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W2085679502 abstract "We have recently described a novel recycling pathway of triglyceride-rich lipoprotein (TRL)-associated apolipoprotein (apo) E in human hepatoma cells. We now demonstrate that not only TRL-derived apoE but also lipoprotein lipase (LPL) is efficiently recycled in vitro and in vivo. Similar recycling kinetics of apoE and LPL in normal and low density lipoprotein receptor-negative human fibroblasts also indicate that the low density lipoprotein receptor-related protein seems to be involved. Intracellular sorting mechanisms are responsible for reduced lysosomal degradation of both ligands after receptor-mediated internalization. Immediately after internalization in rat liver, TRLs are disintegrated, and apoE and LPL are found in endosomal compartments, whereas TRL-derived phospholipids accumulate in the perinuclear region of hepatocytes. Subsequently, substantial amounts of both proteins can be found in purified recycling endosomes, indicating a potential resecretion of these TRL components. Pulse-chase experiments of perfused rat livers with radiolabeled TRLs demonstrated a serum-induced release of internalized apoE and LPL into the perfusate. Analysis of the secreted proteins identified ∼80% of the recycled TRL-derived proteins in the high density lipoprotein fractions. These results provide the first evidence that recycling of TRL-derived apoE and LPL could play an important role in the modulation of lipoproteins in vivo. We have recently described a novel recycling pathway of triglyceride-rich lipoprotein (TRL)-associated apolipoprotein (apo) E in human hepatoma cells. We now demonstrate that not only TRL-derived apoE but also lipoprotein lipase (LPL) is efficiently recycled in vitro and in vivo. Similar recycling kinetics of apoE and LPL in normal and low density lipoprotein receptor-negative human fibroblasts also indicate that the low density lipoprotein receptor-related protein seems to be involved. Intracellular sorting mechanisms are responsible for reduced lysosomal degradation of both ligands after receptor-mediated internalization. Immediately after internalization in rat liver, TRLs are disintegrated, and apoE and LPL are found in endosomal compartments, whereas TRL-derived phospholipids accumulate in the perinuclear region of hepatocytes. Subsequently, substantial amounts of both proteins can be found in purified recycling endosomes, indicating a potential resecretion of these TRL components. Pulse-chase experiments of perfused rat livers with radiolabeled TRLs demonstrated a serum-induced release of internalized apoE and LPL into the perfusate. Analysis of the secreted proteins identified ∼80% of the recycled TRL-derived proteins in the high density lipoprotein fractions. These results provide the first evidence that recycling of TRL-derived apoE and LPL could play an important role in the modulation of lipoproteins in vivo. triglyceride-rich lipoprotein apolipoprotein compartment of uncoupling of receptors and ligands fast performance liquid chromatography high density lipoprotein low density lipoprotein low density lipoprotein receptor lipoprotein lipase low density lipoprotein-related protein multivesicular body receptor recycling compartment very low density lipoprotein 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine SDS-polyacrylamide gel electrophoresis familial hypercholesterolemia receptor-associated protein Triglycerides are transported mainly by two distinct classes of triglyceride-rich lipoproteins (TRLs),1 the chylomicrons and the very low density lipoproteins (VLDLs). After assembly in the intestine, chylomicrons are transported via lymph into the bloodstream, where they are converted at the endothelial surface to remnant lipoproteins through the catalytic action of lipoprotein lipase (LPL) (for review, see Refs. 1Olivecrona G. Olivecrona T. Curr. Opin. Lipidol. 1995; 6: 291-305Crossref PubMed Scopus (182) Google Scholar and 2Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). After lipolysis, LPL remains associated with the chylomicron remnants and, in concert with apolipoprotein (apo) E (3Felts J.M. Itakura H. Crane R.T. Biochem. Biophys. Res. Commun. 1975; 66: 1467-1475Crossref PubMed Scopus (114) Google Scholar, 4Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar, 5Zambon A. Schmidt I. Beisiegel U. Brunzell J.D. J. Lipid Res. 1996; 37: 2394-2404Abstract Full Text PDF PubMed Google Scholar), facilitates their clearance into hepatocytes (6Heeren J. Beisiegel U. Curr. Opin. Lipidol. 2001; 12: 255-260Crossref PubMed Scopus (26) Google Scholar) via LDL receptor (LDLR) and the LDLR-related protein (LRP) (7Beisiegel U. Weber W. Ihrke G. Herz J. Stanley K.K. Nature. 