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 [ 14 C]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 [ 14 C]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.The liver plays a central role in lipoprotein metabolism through secretion of very low density lipoprotein (VLDL) 1 , which carries neutral lipids to all peripheral tissues. VLDL, a large particle containing apolipoprotein B (apoB) and various lipids, including triglyceride (TG), free cholesterol (FC), and cholesteryl ester (CE), is assembled in endoplasmic reticulum (ER) in hepatic cells. Mechanisms of VLDL particle formation are likely to be rigorously regulated, because association of lipids to apoB needs to occur cotranslationally, and poorly lipidated apoB-containing lipoprotein is degraded by proteasome or ER-resident proteases such as ER-60 prior to secretion (1-3). Thus it is thought that the association of lipids to apoB is a rate-limiting step for VLDL secretion, with regulation of this process controlled by as yet unknown mechanisms. One of the factors for this lipid transfer is microsomal triglyceride transfer protein (MTP), a lack of which was identified as the cause of abetalipoproteinemia, an autosomal recessive disease with total loss of apoB-containing lipoprotein in plasma (4, 5). MTP locates in the luminal space of ER by forming heterodimers with protein disulfide isom...

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