©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Secretion of Truncated Apolipoproteins B by Monomethylethanolamine Is Independent of the Length of the Apolipoprotein (*)

Antonio E. Rusiol (§) , Jean E. Vance (¶)

From the (1) Lipid and Lipoprotein Research Group and Department of Medicine, University of Alberta, Edmonton,Alberta T6G 2S2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Translocation of apolipoprotein (apo) B across the endoplasmic reticulum membrane is a likely site for regulation of secretion of very low density lipoproteins from the liver. When primary rat hepatocytes are enriched with the phospholipid phosphatidylmonomethylethanolamine, the secretion of apoB, but not other proteins such as apoprotein A1 and albumin, is disrupted (Vance, J. E.(1991) J. Lipid Res. 32, 1971-1982). Moreover, less apoB enters the microsomal lumen and the intracellular degradation of apoB is increased (Rusiol, A. E., Chan, E. Y. W., and Vance, J. E. (1993a) J. Biol. Chem. 268, 25168-25175). In the present study we have used McArdle 7777 rat hepatoma cells stably transfected with carboxyl-terminal-truncated variants of human apoB100 and have demonstrated that the reduction in apoB secretion induced by phosphatidylmonomethylethanolamine is not a function of assembly of the apoB into a buoyant lipoprotein particle. In addition, inhibition of the intracellular degradation of the apoproteins B does not restore apoB secretion, suggesting that the effect of phosphatidylmonomethylethanolamine enrichment on apoB degradation is secondary to the effect on translocation of the protein into the endoplasmic reticulum lumen. Furthermore, supplementation of the culture medium with oleic acid does not increase apoB secretion, reduce the intracellular degradation of apoB or reverse the effects of phosphatidylmonomethylethanolamine enrichment on these processes. Our data support the hypothesis that translocation of apoB protein across the endoplasmic reticulum membrane, regardless of the association of the apoB with neutral lipids, may be a key regulatory step in very low density lipoprotein secretion.


INTRODUCTION

A major risk factor for the development of atherosclerosis is the presence of high levels of apolipoprotein (apo)() B in the circulation (Sniderman et al., 1980). The plasma concentration of apoB is determined by the balance of its rate of secretion, primarily from the liver, and its rate of removal from the blood via low density lipoprotein receptor-mediated endocytosis (Brown and Goldstein, 1976). ApoB is synthesized in the liver and intestine and is thought to be assembled with lipids into very low density lipoprotein particles in the endoplasmic reticulum (Alexander et al., 1976; Borchardt and Davis, 1987; Boren et al., 1992; Rusiol et al., 1993b). ApoB secretion from the liver is apparently not normally regulated by alteration in the rate of apoB synthesis or in the level of apo-mRNA. Instead, apoB is synthesized constitutively and in excess of its needs for lipoprotein secretion (Pullinger et al., 1989; Dashti et al., 1989; Sorci-Thomas et al., 1989). Superfluous apoB that is not assembled into lipoprotein particles is degraded intracellularly, most likely in the endoplasmic reticulum (Davis et al., 1990; Furukawa et al., 1992; Sato et al., 1990). Two possible scenarios might link the degradation and secretion of apoB. In the first, the intracellular degradation of apoB might limit the secretion of apoB. In the second, another factor, such as translocation of apoB across the endoplasmic reticulum membrane, or the association of apoB with lipids, might control how much apoB is secreted. Consequently, any apoB that is not secreted would be degraded. In the latter case, translocation of newly translated apoB across the endoplasmic reticulum membrane and into the lumen would be a key regulatory process.

As a step toward understanding how the secretion of apoB-containing lipoproteins from the liver is regulated, we have recently developed a model in which the secretion of apoproteins B100 and B48, but not other secretory proteins such as albumin and apoA1, is specifically inhibited when the concentration of the phospholipid, PMME, is increased in rat hepatocyte membranes (Vance, 1991). The defect in apoB secretion upon enrichment of the cells' membranes with PMME was shown to be associated with (i) a disruption in translocation of apoB into the microsomal lumen, (ii) an increased exposure of apoB on the cytosolic surface of the microsomes, and (iii) an increased intracellular degradation of apoB (Rusiol et al., 1993a).

