Very Low Density Lipoprotein Assembly Research Articles

The Role of Factors that Regulate the Synthesis and Secretion of Very-Low-Density Lipoprotein by Hepatocytes

Lipoproteins are particles that contribute to overall metabolic homeo-stasis by transporting hydrophobic lipids in the blood plasma to and from different tissues in the body. Very-low-density lipoprotein (VLDL) is the principal vehicle for the transport of endogenous triglyceride (TG), and, ultimately, through its metabolic product, low-density lipoprotein (LDL), of cholesterol as well. It is synthesized mainly in hepatocytes, with small amounts also being produced by enterocytes in the fasting state. The mechanism of VLDL assembly is complex and is regulated at different levels by a variety of factors. The main structural protein of VLDL is called apolipoprotein B-100 (Apo B). Apo B formation and degradation therefore represent two major points of regulation of VLDL secretion. Hepatic levels of lipids such as phosphatidylcholine (PC), cholesteryl ester (CE), fatty acids (FA), and TG also affect VLDL synthesis. There are different views as to the specific mechanism by which each lipid class affects VLDL particle formation. In general, PC appears to promote the translocation of apo B from the cytosol to the lumen of the endoplasmic reticulum, a step that is crucial in the early stages of VLDL assembly. Apo B degradation is suppressed, and therefore VLDL secretion is enhanced, in the presence of elevated CE levels. For TG to be incorporated into the lipoprotein, it requires the action of a protein called microsomal triglyceride transfer protein (MTP). MTP might have a preference for TG comprised of FA with a certain degree of saturation. It becomes apparent that changes in diet that are accompanied by variations in the type of fats that are ingested affect VLDL formation and secretion. Regulation also occurs post-prandially in response to elevations in plasma insulin levels. Acute elevations in insulin inhibit VLDL secretion by promoting the degradation of apo B. This action is consistent with insulin's anabolic properties as it allows for the hepatic storage of lipid rather than for its distribution in VLDL to other tissues for fuel. Many studies have attempted to unravel the mechanisms of VLDL formation and secretion. The fact that so many factors are involved complicates the issue. The purpose of this article is to describe the relationship between different factors involved in VLDL assembly and secretion so that a better understanding of its metabolic regulation may be achieved.

Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (> 50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes

Our aims were i) to determine which lipids colocalize with newly synthesized apolipoprotein (apo) B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilization and/or translocation of this protein; and ii) to determine the intracellular sites of assembly of lipids into very low density lipoprotein (VLDL). In order to do this, we have developed a new method for the separation of ER-derived microsomes on self-generated gradients of iodixanol. Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles and the heavier peak contained rough vesicles. Each peak was collected in a number of subfractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (smooth endoplasmic reticulum (SER) fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apoB and VLDL lipids determined by ELISA or high performance thin-layer chromatography, respectively. The biosynthetic relationships of apoB and lipids were investigated, in timed or chase experiments, by incubation of isolated rabbit hepatocytes with radiolabeled precursors of apoB or lipids, followed by isolation and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol, and cholesteryl ester co-localize with apoB into the lumen of the RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesized triacyl-glycerol, phospholipid, cholesterol, and cholesteryl ester were also transferred to the lumen of the RER and were chased into the SER lumen. Double-labeling experiments showed that cholesteryl ester produced from newly synthesized cholesterol (labeled with [3H]mevalonate and [14C]oleate) was almost exclusively present in the RER, while cholesteryl ester in the SER was labeled only with [14C]oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.

Apolipoprotein B Sequence Requirements for Hepatic Very Low Density Lipoprotein Assembly: EVIDENCE THAT HYDROPHOBIC SEQUENCES WITHIN APOLIPOPROTEIN B48 MEDIATE LIPID RECRUITMENT

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29-34, AI/B34-37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-beta), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.

Selective recruitment of ApoB-48 for the assembly of VLDL in rat triacylglycerol-enriched hepatocytes.