1989; 341: 162-164Crossref PubMed Scopus (543) Google Scholar, 8Beisiegel U. Heeren J. Proc. Nutr. Soc. 1997; 56: 731-737Crossref PubMed Scopus (32) Google Scholar, 9Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (453) Google Scholar, 10Bradley W.A. Gianturco S.H. J. Lipid Res. 1986; 27: 40-48Abstract Full Text PDF PubMed Google Scholar). The essential role for both receptors in TRL removal in vivo has been demonstrated in gene knockout and gene transfer experiments (Refs. 11Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar and 12Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (252) Google Scholar; for a recent review, see Ref.13Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (185) Google Scholar).Several studies have used different “model particles” to investigate the intracellular processing of TRL constituents. In contrast to the lysosomal degradation of LDL-derived apoB (14Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4308) Google Scholar), β-VLDL-derived apoE was identified in widely distributed vesicles and showed a slow protein degradation in mouse macrophages (15Tabas I. Lim S. Xu X.X. Maxfield F.R. J. Cell Biol. 1990; 111: 929-940Crossref PubMed Scopus (96) Google Scholar, 16Tabas I. Myers J.N. Innerarity T.L. Xu X.X. Arnold K. Boyles J. Maxfield F.R. J. Cell Biol. 1991; 115: 1547-1560Crossref PubMed Scopus (62) Google Scholar). However, in the same cells, β-VLDL-derived lipids were delivered to perinuclear, lysosomal compartments (17Myers J.N. Tabas I. Jones N.L. Maxfield F.R. J. Cell Biol. 1993; 123: 1389-1402Crossref PubMed Scopus (66) Google Scholar). Delayed transport and degradation of TRL proteins were also observed in hepatoma cells (18Lombardi P. Mulder M. van der Boom H. Frants R.R. Havekes L.M. J. Biol. Chem. 1993; 268: 26113-26119Abstract Full Text PDF PubMed Google Scholar,19Jones A.L. Hradek G.T. Hornick C. Renaud G. Windler E.E. Havel R.J. J. Lipid Res. 1984; 25: 1151-1158Abstract Full Text PDF PubMed Google Scholar). In recent studies, we have been able to demonstrate that the altered transport and retarded degradation of internalized TRLs is due to intracellular disintegration and sorting of TRL components in a peripheral cellular compartment. Whereas lipids are directed to lysosomal compartments in human hepatoma cells and fibroblasts, TRL-derived apoE and apoC are recycled back to the cell surface, where resecretion can occur (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). Accumulating evidence indicates that the complex intracellular processing of TRL constituents also exists in vivo. An increased intracellular resistance to lysosomal degradation of apoE compared with cholesteryl oleate was demonstrated in C57Bl/6 mice after hepatic uptake of triglyceride-rich emulsion particles (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Furthermore, Fazio and co-workers (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 23Swift L.L. Farkas M.H. Major A.S. Valyi-Nagy K. Linton M.F. Fazio S. J. Biol. Chem. 2001; 276: 22965-22970Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) identified significant amounts of internalized apoE derived from β-VLDL in Golgi-enriched fractions of mouse liver. These findings indicate that processing of internalized apoE might occur through distinct endosomal compartments.In this study, we addressed the question of whether recycling of TRL-derived apoE and LPL could play a role in hepatic lipoprotein metabolism in vivo. The disintegration of TRL particles within sorting endosomes could be demonstrated in rat liver, where the TRL lipids can be detected in lysosomal compartments, whereas TRL-derived apoE and LPL are found in a peripheral endosomal compartment. Mobilization and subsequent resecretion of TRL-derived apoE and LPL are induced in the presence of serum. These data suggest that apoE and LPL recycling plays an important role in apoE enrichment of HDL precursors and reutilization of LPL in the space of Disse.DISCUSSIONWe have recently proposed a model of intracellular TRL processing that comprises both recycling and degradation of TRL components (6Heeren J. Beisiegel U. Curr. Opin. Lipidol. 2001; 12: 255-260Crossref PubMed Scopus (26) Google Scholar,20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). In the current study, we demonstrated that significant amounts of125I-apoE-TRL and even more apoE-containing125I-LPL-TRL were not degraded but recycled back to the cell surface. Therefore, the association of LPL with apoE-containing TRLs not only stimulates TRL internalization but also reduces its lysosomal degradation compared with apoE-TRL (Figs. 1 and 2). Although internalization of apoE-containing lipoproteins is thought to be mediated in part by cell surface heparan sulfate proteoglycans (2Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar), recent experiments have demonstrated that both LRP and the LDL receptor (11Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar) are essential for hepatic uptake of TRL lipoproteins. However, the possible involvement of the two lipoprotein receptors in the recycling of apoE is a topic of disagreement (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which might be due to the different “model