BIOLOGICAL CHEMISTRY \’ol 255. No I:. Iswe of September IO. pp. H303-8.10;. 19x0 Prulterl m C’ s A T H E ~ O L R N A LOF
Regulation of the Hepatic Uptakeof Triglyceride-rich Lipoproteins in the Rat OPPOSING EFFECTS OF HOMOLOGOUS APOLIPOPROTEIN E AND INDIVIDUAL C APOPROTEINS* (Received for publication, April 14, 1980)
Eberhard WindlerS, Yu-sheng Chaos, and RichardJ. Havela From the Cardiovascular Research Institute and Department of Medicine, Unioersity of California, San Franclsco, California 94143
Incubation wth homologous C apolipoproteins (apo- sequently, as the componenttriglycerides in these lipoproteins proteins C-11, C-111-0, and C-111-3) increased the content are removed by the actionof lipoprotein lipase in extrahepatic of these proteins in rat hepatic very low density lipo- tissues, these apoproteins leave the surface of the partially proteins and caused variable reduction in the content degraded lipoproteins and are returned to HDL. In previous of apolipoprotein E without detectable change in lipid work (2), we showed that the acquisition of C apoproteins composition of the particles. The uptake of each of that occurs when chylomicrons from mesenteric lymph and these modified very low density lipoproteins by the VLDL from liverperfusates of rats are incubatedwith VLDLisolated, perfused rat liver was substantially reduced freeplasma prevents the uptake of these triglyceride-rich as comparedwith that of unincubatedlipoproteins. lipoproteins by the isolated, perfused rat liver. Remnants of Addition of C apolipoproteins to small chylomicrons these lipoproteins, produced in functionally hepatectomized from intestinal lymph also inhibited hepatic uptake. rats, wererapidly taken up and metabolized by the liver. These chylomicrons, obtained from rats made hypoli- Hepatic uptakeof remnant chylomicrons is saturable, consistpidemic by treatment with pharmacological amounts ent with a receptor-dependent process (3, 4). As the major of 17 a-ethinyl estradiol, contained very littleapolipoprotein components of the remnant lipoproteins areapolipoprotein E. Addition of homologous apolipoprotein E to these small chylomicrons resulted in rapid hepaticup- protein (apo) B and apo E (5), and as the rate of uptake by take, similar to that observed previously for chylomi- the perfused liver of low density lipoproteins (LDL), which cron remnants. Taken together with the results of ear- contain only apo B, is much lower than that of remnants (2, lier experiments inwhich the content of C apolipopro- 6), we suggested that apo E comprises an important compoteins of very low density lipoproteins and small chylo- nent of the recognition site for a remnant receptor. In the present research,we have evaluated further therole microns was increased by incubation with rat plasma freed of very low density lipoproteins, the present re- of the C apoproteinsandapo E in the hepatic uptake of The purified proteins were sults indicate that C apolipoproteins and apolipopro- triglyceride-richlipoproteins. tein E have opposingand probably independent effects added individually to hepatic VLDL and small chylomicrons. upon the hepatic uptake of triglyceride-rich lipoproThe chylomicrons, obtained from intestinal lymph of rats teins in the rat. The results are consistent with the made hypolipoproteinemic by administration of large amounts hypothesis that changes in apolipoprotein composition of 17 a-ethinylestradiol (7), containedgreatlyreduced of triglyceride-rich lipoproteins that occur as they are amounts of C apoproteins and apo E, neither of which is secreted from intestine and liver and as they are sub- synthesized at an appreciable rate in the intestinal mucosa sequently metabolized can explainboth thelack of (1).The results provide direct evidence for independent reginitial hepaticuptake and the subsequent rapid hepatic ulation of the hepatic uptakeof triglyceride-rich lipoproteins uptake andcatabolism of remnant lipoproteins. by each of the C apoproteins (apo C-11, apo C-111-0, and apo C-111-3) and by apo E. EXPERIMENTALPROCEDURES When newly secreted chylomicrons and very low density lipoproteins (VLDL)’ enter the lymph and blood, they acquire Animals-Male Sprague-Dawley rats,weighing 300 to 350 g, mainC apoproteins from high density lipoproteins (HDL)(1). Sub- tained on standard Purina rat chow (Ralston Purina Co., St. Louis
