Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2;-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2;-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver.     

Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.

Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.     

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.     

Identification of the proteoglycan binding site in apolipoprotein B48

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.     

Amino acid sequences within the beta1 domain of human apolipoprotein B can mediate rapid intracellular degradation

Apolipoprotein B (apoB)-48 contains a region termed the beta1 domain that is predicted to be composed of extensive amphipathic beta-strands. Analysis of truncated apoB variants revealed that sequences between the carboxyl termini of apoB-37 and apoB-42 governed the secretion efficiency and intracellular stability of apoB. Although apoB-37, apoB-34, and apoB-29 were stable and secreted efficiently, apoB-42 and apoB-100 were secreted poorly and were degraded by an acetyl-leucyl-leucyl-norleucinal (ALLN)-sensitive pathway. Amino acid sequence analysis suggested that a segment between the carboxyl termini of apoB-38 and apoB-42 was 63% homologous to fatty acid binding proteins (FABPs), which contain orthogonal beta-sheets. To test the hypothesis that sequences from the beta1 domain are involved in apoB degradation, fusion proteins were created that contained apoB-29 linked to fragments derived from the beta1 domain of apoB or to liver FABP. Fusion proteins containing the beta1 domain segments apoB-34-42 or apoB-37-42 were degraded rapidly, whereas other fusion proteins were stable and secreted efficiently. Degradation was ALLN-sensitive, and the apoB-34-42 segment increased the association of the apoB protein with the cytosolic surface of the microsomal membrane. Our data suggest that the presence of specific sequences in the beta1 domain of human apoB increases degradation by promoting the cytosolic exposure of the protein, although not all regions of the beta1 domain are functionally equivalent.     

Lipid micelles stimulate the secretion of triglyceride-enriched apolipoprotein B48-containing lipoproteins by Caco-2 cells

Intestinal triglyceride-rich lipoproteins (TRL) are synthesized from dietary lipids. This study was designed to evaluate the effects of lipid micelles, mimicking post-digestive duodenal micelles, on the fate of apolipoprotein B (apoB)48-containing lipoproteins by Caco-2 cells. Such micelles, consisting of oleic acid (OA), taurocholate, 2-monooleoylglycerol (2-MO), cholesterol (Chol), and L-alpha-lysophospatidylcholine, were the most efficient inducers of OA uptake and esterification. The efficiency of TG and apoB48 secretion increased specifically as a function of cell differentiation. PAGE analysis of secreted lipoproteins separated by sequential ultracentrifugation after [35S] labeling revealed differences in the secretion of apoB100- and apoB48-containing lipoproteins. In absence of micelles, apoB48 was secreted mostly in "HDL-like" particles, as observed in enterocytes in vivo. Micelle application increased 2.7-fold the secretion of apoB, resulting in 53 times more apoB48 being recovered as TG-enriched lipoproteins at d < 1.006 g/ml. Electron microscopy revealed the presence of lipid droplets in the secretory pathway and the accumulation of newly synthesized TG in cytoplasmic lipid droplets, as in enterocytes in vivo. We showed that these droplets could be used for secretion. However, apoB48 preferentially bound to newly synthesized TG in the presence of micelles, accounting in part for the functional advantage of apoB editing in the intestine. While Caco-2 cells express both apoB isoforms, our results show that the apical supply of complex lipid micelles favors the physiological route of apoB48-containing TG-enriched lipoproteins.     

Golgi-associated maturation of very low density lipoproteins involves conformational changes in apolipoprotein B, but is not dependent on apolipoprotein E

