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• W2099074546 abstract "Microsomal triglyceride transfer protein (MTP) is an intraluminal protein in the endoplasmic reticulum (ER) that is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins. In this study, we examine how the livers of mice respond to two distinct methods of blocking MTP function: Cre-mediated disruption of the gene for MTP and chemical inhibition of MTP activity. Blocking MTP significantly reduced plasma levels of triglycerides, cholesterol, and apoB-containing lipoproteins in both wild-type C57BL/6 and LDL receptor-deficient mice. While treating LDL receptor-deficient mice with an MTP inhibitor for 7 days lowered plasma lipids to control levels, liver triglyceride levels were increased by only 4-fold. Plasma levels of apoB-100 and apoB-48 fell by >90% and 65%, respectively, but neither apoB isoform accumulated in hepatic microsomes. Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. Levels of microsomal luminal chaperone proteins [e.g., protein disulfide isomerase, glucose-regulated protein 78 (GRP78), and GRP94] and cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) were unaffected by MTP inhibition. These findings show that the liver responds rapidly to inhibition of MTP by degrading apoB and preventing its accumulation in the ER.The rapid degradation of secretion-incompetent apoB in the ER may block the induction of proteins associated with unfolded protein and heat shock responses. Microsomal triglyceride transfer protein (MTP) is an intraluminal protein in the endoplasmic reticulum (ER) that is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins. In this study, we examine how the livers of mice respond to two distinct methods of blocking MTP function: Cre-mediated disruption of the gene for MTP and chemical inhibition of MTP activity. Blocking MTP significantly reduced plasma levels of triglycerides, cholesterol, and apoB-containing lipoproteins in both wild-type C57BL/6 and LDL receptor-deficient mice. While treating LDL receptor-deficient mice with an MTP inhibitor for 7 days lowered plasma lipids to control levels, liver triglyceride levels were increased by only 4-fold. Plasma levels of apoB-100 and apoB-48 fell by >90% and 65%, respectively, but neither apoB isoform accumulated in hepatic microsomes. Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. Levels of microsomal luminal chaperone proteins [e.g., protein disulfide isomerase, glucose-regulated protein 78 (GRP78), and GRP94] and cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) were unaffected by MTP inhibition. These findings show that the liver responds rapidly to inhibition of MTP by degrading apoB and preventing its accumulation in the ER. The rapid degradation of secretion-incompetent apoB in the ER may block the induction of proteins associated with unfolded protein and heat shock responses. VLDLs produced by the liver are the major source of LDLs in the plasma, which are causally related to the development of atherosclerotic cardiovascular disease (1MRC/BHF Heart Protection Study GroupMRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial.Lancet. 2002; 360: 7-22Google Scholar). The importance of the hepatic secretion of apolipoprotein B (apoB) in cardiovascular disease was recognized in early studies of abetalipoproteinemic patients, who lack the ability to secrete apoB-containing lipoproteins (2Kayden H.J. Abetalipoproteinemia.Annu. Rev. Med. 1972; 23: 285-296Google Scholar) and are markedly resistant to cardiovascular disease (3Kayden H.J. Traber M.G. Clinical, nutritional and biochemical consequences of apolipoprotein B deficiency.Adv. Exp. Med. Biol. 1986; 201: 67-81Google Scholar). Subsequent studies showed that specific mutations in the microsomal triglyceride transfer protein (MTP) gene are responsible for abetalipoproteinemia (4Wetterau J.R. Aggerbeck L.P. Bouma M.-E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D. Gregg R.E. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.Science. 1992; 258: 999-1001Google Scholar, 5Gregg R.E. Wetterau J.R. The molecular basis of abetalipoproteineimia.Curr. Opin. Lipidol. 1994; 5: 81-86Google Scholar). MTP exists in a functional complex with protein disulfide isomerase (PDI) (6Wetterau J.R. Zilversmit D.B. Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes.Chem. Phys. Lipids. 