We have now investigated further the mechanism by which PMME inhibits apoB secretion by dissociation of the processes of translocation of the protein into the lumen and acquisition of neutral core lipids by apoB. McArdle rat hepatoma cell lines that had been stably transfected with carboxyl-truncated variants of human apoB100 (apoB15, apoB18, apoB23, and apoB28, representing the amino-terminal 15, 18, 23, and 28% of human apoB100, respectively) were examined for the effect of PMME enrichment on secretion of the truncated apoproteins B. ApoB15, apoB18, and apoB23 are secreted from these hepatoma cells at a density of >1.23 g/ml, implying that essentially no neutral lipids (i.e. triacylglycerols and cholesteryl esters) are associated with the secreted apoB (Yao et al., 1991). ApoB28 is secreted in the form of a lipoprotein particle of size and density corresponding to that of high density lipoprotein (density of 1.17 g/ml). ApoB28 is therefore secreted in association with some neutral ``core'' lipids and presumably also with some surface lipids (phospholipids and cholesterol) (Yao et al., 1991). In the present study, we have found that secretion of all four truncated variants of apoB was inhibited and that the intracellular degradation of the apoproteins B was increased, when the cells were enriched with PMME. These observations indicate that the inhibitory effect of PMME enrichment on apoB secretion is not dependent upon acquisition of the neutral lipid core, but is related to a defect in translocation of newly synthesized apoB across the endoplasmic reticulum membrane. In addition, inhibition of the intracellular degradation of apoB with the cysteine protease inhibitor, ALLN, did not reverse the inhibition of secretion caused by PMME, suggesting that increased degradation of the truncated apoproteins B induced by PMME is secondary to the effect of PMME on translocation of the proteins across the endoplasmic reticulum membrane.


EXPERIMENTAL PROCEDURES

Materials

Stably transfected McArdle 7777 rat hepatoma cells expressing apoB15, apoB18,() apoB23, and apoB28 were generous gifts from Dr. Zemin Yao, University of Alberta. [S]Methionine and Amplify were purchased from Amersham, Canada, and TranS-label was obtained from ICN, Irvine, CA. Polyvinylidene difluoride membranes were from Millipore. Antibodies directed against rat apoB100 and rat apoA1 were prepared and characterized in our laboratory by standard procedures (Rusiol et al., 1993a). The antibody directed against human apoB was purchased from Boehringer Mannheim, as was the calpain inhibitor I, ALLN. Monomethylethanolamine and water-soluble oleic acid (encapsulated in -cyclodextrin) were from Sigma. The reagents used for electrophoresis were supplied by Bio-Rad. All other chemicals and solvents were purchased from either Sigma or Fisher.

Culture of Hepatoma Cells

Transfected McArdle 7777 cells were plated on 60-mm culture dishes (Falcon) at a concentration of approximately 3 10 cells/dish. The cells were cultured in Dulbecco's modified Eagle's medium, DMEM, containing 10% fetal bovine serum (Life Technologies, Inc.) and 10% heat-inactivated horse serum (Life Technologies, Inc.). The cells were grown at 37 °C in an atmosphere of 5% CO. When growth reached 70% confluence, the cells were incubated overnight (16 h) in the presence of 400 or 0 mM MME in DMEM and used the next morning. The culture media were harvested and centrifuged at 10,000 g for 15 min to remove cell debris. Cells were scraped from the dishes and pelleted by centrifugation for 5 min at 1,000 g.

Pulse-Chase Experiments

The monolayer of cells was preincubated for 1 h in methionine-free DMEM, then incubated with 1 ml of the same medium containing 200 µCi/dish of TranS-label for 1 h. Radioactivity was chased for defined periods of time, up to 4 h, in DMEM containing 400 µM unlabeled methionine and various additives, as defined for each experiment. ApoB products were immunoprecipitated from cells and culture medium with polyclonal antibody directed against rat apoB using a published method (Borchardt and Davis, 1987; Rusiol et al., 1993b). After immunoprecipitation the proteins were separated by electrophoresis on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The gel was stained and subsequently impregnated with Amplify for 15 min and dried. Autoradiography of the dried gel was performed at -80 °C for 16-32 h. The apoB-containing bands were excised from the gel, the gel was digested with 20% hydrogen peroxide (Rusiol et al., 1993b), and radioactive incorporation into apoB was measured.