particles” used in these studies. In agreement with the recycling of apoE in LDLR(−/−) mice (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), our results imply that LRP probably participates in the intracellular processing of human TRL-derived apoE and LPL, leading to the recycling of both proteins (Fig. 3). Results presented here indicate that the composition of ligands seems to determine their specific intracellular fate. Thus, the high binding affinities of multivalent ligands on lipoproteins to receptors seem to play an important role in their intracellular metabolism. In support of this hypothesis, the most likely altered ligand binding affinity of apoE3 and E4-enriched β-VLDL has recently been shown to result in different intracellular processing after internalization (33Ji Z.S. Pitas R.E. Mahley R.W. J. Biol. Chem. 1998; 273: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).The main focus of our studies was to characterize TRL processing in vivo. As observed in cell culture experiments (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), the disintegration of TRLs leads to a peripheral, endosomal distribution of apoE, whereas the majority of lipids seem to accumulate in the perinuclear, prelysosomal compartment (Fig. 4). These findings correlate with the relative resistance of apoE against degradation and the concomitant hydrolysis of cholesterol ester from internalized apoE-cholesterol ester-labeled liposomes in mouse liver (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition, Fazio et al. (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) demonstrated the reutilization of internalized apoE for VLDL assembly in Golgi-enriched fractions. In support of these observations, we detected apoE and LPL in three different endosomal preparations from rat liver during analysis of the intracellular fate of internalized TRL protein components in vivo.First of all, CURL represents the early endosomal sorting compartment and contains internalized radiolabeled LDL or TRL that will subsequently be directed to either RRC or MVB vesicles (30Enrich C. Pol A. Calvo M. Pons M. Jackle S. Hepatology. 1999; 30: 1115-1120Crossref PubMed Scopus (13) Google Scholar). RRC endosomal preparations contain substantial amounts of TRL-derived apoE and LPL (Fig. 5). Although RRC fractions are highly enriched for recycling proteins (e.g. transferrin or LDL receptor), it has been demonstrated that these preparations also contain 5′-nucleotidase and sialyl-transferase activity specific for Golgi secretory vesicles (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These findings correlate with the presence of apoE in Golgi-enriched fractions (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and indicate that recycling of apoE and LPL via RRC could be mediated in part by Golgi-derived secretory vesicles. In addition, significant amounts of radiolabeled apoE and LPL were found in MVBs, which represent predominantly late endosomes. However, because LRP and LDL receptor (Fig. 5 c) can be found in the MVB fraction, it can be postulated that a significant portion of recycling endosomes is present in this fraction, as also observed by others (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These vesicles, in addition to RRC endosomes, are likely to be responsible for apoE and LPL recycling.A number of studies have recently postulated the recycling of internalized apoE in mice hepatocytes (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 23Swift L.L. Farkas M.H. Major A.S. Valyi-Nagy K. Linton M.F. Fazio S. J. Biol. Chem. 2001; 276: 22965-22970Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We now provide direct evidence that TRL-derived apoE and LPL are efficiently recycled and resecreted in vivo (Fig. 6 a). Because we have recently described that HDL serves as an extracellular acceptor for the resecretion of apoE (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), HDL was utilized to stimulate apoE and LPL resecretion after TRL internalization in perfused rat livers (Fig. 6 b). Similar to our observations in vitro (Ref.20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar; see also Fig. 2) we determined an ∼60% resecretion of internalized TRL-derived apoE and LPL. Only a minor proportion of radioactivity, representing predominantly LPL, was detected in the VLDL fraction after FPLC and SDS-PAGE analysis (Fig. 6, b and c). In contrast, the majority of resecreted apoE-derived radioactivity was found in HDL, indicating a reutilization of TRL-derived apoproteins for HDL modulation. These apoE-enriched HDL particles would provide a pool of apoE proteins in the plasma, possibly serving as an apoE donor for intravascular transfer to chylomicrons during lipolysis. Although endogenously synthesized apoE might be able to fulfill this function, recycling of apoE would provide a more readily available pool to promote apoE-mediated chylomicron remnant uptake in the postprandial state.In conclusion, we have demonstrated