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• W2069069694 abstract "In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar (∼10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP. In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar (∼10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP. Hepatic lipase (HL) 1The abbreviations used are: HL, hepatic lipase; EEA1, early endosome antigen 1; GM130, Golgi matrix protein of 130 kDa; GST, glutathione S-transferase; HSPGs, heparan sulfate proteoglycans; LPL, lipoprotein lipase; LDL, low density lipoprotein; LRP, LDL receptor-related protein; RAP, receptor-associated protein; RRC, receptor-recycling compartment; α2MG, α2-macroglobulin; ELISA, enzyme-linked immunosorbent assay; LDLR, LDL receptor.1The abbreviations used are: HL, hepatic lipase; EEA1, early endosome antigen 1; GM130, Golgi matrix protein of 130 kDa; GST, glutathione S-transferase; HSPGs, heparan sulfate proteoglycans; LPL, lipoprotein lipase; LDL, low density lipoprotein; LRP, LDL receptor-related protein; RAP, receptor-associated protein; RRC, receptor-recycling compartment; α2MG, α2-macroglobulin; ELISA, enzyme-linked immunosorbent assay; LDLR, LDL receptor. is synthesized by hepatocytes and resides on liver cell surfaces, presumably bound to heparan sulfate proteoglycans (HSPGs) (1Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (952) Google Scholar). HL does not hydrolyze lipids of nascent chylomicrons (2Hamilton R.L. Diss. Abstr. 1965; 26: 1Google Scholar), but it participates in the clearance of chylomicron remnants from the blood and hydrolysis of component triglycerides and phospholipids (3Shafi S. Brady S.E. Bensadoun A. Havel R.J. J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar, 4Qiu S. Bergeron N. Kotite L. Krauss R.M. Bensadoun A. Havel R.J. J. Lipid Res. 1998; 39: 1661-1668Abstract Full Text Full Text PDF PubMed Google Scholar). It also contributes to the conversion of hepatogenous triglyceride-rich lipoproteins to LDL and to remodeling HDL (4Qiu S. Bergeron N. Kotite L. Krauss R.M. Bensadoun A. Havel R.J. J. Lipid Res. 1998; 39: 1661-1668Abstract Full Text Full Text PDF PubMed Google Scholar, 5Demant T. Carlson L.A. Holmquist L. Karpe F. Nilsson-Ehle P. Packard C.J. Shepherd J. J. Lipid Res. 1988; 29: 1603-1611Abstract Full Text PDF PubMed Google Scholar). Chylomicron remnants are efficiently removed from the blood by binding to macromolecules on the basolateral microvilli of hepatocytes, including HL (6Havel R.J. Atherosclerosis. 1998; 141: S1-S7Abstract Full Text Full Text PDF PubMed Google Scholar, 7Chappell D.A. Medh J.D. Prog. Lipid Res. 1998; 37: 393-422Crossref PubMed Scopus (55) Google Scholar). Binding of chylomicron remnants to HL causes a delay in the endocytosis of these particles, which is subsequently mediated by the LDL receptor and LDL receptor-related protein-1 (LRP) (6Havel R.J. Atherosclerosis. 1998; 141: S1-S7Abstract Full Text Full Text PDF PubMed Google Scholar, 8Rohlmann K.A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Investig. 1998; 101: 689-695Crossref PubMed Scopus (393) Google Scholar). It has been postulated that remodeling of chylomicron remnant lipids by HL facilitates receptor binding via apoE (9Brasaemle D.L. Cornely-Moss K. Bensadoun A. J. Lipid Res. 1993; 34: 455-465Abstract Full Text PDF PubMed Google Scholar), leading to efficient endocytosis via coated pits. In the absence of apoE, HL may provide a less efficient pathway for removal of remnant lipoproteins containing apoB-48 (10Bergeron N. Kotite L. Vergés M. Blanche P. Hamilton R.L. Krauss R.M. Bensadoun A. