicron remnant uptake and degradation, correlations between L D L receptor activity and chylomicron remnant uptake could be calculated. As shown in Fig. 3, LDL receptor activity, measured by a 4 h specific degradation assay, correlated significantly
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ANTIBODY GONG /Jg/ml Fig, 4. Effectsof anti LDL receptorantibody on LDL binding to Hep G2 cells. Incubations were started by adding different amounts of anti LDL receptor antibody and I ,ag/ml of [levi] LDL to the dishes and the experiment was carried at 4°C for 2 h. After the incubation period cells were treated as described in Materials and Methods. Each point represents the mean of two dishes.
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