that significant amounts of internalized TRL-derived apoE and LPL escape the lysosomal pathway and are targeted in a new recycling compartment for resecretion. The resecreted apoE and LPL seem to participate in the modulation of VLDL and HDL in vivo. Because apoE recycling depends on the presence of extracellular HDL, future experiments will have to clarify a potential regulatory role of apoE recycling in HDL-induced cholesterol efflux or HDL catabolism. Triglycerides are transported mainly by two distinct classes of triglyceride-rich lipoproteins (TRLs),1 the chylomicrons and the very low density lipoproteins (VLDLs). After assembly in the intestine, chylomicrons are transported via lymph into the bloodstream, where they are converted at the endothelial surface to remnant lipoproteins through the catalytic action of lipoprotein lipase (LPL) (for review, see Refs. 1Olivecrona G. Olivecrona T. Curr. Opin. Lipidol. 1995; 6: 291-305Crossref PubMed Scopus (182) Google Scholar and 2Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). After lipolysis, LPL remains associated with the chylomicron remnants and, in concert with apolipoprotein (apo) E (3Felts J.M. Itakura H. Crane R.T. Biochem. Biophys. Res. Commun. 1975; 66: 1467-1475Crossref PubMed Scopus (114) Google Scholar, 4Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar, 5Zambon A. Schmidt I. Beisiegel U. Brunzell J.D. J. Lipid Res. 1996; 37: 2394-2404Abstract Full Text PDF PubMed Google Scholar), facilitates their clearance into hepatocytes (6Heeren J. Beisiegel U. Curr. Opin. Lipidol. 2001; 12: 255-260Crossref PubMed Scopus (26) Google Scholar) via LDL receptor (LDLR) and the LDLR-related protein (LRP) (7Beisiegel U. Weber W. Ihrke G. Herz J. Stanley K.K. Nature. 1989; 341: 162-164Crossref PubMed Scopus (543) Google Scholar, 8Beisiegel U. Heeren J. Proc. Nutr. Soc. 1997; 56: 731-737Crossref PubMed Scopus (32) Google Scholar, 9Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (453) Google Scholar, 10Bradley W.A. Gianturco S.H. J. Lipid Res. 1986; 27: 40-48Abstract Full Text PDF PubMed Google Scholar). The essential role for both receptors in TRL removal in vivo has been demonstrated in gene knockout and gene transfer experiments (Refs. 11Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar and 12Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (252) Google Scholar; for a recent review, see Ref.13Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (185) Google Scholar). Several studies have used different “model particles” to investigate the intracellular processing of TRL constituents. In contrast to the lysosomal degradation of LDL-derived apoB (14Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4308) Google Scholar), β-VLDL-derived apoE was identified in widely distributed vesicles and showed a slow protein degradation in mouse macrophages (15Tabas I. Lim S. Xu X.X. Maxfield F.R. J. Cell Biol. 1990; 111: 929-940Crossref PubMed Scopus (96) Google Scholar, 16Tabas I. Myers J.N. Innerarity T.L. Xu X.X. Arnold K. Boyles J. Maxfield F.R. J. Cell Biol. 1991; 115: 1547-1560Crossref PubMed Scopus (62) Google Scholar). However, in the same cells, β-VLDL-derived lipids were delivered to perinuclear, lysosomal compartments (17Myers J.N. Tabas I. Jones N.L. Maxfield F.R. J. Cell Biol. 1993; 123: 1389-1402Crossref PubMed Scopus (66) Google Scholar). Delayed transport and degradation of TRL proteins were also observed in hepatoma cells (18Lombardi P. Mulder M. van der Boom H. Frants R.R. Havekes L.M. J. Biol. Chem. 1993; 268: 26113-26119Abstract Full Text PDF PubMed Google Scholar,19Jones A.L. Hradek G.T. Hornick C. Renaud G. Windler E.E. Havel R.J. J. Lipid Res. 1984; 25: 1151-1158Abstract Full Text PDF PubMed Google Scholar). In recent studies, we have been able to demonstrate that the altered transport and retarded degradation of internalized TRLs is due to intracellular disintegration and sorting of TRL components in a peripheral cellular compartment. Whereas lipids are directed to lysosomal compartments in human hepatoma cells and fibroblasts, TRL-derived apoE and apoC are recycled back to the cell surface, where resecretion can occur (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). Accumulating evidence indicates that the complex intracellular processing of TRL constituents also exists in vivo. An increased intracellular resistance to lysosomal degradation of apoE compared with cholesteryl oleate was demonstrated in C57Bl/6 mice after hepatic uptake of triglyceride-rich emulsion particles (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Furthermore, Fazio and co-workers (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 23Swift L.L. Farkas M.H. Major A.S. Valyi-Nagy K. Linton M.F. Fazio S. J. Biol. Chem. 2001; 276: 22965-22970Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) identified significant amounts of internalized apoE derived from β-VLDL in Golgi-enriched fractions of mouse liver. These findings indicate that processing