* This work was supported by grants from the United StatesPublic Health Service(Arteriosclerosis SCOR Grants HL-14237 and HL06285. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Deutsche Forschungsgemeinschaft. Presentaddress, Klinisches Institut fur Herzinfarktforschung an der MedizinischenUniversitatsklinik, BergheimerStr. 58, D 6900 Heidelberg, West Germanv. 3 Supported by National Institutes of Health Training Grant HL07192. 1 To whom reprint requests should be addressed. The abbreviationsused are: VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; apo, apolipoprotein; SDS, sodiumdodecyl sulfate,
’
MO) and tap water,were used, exceptfor preparation of erythrocytes and isolated apoproteins, for which blood plasma was obtained from retired breeders (2). Estrogen-treated rats, used to obtain intestinal lymph, had received five daily injections of 17 a-ethinyl estradiol (5 mg/kg body weight) (7). Preparation of Lipoproteins and Apolipoproteins-Lipoproteins were isolated by preparative ultracentrifugation (2). Apoproteins were obtained from plasma VLDL, prepared by centrifuging rat plasma for IO8 X ge, min a t a density of 1.006 g/ml, and purified by recentrifugation underthesame conditions.VLDLwere delipidated with ethanol/ether, 3:1, and ether (8) and the precipitated apoproteins were dissolved in 0.015 M Tris-HCI, pH 8.2, containing 6 M urea. The soluble proteins were separated by gel permeation chromatography (Sephadex G-200 or G-150, Pharmacia, Piscataway, NJ) in the same buffer (9).Fractions containingE or C apoproteins were pooled, concentrated by vacuum dialysis, and then dialyzed against Krebs7.4 (2). Individual Henseleit buffer, containing noglucose or CaL+, pH
8303
a304
Regulation of Hepatic Uptake
of Triglyceride-rich Lipoproteins
C apoproteins (C-11, C-111-0, and C-111-3) were separated by preparative isoelectric focusing electrophoresis, as described for human C apoproteins (IO). After elution from the gel with 0.05 M Tris-HCI, pH 8.2. the pure proteins containing Ampholines were lyophilized and dissolved in -1 ml of distilled water. Ampholines were removed by gel chromatography (Sephadex C-50) with 0.05 M Tris-HCI, pH 8.2. Purity of the apoproteins was evaluated by anal-ytical isoelectric focusing polyacrylamide gel efectrophoresis in the absenceof reducing agent (11) and SIX-polyacrylamide gel electrophoresis (12). In both systems. the C apoproteins contained no detectable albumin or apo A-I, apo A-IV. or apo E. Apo E appeared to befree of C apoproteins, apo A-I, and apo A-IV. As measured by radioimmunoassay, contamination of apo E by apo A-I was 1.4 2 0.5% (n = 4). VLDL Enriched in C Apoproteins-Perfusates of isolated livers, in which lipoprotein cholesteryl esters were labeled with [''Hlcholesterol, were obtained exactly as described (2). After removal of erythrocytes from the perfusate, perfusateplasma containing 0.3 to 0.5 mg of VLDL-triglyceride/ml was incubated with unfractionated or individual C-apoproteins at a ratio of 130 to 270 p g of protein/mg of triglyceride or with 0.15 M NaCl solution. The mixture was gently shaken at room temperature for 1 h. T h e resultingC apoproteinenriched VLDL were then separated by ultracentrifugation for 10" X g... min (2). C Apoproteins or Apo E-IntesSmall Chylomicrons Enriched in tinal I-yrnph containing [.'H]cholesteryl esters was obtained from normal or estrogen-treated rats during intraduodenal infusion of 10% glucose in 0.15 M NaCl (13). Lymph serum was layered under KrebsHenseleit buffer, without glucose or Ca", pH 7.4 (d = 1.007 g/ml) (2): and small chylomicrons were separated by centrifugation for 3 X 10' X g . min, followed by recentrifugation under the same conditions. To obtain small chylomicrons enriched in C apoproteins or apo E, chylomicrons, obtained by a single centrifugationfrom lymph of estrogen-treated rats (5 to 10 mg of triglyceride/ml), were incubated with unfractionated C apoproteins (50 to 125 pg/mg of triglyceride), with apoprotein E ( 5 0 to 200 pg/mg of triglyceride, or with 0.15 M NaCI. The