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Recent studies indicate that microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) interact physically via two specific binding sites located within the amino-terminal globular region of apoB100. The first site is thought to be within the first 5.8% of the amino-terminal sequence, and the second site is between 9 and 16% of the amino-terminal sequence. It is not clear from prior studies whether these sites have unique or overlapping functions. Furthermore, there are no data differentiating between lipid transfer and potential chaperone functions of MTP. In the present study we have attempted to further characterize the physiologic interaction between apoB and MTP and to determine the relationship between the binding and lipid transfer aspects of the interaction. HepG2 cells were transiently transfected with apoB cDNAs, and MTP binding to apoB polypeptides was determined by two-step immunoprecipitation. MTP bound equally well to apoB polypeptides with (apoB13, 16,beta, apoB34, and apoB42) or without (apoB16, apoB13, and 16 or apoB13, 13, and 16) beta sheet domains. When proteasomal degradation of newly synthesized apoB polypeptides was blocked, MTP binding to all of the apoB polypeptides was only modestly affected by lipid availability and was independent of MTP-associated lipid transfer. Furthermore, MTP did not bind directly to a portion of the first beta sheet domain. We created two apoB constructs (apoB16del and apoB34del) by deleting the first 210 amino acids of apoB16 and apoB34. These apoB polypeptides, therefore, lacked the putative first MTP binding site. MTP binding to apoB16del and apoB34del was decreased significantly. However, the secretion of apoB16del was not different from apoB16, whereas the secretion of apoB34del was impaired significantly. Our results indicate that the interaction between MTP and apoB involves independent binding and lipid transfer activities but that both activities are required for the secretion of apolipoprotein B from liver cells.

In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation.

We have previously demonstrated a close relationship between oleic acid (OA)-stimulated triglyceride (TG) synthesis and apolipoprotein B (apoB) secretion in HepG2 cells. However, other investigators studying the association between glucose-stimulated TG synthesis and apoB secretion have reported variable results. The present study was carried out to answer the question: does TG derived from glucose have different effects on apoB secretion from HepG2 cells compared to TG derived from oleate? We observed that incubations of HepG2 cells for as long as 48 h in 30 mm glucose did not increase apoB secretion. We then demonstrated that the failure of glucose to stimulate apoB secretion from HepG2 cells results from insufficient stimulation of TG synthesis by glucose. Thus, incorporation of [3H]glycerol into [3H]TG in the presence of 30 mm glucose for up till 48 h was not increased compared to basal conditions. The inability of glucose to stimulate TG synthesis was also evidenced by the inability of both 8 -h and 24 -h incubations with 30 mm glucose to increase cell TG mass; similar incubations with OA increased TG mass 50-100%. Additional studies demonstrated that glucose conversion to either glycerol or fatty acids was minimal; this accounted for the lack of stimulation of TG synthesis. We conclude that in HepG2 cells, availability of high glucose levels in the media for as long as 48 h does not stimulate triglyceride synthesis, and that this is the basis for the failure of glucose to stimulate apoB secretion.

Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were co-treated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLN-sensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway.

Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

To differentiate effects of lovastatin on low density lipoprotein (LDL) receptor activity from effects on LDL metabolic properties, LDL apolipoprotein B (apoB) turnover was studied in eight hyperlipidemic subjects during baseline and lovastatin treatment, in the latter case with LDL tracers isolated during both baseline (CLDL) and drug treatment (Rx-LDL) conditions. Lovastatin (40 mg/day) significantly lowered total plasma and LDL cholesterol levels (27% and 25%, respectively) as well as plasma triglyceride levels (30%). Using contemporaneous tracers (C-LDL before and Rx-LDL during treatment), lovastatin caused a modest increase in LDL fractional catabolic rate (FCR) (0.410+/-0.113 vs. 0.339+/-0.108 pools/day, P < 0.04 by paired t). The increase in LDL tracer FCR was higher when C-LDL tracer isolated during the untreated period was injected during lovastatin treatment (0.496+/-0.177 vs. 0.339+/-0.108 pools/day, P < 0.02). These in vivo studies in humans were confirmed by injecting LDL tracers from two patients into five guinea pigs. The C-LDL tracer was cleared consistently faster than the Rx-LDL tracer (0.082+/-0.018 vs. 0.057+/-0.015 pools/h, P< 0.001). The results demonstrate three important outcomes of lovastatin treatment in these subjects: LDL receptor activity increased by 49% (P < 0.02); LDL apoB production rate decreased by 17% (P < 0.03), and LDL particle in vivo affinity for the LDL receptor decreased by 15% (P < 0.01). The decrease in LDL particle affinity partially negated the expected effect of increased LDL receptors on LDL clearance. The present study provides an explanation for earlier observations by several investigators using contemporaneous tracers that treatment with HMG-CoA reductase inhibitors resulted in only modest increases in low density lipoprotein functional catabolic rate.

The lipoprotein lipase (LPL) promoter -93T/G transition has previously been reported as having a triglyceride (Tg)-lowering effect, whereas the D9N variant has been shown to have a Tg-raising effect. These two variants were studied in 66 healthy subjects of Hispanic and 42 subjects of African-American origin, who had participated in a study of postprandial lipemia. While the allele frequency of the -93G was significantly different in the Hispanics and African Americans (0.09: 95% CI 0.04-0.13 and 0.28: 95% CI 0.19-0.38; P=0.0001, respectively), the N9 allele frequency was not different (0.06: 95% CI 0.02-0.1 and 0.05: 95% CI 0.002-0.093, respectively). Linkage disequilibrium between the -93T/G and D9N was highly significant in Hispanics (delta=0.67. P=0.0001), compared to delta=0.09 (NS) in African-Americans. In the combined group, compared to individuals with the common genotype (TT/DD; n=71) with fasting plasma Tg of 1.34 (+/-4.5% SEM) mmol/l, carriers of the G/D haplotype (TG/DD + GG/DD; n=25) had significantly lower plasma Tg levels of 1.08 (+/-10% SEM) mmol/l (P < 0.02). After the fat meal, compared to individuals with neither mutation, TT/DD, the effect of the G/D haplotype was to reduce significantly postprandial Tg (P < 0.036). Retinyl palmitate concentration at 5 hrs was significantly lower in G/D carriers than TT/DD individuals (P < 0.05). The lipid-raising effect of the N9 allele in carriers of the -93G (TG/DN + GG/DN) and effect on postprandial Tg clearance was not significant in this group. Thus carriers of the G/D haplotype have lower fasting plasma Tg and reduced alimentary lipemia. This allele may be associated with reduced risk of coronary artery disease.

A method for determining apoprotein B specific activity in radioiodinated lipoproteins is described and validated. It utilizes organic solvents and tetramethylurea in the isolation of apoprotein B from other radiolabeled contaminants, both lipid and protein, in exogenously labeled VLDL. The contaminants are also removed from those lipoprotein classes subsequently derived from VLDL, namely IDL and LDL. The procedure requires approximately 50 microgram of apoB per analysis, allowing specific activity determinations in triplicate on 3-ml plasma samples with a standard error of less than 6%. Finally, data from a study of apoprotein B turnover in VLDL, IDL, and LDL in a human subject is presented to demonstrate the potential of this method in further elucidating the kinetic interrelationships between these lipoprotein classes.

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.