Studies on the Rate of ApoB Synthesis

McArdle 7777 cells expressing apoproteins B15, B18, and B28 were incubated in the presence of 0 or 400 µM MME for 16 h. Newly synthesized proteins were subsequently radiolabeled by addition of 100 µCi/dish of [S]methionine. After time intervals of from 0.5 to 6 h, apoproteins B were immunoprecipitated from the cells with a polyclonal antibody directed against human apoB100; the proteins were electrophoresed on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate, and the apoB-containing bands were excised from the gels for measurement of radioactivity. In an alternative experimental protocol, apoB18-expressing cells that had been incubated in the presence or absence of MME were labeled with [S]methionine for 5-20 min, and radioactivity in apoB18 was measured after immunoprecipitation and polyacrylamide gel electrophoresis.

Measurement of the Mass of ApoB Variants and ApoA1 in Culture Medium

The amounts of apoproteins B and A1 secreted into the medium were determined by enzyme-linked immunosorbent assays using polyclonal antibodies directed against human apoB and rat apoA1, respectively, as described previously (Rusiol et al., 1993b). The mass of apoproteins B secreted into the medium was also examined by immunoblotting of aliquots of culture medium as described previously using a polyclonal antibody directed against human apoB100 (Rusiol et al., 1993b).

Proteolysis of Microsomal ApoB28, ApoB18, and ApoA1 with Exogenously Added Trypsin

McArdle 7777 cells expressing apoB28 or apoB18 were incubated for 16 h with or without MME (400 µM). Microsomes were isolated as described previously (Rusiol et al., 1993a) and 80 µg of protein was treated with 0.1 volume of trypsin (0.5 mg/ml) for 30 min at 0 °C. Proteolysis was terminated by addition of 0.1 volume of soybean trypsin inhibitor (4 mg/ml), and microsomes were re-isolated by centrifugation for 15 min at 400,000 g in a Beckman TL 100.3 rotor. The membrane pellet was boiled for 5 min in a solution containing 8 M urea and 2% sodium dodecyl sulfate. Aliquots of the extract were subjected to electrophoresis on an 8.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. Proteins were analyzed by immunoblotting using a polyclonal antibody raised against human apoB100 (Boehringer Mannheim) and a polyclonal antibody specific for rat apoA1, raised in our laboratory. The amounts of apoB and apoA1 were estimated by densitometric scanning of the immunoblots.

Other Methods

The protein content of the cells was measured by the method of Lowry et al.(1951).


RESULTS

MME Inhibits the Secretion of Truncated ApoB Variants

We have previously shown that enrichment of microsomal membranes of primary rat hepatocytes with PMME inhibited the secretion of apoproteins B100 and B48 as a result of a defective movement of the apoproteins B into the endoplasmic reticulum lumen (Vance, 1991; Rusiol et al., 1993a). These studies did not, however, distinguish between whether the defect was due to a problem in translocation of the apoB protein across the membrane or whether the defect was the result of the inability of the protein to associate with lipid and assemble into a lipoprotein particle.

We have attempted to distinguish between these two possibilities by examining the effect of PMME enrichment of the endoplasmic reticulum membrane on the translocation and secretion of four carboxyl-truncated variants of human apoB100: apoB15, apoB18, apoB23, and apoB28. McArdle 7777 hepatoma cells expressing these apoproteins were cultured for 16 h in the absence or presence of MME (400 µM). In the MME-treated cells the amount of PMME was 3.18 ± 0.21 nmol/mg of protein and in cells not incubated with MME the PMME content was 0.21 ± 0.08 nmol/mg of protein. The amounts of apoB variants secreted into the culture media were measured by an enzyme-linked immunosorbent assay (Fig. 1A) using a polyclonal antibody directed against human apoB100. As shown in Fig. 1A, the amounts of B15, B18, B23, and B28 secreted were reduced by 55.8, 42.9, 44.8, and 57.3%, respectively, as a result of incubation with MME. Since the mass of apoproteins B in the medium, as shown in Fig. 1A, represents the truncated apoB species, as well as small amounts of endogenous, native rat apoproteins B100 and apoB48, samples of the culture medium isolated after 16-h incubation were examined by immunoblotting specifically for the amount of truncated apoprotein B secreted. The data presented in Fig. 1B, in agreement with the data shown in Fig. 1A, confirm that PMME enrichment significantly reduced the amount of the truncated apoproteins B secreted into the culture medium. These data demonstrate that the inhibitory effect of PMME enrichment previously observed for the secretion of apoB100 and apoB48 from rat hepatocytes is also evident for these truncated apoB species in transfected McArdle 7777 cells. The secretion of native apoB100 and apoB48 from the transfected McArdle cell lines was also inhibited by approximately 50% upon PMME enrichment of the cells (data not shown). Since secreted apoB15 and apoB18 are not appreciably associated with a neutral lipid core (Yao et al., 1991), our observation implies that the effect of PMME on apoB secretion is not related to binding of apoB to neutral lipid. Rather, PMME enrichment of the membranes most likely affects the process of translocation of apoB across the membrane.