mixtures were gently shaken for 1 h a t room temperature and then submitted to thesecond centrifugation as described above. Uptake of Lipoproteins by Perfused Liuers-Liver perfusions, addition of labeledVLDL and chylomicrons, and measurement of ["Hlcholesteryl esters in perfusates wereperformedexactly as described (2). Unincubated and incubated hepatic VLDL and chylomicrons were used within 24 h of preparation. Storage of chylomicrons for more than 3 days resulted in aggregation, detected by agarose gel electrophoresis (chylomicrons failed tomigrate)and by increased optical density. In fresh samples, the ratioof A at 550 nm:triglyceride was the same for control and incubated chylomicrons. Analyses-Protein was determined by a modified procedure of Lowry with bovine serum albumin as standard (14). Apo A-I (15) and apo E (9) were measured by specific radioimmunoassays. Triglycerides in lipoproteins were estimated by an automated procedure (16) and lipid composition of lipoproteins was determined by standard methods (17).Negatively stained lipoproteins were prepared and their diameters quantified as described (18). Electrophoretic mobility of lipoproteins was determined in agarose gel (11). Materials-[ 1,2-"H]cholesterol. 40 to 60 Ci/mmol. was from New England Nuclear, Boston, MA. Calculations-Differences between experimental groups were evaluated by Student's(paired) t test. For multipleexperiments, results are given as the mean of n experiments k S. I). RESULTS
Uptake of VLDL Enriched in C Apoproteins by the Perfused Liver-VLDL incubated with unfractionated C apoproteins or with individual C apoproteins were enriched in the added protein (Figs. 1 and 2), but theirlipid composition was unaltered (Table I) and resembled that reported previously (2).Incubation with unfractionated C apoproteins did not appreciably alter the proportions of the individual components, but enrichmentwith apo C-11, C-111-0,or C-111-3 was at the expense of the other C apoproteins (Fig. 2). In addition, the apo E:triglyceride ratio decreased variably from 29 k 5 ( n = 3) to 1 to 9 pg of apo E/mg of triglyceride and that of apo A-1:triglyceride fell from 0.4 to
Regulation of the Hepatic Uptakeof Triglyceride-rich Lipoproteins in the Rat OPPOSING EFFECTS OF HOMOLOGOUS APOLIPOPROTEIN E AND INDIVIDUAL C APOPROTEINS* (Received for publication, April 14, 1980)
Eberhard WindlerS, Yu-sheng Chaos, and RichardJ. Havela From the Cardiovascular Research Institute and Department of Medicine, Unioersity of California, San Franclsco, California 94143
Incubation wth homologous C apolipoproteins (apo- sequently, as the componenttriglycerides in these lipoproteins proteins C-11, C-111-0, and C-111-3) increased the content are removed by the actionof lipoprotein lipase in extrahepatic of these proteins in rat hepatic very low density lipo- tissues, these apoproteins leave the surface of the partially proteins and caused variable reduction in the content degraded lipoproteins and are returned to HDL. In previous of apolipoprotein E without detectable change in lipid work (2), we showed that the acquisition of C apoproteins composition of the particles. The uptake of each of that occurs when chylomicrons from mesenteric lymph and these modified very low density lipoproteins by the VLDL from liverperfusates of rats are incubatedwith VLDLisolated, perfused rat liver was substantially reduced freeplasma prevents the uptake of these triglyceride-rich as comparedwith that of unincubatedlipoproteins. lipoproteins by the isolated, perfused rat liver. Remnants of Addition of C apolipoproteins to small chylomicrons these lipoproteins, produced in functionally hepatectomized from intestinal lymph also inhibited hepatic uptake. rats, wererapidly taken up and metabolized by the liver. These chylomicrons, obtained from rats made hypoli- Hepatic uptakeof remnant chylomicrons is saturable, consistpidemic by treatment with pharmacological amounts ent with a receptor-dependent process (3, 4). As the major of 17 a-ethinyl estradiol, contained very littleapolipoprotein components of the remnant lipoproteins areapolipoprotein E. Addition of homologous apolipoprotein E to these small chylomicrons resulted in