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15647-15652Crossref PubMed Scopus (30) Google Scholar). Rat HL binds LRP with high affinity (11Kounnas M.Z. Chappell D.A. Wong H. Argraves W.S. Strickland D.K. J. Biol. Chem. 1995; 270: 9307-9312Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In human hepatoma cells, fibroblasts, and Chinese hamster ovary cells, internalization and degradation of HL bound to HSPGs are mediated mainly by LRP (11Kounnas M.Z. Chappell D.A. Wong H. Argraves W.S. Strickland D.K. J. Biol. Chem. 1995; 270: 9307-9312Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Krapp A. Ahle S. Kersting S. Hua Y. Kneser K. Nielsen M. Gliemann J. Beisiegel U. J. Lipid Res. 1996; 37: 926-936Abstract Full Text PDF PubMed Google Scholar). Lipoprotein lipase (LPL) also binds to HSPGs (13Cheng C.-F. Oosta G.M. Bensadoun A. Rosenberg R.D. J. Biol. Chem. 1981; 256: 12893-12898Abstract Full Text PDF PubMed Google Scholar), and its internalization and degradation by human fibroblasts are dependent upon LRP (14Chappell D.A. Fry G.L. Waknitz M.A. Iverius P.-H. Williams S.E. Strickland D.K. J. Biol. Chem. 1992; 267: 25764-25767Abstract Full Text PDF PubMed Google Scholar). Initial binding of LPL to HSPGs is necessary for degradation (14Chappell D.A. Fry G.L. Waknitz M.A. Iverius P.-H. Williams S.E. Strickland D.K. J. Biol. Chem. 1992; 267: 25764-25767Abstract Full Text PDF PubMed Google Scholar, 15Cisar L.A. Hoogewerf A.J. Cupp M. Rapport C.A. Bensadoun A. J. Biol. Chem. 1989; 264: 1767-1774Abstract Full Text PDF PubMed Google Scholar). In light of these observations in cultured cells, we have pursued our earlier observation (16Belcher J.D. Hamilton R.L. Brady S.E. Havel R.J. Circulation. 1988; 28: II-145Google Scholar) that HL is concentrated in hepatocytic endosomes from rat liver, and we report here evidence that LRP contributes in vivo to the endocytosis of HL destined for lysosomal degradation. We also report evidence that lipoprotein lipase (LPL), which is removed from the blood mainly by the rat liver (17Peterson J. Olivecrona T. Bengtsson-Olivecrona G. Biochim. Biophys. Acta. 1985; 4: 262-270Crossref Scopus (83) Google Scholar), is taken up into rat hepatocytes exclusively by this endocytic receptor. Reagents and Antibodies—α2-Macroglobulin (α2MG) (Sigma) was activated with methylamine and radioiodinated (125I) as described (18Mokuno H. Brady S. Kotite L. Herz J. Havel R.J. J. Biol. Chem. 1994; 269: 13238-13243Abstract Full Text PDF PubMed Google Scholar). Rat 39-kDa receptor-associated protein (RAP) was encoded as a bacterial glutathione S-transferase (GST) fusion protein in the plasmid pGEX-DGRAP and prepared as described (19Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). Similarly encoded GST alone was used as a control. Rabbit polyclonal anti-LRP was raised against the C-terminal cytoplasmic tail of the human protein (20Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (733) Google Scholar). Mouse monoclonal anti-LDL receptor (IgG 4A4) was raised against the C-terminal cytoplasmic tail of the human receptor (21van Driel I.R. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1987; 262: 17443-17449Abstract Full Text PDF PubMed Google Scholar). Goat polyclonal anti-asialoglycoprotein receptor was raised against the rat protein (22Harford J. Lowe M. Tsunoo H. Ashwell G. J. Biol. Chem. 1982; 257: 12685-12690Abstract Full Text PDF PubMed Google Scholar). The following antisera against human proteins were generously provided: polymeric immunoglobulin receptor SC166 (J.-P. Kraehenbuhl, ISREC, Lausanne, Switzerland); transferrin receptor H68.4 (I. Trowbridge, Salk Institute, San Diego); cellubrevin (R. Jahn, Yale University, New Haven, CT); endobrevin (W. Hong, Institute of Molecular and Cellular Biology, Singapore); and Rab11 121 (R. Parton, University of Queensland, Australia). Other antibodies were purchased commercially as follows: epidermal growth factor receptor 1005, sc-03 (Santa Cruz Biotechnology, Santa Cruz, CA); Rab5 44-332 (QCB Inc., Hopkinton, MA); early endosome antigen 1 (EEA1) E41120 and Golgi matrix protein of 130-kDa (GM130) clone 35, G65120-050 (Transduction Laboratories, Lexington, KY). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Isolation of Endosomes—Initial experiments to evaluate the presence and distribution of HL in endosomes were carried out in male Sprague-Dawley rats treated with 17-α-ethinyl estradiol, which highly induces LDL receptors in hepatocytes. In these rats, three highly purified endosome fractions, early endosomes, late endosomes (multivesicular bodies), and a receptor-recycling compartment (RRC), can readily be isolated (23Belcher J.D. Hamilton R.L. Brady S.E. Hornick C.A. Jaeckle S. Schneider W.J. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6785-6789Crossref PubMed Scopus (68) Google Scholar). The volume and mass of the multivesicular body fraction characteristically increases 15 min after injection of human LDL. Male Sprague-Dawley rats, weighing 300–350 g and treated with estradiol for 5 days as described (23Belcher J.D. Hamilton R.L. Brady S.E. Hornick C.A. Jaeckle S. Schneider W.J. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6785-6789Crossref PubMed Scopus (68) Google Scholar), were fasted overnight and then anesthetized with Isoflurane (Schering-Plough Animal Health Corp.). Before or 15 min after injection of LDL (1.0–1.5 mg of protein) through an exposed femoral vein, livers were exposed with an abdominal incision. The portal vein was then cannulated. The vena cava was opened below the liver, and the liver was flushed through the portal vein with up to 100 ml of ice-cold 0.15 m NaCl containing 0.1% EDTA. The liver was weighed, and three endosome fractions were prepared exactly as described (23Belcher J.D. Hamilton R.L. Brady S.E. Hornick C.A. Jaeckle S. Schneider W.J. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6785-6789Crossref PubMed Scopus (68) Google Scholar). Their purity was assessed by negative staining electron microscopy (24Hornick C.A. Hamilton R.L. Spaziani E. Enders G.H. Havel R.J. J. Cell Biol. 1985; 100: 1558-1569Crossref PubMed Scopus (57) Google Scholar). To determine the effect of administering RAP upon the endocytosis of HL and LPL, we used untreated male Sprague-Dawley rats weighing 230–270 g, from which we isolated a total endosome fraction from liver homogenates. Rats fasted overnight were anesthetized with a mixture of ketamine (75–95 mg/kg) and xylazine (5 mg/kg), administered intraperitoneally in a volume of ∼0.3 ml 0.15 m NaCl. Additional anesthetic was given as needed to maintain light anesthesia. A midline incision was made over the trachea, and a carotid artery was exposed, cannulated with PE-60 polyethylene tubing advanced ∼1 cm caudad through a small incision, and tied in place. The incision was closed with surgical clips. GST-RAP or GST was injected into the carotid artery as a pulse of 1 ml followed by a constant infusion with a pump at a rate of 4 ml/h. The least amount of GST-RAP required to prevent the hepatic uptake of radioiodinated α2MG over a period of 30 min, determined as described below, was utilized. Control animals received an equimolar amount of GST. Blood samples of 50–100 μl were obtained from the orbital plexus 1 and 15 min after starting the infusions and placed in tubes containing EDTA (final concentration ∼0.2%). At 27 min, the liver was exposed through an abdominal incision, and the portal vein was cannulated with PE-50 polyethylene tubing and tied in place. At 30 min, a final blood sample was obtained from the inferior vena cava, following which the vena cava was opened and the liver flushed as described above. The