Primary rat hepatocyte cultures were enriched in cellular triacylglycerol (TAG) by exposure to extracellular oleate for 3 days. Control cells were cultured for the same time without oleate. The large increase in TAG secretion into the medium of TAG-enriched cells during the final 24 hours (225 +/- 30 versus 40 +/- 10 micrograms/mg cell protein [control cells], P < .01) was not accompanied by a similar change in apolipoprotein B (apoB) secretion (4.22 +/- 0.94 versus 3.72 +/- 0.75 micrograms/mg per 24 hours, respectively). Instead, TAG-enriched cells recruited a larger proportion of apoB for the synthesis of very low density lipoprotein (VLDL), the secretion of which was substantially higher under these circumstances (1.46 +/- 0.39 versus 0.34 +/- 0.06 microgram apoB per milligram cell protein per 24 hours, P < .05). The increase in VLDL assembly was accompanied by a selective 2.5-fold increase (P < .05) in the specific recruitment of apoB-48. There was no significant increase in the amount of apoB-100, which appeared in the VLDL fraction when cells were enriched with TAG. Under these circumstances there was an increase in net cellular synthesis of apoB-48 (5524 +/- 667 versus 2505 +/- 598 disintegrations per minute per milligram protein per hour, P < .05). The net cellular synthesis of apoB-100 was unchanged compared with that observed in control cell cultures (1548 +/- 237 versus 2000 +/- 897 dpm/ mg per hour, respectively). A large proportion of the total secreted apoB was associated with small particles of density higher than VLDL, even when VLDL output was maximally stimulated, suggesting that apoB was oversecreted and in excess of the cells' requirement to transport TAG.

Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B-100-containing lipoproteins

Apolipoprotein B-100 (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL). The mechanism of VLDL assembly was explored by perturbing apoB folding in HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Although apoB contains eight known disulfide bonds, seven of which are positioned in the amino-terminal 21% of the protein, its assembly and secretion was only partially blocked in cells treated with 2 mM DTT, a condition that fully blocks the secretion of other disulfide-bonded proteins. Nonreducing gel electrophoresis of an apoB-derived proteolytic peptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation to a DTT-resistant form after completing synthesis of only the first approximately 20-25% of the protein. If, however, DTT was used under conditions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked. Reduced forms of apoB were extremely labile and, unlike other disulfide-bonded proteins, incapable of achieving secretion competence posttranslationally. These results indicate that disulfide bond formation within the amino-terminus of apoB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascent polypeptide chain is consistent with a model in which the amino-terminal domain of apoB undergoes an independent folding and maturation process, the completion of which may represent an initiation phase of triglyceride-rich lipoprotein assembly.

Brefeldin A Reversibly Inhibits the Assembly of ApoB Containing Lipoproteins in McA-RH7777 Cells

BFA inhibited in a dose dependent way the assembly of apoB-48 very low density lipoprotein (VLDL) but allowed a normal rate of biosynthesis of the apolipoprotein and of the assembly of the dense ("high density lipoprotein (HDL)-like") apoB-48 particle (apoB-48 HDL). The inhibition of the assembly of apoB-48 VLDL occurred at BFA levels that allowed a major secretion of both transferrin and apoB-48 HDL. The assembly of apoB-100 containing lipoproteins was also inhibited by BFA but could be reactivated by a 30-60 min chase in the absence of BFA, which agreed with the time that was estimated to be needed to restore the secretory pathway (approximately 60 min). Also the assembly of apoB-48 VLDL was reversible. Both apoB-48 and apoB-100 that was labeled in the presence of BFA assembled VLDL after removal of the BFA. Both apoB-100 and apoB-48 were associated with the membrane pellet of the microsomes. Virtually all (122 +/- 30%) of the membrane associated pulse-labeled apoB-48 remained in the membrane after a 180-min chase in the presence of BFA, compared to only 21 +/- 2% in normal cells (mean +/- S.D., n = 4). The corresponding figures for apoB-100 was 40 +/- 7% in BFA-treated cells and 9 +/- 7% in normal cells (mean +/- S.D., n = 4). Pulse-chase experiments with BFA offered conditions to selectively follow the turnover of membrane-associated apoB-100. Such experiments indicated that this apoB-100 pool is a precursor to VLDL.