rapid hepaticup- protein (apo) B and apo E (5), and as the rate of uptake by take, similar to that observed previously for chylomi- the perfused liver of low density lipoproteins (LDL), which cron remnants. Taken together with the results of ear- contain only apo B, is much lower than that of remnants (2, lier experiments inwhich the content of C apolipopro- 6), we suggested that apo E comprises an important compoteins of very low density lipoproteins and small chylo- nent of the recognition site for a remnant receptor. In the present research,we have evaluated further therole microns was increased by incubation with rat plasma freed of very low density lipoproteins, the present re- of the C apoproteinsandapo E in the hepatic uptake of The purified proteins were sults indicate that C apolipoproteins and apolipopro- triglyceride-richlipoproteins. tein E have opposingand probably independent effects added individually to hepatic VLDL and small chylomicrons. upon the hepatic uptake of triglyceride-rich lipoproThe chylomicrons, obtained from intestinal lymph of rats teins in the rat. The results are consistent with the made hypolipoproteinemic by administration of large amounts hypothesis that changes in apolipoprotein composition of 17 a-ethinylestradiol (7), containedgreatlyreduced of triglyceride-rich lipoproteins that occur as they are amounts of C apoproteins and apo E, neither of which is secreted from intestine and liver and as they are sub- synthesized at an appreciable rate in the intestinal mucosa sequently metabolized can explainboth thelack of (1).The results provide direct evidence for independent reginitial hepaticuptake and the subsequent rapid hepatic ulation of the hepatic uptakeof triglyceride-rich lipoproteins uptake andcatabolism of remnant lipoproteins. by each of the C apoproteins (apo C-11, apo C-111-0, and apo C-111-3) and by apo E. EXPERIMENTALPROCEDURES When newly secreted chylomicrons and very low density lipoproteins (VLDL)’ enter the lymph and blood, they acquire Animals-Male Sprague-Dawley rats,weighing 300 to 350 g, mainC apoproteins from high density lipoproteins (HDL)(1). Sub- tained on standard Purina rat chow (Ralston Purina Co., St. Louis
* This work was supported by grants from the United StatesPublic Health Service(Arteriosclerosis SCOR Grants HL-14237 and HL06285. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Deutsche Forschungsgemeinschaft. Presentaddress, Klinisches Institut fur Herzinfarktforschung an der MedizinischenUniversitatsklinik, BergheimerStr. 58, D 6900 Heidelberg, West Germanv. 3 Supported by National Institutes of Health Training Grant HL07192. 1 To whom reprint requests should be addressed. The abbreviationsused are: VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; apo, apolipoprotein; SDS, sodiumdodecyl sulfate,
’
MO) and tap water,were used, exceptfor preparation of erythrocytes and isolated apoproteins, for which blood plasma was obtained from retired breeders (2). Estrogen-treated rats, used to obtain intestinal lymph, had received five daily injections of 17 a-ethinyl estradiol (5 mg/kg body weight) (7). Preparation of Lipoproteins and Apolipoproteins-Lipoproteins were isolated by preparative ultracentrifugation (2). Apoproteins were obtained from plasma VLDL, prepared by centrifuging rat plasma for IO8 X ge, min a t a density of 1.006 g/ml, and purified by recentrifugation underthesame conditions.VLDLwere delipidated with ethanol/ether, 3:1, and ether (8) and the precipitated apoproteins were dissolved in 0.015 M Tris-HCI, pH 8.2, containing 6 M urea. The soluble proteins were separated by gel permeation chromatography (Sephadex G-200 or G-150, Pharmacia, Piscataway, NJ) in the same buffer (9).Fractions containingE or C apoproteins were pooled, concentrated by vacuum dialysis, and then dialyzed against Krebs7.4 (2). Individual Henseleit buffer, containing noglucose or CaL+, pH
8303
a304
Regulation of Hepatic Uptake
of Triglyceride-rich Lipoproteins
