Microsomal Triacylglycerol Transfer Protein Is Required for Lumenal Accretion of Triacylglycerol Not Associated with ApoB, as Well as for ApoB Lipidation
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m202015200
ISSN1083-351X
AutoresAgnes Kulinski, Sabina Rustaeus, Jean E. Vance,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranesin vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by ∼45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes. The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranesin vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by ∼45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes. very low density lipoprotein apolipoprotein Dulbecco's modified Eagle's medium endoplasmic reticulum low density lipoprotein receptor microsomal triacylglycerol transfer protein piperazine-N,N′-bis(2-ethanesulfonic acid) triacylglycerol During assembly of very low density lipoproteins (VLDLs)1 in the liver, triacylglycerol (TG) is concentrated within the hydrophobic core of apolipoprotein (apo) B-containing lipoprotein particles. A microsomal lumenal protein, the microsomal triacylglycerol transfer protein (MTP), has been implicated in the acquisition of TG by nascent apoB for assembly and secretion of VLDLs (reviewed in Refs. 1Berriot-Varoqueaux N. Aggerbeck L.P. Samson-Bouma M. Wetterau J.R. Annu. Rev. Nutr. 2000; 20: 663-697Crossref PubMed Scopus (261) Google Scholar, 2Gordon D.A. Jamil H. Biochim. Biophys. Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar, 3Davis R.A. Biochim. Biophys. Acta. 1999; 1440: 1-31Crossref PubMed Scopus (163) Google Scholar, 4Kang S. Davis R.A. Biochim. Biophys. Acta. 2000; 1529: 223-230Crossref PubMed Scopus (66) Google Scholar). Individuals with the rare inherited disease abetalipoproteinemia have a defect in the MTP gene and lack detectable MTP protein and MTP lipid transfer activity (5Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M.-E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Nature. 1993; 365: 65-68Crossref PubMed Scopus (399) Google Scholar). Despite a normal apoB gene, plasma apoB is barely detectable in these patients. MTP has the ability to transfer TG and other lipids, including cholesteryl esters, diacylglycerols, and phospholipids, between membranes in vitro (6Jamil H. Dickson J.K. Chu C.-H. Lago M.W. Rinehart J.K. Biller S.A. Gregg R.E. Wetterau J.R. J. Biol. Chem. 1995; 270: 6549-6554Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and has been proposed to transfer TG to nascent apoB-containing lipoproteins in vivo. This idea is consistent with immunoprecipitation studies showing that apoB and MTP interact physically at early stages of VLDL assembly (7Nicodeme E. Benoist F. McLeod R. Yao Z. Scott J. Shoulders C.C. Grand-Perret T. J. Biol. Chem. 1999; 274: 1986-1993Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Hussain M.M. Bakillah A. Nayak N. Shelness G.S. J. Biol. Chem. 1998; 273: 25612-25615Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 9Wu X. Zhou M. Huang L.-S. Wetterau J. Ginsberg H.N. J. Biol. Chem. 1996; 271: 10277-10281Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). MTP is expressed primarily in the liver and intestine as a soluble heterodimer with protein-disulfide isomerase (55 kDa) (10Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar), a ubiquitous protein of the endoplasmic reticulum (ER) lumen that catalyzes disulfide bond formation during protein folding (11Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The 97-kDa MTP subunit confers all lipid transfer activity to the heterodimer (12Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (154) Google Scholar). In in vitro assays, the lipid transfer activity of MTP displays Ping Pong Bi Bi kinetics implying that MTP transfers lipids between membranes via a “shuttle” mechanism (13Atzel A. Wetterau J.R. Biochemistry. 1993; 32: 10444-10450Crossref PubMed Scopus (80) Google Scholar). The tissue and subcellular location of MTP, and its preference for transferring neutral lipids between membranes in vitro, have suggested that MTP participates in the loading of apoB with TG.Specific inhibitors of the MTP lipid transfer activity have been developed that lower plasma cholesterol levels by up to 80% in rabbits, hamsters, and rats (14Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C.-H. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S.-J. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan V.J.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. Science. 1998; 282: 751-754Crossref PubMed Scopus (251) Google Scholar). Complete elimination of theMTP gene in mice is embryonically lethal, probably because apoB-containing lipoproteins are required for transferring lipids from the yolk sac to the developing embryo (15Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8686-8691Crossref PubMed Scopus (220) Google Scholar). However, viable mice were generated in which the MTP gene was specifically inactivated in the liver (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar, 17Chang B.H.