Figure 1: Secretion of truncated apoB variants, but not apoA1, from McArdle 7777 cells is inhibited by enrichment of membranes in PMME. Stably transfected McArdle 7777 cells expressing the amino-terminal 15, 18, 23, and 28% of human apoB100 (i.e. apoB15, apoB18, apoB23, and apoB28, respectively) were cultured for 16 h in medium containing no or 400 µM MME. The amount of apoB variants and native apoA1 secreted into the culture medium was determined by enzyme-linked immunosorbent assays (A) and immunoblotting (B). The data in A represent averages ± S.D. of six individual experiments. Open bars represent cells incubated without MME, solid bars represent cells incubated with 400 µM MME. For the immunoblotting experiment (B) the cells were incubated for 16 h, and medium equivalent to the following amounts of cell protein was applied to the 3-15% polyacrylamide gel: apoB15, 75 µg; apoB18, 30 µg; apoB23, 47 µg; apoB28, 20 µg. The locations of endogenous apoproteins B100 and B48 on the gel are indicated by the notations at the left-hand side.



The defect in secretion induced by PMME appeared to be specific for apoB. In primary rat hepatocytes PMME enrichment did not impair the translocation/secretion of other secretory proteins, such as albumin and apoAI (Vance, 1991). Furthermore, in the McArdle cells expressing the truncated apoproteins B we measured the secretion of apoA1 into the culture medium by an enzyme-linked immunosorbent assay. PMME enrichment of these cells did not inhibit the secretion of apoA1 (Fig. 1A).

Intracellular Degradation of Truncated ApoB Variants Is Increased upon MME Treatment

ApoB is constitutively synthesized by hepatocytes (Pullinger et al., 1989) in excess of the requirement for lipoprotein secretion. ApoB that is not assembled into lipoprotein particles for secretion is apparently degraded intracellularly, most likely in the endoplasmic reticulum (Davis et al., 1990; Sato et al., 1990; Dixon et al., 1991; Furukawa et al., 1992). The intracellular degradation of apoB15, apoB18, and apoB28 was examined in transfected McArdle 7777 hepatoma cells. Cells were pulse-labeled with [S]methionine for 1 h, after which the radioactive medium was removed, and the incubation was continued for a chase period of up to 4 h. ApoB proteins were immunoprecipitated from cells and culture medium, separated by polyacrylamide gel electrophoresis and the apoB-containing bands were excised from the gel for measurement of radioactivity (Fig. 2). In cells incubated without MME, radioactivity in all three cellular apoproteins B decayed as a function of time with a concomitant increase in radioactivity in secreted apoB. Almost all of the decrease in radioactivity in the intracellular apoproteins B (94.5% for apoB15, 80.9% for apoB18, and 84.5% for apoB28) was recovered in the secreted apoproteins B. Therefore, only 5-19% of the newly synthesized, truncated apoproteins B was degraded intracellularly, in agreement with the report of Yao et al. (1991). The experiment was repeated with cells that had been preincubated with MME for 16 h. When these data were compared with those from cells incubated in the absence of MME, a smaller percentage of radioactive apoB present in the cells at the end of the 1-h pulse was secreted from cells treated with MME (Fig. 2). Of the radioactivity present in apoB15, apoB18, and apoB28 at the end of the 1-h pulse, 40.3, 50.1, and 55.0%, respectively, had been degraded intracellularly by the end of the 4-h chase period. These data demonstrate that the intracellular degradation of the apoB variants was increased upon enrichment of cells with PMME and that the efficiency of secretion was decreased.