The major protein component in secreted very low density lipoproteins (VLDL) is apoB, and it is established that these particles can reach sizes approaching 100 nm. We previously employed a cell-free system to investigate the nature of the vesicles in which this large cargo exits the endoplasmic reticulum (ER) (Gusarova, V., Brodsky, J. L., and Fisher, E. A. (2003) J. Biol. Chem. 278, 48051-48058). We found that apoB-containing lipoproteins exit the ER as dense lipid-protein complexes regardless of the final sizes of the particles and that further expansion occurs via post-ER lipidation. Here, we focused on maturation in the Golgi apparatus. In three separate approaches, we found that VLDL maturation (as assessed by changes in buoyant density) was associated with conformational changes in apoB. In addition, as the size of VLDL expanded, apoE concentrated in a subclass of Golgi microsomes or Golgi-derived vesicles that co-migrated with apoB-containing microsomes or vesicles, respectively. A relationship between apoB and apoE was further confirmed in co-localization studies by immunoelectron microscopy. These combined results are consistent with previous suggestions that apoE is required for VLDL maturation. To our surprise, however, we observed robust secretion of mature VLDL when apoE synthesis was inhibited in either rat hepatoma cells or apoE(-/-) mouse primary hepatocytes. We conclude that VLDL maturation in the Golgi involves apoB conformational changes and that the expansion of the lipoprotein does not require apoE; rather, the increase in VLDL surface area favors apoE binding.     

Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.     

Defining lipid-interacting domains in the N-terminal region of apolipoprotein B

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted alpha-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogeneous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC-dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous with lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.     

The assembly and secretion of ApoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins.

The possibility that apoB 100 is cotranslationally translocated to the endoplasmic reticulum lumen and integrated into lipoproteins has been investigated. ApoB 100 nascent polypeptides were shown to be secreted from pulse-labeled Hep G2 cells after treatment with puromycin and chase for 1 or 2 h in the presence of puromycin and cycloheximide. These nascent polypeptides banded during sucrose gradient ultracentrifugation between the position of the high (HDL) and the low (LDL) density lipoproteins, revealing an inverse relationship between the length of the polypeptide and the density of the fraction. ApoB 100 occurred in the position of LDL and very low density lipoproteins (VLDL). Electronmicroscopy studies of the apoB-containing particles from the gradient indicated an increase in size with increasing length of the polypeptide. Furthermore, labeling studies indicated that the triglyceride load increased with the length of the polypeptide. An inverse relationship between the size of C-terminally truncated apoB polypeptides and the density of the assembled lipoproteins was also observed in experiments with transfected minigenes coding for apoB 41, apoB 29, and apoB 23. These proteins appeared on HDL particles. Pulse-chase experiments indicated that 80-200-kDa apoB nascent polypeptides on particles with HDL density, with time, were converted into larger polypeptides on lighter particles, to be fully replaced by apoB 100 on LDL-VLDL particles. The formation of these LDL-VLDL particles could be blocked by cycloheximide. Sixty-five percent of pulse-labeled apoB nascent polypeptides present in the microsomal fraction was released by sodium carbonate treatment, and 77% of these polypeptides could be recovered on the immature particles (banding between HDL and LDL) after sucrose gradient ultracentrifugation. Pulse-chase experiments indicated that these nascent polypeptides, on the immature lipoproteins, had the capacity to be precursors for all the apoB 100-containing LDL and VLDL particles formed in the cell. The obtained results indicate that a major portion of the apoB nascent polypeptides in the cell form lipoproteins cotranslationally during the translocation to the lumen of the endoplasmic reticulum.     

Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line,LMH-2A

The synthesis and secretion of apolipoprotein A-I (apoA-I) in response to the treatment with estrogen were investigated in the chicken hepatoma cell line, LMH-2A. Exposure of these cells to exogenous estrogen for up to 48 h results in a decrease of apoA-I production, as evident from Western blotting, immunoprecipitation, and immunofluorescence experiments. Likewise, the secretion of apoA-I is also decreased in estrogen-treated cells when compared to controls. However, under both conditions, the disappearance of the apoprotein from the cells occurs very rapidly and with similar kinetics. The bulk of apoA-I secreted from LMH-2A cells is recovered on lipoprotein particles with a buoyant density of > or =1.10 g/ml, corresponding to HDL and heavy LDL. Interestingly, apoA-I is detectable on apoB-containing lipoproteins by sequential immunoprecipitation, suggesting that the two apoproteins co-reside at least on a subfraction of the secreted particles, or that apoB- and apoA-I-containing particles interact. These interactions are more pronounced in estrogen-treated cells, most likely due to the dramatic estrogen-mediated induction of apoB synthesis and secretion.     