1985; 38: 205-222Google Scholar, 7Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.J. Biol. Chem. 1990; 265: 9801-9807Google Scholar). The loss of MTP function blocks the secretion of apoB-containing lipoproteins from both the liver and the intestine (5Gregg R.E. Wetterau J.R. The molecular basis of abetalipoproteineimia.Curr. Opin. Lipidol. 1994; 5: 81-86Google Scholar). Apart from neuropathy, which can be prevented by vitamin E supplements (8Iannaccone S.T. Sokol R.J. Vitamin E deficiency in neuropathy of abetalipoproteinemia.Neurology. 1986; 361009Google Scholar) and moderate steatosis in enterocytes and hepatocytes, abetalipoproteinemia is not usually associated with liver failure or cirrhosis (3Kayden H.J. Traber M.G. Clinical, nutritional and biochemical consequences of apolipoprotein B deficiency.Adv. Exp. Med. Biol. 1986; 201: 67-81Google Scholar). The absence of severe symptoms has suggested that inactivation of MTP might be a useful strategy for combating hyperlipidemia and cardiovascular disease. Several chemical inhibitors of the lipid transfer activity of MTP have been developed (9Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.J. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek C.J. Biller S.A. Gregg R.E. Wetterau J.R. An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 11991-11995Google Scholar, 10Bakillah A. Nayak N. Saxena U. Medford R.M. Hussain M.M. Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist.Biochemistry. 2000; 39: 4892-4899Google Scholar, 11Ksander G.M. deJesus R. Yuan A. Fink C. Moskal M. Carlson E. Kukkola P. Bilci N. Wallace E. Neubert A. Feldman D. Mogelesky T. Poirier K. Jeune M. Steele R. Wasvery J. Stephan Z. Cahill E. Webb R. Navarrete A. Lee W. Gibson J. Alexander N. Sharif H. Hospattankar A. Diaminoindanes as microsomal triglyceride transfer protein inhibitors.J. Med. Chem. 2001; 44: 4677-4687Google Scholar, 12Chang G. Ruggeri R.B. Harwood Jr., H.J. Microsomal triglyceride transfer protein (MTP) inhibitors: discovery of clinically active inhibitors using high-throughput screening and parallel synthesis paradigms.Curr. Opin. Drug Discov. Devel. 2002; 5: 562-570Google Scholar). Many of these inhibitors lower plasma lipid levels in animal models (11Ksander G.M. deJesus R. Yuan A. Fink C. Moskal M. Carlson E. Kukkola P. Bilci N. Wallace E. Neubert A. Feldman D. Mogelesky T. Poirier K. Jeune M. Steele R. Wasvery J. Stephan Z. Cahill E. Webb R. Navarrete A. Lee W. Gibson J. Alexander N. Sharif H. Hospattankar A. Diaminoindanes as microsomal triglyceride transfer protein inhibitors.J. Med. Chem. 2001; 44: 4677-4687Google Scholar, 12Chang G. Ruggeri R.B. Harwood Jr., H.J. Microsomal triglyceride transfer protein (MTP) inhibitors: discovery of clinically active inhibitors using high-throughput screening and parallel synthesis paradigms.Curr. Opin. Drug Discov. Devel. 2002; 5: 562-570Google Scholar, 13Wetterau 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 J.V. 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. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Especially encouraging results were obtained in Watanabe-heritable hyperlipidemic rabbits, a model of homozygous familial hypercholesterolemia (13Wetterau 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 J.V. 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. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Administration of an MTP inhibitor to Watanabe-heritable hyperlipidemic rabbits reduced plasma lipid and lipoprotein levels to normal (13Wetterau 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 J.V. 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. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Since many homozygous familial hypercholesterolemias are resistant to statin therapies (14Uauy R. Vega G.L. Grundy S.M. Bilheimer D.M. Lovastatin therapy in receptor-negative homozygous familial hypercholesterolemia: lack of effect on low-density lipoprotein concentrations or turnover.J. Pediatr. 1988; 113: 387-392Google Scholar), MTP inhibitors might provide a more attractive treatment to liver transplantation, which effectively reduces atherosclerosis in these patients (15Bilheimer D.W. Goldstein J.L. Grundy S.M. Starzl T.E. Brown M.S. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia.N. Engl. J. Med. 1984; 311: 1658-1664Google Scholar). MTP inhibitor drugs have provided the opportunity to examine the processes regulating the assembly, secretion, and degradation of apoB-containing lipoproteins. Studies of cultured cells suggest that the cotranslational translocation of apoB into the lumen of the endoplasmic reticulum (ER) governs the fate of newly synthesized apoB [reviewed in refs. (16Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1997; 38: 1937-1953Google Scholar, 17Davis R.A. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim. Biophys. Acta. 1999; 1440: 1-31Google Scholar, 18Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Google Scholar, 19Davidson N.O. Shelness G.S. Apolipoprotein B: mRNA editing, lipoprotein assembly, and presecretory degradation.Annu. Rev. Nutr. 2000; 20: 169-193Google Scholar, 20Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins.J. Biol. Chem. 2002; 277: 17377-17380Google Scholar)]. Unlike other proteins secreted by the liver, apoB-100 cannot be completely translocated into the ER lumen without MTP (21Du E. Kurth J. Wang S-L. Humiston P. Davis R.A. Proteolysis-coupled secretion of the N-terminus of apolipoprotein B: characterization of a transient, translocation arrested intermediate.J. Biol. Chem. 1994; 269: 24169-24176Google Scholar, 22Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Google Scholar, 23Chang B.H. Liao W. Li L. Nakamuta M. Mack D. Chan L. Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse.J. Biol. Chem. 1999; 274: 6051-6055Google Scholar). MTP appears to facilitate both the folding and lipidation of apoB during translocation (24Du E.Z. Wang S-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in abetalipoproteinemia.J. Lipid Res. 1996; 37: 1309-1315Google Scholar, 25Benoist F. Grand P.T. Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein.J. Biol. Chem. 1997; 272: 20435-20442Google Scholar, 26Wang L. Fast D.G. Attie A.D. The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion.J. Biol. Chem. 1997; 272: 27644-27651Google Scholar, 27Hussain M.M. Bakillah A. Jamil H. Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis.Biochemistry. 1997; 36: 13060-13067Google Scholar, 28Liang J.S. Wu X. Jiang H. Zhou M. Yang H. Angkeow P. Huang L.S. Sturley S.L. Ginsberg H. Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains.J. Biol. Chem. 1998; 273: 35216-35221Google Scholar, 29Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells.J. Biol. Chem. 1998; 273: 5196-5203Google Scholar, 30Mitchell D.M. Zhou M. Pariyarath R. Wang H. Aitchison J.D. Ginsberg H.N. Fisher E.A. Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence.Proc. Natl. Acad. Sci. USA. 1998; 95: 14733-14738Google Scholar, 31Nicodeme E. Benoist F. McLeod R. Yao Z. Scott J. Shoulders C.C. Grand P.T. Identification of domains in apolipoprotein B100 that confer a high requirement for the microsomal triglyceride transfer protein.J. Biol. Chem. 1999; 274: 1986-1993Google Scholar, 32Kulinski A. Rustaeus S. Vance J.E. Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB, as well as for ApoB lipidation.J. Biol. Chem. 2002; 277: 31516-31525Google Scholar). In the absence of MTP, apoB translocation is abrogated, resulting in the rapid cotranslational degradation of the cytoplasmic, translocation-arrested C terminus of apoB (33Davis R.A. Thrift R.N. Wu C.C. Howell K.E. Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools.J. Biol. Chem. 1990; 265: 10005-10011Google Scholar) via the proteasome (34Yeung S.J. Chen S.H. Chan L. Ubiquitin-proteasome pathway mediates intracellular degradation of apolipoprotein B.Biochemistry. 1996; 35: 13843-13848Google Scholar, 35Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70.J. Biol. Chem. 1997; 272: 20427-20434Google Scholar, 36Liang J. Wu X. Fisher E.A. Ginsberg H.N. The amino-terminal domain of apolipoprotein B does not undergo retrograde translocation from the endoplasmic reticulum to the cytosol. Proteasomal degradation of nascent apolipoprotein b begins at the carboxyl terminus of the protein, while apolipoprotein b is still in its original translocon.J. Biol. Chem. 2000; 275: 32003-32010Google Scholar). Approximately 85 kDa of the N-terminal portion of translocation-arrested apoB is released from the ER membrane, enters the lumen, and can be secreted or degraded (21Du E. Kurth J. Wang S-L. Humiston P. Davis R.A. Proteolysis-coupled secretion of the N-terminus of apolipoprotein B: characterization of a transient, translocation arrested intermediate.J. Biol. Chem. 1994; 269: 24169-24176Google Scholar, 24Du E.Z. Wang S-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in abetalipoproteinemia.J. Lipid Res. 1996; 37: 1309-1315Google Scholar, 37Bonnardel J.A. Davis R.A. In HepG2 cells, translocation, not degradation, determines the fate of de novo