Assembly of very low density lipoproteins in rat liver: a study of nascent particles recovered from the rough endoplasmic reticulum.

To investigate the assembly pathway for hepatic very low density lipoproteins (VLDL), nascent lipoproteins were recovered from a purified, intact rough endoplasmic reticulum (ER) fraction isolated from rat liver. Two fractions were recovered by ultracentrifugation. Particles isolated at d 1.006 g/ml were triglyceride-rich particles containing apolipoprotein (apo)B-100 or apoB-48, and apoE with very small amounts of apoA-I. Compared with VLDL recovered from the Golgi apparatus, the particles from the rough ER had less triglyceride, but more cholesteryl ester and phospholipid. The second class of particles isolated between d 1.006 and 1.210 g/ml were phospholipid-rich and contained apoB-48, apoE, and apoA-I. ApoB-100 was a minor component. Radioisotope incorporation studies utilizing [3H]leucine revealed differential rates of labeling of the apoproteins in these two lipoprotein fractions. ApoB-100 and apoE followed similar patterns in both fractions with peak incorporation occurring within 15 min of isotope injection. Incorporation of [3H]leucine into apoB-48 in the dense fraction peaked within 15 min of isotope administration, but peak incorporation in the d 1.006 g/ml fraction did not occur until approximately 30 min after injection. We propose that the two lipoprotein fractions recovered from the rough ER are intermediates in the assembly of VLDL by the liver. Comparison of the composition of these two particles with that of Golgi VLDL supports the sequential assembly of VLDL by the liver. Furthermore, we propose that the initial steps in the assembly of apoB-100- and apoB-48-containing lipoproteins are different with nascent apoB-100-containing particles being formed through the cotranslational association of this apoprotein with lipid while nascent apoB-48-containing VLDL are formed in the rough ER through a two-step process.

Intracellular triacylglycerol lipase: Its role in the assembly of hepatic very-low-density lipoprotein (VLDL)

Extracellular fatty acids entering the hepatocyte are either esterified to cytosolic TAG or oxidized to ketone bodies. Very little is esterified and secreted directly in association with VLDL. Thus, even when extracellular fatty acids are available, the major, direct source of VLDL TAG is the cytosolic pool. The recruitment of cytosolic TAG for VLDL assembly involves lipolysis followed by re-esterification. At least 70% of the secreted TAG is derived via this route. Fatty acids released at this lipolytic step are utilized exclusively for VLDL TAG synthesis and are not available for ketogenesis. Substantially more cytosolic TAG undergoes lipolysis than is required to meet the needs of VLDL assembly. The remaining fatty acids are re-esterified and re-cycled to the cell cytosol. From a physiological viewpoint, the presence of this indirect route for VLDL TAG recruitment would provide a means of regulation of VLDL secretion which is independent of the plasma fatty acid concentration. In this respect, several pathophysiological conditions are known in which there is a negative association between plasma fatty acid concentration and the rate of VLDL secretion. These are: (a) insulin-dependent diabetes, (b) starvation, (c) fat-feeding. Lipolysis of cytosolic TAG and transfer of fatty acids into the ER lumen may provide a regulatory focus for the control of hepatic VLDL output.

Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.