Chinese hamster ovary cells require the coexpression of microsomal triglyceride transfer protein and cholesterol 7alpha-hydroxylase for the assembly and secretion of apolipoprotein B-containing lipoproteins

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.     

Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.     

Identification of the lipoprotein initiating domain of apolipoprotein B

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first approximately 884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.     

An expression system for human apolipoprotein B100 in a rat hepatoma cell line

We have designed an in vitro expression system for human apolipoprotein (apo) B. A full-length human apoB minigene was constructed from cDNA and genomic apoB clones and inserted into a vector where its expression was directed by the cytomegalovirus promoter. The apoB minigene was expressed in a rat hepatoma cell line, McA-RH7777. Human apoB100, which is the ligand for the low density lipoprotein receptor, was secreted in low density lipoprotein or very low density lipoprotein particles, depending on the composition of the medium. A protein with the mobility of apoB48, a structurally related protein involved in cholesterol metabolism, was also produced from the human apoB minigene. This in vitro expression system for human apoB will enable investigators to identify which domains of this protein are involved in processes such as lipoprotein assembly and secretion. This system should also allow investigators to identify definitively the domain in apoB that enables the protein to bind to the low density lipoprotein receptor.     

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.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

Inhibition of apolipoprotein B secretion by taurocholate is controlled by the N-terminal end of the protein in rat hepatoma McArdle-RH7777 cells

Bile salts (BS) inhibit the secretion of apolipoprotein B (apoB) and triacylglycerol (TG) in primary rat, mouse and human hepatocytes and in mice in vivo. We investigated whether lipidation of apoB into a lipoprotein particle is required for this inhibitory action of BS. The sodium/taurocholate co-transporting polypeptide (Ntcp) was co-expressed in McArdle-RH7777 (McA-RH7777) cells stably expressing the full-length human apoB100 (h-apoB100, secreted as TG-rich lipoprotein particles) or carboxyl-truncated human apoB18 (h-apoB18, secreted in lipid-free form). The doubly transfected cell lines (h-apoB/r-Ntcp) effectively accumulated taurocholic acid (TC). TC incubation decreased the secretion of endogenous rat apoB100 (-50%) and h-apoB18 (-35%), but did not affect secretion of rat apoA-I. Pulse-chase experiments (35S-methionine) indicated that the impaired secretion of radiolabeled h-apoB18 and h-apoB100 was associated with accelerated intracellular degradation. The calpain protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) partially inhibited intracellular apoB degradation but did not affect the amount of either h-apoB18 or h-apoB100 secreted into the medium, indicating that inhibition of apoB secretion by TC is not due to calpain-dependent proteasomal degradation. We conclude that TC does not inhibit apoB secretion by interference with its lipidation, but rather involves a mechanism dependent on the N-terminal end of apoB.     

Charged amino acid residues 997-1000 of human apolipoprotein B100 are critical for the initiation of lipoprotein assembly and the formation of a stable lipidated primordial particle in McA-RH7777 cells

We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpin-bridge mechanism. We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model. To identify the specific motif within residues 931 and 1000 that is critical for apoB particle assembly, apoB:956 and apoB:986 were produced. To test the hairpin-bridge hypothesis, the following mutations were made: 1) residues 997-1000 deletion (apoB:996), 2) residues 717-720 deletion (apoB:1000Delta717-720), and 3) substitution of charged residues 997-1000 with alanines (apoB:996 + 4Ala). Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. 1) ApoB:956 did not form stable particles and was secreted as large lipid-rich aggregates. 2) ApoB:986 formed both a lipidated particle that was denser than HDL(3) and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000Delta717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) In striking contrast to wild-type apoB:1000, (i) apoB:996 and apoB:996 + 4Ala were highly susceptible to intracellular degradation, (ii) only a small proportion of the secreted proteins formed stable HDL(3)-like lipoproteins, and (iii) a majority of the secreted proteins formed large lipid-rich aggregates. We conclude that the first 1000 amino acid residues of human apoB100 are required for the initiation of nascent apoB-containing lipoprotein assembly, and residues 717-720 and 997-1000 play key roles in this process, perhaps via a hairpin-bridge mechanism.