chest was then opened to ensure cessation of breathing. Livers were removed, weighed, and immediately placed in ice-cold 0.15 m NaCl. Routinely, four rats were studied in a given day, receiving GST-RAP and GST alternately, and two livers were used from each group to isolate endosomes. Livers were homogenized within 2 h of harvesting. The initial stages of endosome isolation from homogenates were identical to those used for estradiol-treated rats. At the final step, however, only two rather than four sucrose solutions were used, with densities of 1.033 and 1.13 g/ml, to create a discontinuous gradient with a single interface at which a single “total” endosome fraction accumulated after centrifugation in a Beckman SW 41 rotor at 197,500 gav for 90 min. This procedure yielded sufficient material (1.0–1.5 mg of endosomal protein) for all of the analyses described below. Negative stains confirmed that this fraction was composed of structures resembling early, late, and receptor-recycling endosomes, with no apparent differences between rats given GST-RAP or GST. Selection of Dose of GST-RAP—Untreated rats, anesthetized as described above and with cannulated carotid arteries, were injected with various amounts of GST-RAP, administered as a pulse followed by a constant infusion. Immediately after the pulse, the rats received 50 μg (2.5–5 × 107 cpm) of 125I-labeled, activated α2MG in a volume of 0.5–1 ml, injected into an exposed femoral vein. Blood samples of 50–100 μl were obtained at 1, 5, 10, and 20 min later from the orbital plexus and at 30 min from the exposed inferior vena cava, followed by flushing the liver and opening the chest as described above. The liver was removed and weighed, and a small portion was taken for assay of 125I. Analyses—Protein was quantified (25Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7065) Google Scholar) in liver fractions (homogenate and endosomes). Total cholesterol (26Allain C.C. Poon L.S. Chan C.S. Richmond W. Fu P.C. Clin. Chem. 1974; 20: 470-475Crossref PubMed Scopus (7242) Google Scholar) and triglycerides (27Bucolo G. David H. Clin. Chem. 1973; 19: 476-482Crossref PubMed Scopus (2530) Google Scholar) were quantified in plasma (mg/dl) and liver fractions (mg/mg protein). Rat HL (28Cisar L.A. Bensadoun A. J. Lipid Res. 1985; 26: 380-386Abstract Full Text PDF PubMed Google Scholar) and LPL (29van Vlijmen B.J. Rohlmann A. Page S.T. Bensadoun A. Bos I.S. van Berkel T.J.C. Havekes L.M. Herz J. J. Biol. Chem. 1999; 274: 35219-35226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) masses were estimated by specific ELISAs in plasma (ng/ml) and liver fractions (ng/mg protein). In estradiol-treated rats, HL activity against an emulsified triglyceride substrate was also assayed in homogenate and endosome fractions (30Belcher J.D. Sisson P.J. Waite M. Biochem. J. 1985; 229: 343-351Crossref PubMed Scopus (12) Google Scholar). Values for these enzymes in endosomes are expressed as fold enrichment over homogenate. Enzyme masses in total plasma were calculated for a plasma volume of 4.5% of body weight and compared with corresponding values for total liver (ng/mg protein × total liver protein). Proteins in liver fractions were separated by SDS-PAGE and subjected to Western blotting as described (31Enrich C. Jäckle S. Havel R.J. Hepatology. 1996; 24: 226-232Crossref PubMed Google Scholar). Components were visualized by enhanced chemiluminescence (ECL, PerkinElmer Life Sciences). Band intensities were quantified with the IPlab Gel program (Signal Analytics Corp., Vienna, VA) and expressed as volume units/mg of protein. All estimates of enrichment in endosomes/homogenate were from the same exposure of a given gel. Statistical Analysis—The number of experiments is indicated in the legend of each figure and table (n). Student’s paired t test was used to estimate the significance of difference between experimental and control conditions. HL Is Enriched in a Characteristic Pattern in Hepatocytic Endosomal Fractions—In rat liver, ligands taken up from the blood by high affinity receptors rapidly enter endosomal compartments in hepatocytes (32Havel R.J. Hamilton R.L. Hepatology. 