of internalized apoE might occur through distinct endosomal compartments. In this study, we addressed the question of whether recycling of TRL-derived apoE and LPL could play a role in hepatic lipoprotein metabolism in vivo. The disintegration of TRL particles within sorting endosomes could be demonstrated in rat liver, where the TRL lipids can be detected in lysosomal compartments, whereas TRL-derived apoE and LPL are found in a peripheral endosomal compartment. Mobilization and subsequent resecretion of TRL-derived apoE and LPL are induced in the presence of serum. These data suggest that apoE and LPL recycling plays an important role in apoE enrichment of HDL precursors and reutilization of LPL in the space of Disse. DISCUSSIONWe have recently proposed a model of intracellular TRL processing that comprises both recycling and degradation of TRL components (6Heeren J. Beisiegel U. Curr. Opin. Lipidol. 2001; 12: 255-260Crossref PubMed Scopus (26) Google Scholar,20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). In the current study, we demonstrated that significant amounts of125I-apoE-TRL and even more apoE-containing125I-LPL-TRL were not degraded but recycled back to the cell surface. Therefore, the association of LPL with apoE-containing TRLs not only stimulates TRL internalization but also reduces its lysosomal degradation compared with apoE-TRL (Figs. 1 and 2). Although internalization of apoE-containing lipoproteins is thought to be mediated in part by cell surface heparan sulfate proteoglycans (2Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar), recent experiments have demonstrated that both LRP and the LDL receptor (11Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar) are essential for hepatic uptake of TRL lipoproteins. However, the possible involvement of the two lipoprotein receptors in the recycling of apoE is a topic of disagreement (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which might be due to the different “model particles” used in these studies. In agreement with the recycling of apoE in LDLR(−/−) mice (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), our results imply that LRP probably participates in the intracellular processing of human TRL-derived apoE and LPL, leading to the recycling of both proteins (Fig. 3). Results presented here indicate that the composition of ligands seems to determine their specific intracellular fate. Thus, the high binding affinities of multivalent ligands on lipoproteins to receptors seem to play an important role in their intracellular metabolism. In support of this hypothesis, the most likely altered ligand binding affinity of apoE3 and E4-enriched β-VLDL has recently been shown to result in different intracellular processing after internalization (33Ji Z.S. Pitas R.E. Mahley R.W. J. Biol. Chem. 1998; 273: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).The main focus of our studies was to characterize TRL processing in vivo. As observed in cell culture experiments (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), the disintegration of TRLs leads to a peripheral, endosomal distribution of apoE, whereas the majority of lipids seem to accumulate in the perinuclear, prelysosomal compartment (Fig. 4). These findings correlate with the relative resistance of apoE against degradation and the concomitant hydrolysis of cholesterol ester from internalized apoE-cholesterol ester-labeled liposomes in mouse liver (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition, Fazio et al. (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) demonstrated the reutilization of internalized apoE for VLDL assembly in Golgi-enriched fractions. In support of these observations, we detected apoE and LPL in three different endosomal preparations from rat liver during analysis of the intracellular fate of internalized TRL protein components in vivo.First of all, CURL represents the early endosomal sorting compartment and contains internalized radiolabeled LDL or TRL that will subsequently be directed to either RRC or MVB vesicles (30Enrich C. Pol A. Calvo M. Pons M. Jackle S. Hepatology. 1999; 30: 1115-1120Crossref PubMed Scopus (13) Google Scholar). RRC endosomal preparations contain substantial amounts of TRL-derived apoE and LPL (Fig. 5). Although RRC fractions are highly enriched for recycling proteins (e.g. transferrin or LDL receptor), it has been demonstrated that these preparations also contain 5′-nucleotidase and sialyl-transferase activity specific for Golgi secretory vesicles (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These findings correlate with the presence of apoE in Golgi-enriched fractions (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and indicate that recycling of apoE and LPL via RRC could be mediated in part by Golgi-derived secretory vesicles. In addition, significant amounts of radiolabeled apoE and LPL were found in MVBs, which represent predominantly late endosomes. However, because LRP and