Figure 2: Intracellular degradation of truncated apoB variants is increased upon MME treatment. McArdle 7777 cells harboring truncated apoB constructs (apoB15, apoB18, and apoB28) were cultured for 16 h in the presence of 400 µM (closed symbols) or 0 µM (open symbols) MME. Methionine-free medium without or with 400 µM MME, and containing 200 µCi of [S]methionine/60-mm dish, was added for 1 h. The medium was removed, and cells were incubated for the indicated times with medium containing unlabeled methionine. Apoproteins B were immunoprecipitated from cells (circles) and media (triangles) and separated by polyacrylamide gel electrophoresis on 10% gradient gels containing 0.1% sodium dodecyl sulfate. Data are plotted as radioactivity in apoB as a percentage of total radioactivity in cellular apoB at the end of the 1-h pulse period (T). Radioactivity in cellular apoB15, apoB18, apoB23, and apoB28 at the end of the pulse of cells incubated without MME was: 16,328, 17,532, and 20,023 dpm/dish, respectively, and in cells incubated with MME was 17,415, 18,160, and 21,394 dpm/dish, respectively. Similar results were obtained when the experiment was repeated.



As confirmation that the truncated apoproteins B were not being degraded in the culture medium, apoB28-expressing cells that had been enriched in PMME were incubated for 16 h with [S]methionine. One-half of the medium was used directly for immunoprecipitation of apoB28. The other half of the medium was added to unlabeled cells, then re-isolated after 4 h incubation. ApoB was immunoprecipitated and separated by polyacrylamide gel electrophoresis. The recovery of radioactivity in apoB28 from medium incubated with cells for 4 h was 89.3%, indicating that little degradation of apoB had occurred in the culture medium.

We have previously demonstrated that incubation of primary rat hepatocytes with 400 µM MME overnight did not inhibit the synthesis of apoproteins B100 or B48 (Rusiol et al., 1993a). Since the amount of intracellular apoB reflects both synthesis and removal (by intracellular degradation and secretion), we examined whether or not the decreased rate of secretion of the truncated apoB variants in the presence of MME could be explained by a decreased rate of synthesis. In a continuous labeling experiment McArdle cells expressing apoB28 were incubated for 16 h in the presence of 0 or 400 µM MME, then 100 µCi/dish of [S]methionine was added. After time intervals of 30 min to 6 h apoB28 was immunoprecipitated from the cells, the proteins were separated by polyacrylamide gel electrophoresis, and the apoB28-containing band was excised for measurement of radioactivity. The labeling curves for apoB28 were essentially identical for cells incubated in the presence and absence of MME (data not shown). We also examined the synthesis of apoB18 by labeling the cells with [S]methionine for 5-20 min. At all times, the radioactivity in apoB18 in MME-treated cells was approximately 20% higher than in control cells (data not shown). We conclude, therefore, that the synthesis of the truncated apoBs is not inhibited by MME treatment. Rather, for apoB18, the synthetic rate appears to be slightly enhanced in MME-treated cells compared with untreated cells.

Sensitivity of ApoB18 and ApoB28 to Exogenously Added Trypsin Is Increased in PMME-enriched Microsomes

In primary rat hepatocytes (Thrift et al., 1992) and in Hep G2 human hepatoma cells (Dixon et al., 1991) a pool of microsomal apoB is apparently exposed to the cytosolic surface and is consequently susceptible to degradation upon treatment of intact microsomes with exogenously added proteases. In primary rat hepatocytes treated with MME, microsomal apoB100 and apoB48 are more susceptible to degradation by exogenous trypsin than is apoB in microsomes from hepatocytes not treated with MME (Rusiol et al., 1993a). These data indicated that PMME enrichment of microsomal membranes impaired the translocation of apoB into the microsomal lumen so that a smaller fraction of the apoB was protected from degradation by the exogenously added protease. Microsomes were isolated from apoB28- and apoB18-expressing McArdle 7777 cells that had been incubated for 16 h with or without MME. The sensitivity of apoB28 and apoB18 in these microsomes to digestion by exogenously added trypsin was examined. The results presented in Fig. 3 revealed that in cells not treated with MME essentially all of the microsomal apoB28 (83.6 ± 2.9%) and apoB18 (99.0 ± 3.6%) was protected from proteolysis. In contrast, in PMME-enriched microsomes the majority of microsomal apoB28 (89.4 ± 1.6%), and 39.4 ± 9.1% of apoB18 (according to densitometric scanning of the immunoblots), was degraded by trypsin, apparently as a result of being exposed on the cytosolic surface of the microsomes. The resistance of the microsomal apoproteins B to trypsin digestion was not due to an inherent inability of trypsin to degrade the proteins, because when the microsomes were disrupted with the detergent Triton X-100, all the microsomal apoB28 and apoB18 was proteolyzed (Fig. 3). As a note of caution when interpreting these results, some investigators have questioned whether or not the susceptibility of apoB to exogenous protease is indeed due to exposure of the protein on the cytoplasmic side of the membrane (Pease et al., 1991; Shelness et al., 1994). However, the accessibility of apoB, but not apoA1, to exogenous protease is clearly different in PMME-enriched cells compared with that in control cells. We did not examine the protease sensitivity of apoB15 in transfected McArdle cells. However, in other studies we have shown that protection of in vitro translated apoB15 from protease is decreased upon PMME enrichment of microsomes.()


