title: Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum.
abstract: Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.
authors.affiliation: Department of Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-0195, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Higashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Itabe
authors.forename: Hironaga
authors.initials: H
authors.lastname: Fukase
authors.forename: Masahiro
authors.initials: M
authors.lastname: Mori
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Fujimoto
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Takano
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D001055:Apolipoproteins B / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D050356:Lipid Metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D013997:Time Factors; D014280:Triglycerides / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000085:Acetates; D000418:Albumins; D001055:Apolipoproteins B; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008079:Lipoproteins, VLDL; D014280:Triglycerides; C063261:acetylleucyl-leucyl-norleucinal; D002784:Cholesterol; D014700:Verapamil