synthesized apolipoprotein B.J. Biol. Chem. 1995; 270: 28892-28896Google Scholar). The finding that plasma from abetalipoproteinemic patients was enriched with an 85 kDa N-terminal peptide compared with plasma from unaffected family members suggested that MTP-facilitated translocation occurs in the human liver (24Du E.Z. Wang S-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in abetalipoproteinemia.J. Lipid Res. 1996; 37: 1309-1315Google Scholar). Several additional degradative processes act to ensure that secretion-incompetent apoB does not accumulate [reviewed in refs. (16Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1997; 38: 1937-1953Google Scholar, 17Davis R.A. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim. Biophys. Acta. 1999; 1440: 1-31Google Scholar, 18Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Google Scholar, 19Davidson N.O. Shelness G.S. Apolipoprotein B: mRNA editing, lipoprotein assembly, and presecretory degradation.Annu. Rev. Nutr. 2000; 20: 169-193Google Scholar, 20Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins.J. Biol. Chem. 2002; 277: 17377-17380Google Scholar)]. Preventing the accumulation of apoB within the secretory pathway may be essential in preventing an induction of unfolded-protein response (38Hampton R.Y. ER stress response: getting the UPR hand on misfolded proteins.Curr. Biol. 2000; 10: R518-R521Google Scholar, 39Urano F. Bertolotti A. Ron D. IRE1 and efferent signaling from the endoplasmic reticulum.J. Cell Sci. 2000; 113: 3697-3702Google Scholar, 40Patil C. Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals.Curr. Opin. Cell Biol. 2001; 13: 349-355Google Scholar). To our knowledge, there have been no published studies that have examined how the liver in vivo responds to loss of MTP in regard to the accumulation of apoB and possible induction of unfolded protein and/or heat shock responses. Our studies revealed that the inactivation of MTP (by gene disruption or chemical inactivation) led to a block in the secretion of apoB and prevented its accumulation in the ER, as well as the induction of unfolded protein and/or heat shock responses. The MTP inhibitor 8aR (11Ksander G.M. deJesus R. Yuan A. Fink C. Moskal M. Carlson E. Kukkola P. Bilci N. Wallace E. Neubert A. Feldman D. Mogelesky T. Poirier K. Jeune M. Steele R. Wasvery J. Stephan Z. Cahill E. Webb R. Navarrete A. Lee W. Gibson J. Alexander N. Sharif H. Hospattankar A. Diaminoindanes as microsomal triglyceride transfer protein inhibitors.J. Med. Chem. 2001; 44: 4677-4687Google Scholar) was kindly provided by Dr. Gary Ksander (Novartis, Summit, NJ). Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Mice homozygous for both a conditional Mttp allele (Mttpflox) and the Mx1-Cre transgene have been described (22Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Google Scholar). Wild-type C57BL/6 mice and C57BL/6 LDL receptor knockout mice (Ldlr –/–) were obtained from The Jackson Laboratory (http://www.jax.org). Mice were maintained under standard conditions on a 12 h light-dark cycle (lights on from 0600 to 1800). To eliminate MTP expression in the liver, 8-week-old Mttpflox/floxMx1-Cre+/+ mice were injected with polyinosinic-polycytidylic ribonucleic acid (polyIC; Sigma; 300 μg every two days for 5 times). Control mice received vehicle only. Three or six weeks after last injection, blood was drawn from the retro-orbital plexus into tubes containing EDTA, and plasma was separated by centrifugation. The mice were killed by cervical dislocation, and the livers were removed immediately and used to prepare microsomes (33Davis R.A. Thrift R.N. Wu C.C. Howell K.E. Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools.J. Biol. Chem. 1990; 265: 10005-10011Google Scholar). All protocols were approved by the Animal Care and Use Committee of San Diego State University. The inhibitor 8aR was prepared as a water suspension in 3% cornstarch, and was administered orally at a dose of 50 mg/kg daily for 7 days, or using as a single dose of 100 mg/kg. The liver was homogenized in 250 mM sucrose and 10 mM Hepes (pH 7.4) containing 1 mM phosphomethylsulfonylfluoride, 0.1 mM acetylated leucine, leucine-norleucal (ALLN), and 5 mM N-ethylmaleimide. Microsomes were obtained by ultracentrifugation and washing of fractions, as described (33Davis R.A. Thrift R.N. Wu C.C. Howell K.E. Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools.J. Biol. Chem. 1990; 265: 10005-10011Google Scholar). The protein concentration in the samples was measured using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of proteins from liver homogenates, microsomes, or plasma were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies as described (21Du E. Kurth J. Wang S-L. Humiston P. Davis R.A. Proteolysis-coupled secretion of the N-terminus of apolipoprotein B: characterization of a transient, translocation arrested intermediate.J. Biol. Chem. 1994; 269: 24169-24176Google Scholar). Blots were detected by enhanced chemiluminescence (ECL kit, Amersham). The relative intensity of the immunoblot bands was quantified with a Storm PhosphoImager, (Amersham Pharmacia Biotech, Piscataway, NJ). Equal volumes of plasma from each mouse in each group were pooled (0.2 ml) and loaded onto a fast protein liquid chromatography (FPLC) system with two Superose 6B columns connected in series (HR10/30, Pharmacia FPLC System, Amersham Pharmacia Biotech) (41Miyake J.H. Duong X.D-T. Taylor J.M. Du E.Z. Castellani L.W. Lusis A.J. Davis R.A. Transgenic expression of cholesterol-7a-hydroxylase prevents atherosclerosis in C57BL/6J mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 121-126Google Scholar). Fractions (1 ml) were collected at a flow rate of 0.5 ml/min with an elution buffer (15 mM NaCl, 0.01% EDTA, 0.02% sodium azide, pH 7.3). Cholesterol and triglycerides in plasma and FPLC fractions were assayed with commercial kits from Sigma, as described (41Miyake J.H. Duong X.D-T. Taylor J.M. Du E.Z. Castellani L.W. Lusis A.J. Davis R.A. Transgenic expression of cholesterol-7a-hydroxylase prevents atherosclerosis in C57BL/6J mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 121-126Google Scholar). Results are given as the mean ± SD. Statistical significance was determined by Student’s t-test with two-tailed P values. Differences were considered to be significant at P < 0.05. For our studies, we used Mttpflox/floxMx1-Cre+/+ mice. Cre expression in these mice can be induced with polyIC, which eliminates exon 1 of Mttp and prevents the formation of a functional Mttp transcript (22Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Google Scholar). The absence of MTP expression by the liver did not affect body weight, but increased liver weight (Fig. 1A)and reduced the plasma levels of triglycerides (45%) and cholesterol (60%) (Fig. 1B). As a result, VLDL, IDLs, and LDLs were almost undetectable, and HDL levels were reduced by ∼50% (Fig. 1B). Hepatic triglycerides were increased 2.5-fold, and hepatic cholesterol was increased by 65% (Fig. 1C). ApoB-100 was reduced to undetectable levels in plasma, and plasma levels of apoB-48 were reduced by ∼65% (Fig. 1D). Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. The amount of apoB-48 was unaffected (Fig. 1E). The lack of MTP expression did not affect the levels of microsomal luminal chaparone proteins (e.g., PDI, glucose-regulated protein (GRP) 78 and GRP94) or cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) (Fig. 1F). Hepatic microsomes from mice treated for 6 weeks with polyIC contained no detectable MTP protein (Fig. 1F). Northern blots showed that apoB mRNA levels were unchanged by polyIC treatment (not shown). Thus, changes in apoB were the result of the absence of hepatic MTP expression. These data suggest that deletion of hepatic MTP expression blocks the assembly and secretion of apoB-containing lipoproteins without causing apoB-100 or apoB-48 to accumulate in the ER. This experiment was repeated twice with similar results (not shown). We next examined how mice responded to chemical inhibition of MTP. After 7 days of treatment with 8aR, the phenotype of C57BL/6 mice was essentially the same as that of mice lacking hepatic MTP. The liver weight increased (Fig. 2A), the plasma levels of triglyceride (50%) and cholesterol (70%) decreased (Fig. 2B), and the liver content of triglyceride (4-fold) and cholesterol (35%) increased (Fig. 2C). The MTP inhibitor markedly decreased plasma levels of apoB-48 (70%) and apoB-100 (95%) (Fig. 2D). Hepatic microsomes from 8aR-treated mice contained almost no apoB-100, and there was no accumulation of apoB-48 (Fig. 2E). The MTP inhibitor did not affect the levels of microsomal luminal chaperone proteins (Fig. 2F) or of cytosolic HSPs (Fig. 2F). These findings suggest that the liver adapts to inhibition of apoB secretion and prevents the accumulation of apoB in the ER. We next examined the hepatic response to MTP inhibition in extremely hyperbetalipoproteinemic Ldlr –/– mice. Chemical inhibition