-J. Liao W. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In these mice, compared with wild-type mice, plasma apoB100 levels were reduced by >90% but apoB48 levels were reduced by only ∼20% (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar). In support of the hypothesis that MTP provides TG for VLDL assembly, the majority of apoB48 in the plasma of these MTP-deficient mice was present in lipoprotein particles that were only partially lipidated (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar). Moreover, apoB100 secretion from cultured MTP−/− hepatocytes was greatly reduced, whereas apoB48 secretion was only slightly reduced compared with that in MTP+/+ hepatocytes (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar). Ultrastructural analyses revealed that compared with MTP+/+ hepatocytes, MTP−/−hepatocytes contained very few VLDL-sized particles within the ER and Golgi lumina (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar). In addition, in mice unable to synthesize apoB in the intestine, chylomicron-sized, lipid particles (without apoB) accumulated in the ER lumen of enterocytes (18Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar) suggesting that ER lumenal TG droplets might exist as a source of TG for assembly with apoB. The importance of MTP for apoB secretion is underscored by the demonstration that overexpression of MTP activity in mouse liver increased plasma levels of apoB100 and -B48, as well as TG (19Tietge U.J.F. Bakillah A. Maugeais C. Tsukamoto K. Hussain M. Rader D.J. J. Lipid Res. 1999; 40: 2134-2139Abstract Full Text Full Text PDF PubMed Google Scholar).MTP has been reported to play several roles in VLDL assembly: (i) as a chaperone that mediates the translocation of newly synthesized apoB across the ER membrane (20Du E.Z. Wang S.-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. J. Lipid Res. 1996; 37: 1309-1315Abstract Full Text PDF PubMed Google Scholar, 21Liang J.S. Ginsberg H.N. J. Biol. Chem. 2001; 276: 28606-28612Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar); (ii) in the co-translational lipidation of apoB during its translocation into the ER lumen (22Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 23Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Mitchell D.M. Zhou M. Pariyarath R. Wang H. Aitchison J.D. Ginsberg H.N. Fisher E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14733-14738Crossref PubMed Scopus (108) Google Scholar); (ii) in the transfer of the bulk of TG to poorly lipidated apoB in the ER lumen (25Wang Y. Tran K. Yao Z. J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 26Wang S. McLeod R.S. Gordon D.A. Yao Z. J. Biol. Chem. 1996; 271: 14124-14133Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); and (iv) in the transfer of TG into the ER lumen for assembly into VLDLs (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar, 18Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar). The majority of studies on the role of MTP have been performed in cultured hepatoma cells, either HepG2 or McArdle 7777 cells, that compared with primary hepatocytes, inefficiently secrete VLDLs (21Liang J.S. Ginsberg H.N. J. Biol. Chem. 2001; 276: 28606-28612Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 23Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 27Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.K. Chen Y. Ricci B. Chu C.-H. Harrity T.W. Ciosek C.P. Biller S.A. Gregg R.E. Wetterau J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11991-11995Crossref PubMed Scopus (144) Google Scholar, 28Wang Y.W. McLeod R.S. Yao Z.M. J. Biol. Chem. 1997; 272: 12272-12278Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 29Blackhart B.D. Yao Z. McCarthy B.J. J. Biol. Chem. 1990; 265: 8358-8360Abstract Full Text PDF PubMed Google Scholar, 30Macri J. Kazemian P. Kulinski A. Rudy D. Aiton A. Thibert R.J. Adeli K. Biochem. Biophys. Res. Commun. 2000; 276: 1035-1047Crossref PubMed Scopus (10) Google Scholar). Moreover, in hepatoma cells, the activities of several enzymes involved in lipid metabolism are either absent or impaired. The function of MTP has also been investigated in HeLa and COS cells that do not normally secrete lipoproteins but in which apoB secretion was induced by transfection with cDNAs encoding MTP and apoB variants (31Thrift R.N. Drisko J. Dueland S. Trawick J.D. Davis R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9161-9165Crossref PubMed Scopus (81) Google Scholar, 32Fleming J.F. Spitsen G.M. Hui T.Y. Olivier L., Du, E.Z. Raabe M. Davis R.A. J. Biol. Chem. 1999; 274: 9509-9514Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 33Shelness G.S. Morris-Rogers K.C. Ingram M.F. J. Biol. Chem. 1994; 269: 9310-9318Abstract Full Text PDF PubMed Google Scholar). Some of the conflicting data on the role of MTP in VLDL assembly might, therefore, be related to the different model systems used. Only a few studies have been performed on the function of MTP in primary hepatocytes (34Hebbachi A.