Phospholipid transfer protein deficiency impairs apolipoprotein-B secretion from hepatocytes by stimulating a proteolytic pathway through a relative deficiency of vitamin E and an increase in intracellular oxidants

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ER-to-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wild-type cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.     

Assembly and secretion of very low density lipoproteins containing apolipoprotein B48 in transfected McA-RH7777 cells. Lack of evidence that palmitoylation of apolipoprotein B48 is required for lipoprotein secretion

We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys(1085), Cys(1396), Cys(1478), and Cys(1635)) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.     

Apolipoprotein B overproduction by the perfused liver of the St. Thomas' mixed hyperlipidemic (SMHL) rabbit

The St. Thomas' mixed hyperlipidemic (SMHL) rabbit (previously St. Thomas' Hospital rabbit) is a putative model of familial combined hyperlipidemia (FCH). When fed a low (0.08%) cholesterol diet, it exhibits elevations in both plasma cholesterol and triglyceride compared to New Zealand White (NZW) controls. To determine the mechanism for this hyperlipidemia we studied the secretion of apolipoprotein B (apoB)-containing lipoproteins from perfused livers of both young and mature rabbits. During a 3-h perfusion we measured the total cholesterol and triglyceride content of the medium and the cholesterol, triglyceride, and apoB content of very low density lipoprotein (VLDL)(1) (S(f) 60;-400), VLDL(2) (S(f) 20;-60), intermediate (S(f) 12;-20), and low (S(f) 0;-12) density lipoproteins (IDL, LDL). Lipoprotein concentrations increased linearly throughout the perfusion period. The rate of cholesterol output was 3-fold higher (459 vs. 137 ng/g liver/min, P = 0.003) in SMHL versus NZW rabbits whilst that of triglyceride was similar (841 vs. 662 ng/g liver/min, NS). VLDL(1) cholesterol output was elevated 2-fold (232 vs. 123 ng/g liver/min, P < 0.05) and VLDL(2) + IDL + LDL cholesterol output, 4.5-fold (106 vs. 23 ng/g liver/min, P < 0. 005) in SMHL versus NZW rabbits. ApoB output in VLDL1 was 38 ng/g liver per min in SMHL and 14 ng/g liver per min in NZW (NS). In SMHL VLDL(2) + IDL + LDL apoB was increased 9-fold at 53 versus 6 ng/g liver per min in NZW (P < 0.001). We conclude that the SMHL rabbit overproduces apoB-containing lipoproteins particularly in the VLDL(2) + IDL + LDL fraction, a characteristic consistent with its use as a model of FCH.     

The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways

We previously showed that Omega-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that Omega-3-induced degradation preferentially reduces the secretion of large, assembled apoB-lipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but Omega-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that Omega-3 fatty acids induced a striking loss of apoB100 from the Golgi, while sparing apoB100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the Omega-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.     

The degradation of apolipoprotein B100: multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways

Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.     

Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER

Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.     

An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt(-/-)) compared with Pemt(+/+) mice. Hepatocytes were incubated with 0.75 mm oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt(+/+) mice, hepatocytes from Pemt(-/-) mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by approximately 70% in VLDLs/low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.     

Direct evidence for a two-step assembly of ApoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes.

The aim of this study was to investigate the types and characteristics of chylomicron precursors in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized, and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.     

ACTH decreases the expression and secretion of apolipoprotein B in HepG2 cell cultures

Administration of adrenocorticotropic hormone (ACTH) has been shown to decrease plasma concentrations of apolipoprotein B (apoB) containing lipoproteins, including lipoprotein(a), in man. However, the mechanism behind this hypolipidemic effect is unknown. This study aimed at distinguishing between the main possibilities (increased elimination or decreased production of lipoproteins) using HepG2 cell cultures. Addition of ACTH to the cell culture medium selectively down-regulated apoB mRNA expression and apoB secretion in a dose-dependent manner. At 100 pmol/liter ACTH, the apoB mRNA level was about 40% lower than in the untreated cells, and the secretion of apoB into the medium was decreased to a similar extent. The expression and secretion of other apolipoproteins (apoA-I, apoE, and apoM), however, were not affected by ACTH. Under normal culture conditions the level of secretion of apoB from HepG2 cells is quite low. In the presence of 0.4 mmol/liter oleic acid secretion of apoB increased 3-fold, but this phenomenon was not seen in ACTH-treated cells. Binding and internalization of radiolabeled low density lipoprotein (LDL) by HepG2 cell, as well as LDL-receptor mRNA and scavenger receptor B-I mRNA levels, were not influenced by ACTH. In conclusion, ACTH directly and selectively down-regulated the production and secretion of apoB in HepG2 cell cultures, suggesting that a principal mechanism behind the cholesterol-lowering effect of ACTH in vivo may be a decreased production rate of apoB-containing lipoproteins from the liver.     

Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools

Apolipoprotein B (apoB), a protein containing several hydrophobic beta-sheet structures, is essential for the assembly of triglyceride-rich lipoproteins. Previously, we found that only a fraction of de novo synthesized apoB is secreted; the remainder is retained in the endoplasmic reticulum where it is degraded. To understand the basis for these observations, translocation, the first step in the secretory pathway, was examined. Translocation of apoB was determined by its sensitivity to degradation by the exogenous protease, trypsin. In rough microsomes, about half of the apoB was degraded by trypsin. In contrast, in Golgi fractions little (if any) apoB was accessible to trypsin. Essentially all of the apoB that was degraded was membrane bound. Monoclonal IgGs against either the N-terminal or C-terminal halves of apoB were bound to magnetic beads and used to immunoisolate microsomes. In contrast to the specific ability of the IgGs against apoB to isolate microsomes, little or no microsomes were isolated using nonimmune IgG and IgG against albumin. Since microsomes remained intact and oriented right-side out as demonstrated by the inability of trypsin both to degrade albumin and to affect the capacity of the intralumenal enzyme glucose-6-phosphatase to dephosphorylate mannose 6-phosphate, the data suggest that a pool of apoB is exposed on the cytoplasmic surface of the endoplasmic reticulum membrane. To determine if the trypsin-accessible pool of apoB is a transient form, pulse-chase experiments were performed. The results show that the percent of apoB that was trypsin accessible increased during the first 20 min of the chase, suggesting that during this time the trypsin-accessible pool of apoB is not translocated (it does not become trypsin insensitive). Thus, in two in vivo models (cultured cells and rat liver) translocation of apoB is not quantitative. We propose that apoB translocation across the endoplasmic reticulum determines its entry into two functionally distinct pools. The intralumenal trypsin-insensitive pool participates in the assembly of very low density lipoprotein; the trypsin-accessible nontranslocated cytoplasmic pool is shunted into a degradative pathway. Regulated translocation of apoB may provide a unique mechanism with which to determine the rate of very low density lipoprotein assembly/secretion.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Nascent lipoproteins from recirculating and nonrecirculating liver perfusions and from the hepatic Golgi apparatus of African green monkeys

Perfusate apoB-100-containing lipoproteins from the isolated, perfused livers of African green monkeys consist of significant amounts of d greater than 1.006 g/ml particles in addition to very low density lipoproteins (VLDL). Distinguishing characteristics of these perfusate lipoproteins are the relative abundance of surface lipids and deficiency of core lipids. The present studies were performed to determine the likelihood that the d greater than 1.006 g/ml perfusate lipoproteins are secretion products instead of products of post-secretory modification (e.g., lipolysis) of secreted VLDL. [14C]Leucine from the perfusate became incorporated into the apoB of each of the perfusate lipoprotein classes to a similar extent in both recirculating and nonrecirculating perfusions. When endogenously radiolabeled perfusate VLDL from one liver was recirculated through a second liver, only about 15% of the radiolabeled protein appeared in the d greater than 1.006 g/ml fraction. The particle morphology and the cholesterol and apoB distribution between VLDL and d greater than 1.006 g/ml fractions were similar in recirculating and nonrecirculating perfusions. A Golgi apparatus-rich fraction was isolated from the homogenates of fresh liver samples and the isolated Golgi VLDL and d greater than 1.006 g/ml lipoproteins exhibited morphologic evidence of extra surface material analogous to that seen in perfusate. Taken together, these data support the possibility that significant amounts of d greater than 1.006 g/ml lipoproteins, many with surface-rich properties, are nascent, secretory products of the primate liver. The low level of lecithin:cholesterol acyltransferase (LCAT) in this perfusion system appears to permit detection of these secretion products and it is significant to note that the perfusate lipoprotein profile, which is unlike that of normal plasma, is similar to that of LCAT-deficient patients.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.     

Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference.

Apolipoprotein B (apoB) is an essential structural protein for the two triglyceride-rich lipoproteins synthesized by humans: chylomicrons and very low density lipoproteins. Although much is known about the role of apoB in clearance of lipoproteins from the circulation, relatively little is known about its role in the assembly of nascent lipoproteins. Therefore, we have investigated the relationship between the length of various N-terminal apoB fragments and the characteristics of the lipoproteins with which these fragments were associated. After the addition of puromycin, HepG2 cells secreted a discrete series of C-terminally truncated apoB fragments on lipoprotein particles including apoB25, apoB29, apoB31, apoB33, apoB36, apoB38, apoB42, apoB45, apoB49, apoB51, apoB55, apoB70, and apoB80. Also, using plasmids encoding apoB26, apoB33, apoB37, apoB42, and apoB48, C-terminally truncated apoB fragments were expressed and secreted after transient transfection of HepG2 cells. Lipoproteins bearing the metabolically labeled apoB fragments were isolated from the cell culture media and characterized in terms of size, density, flotation coefficient, and composition. Lipoprotein radii, calculated from their flotation coefficients and buoyant densities, were used to derive the circumference of the non-polar core of each lipoprotein species. When plotted as a function of apoB size, core circumference defined a straight line of near-zero intercept. The slope of this line was approximately 1 A of core circumference/1 kDa of apoB molecular mass. A model for the mechanism of lipoprotein assembly in HepG2 cells, consistent with the concept that apoB size determines lipoprotein core circumference, is proposed.     

The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.     

Regulation of intestinal apolipoprotein B synthesis and secretion by Caco-2 cells. Lack of fatty acid effects and control by intracellular calcium ion

To investigate the mechanism of control of intestinal apolipoprotein B (apoB) secretion, we studied the effects of fatty acids and calcium ionophores on the human intestinal model cell line Caco-2. Although treatment with various fatty acids (18:1w9, 18:2w6, and 20:5w3) complexed to bovine serum albumin resulted in a dramatic redistribution of apoB-100 from the low density and high density lipoproteins to the very low density lipoprotein fraction, there was no effect of any of the fatty acids on the overall rate of total apoB (apoB-100 and apoB-48) secretion. Treatment of differentiated monolayers with calcium ionophores A23187 or ionomycin caused dose-specific increases (125% at 1 microM) in the accumulation of total apoB, but not apoA-I, in conditioned medium as measured by specific immunoassays. Incubation studies with 35S-labeled Caco-2 apoB,E-containing low density lipoprotein particles revealed that treatment with ionomycin over a broad concentration range had no effect on the reuptake of secreted apoB-100. The effect on A23187 on total apoB secretion was blocked by prior chelation of medium calcium and was significantly enhanced by the addition of calcium (up to 50 mM) to the medium. The effect of A23187 was significantly blunted by treatment with the calmodulin antagonist trifluoperazine (10 microM). The time course of A23187 action on Caco-2 apoB secretion required at least 6 h to occur. In contrast to the concentration of apoB in the medium, cellular apoB content was not influenced by treatment with ionophore. Pulse-chase experiments demonstrated a significant reduction in the synthesis-secretion interval for apoB-100 and apoB-48 after 24 h of exposure to ionomycin. Neither fatty acid treatment nor stimulation with ionophore affected the ratio of apoB-100 to apoB-48 produced by the cells. These findings with calcium ionophores implicate the involvement of calcium ion in the mechanism of intestinal apoB secretion. A role for calcium-dependent processes in apoB production raises the possibility that, rather than fatty acid flux, calcium-evoked or calcium-dependent hormones may be important regulators of apoB secretion.     

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.