of MTP did not increase liver weight (Fig. 3A), perhaps because Ldlr –/– mice already have enlarged livers; however, there were decreases in the plasma levels of cholesterol (85%), triglyceride (65%) (Fig. 3B), apoB-100 (70%), and apoB-48 (80%) (Fig. 3D). As a result, plasma VLDL, IDL, and LDL essentially disappeared, and HDL levels decreased by 65% (Fig. 3B). In the livers of mice, the stores of triglycerides increased 4-fold and cholesterol increased 2-fold (Fig. 3C). Despite the marked decrease in apoB secretion, hepatic microsomes contained almost no apoB-100, and there was no accumulation of apoB-48 (Fig. 3E). Microsomes from Ldlr –/– mice not treated with the MTP inhibitor contained markedly more apoB-100 than microsomes from untreated wild-type mice (Fig. 3F). Thus, the absence of the LDL receptor caused apoB-100 to accumulate in the ER of mice not treated with MTP. The MTP inhibitor did not affect the content of any microsomal luminal chaperone proteins (Fig. 3G) or the content of cytosolic HSPs (Fig. 3G). These data show that inhibition of MTP ameliorates severe hypercholesterolemia without causing accumulation of apoB in the ER, hepatic inflammation, or massive fatty liver. To determine how rapidly apoB degradation is induced in response to MTP inhibition, we assessed the effects of a single dose of the MTP inhibitor. A single dose of 8aR did not alter liver weight (Fig. 4A). It did, however, reduce plasma levels of triglycerides (43%) and cholesterol (22%) (Fig. 4B), apoB-100 (95%), and apoB-48 (88%) (Fig. 4D). Neither apoB isoform accumulated in the microsomal fractions (Fig. 4E). Hepatic levels of markers of the HSP and unfolded protein responses were also unaffected (Fig. 4F). These findings show that the hepatic response to MTP inhibition and impaired apoB secretion is sufficiently rapid to prevent the apoB accumulation in the ER. This rapid response may help to explain the absence of an accumulation of misfolded proteins in the ER and an associated inflammatory response. This study shows, for the first time, that blocking hepatic apoB secretion in vivo in mice results in a rapid homeostatic degradation pathway that prevents the accumulation of apoB in the ER without inducing heat shock or unfolded protein responses. Blocking MTP function by Cre-mediated gene disruption or chemical inhibition also reduced plasma lipid levels markedly without causing massive fatty liver. These findings suggest that MTP could be an effective therapeutic target for hyperlipidemia. The processes through which apoB is assembled into lipoproteins and secreted by the liver are complex and controlled at many different levels throughout the secretory pathway [reviewed in refs. (16Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1997; 38: 1937-1953Google Scholar, 17Davis R.A. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim. Biophys. Acta. 1999; 1440: 1-31Google Scholar, 18Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Google Scholar, 19Davidson N.O. Shelness G.S. Apolipoprotein B: mRNA editing, lipoprotein assembly, and presecretory degradation.Annu. Rev. Nutr. 2000; 20: 169-193Google Scholar, 20Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins.J. Biol. Chem. 2002; 277: 17377-17380Google Scholar)]. Since MTP is localized to the proximal portion of the secretory pathway (i.e., the ER) (42Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Google Scholar), we have focused these studies on examining how blocking MTP affects the ER content of apoB and of lumenal proteins that participate in the unfolded protein response. Our findings are the first to show that in vivo deletion of MTP function (either by Cre-mediated gene disruption or chemical inhibition) blocked hepatic secretion of apoB-100 without causing apoB to accumulate in the ER. These findings are consistent with the notion that while loss of MTP function blocks apoB translocation, apoB does not accumulate in the ER (21Du E. Kurth J. Wang S-L. Humiston P. Davis R.A. Proteolysis-coupled secretion of the N-terminus of apolipoprotein B: characterization of a transient, translocation arrested intermediate.J. Biol. Chem. 1994; 269: 24169-24176Google Scholar, 25Benoist F. Grand P.T. Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein.J. Biol. Chem. 1997; 272: 20435-20442Google Scholar, 43Thrift R.N. Drisko J. Dueland S. Trawick J.D. Davis R.A. Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in a nonhepatic cell line.Proc. Natl. Acad. Sci. USA. 1992; 89: 9161-9165Google Scholar, 44Du E.Z. Fleming J.F. Wang S-L. Spitzen G.M. Davis R.A. Translocation-arrested apolipoprotein B evades proteasome degradation via a sterol-sensitive block in ubiquitin conjugation.J. Biol. Chem. 1999; 274: 1856-1862Google Scholar, 45Wang S. McLeod R.S. Gordon D.A. Yao Z. The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells.J. Biol. Chem. 1996; 271: 12124-12133Google Scholar) because it is cotranslationally degraded (30Mitchell D.M. Zhou M. Pariyarath R. Wang H. Aitchison J.D. Ginsberg H.N. Fisher E.A. Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence.Proc. Natl. Acad. Sci. USA. 1998; 95: 14733-14738Google Scholar, 37Bonnardel J.A. Davis R.A. In HepG2 cells, translocation, not degradation, determines the fate of de novo synthesized apolipoprotein B.J. Biol. Chem. 1995; 270: 28892-28896Google Scholar, 46Liao W. Yeung S. Chan L. Proteasome-mediated degradation of apolipoprotein B targets both nascent peptides cotranslationally before translocation and full-length apolipoprotein B after translocation into the endoplasmic reticulum.J. Biol. Chem. 1998; 273: 27225-27230Google Scholar, 47Pariyarath R. Wang H. Aitchison J.D. Ginsberg H.N. Welch W.J. Johnson A.E. Fisher E.A. Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome.J. Biol. Chem. 2001; 276: 541-550Google Scholar). Our finding that interruption of MTP function results in the rapid and efficient degradation of secretion-incompetent apoB in vivo explains why apoB does not accumulate in hepatic ER. Our findings may also explain why blocking the secretion of apoB-containing lipoproteins by the liver in mice is not associated with the inflammatory responses associated with the accumulation of secretion-incompetent protein in the secretory pathway (unfolded-protein and heat shock responses) (38Hampton R.Y. ER stress response: getting the UPR hand on misfolded proteins.Curr. Biol. 2000; 10: R518-R521Google Scholar, 39Urano F. Bertolotti A. Ron D. IRE1 and efferent signaling from the endoplasmic reticulum.J. Cell Sci. 2000; 113: 3697-3702Google Scholar, 40Patil C. Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals.Curr. Opin. Cell Biol. 2001; 13: 349-355Google Scholar). This proposal is further supported by the recent observation that deletion by itself of MTP expression in the livers of mice is not associated with hepatic inflammation or dysfunction (48Bjorkegren J. Beigneux A. Bergo M.O. Maher J.J. Young S.G. Blocking the secretion of hepatic very low density lipoproteins renders the liver more susceptible to toxin-induced injury.J. Biol. Chem. 2002; 277: 5476-5483Google Scholar). The livers of Ldlr –/– mice contained more apoB-100 in the ER than livers of C57BL/6 mice (Fig. 3F). Since apoB mRNA expression was similar in the two groups of mice, this difference probably reflects differences in the degradation of apoB in the ER. Our findings are consistent with studies showing that hepatocytes from Ldlr –/– mice secrete more apoB-100 and degrade less of it in the ER than do hepatocytes from wild-type mice (49Twisk J. Gillian-Daniel D.L. Tebon A. Wang L. Barrett P.H. Attie A.D. The role of the LDL receptor in apolipoprotein B secretion.J. Clin. Invest. 2000; 105: 521-532Google Scholar). Apparently, the association of the LDL receptor with nascent apoB-100 facilitates its removal from the ER or its degradation (50Gillian-Daniel D.L. Bates P.W. Tebon A. Attie A.D. Endoplasmic reticulum localization of the low density lipoprotein receptor mediates presecretory degradation of apolipoprotein B.Proc. Natl. Acad. Sci. USA. 2002; 99: 4337-4342Google Scholar). The ligand-binding domain of the LDL receptor in the ER is in the lumen (51Brown M.S. Anderson R.G. Goldstein J.L. Recycling receptors: the round-trip itinerary of migrant membrane proteins.Cell. 1983; 32: 663-667Google Scholar). Since the LDL receptor binding domain of apoB is located in the C terminus terminus (52Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Complete protein sequence and identification of structural domains of human apolipoprotein B.Nature. 1986; 323: 734-738Google Scholar, 53Yang C-Y. Chen S-H. Gianturco S.H. Bradley W.A. Sparrow J.T. Tanimura M. Li W-H. Sparrow D.A. DeLoof H. Rosseneu M. Lee F. Gu Z-W. Gotto A.M. Chan L. Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100.Nature. 