The mechanisms by which apolipoprotein B-100 (apoB-100) and apoB-48 assemble lipoproteins have been studied in the McA-RH7777 cell line. After incubation for 2 h with 360 microM oleic acid, the McA-RH7777 cells secreted apoB-100 mainly on very low density lipoprotein (VLDL) particles, as judged from sucrose gradient and sequential ultracentrifugation. ApoB-48 was secreted on both VLDL and on denser, high density lipoprotein (HDL)-like lipoproteins. Both apoB-48 and apoB-100 occurred on VLDL particles in the luminal content of the total microsomal fraction. In addition, both proteins were present on denser particles, in particular, those that banded in the HDL region. The denser particles containing apoB-48 were secreted from the cells, whereas those containing apoB-100 were retained in the cell and degraded. Pulse-chase experiments showed that apoB-100 on VLDL was present in the secretory pathway already after a labeling period as short as 3 min. Thus, this particle was the first apoB-100-containing lipoprotein that could be detected in the microsomal fraction of the cell. The assembly of labeled apoB-100 VLDL was acutely (within min) and completely blocked by cycloheximide, if the cycloheximide was added after the pulse labeling period. If, on the other hand, a 15-min chase was introduced after the labeling period, there was no effect of cycloheximide on the assembly of apoB-100 VLDL. A 15-min chase is enough to allow the pulse-labeled apoB nascent polypeptides to be completed to apoB-100. These results indicated that the assembly of apoB-100-containing VLDL is dependent upon ongoing protein biosynthesis during the time when the nascent polypeptide elongate. After this period, the assembly and secretion of the particles are independent of ongoing protein biosynthesis. The first apoB-48-containing particle seen in the luminal content of the microsomal fraction was the HDL-like particle. Pulse-chase experiments as well as experiments with different lengths of the radioactive pulse indicated that the formation of apoB-48-containing VLDL was delayed compared with the formation of the apoB-48-containing HDL-like particle and also in relation to the assembly of the different apoB-100-containing particles, including VLDL. After a 30-min pulse with [35S]methionine followed by a 120-min chase the secretory pathway of the cell was depleted of almost all lipoproteins except the HDL-like apoB-48-containing particle.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

Extracellular fatty acids are not utilized directly for the synthesis of very-low-density lipoprotein in primary cultures of rat hepatocytes.

In hepatocytes cultured in the presence of oleate (initial concn. 0.75 mM), the secretion of very-low-density lipoprotein (VLDL) triacylglycerol and, to a lesser extent, apoprotein B (apoB) increased with time, whereas there was a large decline in the extracellular concentration of fatty acid. There was thus no synchronous relationship between the extracellular fatty acid concentration and the secretion of VLDL. Rather, the appearance of VLDL in the medium was dependent on the intracellular triacylglycerol concentration. At a given concentration of extracellular fatty acid, cells depleted of triacylglycerol secreted less VLDL triacylglycerol and apoB than did control cells. A similar pattern was observed for triacylglycerol newly synthesized from extracellular [3H]oleate. By contrast, the synthesis and output of ketone bodies were directly dependent on the fatty acid concentration of the medium. These results suggest that, at least for oleic acid, extracellular fatty acids are not utilized directly for VLDL assembly, but first enter a temporary intracellular storage pool of triacylglycerol, which is the immediate precursor of secreted triacylglycerol. The size of this pool then determines the rate of secretion of VLDL triacylglycerol apoB. Ketogenesis, on the other hand, relies mainly on the direct utilization of extracellular fatty acids.

The lipolysis/esterification cycle of hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones and sulphonylureas.

In hepatocyte cultures maintained in the absence of extracellular fatty acids, at least 70% of the secreted very-low-density lipoprotein (VLDL) triacylglycerol was derived via lipolysis of intracellular triacylglycerol. This proportion was unchanged when the cells were exposed for 24 h to insulin or glucagon, hormones which decreased the overall secretion of intracellular triacylglycerol, or to chloroquine or tolbutamide, agents which inhibit lysosomal lipolysis. The rate of intracellular lipolysis was 2-3-fold greater than that required to maintain the observed rate of triacylglycerol secretion. Most of the fatty acids released were returned to the intracellular pool. Neither insulin nor glucagon had any significant effect on the overall lipolysis and re-esterification of intracellular triacylglycerol. In these cases a greater proportion of the released fatty acids re-entered the cellular pool, rather than being recruited for VLDL assembly. Tolbutamide inhibited intracellular lipolysis, but suppressed VLDL secretion to a greater extent. 3,5-Dimethylpyrazole did not affect lipolysis or VLDL secretion. The increased secretion of VLDL triacylglycerol observed after exposure of cells to insulin for 3 days was not accompanied by an increased rate of intracellular lipolysis. However, a larger proportion of the triacylglycerol secreted under these conditions may not have undergone prior lipolysis.