LDL receptor (Fig. 5 c) can be found in the MVB fraction, it can be postulated that a significant portion of recycling endosomes is present in this fraction, as also observed by others (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These vesicles, in addition to RRC endosomes, are likely to be responsible for apoE and LPL recycling.A number of studies have recently postulated the recycling of internalized apoE in mice hepatocytes (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 23Swift L.L. Farkas M.H. Major A.S. Valyi-Nagy K. Linton M.F. Fazio S. J. Biol. Chem. 2001; 276: 22965-22970Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We now provide direct evidence that TRL-derived apoE and LPL are efficiently recycled and resecreted in vivo (Fig. 6 a). Because we have recently described that HDL serves as an extracellular acceptor for the resecretion of apoE (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), HDL was utilized to stimulate apoE and LPL resecretion after TRL internalization in perfused rat livers (Fig. 6 b). Similar to our observations in vitro (Ref.20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar; see also Fig. 2) we determined an ∼60% resecretion of internalized TRL-derived apoE and LPL. Only a minor proportion of radioactivity, representing predominantly LPL, was detected in the VLDL fraction after FPLC and SDS-PAGE analysis (Fig. 6, b and c). In contrast, the majority of resecreted apoE-derived radioactivity was found in HDL, indicating a reutilization of TRL-derived apoproteins for HDL modulation. These apoE-enriched HDL particles would provide a pool of apoE proteins in the plasma, possibly serving as an apoE donor for intravascular transfer to chylomicrons during lipolysis. Although endogenously synthesized apoE might be able to fulfill this function, recycling of apoE would provide a more readily available pool to promote apoE-mediated chylomicron remnant uptake in the postprandial state.In conclusion, we have demonstrated that significant amounts of internalized TRL-derived apoE and LPL escape the lysosomal pathway and are targeted in a new recycling compartment for resecretion. The resecreted apoE and LPL seem to participate in the modulation of VLDL and HDL in vivo. Because apoE recycling depends on the presence of extracellular HDL, future experiments will have to clarify a potential regulatory role of apoE recycling in HDL-induced cholesterol efflux or HDL catabolism. We have recently proposed a model of intracellular TRL processing that comprises both recycling and degradation of TRL components (6Heeren J. Beisiegel U. Curr. Opin. Lipidol. 2001; 12: 255-260Crossref PubMed Scopus (26) Google Scholar,20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). In the current study, we demonstrated that significant amounts of125I-apoE-TRL and even more apoE-containing125I-LPL-TRL were not degraded but recycled back to the cell surface. Therefore, the association of LPL with apoE-containing TRLs not only stimulates TRL internalization but also reduces its lysosomal degradation compared with apoE-TRL (Figs. 1 and 2). Although internalization of apoE-containing lipoproteins is thought to be mediated in part by cell surface heparan sulfate proteoglycans (2Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar), recent experiments have demonstrated that both LRP and the LDL receptor (11Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar) are essential for hepatic uptake of TRL lipoproteins. However, the possible involvement of the two lipoprotein receptors in the recycling of apoE is a topic of disagreement (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which might be due to the different “model particles” used in these studies. In agreement with the recycling of apoE in LDLR(−/−) mice (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), our results imply that LRP probably participates in the intracellular processing of human TRL-derived apoE and LPL, leading to the recycling of both proteins (Fig. 3). Results presented here indicate that the composition of ligands seems to determine their specific intracellular fate. Thus, the high binding affinities of multivalent ligands on lipoproteins to receptors seem to play an important role in their intracellular metabolism. In support of this hypothesis, the most likely altered ligand binding affinity of apoE3 and E4-enriched β-VLDL has recently been shown to result in different intracellular processing after internalization (33Ji Z.S. Pitas R.E. Mahley R.W. J. Biol. Chem. 1998; 273: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The main focus of our studies was to characterize TRL processing in vivo. As observed in cell culture experiments (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), the disintegration of TRLs leads to a peripheral, endosomal distribution of apoE, whereas the majority of lipids seem to accumulate in the perinuclear, prelysosomal compartment (Fig. 4). These findings correlate