1988; 8: 1689-1704Crossref PubMed Scopus (97) Google Scholar). Little HL circulates in the blood of rats, and most is present in the extracellular space of the liver (space of Disse), presumably bound to proteoglycans (3Shafi S. Brady S.E. Bensadoun A. Havel R.J. J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar, 33Schoonderwoerd K. Verhoeven A.J. Jansen H. Biochem. J. 1994; 302: 717-722Crossref PubMed Scopus (12) Google Scholar). We found, however, that this protein, evaluated by its activity against triglyceride substrate and mass (estimated immunochemically), was enriched in three endosome fractions from estradiol-treated rats in a distinctive pattern: highest in multivesicular bodies (late endosomes), intermediate in early endosomes, and lowest in receptor-recycling endosomes (Table I). This pattern is characteristic of ligands and receptors destined for lysosomal degradation in hepatocytes, and the opposite of that was observed for receptors and ligands, such as the LDL receptor and transferrin, that recycle to the cell surface (23Belcher J.D. Hamilton R.L. Brady S.E. Hornick C.A. Jaeckle S. Schneider W.J. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6785-6789Crossref PubMed Scopus (68) Google Scholar, 34Jäckle S. Runquist E. Brady S. Hamilton R.L. Havel R.J. J. Lipid Res. 1991; 32: 485-498Abstract Full Text PDF PubMed Google Scholar, 35Jäckle S. Runquist E.A. Miranda-Brady S. Havel R.J. J. Biol. Chem. 1991; 266: 1396-1402Abstract Full Text PDF PubMed Google Scholar). Total HL activity recovered in multivesicular bodies increased from 0.31 ± 0.06 to 0.77 ± 0.27% of total liver homogenate (mean and S.D., n = 5) 15 min after loading the liver with human LDL, with no change in enrichment. Endosomal HL actively hydrolyzed triglycerides in endocytosed chylomicron remnants, and its activity in homogenate and endosomes was completely inhibited by goat antiserum against dog HL (data not shown). The extent of enrichment of HL in multivesicular bodies over liver homogenate (20–30-fold) was lower than that observed for ligands such as LDL (∼200-fold). This presumably reflects the relatively large pool of HL on liver cell surfaces (3Shafi S. Brady S.E. Bensadoun A. Havel R.J. J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar, 33Schoonderwoerd K. Verhoeven A.J. Jansen H. Biochem. J. 1994; 302: 717-722Crossref PubMed Scopus (12) Google Scholar). These results are consistent with the hypothesis that HL may leave this pool by binding to receptors that enter hepatocytes via coated pits.Table IEnrichment of hepatic lipase in endosomes isolated from livers of estradiol-treated ratsEndosome fractionActivityaFold enrichment over liver homogenate (n = 3; mean and S.E.); homogenate activity = 5.28 ± 0.82 nmol min—1 mg protein—1MassbFold enrichment over homogenate (n = 1); homogenate mass = 28 ng/mg proteinEarly endosomes13.5 ± 5.712.2Multivesicular bodies30.1 ± 8.919.7Receptor-recycling compartment4.1 ± 2.72.6a Fold enrichment over liver homogenate (n = 3; mean and S.E.); homogenate activity = 5.28 ± 0.82 nmol min—1 mg protein—1b Fold enrichment over homogenate (n = 1); homogenate mass = 28 ng/mg protein Open table in a new tab Selection of Dose of GST-RAP to Inhibit Binding of Lipases to LRP—RAP prevents binding of all high affinity ligands, such as HL and LPL, to LRP. GST-RAP has manyfold higher affinity for LRP