Effect of nutritional state on the utilization of fatty acids for hepatitic triacylglycerol synthesis and secretion as very-low-density lipoprotein

The mass of very-low-density-lipoproteins (VLDL) triacylglycerol secreted from isolated hepatocytes was dependent on the nutritional state of the donor rats, and declined in the order sucrose-fed greater than chow-fed greater than polyunsaturated-fat-fed greater than starved. This was the case irrespective of the presence or absence of exogenous oleate. The contribution of newly synthesized fatty acids to the total mass of VLDL triacylglycerol also declined in the above order, and reflected the relative rates of fatty acid synthesis de novo in each of the groups. The contribution of exogenous oleate to VLDL triacylglycerol varied in a manner similar to that for newly synthesized fatty acid. However, the contribution either of exogenous oleate or of newly synthesized fatty acid never exceeded 17-20% of the total VLDL triacylglycerol fatty acid even in the sucrose-fed animals. The increased contribution of newly synthesized fatty acids in the sucrose-fed group was not sufficient to account for the increase in the total mass of VLDL triacylglycerol secreted. These results suggest that: (a) changes in the rate of triacylglycerol secretion are not a direct consequence of variations in the rate of fatty acid synthesis de novo; (b) in the short term, most of the triacylglycerol required for VLDL assembly and secretion is derived from an intracellular storage source: (c) the distribution of newly synthesized triacylglycerol between the cytosolic and secretory pools was similar irrespective of the source of fatty acids (i.e. synthesized de novo or exogenous).

The distribution of apolipoprotein B in endoplasmic reticulum and Golgi subfractions of rabbit liver

The liver is the site of synthesis and secretion of very-lowdensity lipoproteins (VLDL), the vehicle for the transport of endogenous triacylglycerol (TAG) and the precursor of lowdensity lipoproteins (LDL). Elevated plasma levels of VLDL and LDL are recognized risk factors in the development of atherosclerosis. The production of VLDL by the liver is considered to be the most appropriate therapeutic target for the reduction of circulating lipoprotein levels. An understanding of the molecular and topographical events in the assembly of VLDL is thus of fundamental importance. Apolipoprotein B (apo B) is the major non-exchangeable protein of VLDL, and is essential for their secretion. Studies in HEP-G2 cells have indicated that apo B is synthesized by membrane-bound polysomes [I]. Newly synthesized apo B remains associated with the endoplasmic reticulum (ER) membrane for a period of time before being transferred to the cisternal space [2]. Consistent with this, we have previously shown that apo B does not associate with TAG-richVLDL precursors in the ER cisternae, and that the main site of packaging of VLDL lipid and protein is the Golgi cisternae [3]. To examine the intracellular events in apo B synthesis and translocation in more detail, we have determined the intracellular location and amount of apo B within the secretory pathway. The intracellular location of apo B was determined by SDS/PAGE and immunoblotting of subcellular fractions. ER and Golgi fractions were prepared from rabbit liver, and separated into membrane and cisternal content fractions by sodium carbonate treatment 14, 51. Fractions were delipidated and separated on 3-20% (w/v) polyacrylamidegradient gels [6], and then electroblotted on to nitrocellulose membrane [7]. Apo B- 100, the only apparent species of apo B secreted by rabbit liver, was detected by immunoblotting using sheep anti-rabbit apo B (primary antibody), and biotinylated anti-sheep IgG (secondary antibody). The secondary antibody was detected colorimetrically by the highly sensitive Vector ABC-alkaline phosphatase system. Apo €3- 100 was detectable in both ER and Golgi fractions (Fig. 1 ). When ER was separated into membranes and cisternal contents, apo B-100 was found in both fractions at apparently similar levels. When Golgi was separated into membranes and cisternal contents, although apo B- 100 could be detectcd in membranes, most of the protein appeared to be located in the contents. Apo €3- 100 in subcellular fractions was quantified by radioimmunoassay. LDL were isolated from rabbit plasma (d 1 .O 19-d 1.063). and radiolabclled with lz51 [ 8, 01. Apo B-I00 was the only protein detectable in LDL by SDS/ PAGE. and was found to be 'z51-labelled. Using a fixed concentration of labelled LDL with varying concentrations of

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes.