-M. Gibbons G.F. Biochim. Biophys. Acta. 1999; 1441: 36-50Crossref PubMed Scopus (18) Google Scholar, 35Hebbachi A.-M. Brown A. Gibbons G.F. J. Lipid Res. 1999; 40: 1758-1768Abstract Full Text Full Text PDF PubMed Google Scholar, 36Hebbachi A.-M. Gibbons G.F. J. Lipid Res. 2001; 42: 1609-1617Abstract Full Text Full Text PDF PubMed Google Scholar, 37Boren J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Although the role of MTP has been investigated in liver-specific MTP knock-out mice (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar, 17Chang B.H.-J. Liao W. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), detailed studies were not reported on the function of MTP in hepatocytes from these mice. Because many types of genetically modified mice are now frequently used as models of human lipoprotein metabolism it is important to understand the mechanism of lipoprotein assembly in murine hepatocytes.We have investigated the requirement of MTP for VLDL assembly and secretion in murine primary hepatocytes. Our data show that secretion of apoB100 is markedly more sensitive to inhibition of MTP than is apoB48, and that when MTP is inhibited the apoB48 particles produced are lipid-poor compared with those made in hepatocytes with normal MTP activity. We have also isolated from the microsomal lumen a pool of TG that is not associated with apoB and show that inhibition of MTP reduces the amount of TG within this pool. However, inhibition of MTP does not directly impair the transfer of TG from this pool to lipid-poor apoB-containing particles.DISCUSSIONWe have studied the role of MTP in the hepatic assembly of apoB-containing lipoproteins in murine hepatocytes. Our experiments show that inhibition of MTP (as monitored by inhibition of TG secretion) abolished the secretion of apoB100, whereas the amount of apoB48 secreted was unaffected, although the lipidation of apoB48 was markedly diminished. Parallel differences were found in the amounts, and degree of lipidation, of apoB100 and apoB48 in the microsomal lumen. The difference in sensitivity of apoB100 and apoB48 to inhibition of MTP was independent of the presence of LDLRs. We isolated a microsomal lumenal pool of TG that was not associated with apoB and showed that the size of this pool was ∼3 times larger than the pool of TG associated with apoB. Although the MTP inhibitor decreased by 40–45% the amount of both apoB-free and apoB-associated TG in the microsomal lumen, inhibition of MTP did not impair the transfer of TG to poorly lipidated apoB for VLDL formation.Inhibition of MTP Differentially Impairs the Assembly of ApoB100- and ApoB48-containing LipoproteinsSome of the apparently conflicting reports on the role of MTP in lipoprotein assembly can probably be ascribed to the different cell models used to study this process. MTP has been proposed to act as a “chaperone” that assists in translocating newly synthesized apoB across the ER membrane and into the lumen (20Du E.Z. Wang S.-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. J. Lipid Res. 1996; 37: 1309-1315Abstract Full Text PDF PubMed Google Scholar, 21Liang J.S. Ginsberg H.N. J. Biol. Chem. 2001; 276: 28606-28612Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 32Fleming J.F. Spitsen G.M. Hui T.Y. Olivier L., Du, E.Z. Raabe M. Davis R.A. J. Biol. Chem. 1999; 274: 9509-9514Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Our data demonstrate that when TG secretion is inhibited by 85% the amount of apoB100 in the microsomal lumen is reduced by ∼90%, whereas the amount of apoB48 is unaffected. This observation is consistent with studies in which the murine MTP gene had been inactivated specifically in the liver. In hepatocytes from these mice apoB100 secretion was abolished, whereas apoB48 secretion was reduced only slightly (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar). In contrast, in another study using hepatocytes from a different line of liver-specific MTP-deficient mice, the secretion of both apoB100 and apoB48 was undetectable (17Chang B.H.-J. Liao W. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The reason for the different results obtained from these two lines of genetically modified mice is not clear. Small amounts of apoB, particularly apoB48 have been detected in the serum of individuals with abetalipoproteinemia (20Du E.Z. Wang S.