C apoproteins (C-11, C-111-0, and C-111-3) were separated by preparative isoelectric focusing electrophoresis, as described for human C apoproteins (IO). After elution from the gel with 0.05 M Tris-HCI, pH 8.2. the pure proteins containing Ampholines were lyophilized and dissolved in -1 ml of distilled water. Ampholines were removed by gel chromatography (Sephadex C-50) with 0.05 M Tris-HCI, pH 8.2. Purity of the apoproteins was evaluated by anal-ytical isoelectric focusing polyacrylamide gel efectrophoresis in the absenceof reducing agent (11) and SIX-polyacrylamide gel electrophoresis (12). In both systems. the C apoproteins contained no detectable albumin or apo A-I, apo A-IV. or apo E. Apo E appeared to befree of C apoproteins, apo A-I, and apo A-IV. As measured by radioimmunoassay, contamination of apo E by apo A-I was 1.4 2 0.5% (n = 4). VLDL Enriched in C Apoproteins-Perfusates of isolated livers, in which lipoprotein cholesteryl esters were labeled with [''Hlcholesterol, were obtained exactly as described (2). After removal of erythrocytes from the perfusate, perfusateplasma containing 0.3 to 0.5 mg of VLDL-triglyceride/ml was incubated with unfractionated or individual C-apoproteins at a ratio of 130 to 270 p g of protein/mg of triglyceride or with 0.15 M NaCl solution. The mixture was gently shaken at room temperature for 1 h. T h e resultingC apoproteinenriched VLDL were then separated by ultracentrifugation for 10" X g... min (2). C Apoproteins or Apo E-IntesSmall Chylomicrons Enriched in tinal I-yrnph containing [.'H]cholesteryl esters was obtained from normal or estrogen-treated rats during intraduodenal infusion of 10% glucose in 0.15 M NaCl (13). Lymph serum was layered under KrebsHenseleit buffer, without glucose or Ca", pH 7.4 (d = 1.007 g/ml) (2): and small chylomicrons were separated by centrifugation for 3 X 10' X g . min, followed by recentrifugation under the same conditions. To obtain small chylomicrons enriched in C apoproteins or apo E, chylomicrons, obtained by a single centrifugationfrom lymph of estrogen-treated rats (5 to 10 mg of triglyceride/ml), were incubated with unfractionated C apoproteins (50 to 125 pg/mg of triglyceride), with apoprotein E ( 5 0 to 200 pg/mg of triglyceride, or with 0.15 M NaCI. The mixtures were gently shaken for 1 h a t room temperature and then submitted to thesecond centrifugation as described above. Uptake of Lipoproteins by Perfused Liuers-Liver perfusions, addition of labeledVLDL and chylomicrons, and measurement of ["Hlcholesteryl esters in perfusates wereperformedexactly as described (2). Unincubated and incubated hepatic VLDL and chylomicrons were used within 24 h of preparation. Storage of chylomicrons for more than 3 days resulted in aggregation, detected by agarose gel electrophoresis (chylomicrons failed tomigrate)and by increased optical density. In fresh samples, the ratioof A at 550 nm:triglyceride was the same for control and incubated chylomicrons. Analyses-Protein was determined by a modified procedure of Lowry with bovine serum albumin as standard (14). Apo A-I (15) and apo E (9) were measured by specific radioimmunoassays. Triglycerides in lipoproteins were estimated by an automated procedure (16) and lipid composition of lipoproteins was determined by standard methods (17).Negatively stained lipoproteins were prepared and their diameters quantified as described (18). Electrophoretic mobility of lipoproteins was determined in agarose gel (11). Materials-[ 1,2-"H]cholesterol. 40 to 60 Ci/mmol. was from New England Nuclear, Boston, MA. Calculations-Differences between experimental groups were evaluated by Student's(paired) t test. For multipleexperiments, results are given as the mean of n experiments k S. I). RESULTS
Uptake of VLDL Enriched in C Apoproteins by the Perfused Liver-VLDL incubated with unfractionated C apoproteins or with individual C apoproteins were enriched in the added protein (Figs. 1 and 2), but theirlipid composition was unaltered (Table I) and resembled that reported previously (2).Incubation with unfractionated C apoproteins did not appreciably alter the proportions of the individual components, but enrichmentwith apo C-11, C-111-0,or C-111-3 was at the expense of the other C apoproteins (Fig. 2). In addition, the apo E:triglyceride ratio decreased variably from 29 k 5 ( n = 3) to 1 to 9 pg of apo E/mg of triglyceride and that of apo A-1:triglyceride fell from 0.4 to