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.

Modulation of apolipoprotein B-100 mRNA editing: effects on hepatic very low density lipoprotein assembly and intracellular apoB distribution in the rat.

We have studied the consequences of alterations to hepatic apoB mRNA editing on the biosynthesis and intracellular distribution of newly synthesized apoB variants together with their mass distribution in nascent Golgi very low density lipoproteins (VLDL). Radiolabeled liver membrane fractions were prepared from control or hypothyroid animals and separated by discontinuous sucrose gradient centrifugation. Hepatic apoB-100 synthesis in these groups accounted for 93-100% of total newly synthesized apoB species of Golgi fractions recovered from the sucrose gradients (G1 and G2). The analogous fractions isolated from the livers of hyperthyroid (treated with 3,3',5-triiodo-L-thyronine, T3) animals revealed that newly synthesized apoB-100 accounted for only 46 +/- 10% (G1) and 24 +/- 11% (G2), respectively, of total newly synthesized apoB. ApoB-100 mass in nascent Golgi VLDL from control and hypothyroid G1 fractions represented 70-78% total apoB as determined by Western blot analysis. By contrast, Golgi VLDL from hyperthyroid animals contained predominantly (greater than 78%) apoB-48 as the apoB species. Electron microscopy revealed that the morphology and size distribution of hyperthyroid G1 VLDL were similar to particles isolated from control animals. Thus, despite a profound reduction in the proportion of apoB-100 mRNA species containing an unmodified codon (CAA, B-GLN) at position 2153 in hyperthyroid animals (6 +/- 1% vs 50-61% in control and hypothyroid animals) apoB-100 biosynthesis was detectable in a defined membrane fraction isolated by discontinuous sucrose gradient centrifugation. However, no apoB-100 synthesis was detectable in liver samples prepared by Polytron disruption in Triton-containing buffers. These data suggest that effective hepatic VLDL assembly and secretion in the T3-treated rat continues despite a profound reduction in apoB-100 biosynthesis and implies that apoB-48 contains the requisite domains to direct this process, a situation analogous to that in the intestine.

Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins.

The transfer of triglyceride from sites of synthesis in the endoplasmic reticulum to cytoplasmic lipid droplets and nascent VLDL (very low density lipoproteins) in rat liver in vivo has been examined with [3H]glycerol, cell fractionation, and electron microscopy. Rates of mass transfer of newly synthesized triglyceride were estimated from the specific radioactivity of triglyceride present in microsomal membranes and the radioactivity observed in recipient triglyceride pools. Fasting decreased the transfer of triglyceride to nascent VLDL without affecting transfer to lipid droplets. Stimulation of triglyceride synthesis with 2-tetradecylglycidic acid (TDGA) increased transfer of triglyceride to nascent VLDL 5-fold, and to lipid droplets 14-fold, 1 hr after TDGA administration. Triglyceride transfer to nascent VLDL was increased 6-fold, and to lipid droplets 37-fold, above control rates 6 hr following TDGA treatment, indicative of saturation of triglyceride assembly into nascent VLDL and storage of excess triglyceride in lipid droplet reservoirs. These liver triglyceride pools were concurrently expanded and electron microscopy demonstrated more abundant VLDL particles in the endoplasmic reticulum together with a proliferation of lipid droplets in hepatocytes. TDGA progressively decreased hepatic sn-glycerol-3-phosphate in fasting rats while triglyceride synthesis increased, indicating that sn-glycerol-3-phosphate does not limit the rate of triglyceride synthesis in this metabolic state. Results implicate triglyceride transfer from endoplasmic reticulum membranes to nascent VLDL as a regulated determinant of hepatic VLDL assembly and VLDL triglyceride secretion in vivo.

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution.

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.