than for the LDL receptor (18Mokuno H. Brady S. Kotite L. Herz J. Havel R.J. J. Biol. Chem. 1994; 269: 13238-13243Abstract Full Text PDF PubMed Google Scholar, 36Medh J.D. Fry G.L. Bowen S.L. Pladet M.W. Stickland D.K. Chappell D.A. J. Biol. Chem. 1995; 270: 536-540Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Previous studies (18Mokuno H. Brady S. Kotite L. Herz J. Havel R.J. J. Biol. Chem. 1994; 269: 13238-13243Abstract Full Text PDF PubMed Google Scholar) showed that GST-RAP is rapidly endocytosed into rat hepatocytes after intravenous injection. Accordingly, to avoid appreciable binding of GST-RAP to the LDL receptor, we first determined the smallest dose of GST-RAP, given as a pulse intravenously and followed by a continuous infusion, that would completely prevent binding of a known high affinity ligand for LRP in fasted male Sprague-Dawley rats. As shown in Fig. 1, a pulse of 2 mg of GST-RAP, followed by infusion at a rate of 0.13 mg/min for 30 min, almost completely eliminated the clearance of 50 μg of activated α2MG, whereas a 50% lower amount of GST-RAP yielded only partial inhibition of clearance as compared with infusion of GST. The effective dose was utilized in all subsequent experiments. Effect of GST-RAP on the Concentration of HL and LPL in Plasma and Liver and Their Enrichment in Hepatocytic Endosomes—During the 30-min infusion of GST-RAP, the concentration of HL increased progressively in blood plasma (Fig. 2A and Table II). After 30 min, the concentration of HL in plasma had increased significantly by 20% in animals given GST-RAP, whereas no change occurred in control animals given GST. However, the liver still contained 99% of the total mass of HL in liver + plasma in rats given GST-RAP, as it did in those given GST (Table II). The increasing concentration of plasma HL in the GST-RAP group is consistent with reduced internalization of hepatic HL, which is mainly extracellular (3Shafi S. Brady S.E. Bensadoun A. Havel R.J. J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar, 33Schoonderwoerd K. Verhoeven A.J. Jansen H. Biochem. J. 1994; 302: 717-722Crossref PubMed Scopus (12) Google Scholar). Indeed, as shown in Fig. 2B, enrichment of HL in the hepatocytic total endosome fraction was about 50% lower in animals given GST-RAP than in those given GST (p < 0.05). The effect of GST-RAP infusion on the concentration of LPL in plasma and its enrichment in endosomes was more pronounced than that observed for HL. As shown in Fig. 2C and Table III, the concentration of LPL in plasma was 2.5-fold higher in rats given GST-RAP than in those given GST, and LPL became undetectable in hepatocytic endosomes in the former group. Given the sensitivity of the ELISA for LPL, we estimate that endosomal enrichment of LPL was reduced by at least 95%. Enrichment of LPL in endosomes of control animals given GST (10-fold) was similar to that of HL in corresponding controls (8-fold), even though the mass of LPL in liver was less than 1% that of HL (compare Tables II and III). Livers contained 96.5% of the combined mass of LPL in liver + plasma in GST-infused control animals. This value was lower, 93.8%, in animals given GST-RAP (Table III). These results, taken together, suggest that HL is internalized into hepatocytes in part, and LPL is internalized almost entirely by a RAP-sensitive process or processes.Table IIConcentration and distribution of HL in liver and plasma during infusions of GST or GST-RAP Total HL mass in plasma was calculated for a plasma volume of 4.5% of body weight. Total HL mass in liver was calculated by multiplying the concentration values by liver total protein. Values are mean ± S.E.Duration of infusionPlasma HLGSTGST-RAPminng/ml%ng/ml%170.6 ± 8.21.0 ± 0.177.9 ± 8.51.1 ± 0.11571.8 ± 