The Late Addition of Core Lipids to Nascent Apolipoprotein B100, Resulting in the Assembly and Secretion of Triglyceride-rich Lipoproteins, Is Independent of Both Microsomal Triglyceride Transfer Protein Activity and New Triglyceride Synthesis

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. Apolipoprotein B (apoB)2 is synthesized primarily in hepatocytes and enterocytes and has a fundamental role in the transport and metabolism of plasma triacylglycerols (TAG) and cholesterol (1, 2). ApoB is a predominant protein component of very low density lipoproteins (VLDL) and intermediate density lipoproteins and is essentially the only apoprotein component of low density lipoproteins (LDL 2 ) (3, 4). ApoB100 (the fulllength protein) is one of the largest monomeric proteins known with 4536 amino acid residues (2). It is expressed primarily in mammalian liver, is an essential structural component for the formation and secretion of VLDL, and serves as a ligand for the LDL receptor (2). ApoB is present as a single molecule per lipoprotein particle (5); and therefore, its concentration in the plasma approximates the number of potential atherogenic lipoprotein particles.The processes involved in the assembly of apoB-containing lipoproteins in the liver are complex and are regulated at multiple levels throughout the secretory pathway. The assembly of apoB-containing lipoproteins occurs co-translationally (1), i.e. while the C-terminal portion is still being synthesized on the ribosome of the endoplasmic reticulum (ER), the N-terminal portion is translocated across the ER and is assembled as a small lipoprotein particle. The addition of lipids to apoB is widely believed to occur in two steps (2, 6, 7). The first step involves the addition of small amounts of lipids to apoB, as it is translated and translocated into the lumen of ER preventing its degradation and formation of a partially lipidated small pre-VLDL particle in the high density lipoprotein (HDL) density range (2,7,8). In the second step, this pre-VLDL particle is believed to acquire the bulk of its core lipids and is converted to bona fide VLDL (2, 7, 9), presumably by fusing with a large, VLDL-sized, apoB-free TAG particle (9). Biochemical studies of VLDL assembly support the concept that the bulk of neutral lipids are added in the second step after apoB translation is completed (10).* This work was supported by the National Institutes of Health Grants HL084685 and PO1 HL34343. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 2 The abbreviations used are: apoB, apolipoprotein B; apoB:1000, N-terminal 22.05% (residues 1-1000) of the mature protein; BSA, bovine serum albu-

Section: Discussionmentioning

confidence: 99%

Section: * This Work Was Supported By the National Institutes Of Healmentioning

confidence: 99%

Microsomal Triglyceride Transfer Protein Activity Is Not Required for the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in McA-RH7777 Cells

Dashti

Manchekar

Sun

et al. 2007

“…Rather, they combine with bulk neutral lipids (the second step) to form buoyant low density lipoproteins (LDL), intermediate density lipoproteins (IDL), or VLDL that are detectable in the lumen of microsomes (2,12). Acquisition of bulk neutral lipids in the second step appears to be independent of the MTP activity (10,11,15).…”

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confidence: 99%

Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Tran¹,

Thorne-Tjomsland²,

DeLong³

et al. 2002

109

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatmentinduced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulsechase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/ medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.

“…We previously demonstrated in cultured HepG2 and Mc RH7777 cells that OA could stimulate the assembly and secretion of VLDL even though TG synthesis and MTP activity were inhibited (36), leading us to conclude that OA acted as a signaling molecule to allow completion of the translocation of lipid-poor apoB and its subsequent fusion with a pre-existing lipid droplet in the secretory compartment (1,36,58,59). Our present data support a signaling role for OA in vivo but differ from the tissue culture results in that OA was unable to stimulate the concomitant secretion of TG.…”

Section: Discussionmentioning

confidence: 99%

“…2) Do FA only act as a substrate for core and surface lipids of apoB-lipoproteins, or can they act as signaling molecules, stimulating specific steps in the transport of apoB through the secretory pathway? Our recent work in cultured liver cell lines supports a signaling role for FA (36). 3) What are the mechanisms whereby the liver maintains lipid homeostasis in the face of increasing or decreasing demands related to the secretion of TG-rich apoB-lipoproteins?…”

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confidence: 99%

Regulation of Hepatic Apolipoprotein B-lipoprotein Assembly and Secretion by the Availability of Fatty Acids

Zhang

Hernandez‐Ono

et al. 2004

Self Cite

The in vivo effects of increased delivery of fatty acids (FA) to the liver are poorly defined. Therefore, we compared the effects of infusing either 6 mM oleic acid (OA) bound to albumin, 0.5-20% Intralipid, or saline for 3 or 6 h into male C57BL/6J mice. Infusions were followed by studies of triglyceride (TG) and apoB secretion. Although plasma FA levels increased similarly after either 20% Intralipid or 6 mM OA, TG secretion increased only after infusion of 4 -20% Intralipid; TG secretion was unchanged by 6 mM OA. By contrast, 6-h infusions of either 6 mM OA or 4 -20% Intralipid increased apoB secretion. 6 mM OA and 20% Intralipid each increased secretion of apoB from primary hepatocytes ex vivo. Importantly, 0.5-2% Intralipid, which delivered more FA to the liver than 6 mM OA, did not stimulate apoB secretion. Hepatic apoB mRNA levels were unaffected by either 6 mM OA or 20% Intralipid, but microsomal triglyceride transfer protein mRNA was significantly lower after 6-h infusions with 6 mM OA versus either saline or 20% Intralipid. Lower microsomal triglyceride transfer protein mRNA levels were associated with reduced hepatic TG mass after 6-h infusions of 6 mM OA. We conclude that 1) increased FA delivery to the liver in vivo increases secretion of apoBlipoproteins via post-transcriptional mechanisms, 2) OA-induced apoB-lipoprotein secretion occurred at least in part via mechanisms other than by providing substrate for TG synthesis, and 3) the route of delivery of FA is important for its effects on apoB secretion.Regulation of the assembly and secretion of apolipoprotein B (apoB)-lipoproteins and, in particular, very low density lipoprotein (VLDL), 1 by the liver has been studied intensively for many years in cultured hepatocytes (1-4). Many laboratories have also conducted in vivo studies of apoB-lipoprotein secretion in humans and animal models (5-7). However, despite the presence of a large body of work, several questions remain incompletely answered.First and foremost is the question of whether increased flux of plasma fatty acids (FA) to the liver can stimulate VLDL secretion. Studies in cultured liver cell lines, primary hepatocytes, and perfused livers have provided conflicting results relative to the role of FA delivery in VLDL secretion. Thus, several (8 -13), but not all (14 -16) studies in cultured liver cell lines support the proposal that apoB secretion is increased by increased FA uptake. Studies in primary hepatocytes from fed rats (16 -18) or hamsters (19,20) have failed to show that exogenous FA stimulate secretion of apoB. However, we were able recently to demonstrate oleic acid (OA)-induced apoB secretion in primary hepatocytes from fasted mice (21). Results from perfused rat livers have also been mixed; OA had no effect on apoB secretion in chow-fed rats (22) but increased apoB secretion in fasted (22) or high carbohydrate diet-fed (23) rats. In vivo studies in humans have also been inconclusive. Lewis et al. (24,25) demonstrate that, when they increased the plasma levels of FA by infusin...