-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. J. Lipid Res. 1996; 37: 1309-1315Abstract Full Text PDF PubMed Google Scholar, 46Herbert P.N. Hyams J.S. Bernier D.N. Berman M.M. Saritelli A.L. Lynch K.M. Nichols A.V. Forte T.M. J. Clin. Invest. 1985; 76: 403-412Crossref PubMed Scopus (43) Google Scholar).The finding that some newly synthesized apoB100 was translocated across the ER membrane and protected from trypsin digestion in the presence of the inhibitor (Fig. 2), particularly after the 15-min labeling period, might be explained by residual MTP activity. Alternatively, because no buoyant apoB100-containing lipoproteins were detected in the lumen of inhibitor-treated cells (Fig. 3), the protease-protected apoB100 might represent apoB100 that is associated with the lumenal surface of the ER membrane because the protease protection assay does not distinguish between apoB that is associated with the lumenal leaflet of the membrane and apoB that has been released into the lumen.Our data using hepatocytes from LDLR−/− and LDLR+/+ mice show that the preferential reduction in secretion of apoB100, compared with apoB48, upon inhibition of MTP cannot be attributed to LDLRs. An alternative explanation might be that apoB100 is preferentially degraded either during, or shortly after, its translocation into the lumen because apoB that is misfolded and/or underlipidated is rapidly degraded by the ubiquitin-proteasome pathway (47Fisher E.A. Pan M. Chen X., Wu, X. Wang H. Jamil H. Sparks J.D. Williams K.J. J. Biol. Chem. 2001; 276: 27855-27863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 48Yeung S.J. Chen S.H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (169) Google Scholar). We speculate that because of its large size, apoB100 requires more lipids to attain a stable conformation than does the smaller apoB48. Thus, if lipid availability were reduced by the MTP inhibitor, apoB100 might preferentially be targeted for degradation. We attempted, unsuccessfully, to test this hypothesis by incubating hepatocytes with the proteasome inhibitors lactacystin, N-acetyl-Leu-Leu-norleucinal, or MG132. However, although these inhibitors have been successfully used in other cell types, the viability of the murine hepatocytes was compromised by equivalent doses of the inhibitors (data not shown).MTP Is Required for Accumulation of Lumenal ApoB-free TG as Well as for ApoB LipidationTo our knowledge, our study is the first report of isolation of an apoB-free pool of TG from the microsomal lumen of hepatocytes. We consider it unlikely that this pool of TG is the result of contamination by the cytosolic TG pool for the following reasons. (i) Before the lumenal contents were isolated, microsomes were thoroughly washed with 0.5 m KCl, which removes loosely bound proteins and lipid droplets adhering to membranes. (ii) When hepatocytes were incubated with the MTP inhibitor, the amount of cytosolic [3H]TG was unaltered, whereas the amount of apoB-free [3H]TG in the lumenal contents was decreased by 45%. Thus, the pattern of labeling of the lumenal TG did not reflect that of cytosolic TG (3Davis R.A. Biochim. Biophys. Acta. 1999; 1440: 1-31Crossref PubMed Scopus (163) Google Scholar). When cytosolic [3H]TG was mixed with unlabeled microsomes, the amount of [3H]TG in the lumenal apoB-independent pool was only 4% of that found when this TG pool was isolated from radiolabeled cells. Our isolation of the pool of apoB-free TG from the microsomal lumen is consistent with an ultrastructural analysis of mice that were unable to synthesize apoB in the intestine (18Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar). In these mice, chylomicron-sized, lipid-staining particles (that did not contain apoB) accumulated in the ER lumen of enterocytes. In another study (49Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar), lipid-staining particles, the size of VLDLs but lacking immunoreactive apoB, were detected in the lumen of the rough ER of rat hepatocytes. From these studies, a model for VLDL assembly has been proposed in which a TG droplet in the rough ER lumen fuses with a small apoB-containing particle at the junction of rough and smooth ER (49Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar).A clue to the function of MTP is our observation that the MTP inhibitor decreased by 45% the amount of microsomal lumenal TG not associated with apoB. This finding, combined with our observation that the MTP inhibitor reduced the amount of TG associated with apoB by 40%, supports the electron microscopy studies of Raabe et al. (16Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar) in which disruption of the MTP gene in liver essentially eliminated VLDL-sized particles from the ER and Golgi lumina. Thus, our data suggest that an important function of MTP is to mediate TG accumulation in the ER lumen in a pool not associated with apoB. In light of published data (50Wiggins D. Gibbons G.F. Biochem. J. 1992; 284: 457-462Crossref PubMed Scopus (199) Google Scholar, 51Yang L.