6.81.2 ± 0.280.8 ± 11.81.1 ± 0.23073.8 ± 8.11.1 ± 0.289.4 ± 8.81.2 ± 0.2Hepatic HLGSTGST-RAPng/mg%ng/mg%78.4 ± 5.998.9 ± 0.286.1 ± 5.698.8 ± 0.2 Open table in a new tab Table IIIConcentration and % distribution of LPL in plasma and liver after 30-min infusions of GST or GST-RAP Percentages were calculated as in Table II. Values are mean ± S.E. (n = 6).Plasma LPLGSTGST-RAPng/ml%ng/ml%1.0 ± 0.33.5 ± 0.92.4 ± 0.46.2 ± 0.8Hepatic LPLGSTGST-RAPng/mg protein%ng/mg protein%0.3 ± 0.096.5 ± 0.90.4 ± 0.093.8 ± 0.8 Open table in a new tab Effect of GST-RAP Upon Endosomal Enrichment of LRP and Other High Affinity Endocytic Receptors—The selected dose of GST-RAP used in these experiments was sufficient to block completely the clearance of α2-MG from plasma and, by inference, its endocytosis into hepatocytes by LRP (18Mokuno H. Brady S. Kotite L. Herz J. Havel R.J. J. Biol. Chem. 1994; 269: 13238-13243Abstract Full Text PDF PubMed Google Scholar). LRP is enriched in hepatocytic endosome fractions from untreated as well as estradiol-treated rats in a pattern consistent with extensive receptor recycling (37Lund H. Takahashi K. Hamilton R.L. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9318-9322Crossref PubMed Scopus (76) Google Scholar). GST-RAP is known to block similarly the endocytosis of all other high affinity ligands for LRP (38Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1054) Google Scholar). We found no effect of the 30-min infusion of GST-RAP upon the mass of LRP in liver homogenates, as estimated by Western blotting (Fig. 3A). As shown in Fig. 3, A and B, however, endosomal enrichment was about 40% lower in animals given GST-RAP than in those given GST (p < 0.05). As in estradiol-treated rats (23Belcher J.D. Hamilton R.L. Brady S.E. Hornick C.A. Jaeckle S. Schneider W.J. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6785-6789Crossref PubMed Scopus (68) Google Scholar), the LDL receptor was also comparably enriched in endosome fractions from untreated rats in a pattern consistent with receptor recycling. 2M. Vergés and R. Havel, unpublished observations. GST-RAP infusion had no effect upon its endosomal enrichment, however. Furthermore, enrichment of other high affinity receptors studied previously in untreated or estradiol-treated rats (35Jäckle S. Runquist E.A. Miranda-Brady S. Havel R.J. J. Biol. Chem. 1991; 266: 1396-1402Abstract Full Text PDF PubMed Google Scholar, 39Vergés M. Havel R.J. Mostov K.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10146-10151Crossref PubMed Scopus (39) Google Scholar) was unaffected by GST-RAP infusion (Fig. 3B). Effect of GST-RAP Upon Endosomal Purity and Endosomal Trafficking Machinery—To determine whether GST-RAP affected the purity of our endosome fractions, we first addressed Golgi contamination by probing for GM130, a cis-Golgi marker (40Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (663) Google Scholar). GM130 was only slightly enriched in endosomes from GST-infused animals (∼3-fold over homogenate), and no difference was found in endosomes from animals given GST-RAP (data not shown). Enrichment of total cholesterol and triglycerides in endosomes was also unaffected by GST-RAP infusion (Table IV). Plasma concentrations of total cholesterol and triglycerides in blood samples taken at the end of the infusions were also comparable in animals given GST (cholesterol 57 ± 3.7 mg/dl and triglycerides 66 ± 7.4 mg/dl) and GST-RAP (cholesterol 49 ± 4.4 mg/dl and triglycerides 55 ± 6.4 mg/dl, n = 6).Table IVTotal cholesterol and triglycerides in hepatocytic endosomes after infusion of GST or GST-RAP Fold enrichment for total cholesterol (TC) and triglycerides (TG) was determined as describe" @default.
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