1986; 323: 738-742Google Scholar), it is likely that the LDL receptor associates with apoB after its translocation into the ER lumen is completed. Our finding that blocking MTP caused a similar decrease in the apoB-100 content in the ER indicates that the presence of the LDL receptor did not affect the degradation of apoB-100 induced in response to blocked secretion. These combined findings suggest the possibility that the absence of MTP function causes increased degradation of cytoplasmic (translocation-arrested) apoB-100, and the absence of the LDL receptor reduces the degradation of luminal (fully translocated) apoB. The observation that some (54Collins J.C. Scheinberg I.H. Giblin D.R. Sternlieb I. Hepatic peroxisomal abnormalities in abetalipoproteinemia.Gastroenterology. 1989; 97: 766-770Google Scholar) [but not all (2Kayden H.J. Abetalipoproteinemia.Annu. Rev. Med. 1972; 23: 285-296Google Scholar, 8Iannaccone S.T. Sokol R.J. Vitamin E deficiency in neuropathy of abetalipoproteinemia.Neurology. 1986; 361009Google Scholar, 55Ohashi K. Ishibashi S. Osuga J. Tozawa R. Harada K. Yahagi N. Shionoiri F. Iizuka Y. Tamura Y. Nagai R. Illingworth D.R. Gotoda T. Yamada N. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia.J. Lipid Res. 2000; 41: 1199-1204Google Scholar)] abetalipoproteinemic patients develop liver disease implies that the response of the human liver to MTP inhibition may be influenced by genetic and environmental factors. In the same mice we used in this study, deletion of MTP did not, by itself, cause liver dysfunction, but it did increase susceptibility to toxin-induced liver injury (48Bjorkegren J. Beigneux A. Bergo M.O. Maher J.J. Young S.G. Blocking the secretion of hepatic very low density lipoproteins renders the liver more susceptible to toxin-induced injury.J. Biol. Chem. 2002; 277: 5476-5483Google Scholar). This increased susceptibility may be caused by impaired transport of essential lipid nutrients, such as vitamin E (8Iannaccone S.T. Sokol R.J. Vitamin E deficiency in neuropathy of abetalipoproteinemia.Neurology. 1986; 361009Google Scholar, 55Ohashi K. Ishibashi S. Osuga J. Tozawa R. Harada K. Yahagi N. Shionoiri F. Iizuka Y. Tamura Y. Nagai R. Illingworth D.R. Gotoda T. Yamada N. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia.J. Lipid Res. 2000; 41: 1199-1204Google Scholar, 56Saito K. Matsumoto S. Yokoyama T. Okaniwa M. Kamoshita S. Pathology of chronic vitamin E deficiency in fatal familial intrahepatic cholestasis (Byler disease).Virchows Arch. 1982; 396: 319-330Google Scholar). The previous studies suggesting that MTP inhibitors corrected the marked hypercholesterolemia in Watanabe-heritable hyperlipidemic rabbits (13Wetterau 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 J.V. 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. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar) provided support suggesting a possible alternative treatment for homozygous familial hypercholesterolemia, a lethal disorder corrected by liver transplantation (15Bilheimer D.W. Goldstein J.L. Grundy S.M. Starzl T.E. Brown M.S. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia.N. Engl. J. Med. 1984; 311: 1658-1664Google Scholar). The studies in Watanabe-heritable hyperlipidemic rabbits did not provide data concerning liver function or inflammatory response (13Wetterau 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 J.V. 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. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Our studies reported herein clearly show that inhibition of MTP corrected the hyperlipidemia in a mouse model of homozygous familial hypercholesterolemia without causing massive fatty liver or induction of heat shock or unfolded protein responses. Our findings provide further evidence that MTP may be a therapeutically useful target for reducing plasma levels of atherogenic lipoproteins in homozygous familial hypercholesterolemia. The authors thank S. Ordway and G. Howard for comments on the manuscript. The authors are most grateful to Dr. Gary Ksander and Novartis, Inc. for their gift of 8aR MTP inhibitor. This project was supported by National Institutes of Health Grants HL-51648, HL-57974, and HL-41633; an American Heart Association Scientist Development Grant; and a grant from the University of California Tobacco-Related Disease Program. W.L. was an American Liver Foundation Clarence A. Kruse Liver Scholar. acetylated leucine, leucine, norleucal endoplasmic reticulum glucose-regulated protein heat shock protein microsomal triglyceride transfer protein protein disulfide isomerase" @default.
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