Microsomal Triacylglycerol Transfer Protein Is Required for Lumenal Accretion of Triacylglycerol Not Associated with ApoB, as Well as for ApoB Lipidation

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...

Section: Verapamil Specifically Inhibits the Secretion Of Apob-contaimentioning

confidence: 70%

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confidence: 99%

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confidence: 99%

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Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

Higashi

Itabe

Fukase

et al. 2003

“…In addition, we observed functional and structural differences between CG9342 and human MTP, possibly reflecting known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism. The identification of invertebrate orthologs of human MTP may enable structural and functional dissection of the multiple roles of human MTP in apoB assembly as well as apoB-independent effects on intracellular lipid trafficking (23)(24)(25) (26) as implemented by the SignalIP V2.0 server on the web. The amino terminus of mature human MTP was based on data reported by Shoulders et al (27).…”

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confidence: 99%

A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B

Sellers

Athar

et al. 2003

The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of ϳ1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism.

“…The production of apolipoprotein B (apoB)-containing lipoproteins by the liver is mainly regulated by the relative amount of de novo synthesized apoB that is translocated into the endoplasmic reticulum and assembled into a secretion-competent lipoprotein particle (7)(8)(9)(10). MTP, an intraluminal protein in the endoplasmic reticulum (11), plays an important role in regulating the assembly and secretion of apoB-containing lipoproteins (12)(13)(14)(15)(16). In humans, the loss of MTP function blocks the production of apoB-containing lipoproteins by both the intestine and the liver (11).…”

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confidence: 99%

ARP-1/COUP-TF II Determines Hepatoma Phenotype by Acting as Both a Transcriptional Repressor of Microsomal Triglyceride Transfer Protein and an Inducer of CYP7A1

Kang

Spann

Hui

et al. 2003

Individual liver cells, distinguished by their anatomical localization within the portal triad and their relative expression of genes, allow the liver to provide diverse anabolic and catabolic functions. Hepatocytes localized near the portal vein express high levels of lipogenic (1) and gluconeogenic (2) enzymes, whereas those localized near the central vein express high levels of catabolic (CYP7A1) (3, 4) and glycolytic (2) enzymes. Environmental differences caused by hormonal, nutritional, and xenobiotic variations in portal versus central blood result in zonal distinctions in the gene expression of transcription factors within the hepatic acinus (5). The molecular mechanisms responsible for the distinct subsets of genes expressed by individual liver cells remain unknown. We have derived and characterized stable lines of rat hepatoma cells in order to identify the molecular mechanisms responsible for the phenotypic expression of the anabolic lipoprotein assembly pathway regulated by microsomal triglyceride transfer protein (MTP) 1 and the catabolic pathway through which cholesterol is converted into bile acids by the rate-limiting enzyme CYP7A1.The liver is the major site for both the production of plasma lipoproteins and their uptake from plasma and catabolism (reviewed in Ref. 6). The production of apolipoprotein B (apoB)-containing lipoproteins by the liver is mainly regulated by the relative amount of de novo synthesized apoB that is translocated into the endoplasmic reticulum and assembled into a secretion-competent lipoprotein particle (7-10). MTP, an intraluminal protein in the endoplasmic reticulum (11), plays an important role in regulating the assembly and secretion of apoB-containing lipoproteins (12-16). In humans, the loss of MTP function blocks the production of apoB-containing lipoproteins by both the intestine and the liver (11). On the other hand, the catabolic bile acid biosynthetic pathway, regulated by CYP7A1, is the major route through which hepatic (and total body) cholesterol homeostasis is maintained (17-20). Hepatic cholesterol levels indirectly regulate the expression of low density lipoprotein receptors via changes in serum response element (SRE)-binding proteins (21). Thus, the expression of CYP7A1 indirectly regulates the expression of low density lipoprotein receptors (22,23). Bile acids, which are the end products of the CYP7A1 reaction, stimulate the secretion of phospholipids and cholesterol into bile (24). Thus, the relative expression levels of MTP and CYP7A1 by individual hepatic parenchymal cells are important determinants of whether phospholipids and cholesterol are targeted into plasma (lipoproteins) or bile (biliary lipids).We have isolated stable cell clones from rat hepatoma cells as