with the relative resistance of apoE against degradation and the concomitant hydrolysis of cholesterol ester from internalized apoE-cholesterol ester-labeled liposomes in mouse liver (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition, Fazio et al. (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) demonstrated the reutilization of internalized apoE for VLDL assembly in Golgi-enriched fractions. In support of these observations, we detected apoE and LPL in three different endosomal preparations from rat liver during analysis of the intracellular fate of internalized TRL protein components in vivo. First of all, CURL represents the early endosomal sorting compartment and contains internalized radiolabeled LDL or TRL that will subsequently be directed to either RRC or MVB vesicles (30Enrich C. Pol A. Calvo M. Pons M. Jackle S. Hepatology. 1999; 30: 1115-1120Crossref PubMed Scopus (13) Google Scholar). RRC endosomal preparations contain substantial amounts of TRL-derived apoE and LPL (Fig. 5). Although RRC fractions are highly enriched for recycling proteins (e.g. transferrin or LDL receptor), it has been demonstrated that these preparations also contain 5′-nucleotidase and sialyl-transferase activity specific for Golgi secretory vesicles (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These findings correlate with the presence of apoE in Golgi-enriched fractions (22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and indicate that recycling of apoE and LPL via RRC could be mediated in part by Golgi-derived secretory vesicles. In addition, significant amounts of radiolabeled apoE and LPL were found in MVBs, which represent predominantly late endosomes. However, because LRP and LDL receptor (Fig. 5 c) can be found in the MVB fraction, it can be postulated that a significant portion of recycling endosomes is present in this fraction, as also observed by others (29Pol A. Ortega D. Enrich C. Biochem. J. 1997; 323: 435-443Crossref PubMed Scopus (35) Google Scholar). These vesicles, in addition to RRC endosomes, are likely to be responsible for apoE and LPL recycling. A number of studies have recently postulated the recycling of internalized apoE in mice hepatocytes (21Rensen P.C. Jong M.C. van Vark L.C. van der Boom H. Hendriks W.L. van Berkel T.J. Biessen E.A. Havekes L.M. J. Biol. Chem. 2000; 275: 8564-8571Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Fazio S. Linton M.F. Hasty A.H. Swift L.L. J. Biol. Chem. 1999; 274: 8247-8253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 23Swift L.L. Farkas M.H. Major A.S. Valyi-Nagy K. Linton M.F. Fazio S. J. Biol. Chem. 2001; 276: 22965-22970Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We now provide direct evidence that TRL-derived apoE and LPL are efficiently recycled and resecreted in vivo (Fig. 6 a). Because we have recently described that HDL serves as an extracellular acceptor for the resecretion of apoE (20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar), HDL was utilized to stimulate apoE and LPL resecretion after TRL internalization in perfused rat livers (Fig. 6 b). Similar to our observations in vitro (Ref.20Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar; see also Fig. 2) we determined an ∼60% resecretion of internalized TRL-derived apoE and LPL. Only a minor proportion of radioactivity, representing predominantly LPL, was detected in the VLDL fraction after FPLC and SDS-PAGE analysis (Fig. 6, b and c). In contrast, the majority of resecreted apoE-derived radioactivity was found in HDL, indicating a reutilization of TRL-derived apoproteins for HDL modulation. These apoE-enriched HDL particles would provide a pool of apoE proteins in the plasma, possibly serving as an apoE donor for intravascular transfer to chylomicrons during lipolysis. Although endogenously synthesized apoE might be able to fulfill this function, recycling of apoE would provide a more readily available pool to promote apoE-mediated chylomicron remnant uptake in the postprandial state. In conclusion, we have demonstrated that significant amounts of internalized TRL-derived apoE and LPL escape the lysosomal pathway and are targeted in a new recycling compartment for resecretion. The resecreted apoE and LPL seem to participate in the modulation of VLDL and HDL in vivo. Because apoE recycling depends on the presence of extracellular HDL, future experiments will have to clarify a potential regulatory role of apoE recycling in HDL-induced cholesterol efflux or HDL catabolism. We are grateful to J. Hoeppner and W. Tauscher for excellent technical assistance. We thank Drs. G. Olivecrona, S. K. Moestrup, J. Herz, and C. Enrich for generously providing bovine LPL, recombinant proteins, antibodies, and technical advice." @default.
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W2085679502 title "Recycling of Apolipoprotein E and Lipoprotein Lipase through Endosomal Compartments in Vivo" @default.
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