Heat shock protein 72/73 (Hsp70) is a cytosolic molecular chaperone that carries out fundamental roles under both normal and stress situations. There is great interest in delineating the mechanisms whereby Hsp70 levels are regulated. We observed that N-acetyl-leucyl-leucyl-norleucinal (ALLN), a synthetic aldehydic tripeptide that inhibits proteasomes, markedly induced Hsp70 levels (up to 30-fold above base line in HepG2 cells and human endothelial cells). Induction of Hsp70 by ALLN was dose-dependent and not related to cell toxicity. ALLN selectively increased Hsp70 levels without affecting Hsp25, Hsp27, Hsp60, Hsp86, Hsp90, Hsp104, or Bip (immunoglobulin heavy chain binding protein) in HepG2 cells. ALLN induced Hsp70 not only by stabilizing the protein but also by dramatically increasing its synthesis. The modulation of Hsp70 synthesis by ALLN resulted from a rapid and marked increase in transcription of the hsp72 gene, since the induction of hsp72 mRNA was blocked in cells co-treated with actinomycin D. hsp72 mRNA levels were affected in a time-dependent manner by exposure to ALLN; significant elevations occurred within 60 min of treatment, and a decline to background levels was observed by 7 h of recovery. The ALLN-induced increase in hsp72 gene expression was associated with trimerization of the heat shock transcriptional factor (HSF1). ALLN did not affect the steady-state level of HSF1 protein. The effects of ALLN appeared to require de novo protein synthesis, since the induction of both HSF1 trimerization and hsp72 transcription was blocked by co-treatment with cycloheximide. When we tested a series of protease inhibitors, only the related aldehydic tripeptides, N-acetyl-leucyl-leucyl-methioninal and the proteasome inhibitor, Cbz-leucyl-leucyl-leucinal, induced Hsp70 levels. The specific proteasome inhibitor, lactacystin, which has a different structure, also induced Hsp70 levels. Overall, our results suggest that a rapidly turning over protein that is normally degraded by proteasomes may be involved in the regulation of Hsp70 synthesis via effects on the hsp70 transcriptional factor, HSF1.

Apoprotein B100 (apoB) is a secretory protein that appears to be constitutively translated but inefficiently translocated into the lumen of the endoplasmic reticulum. Using several experimental approaches, we found that apoB is bound to the cytosolic chaperone protein, heat shock protein 72/73 (commonly referred to as Hsp70). Similar to other chaperone-protein interactions, this binding was transient and ATP-sensitive. The binding of apoB to Hsp70 in HepG2 cells was decreased by treatment with oleic acid, which increases both translocation and secretion of apoB, and was increased by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor which efficiently protects apoB from cellular degradation without affecting translocation. The N-terminal 16% of apoB, which is efficiently translocated into the endoplasmic reticulum lumen in stably transfected Chinese hamster ovary (CHO) cells, showed minimal, if any, binding to Hsp70. The N-terminal 50% of apoB, which is very poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggest that domains of nascent apoB localized on the C-terminal regions of the molecule are transiently exposed to the cytosol during translation and/or translocation, and that Hsp70 functions as a molecular chaperone to maintain apoB in a translocational competent conformation until translocation is completed.

Presentation of a wild-type secretory protein, apolipoprotein B100 (apoB), to the cytosol for ubiquitin-proteasome proteolysis has been observed in HepG2 cells. A currently accepted model for proteasomal degradation of secretory proteins is retrograde translocation of the substrate polypeptides from the lumen of endoplasmic reticulum (ER) back to the cytosol. In this report, we present evidence that newly synthesized apoB becomes exposed to the cytosol and targeted to the proteasomes in a co-translational manner. Thus, after protein translation was synchronized with puromycin, partially synthesized apoB polypeptides were found to be conjugated to ubiquitin. The magnitude of co-translational ubiquitination and subsequent degradation of apoB was increased when cells were pretreated with either herbimycin A to induce cytosolic Hsp70 or with an inhibitor of microsomal triglyceride transfer protein; both treatments impede translocation of nascent apoB across the ER membrane. These treatments also decreased secretion of apoB and increased its degradation via the ubiquitin-proteasome pathway. We suggest that translocation arrest with subsequent co-translational exposure to the cytosol provides an alternative model to explain how mammalian secretory proteins can overcome topological segregation by the ER membrane and undergo degradation by the ubiquitin-proteasome pathway.