-Y. Kuksis A. Myher J.J. Steiner G. J. Lipid Res. 1995; 36: 125-136Abstract Full Text PDF PubMed Google Scholar, 52Lehner R. Cui Z. Vance D.E. Biochem. J. 1999; 338: 761-768Crossref PubMed Scopus (86) Google Scholar, 53Lehner R. Vance D.E. Biochem. J. 1999; 343: 1-10Crossref PubMed Scopus (115) Google Scholar), it seems likely that this TG is derived primarily from the cytosolic TG pool.Our experiments do not distinguish between the apoB-independent TG being loosely associated with the lumenal surface of the membrane (such a pool would have been released upon sodium carbonate treatment) or floating freely in the lumen. Our laboratory has previously concluded that VLDLs are completely assembled with their full complement of lipid within the rough ER of rat hepatocytes (54Rusiñol A. Verkade H. Vance J.E. J. Biol. Chem. 1993; 268: 3555-3562Abstract Full Text PDF PubMed Google Scholar). Analysis of the apoB structure suggests that apoB contains a “lipid pocket” that expands as lipids are added (55Segrest J.P. Jones M.K. Dashti N. J. Lipid Res. 1999; 40: 1401-1416Abstract Full Text Full Text PDF PubMed Google Scholar). Based on evidence that MTP transfers TG between membranes via a shuttle mechanism (13Atzel A. Wetterau J.R. Biochemistry. 1993; 32: 10444-10450Crossref PubMed Scopus (80) Google Scholar), one possibility is that a sequential addition of TG to apoB occurs on the lumenal surface of the ER membrane. Our observation that apoB48 is distributed among lipoprotein particles with a wide range of densities in the microsomal lumen (Fig. 3) and in the medium (Fig. 7) is also consistent with a sequential addition of TG to apoB. Alternatively, the wide spectrum of densities of apoB48 particles might be the consequence of lipid-poor apoB particles “fusing” with TG droplets of different sizes.The mechanism by which MTP regulates the supply of TG within the ER lumen for lipoprotein assembly remains to be determined. Mobilization of cytosolic TG via a lipolysis re-esterification cycle is thought to provide the majority (∼70%) of TG that is assembled with apoB (50Wiggins D. Gibbons G.F. Biochem. J. 1992; 284: 457-462Crossref PubMed Scopus (199) Google Scholar, 51Yang L.-Y. Kuksis A. Myher J.J. Steiner G. J. Lipid Res. 1995; 36: 125-136Abstract Full Text PDF PubMed Google Scholar) but the enzymes involved in this lipolysis-esterification cycle have not yet been fully characterized. A microsomal TG hydrolase has been recently implicated in this TG lipolysis re-esterification cycle (52Lehner R. Cui Z. Vance D.E. Biochem. J. 1999; 338: 761-768Crossref PubMed Scopus (86) Google Scholar, 53Lehner R. Vance D.E. Biochem. J. 1999; 343: 1-10Crossref PubMed Scopus (115) Google Scholar, 56Lehner R. Verger R. Biochemistry. 1997; 36: 1861-1868Crossref PubMed Scopus (98) Google Scholar). Interestingly, mice with targeted disruption of the diacylglycerol acyltransferase-1 gene have normal plasma TG levels (57Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese R.V. Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (724) Google Scholar), indicating that a different TG-synthesizing enzyme makes the TG utilized for VLDL secretion. A candidate enzyme is diacylglycerol acyltransferase-2, which is expressed primarily in liver and adipose. The gene encoding this enzyme has recently been identified (58Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar) but its requirement for providing TG for VLDL secretion has not yet been established.In conclusion, our data demonstrate that MTP is required for TG accumulation within the ER lumen, as well as for apoB lipidation. However, in agreement with studies in hepatoma cells (2Gordon D.A. Jamil H. Biochim. Biophys. Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar), MTP does not appear to transfer the majority of TG to apoB. We, therefore, propose a model (Fig. 9) for the role of MTP in VLDL assembly that incorporates our new findings. When MTP is inhibited, the accumulation of apoB-independent TG in the ER lumen is compromised, resulting in the availability of less TG for assembly with apoB. We speculate that the decreased lipidation of apoB induced by inhibition of MTP is a consequence of the decreased availability of TG in the apoB-independent lumenal TG pool. During assembly of very low density lipoproteins (VLDLs)1 in the liver, triacylglycerol (TG) is concentrated within the hydrophobic core of apolipoprotein (apo) B-containing lipoprotein particles. A microsomal lumenal protein, the microsomal triacylglycerol transfer protein (MTP), has been implicated in the acquisition of TG by nascent apoB for assembly and secretion of VLDLs (reviewed in Refs. 1
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