Figure 3: Protease digestion of apoB28 and apoB18, but not ApoA1, is increased in PMME-enriched microsomes. Microsomes were prepared from apoB28- and apoB18-expressing McArdle 7777 cells that had been incubated for 16 h in the presence or absence of MME (400 µM). Aliquots of microsomal protein were incubated with trypsin, and proteolysis was terminated by addition of trypsin inhibitor. Microsomes were re-isolated by centrifugation, and microsomal proteins (20 µg/lane) were subjected to polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane. ApoB28 (A) and apoB18 (B) on the membranes were probed with anti-human apoB polyclonal antibody. ApoA1 was detected with a polyclonal antibody directed against rat apoA1. In some samples, as indicated, trypsin digestion was performed in the presence of 0.5% Triton X-100. The amounts of apoproteins B and A1 were estimated by densitometric scanning of the immunoblots from three independent experiments for apoB18 and apoB28 and from six independent experiments for apoA1. Data given in the text are averages ± S.D.



The secretion of endogenous, native apoA1 from the McArdle cells was not affected by PMME enrichment (Fig. 1A). Moreover, microsomal apoA1 in the transfected McArdle cells was almost completely protected from exogenous trypsin in both control and PMME-enriched microsomes (by 94.3 ± 7.5% and 87.3 ± 2.3%, respectively) (Fig. 3, A and B). These observations indicate that the increased degradation of apoB18 and apoB28 observed in PMME-enriched microsomes was not the result of ``leaky'' microsomes, but was due to an increased exposure of the apoproteins B on the cytosolic surface of the microsomes.

Inhibition of Intracellular Degradation of ApoB18 and ApoB28 Does Not Reverse the Inhibitory Effect of MME on Secretion

Two alternative explanations might account for the increased degradation and decreased secretion of apoproteins B in PMME-enriched cells. First, the increased content of PMME in the endoplasmic reticulum membrane might have stimulated apoB degradation making less apoB available for secretion. In this case, the degradation of apoB would have limited its secretion. Alternatively, enrichment of PMME in the membranes might have impaired the translocation process per se, resulting in less apoB being secreted. As a consequence of the blocked translocation process, apoB would reside in the translocation channel, or become associated with the membrane, for an increased length of time, and the susceptibility of apoB to degradation would be increased.

In an attempt to examine a cause and effect relationship between translocation and degradation of the truncated apoproteins B, we investigated whether or not inhibition of apoB degradation would promote the secretion of truncated apoproteins B and thereby reverse the impact of PMME enrichment on this process. The cysteine protease inhibitor ALLN (calpain inhibitor I), a tripeptide which blocks the intracellular degradation of apoB in Chinese hamster ovary cells (Thrift et al., 1992) and Hep G2 cells (Sakata et al., 1993), was used as an inhibitor of apoB degradation. McArdle 7777 cell lines expressing apoB28 were incubated for 1 h in the presence or absence of ALLN. The cells were subsequently pulse-labeled for 1 h with [S]methionine in the presence or absence of ALLN, then chase medium containing 40 µg/ml of ALLN was added. After 1 h, the medium was replaced with fresh medium containing ALLN (40 µg/ml), and the incubation was continued for an additional 3 h. ApoB28 was immunoprecipitated from cells and culture medium, separated by polyacrylamide gel electrophoresis, and excised from the gel for measurement of radioactivity. In cells not exposed to MME, of the radioactivity present in intracellular apoB28 at the end of the 1-h pulse period, approximately 20% had been degraded intracellularly by the end of the 4-h chase period (i.e. the total radioactivity in cells plus medium decreased by approximately 20%) (Fig. 4B), in agreement with the data shown in Fig. 2 . ALLN did not significantly influence either the rate of secretion of apoB28 into the medium or the rate of decay of radiolabeled intracellular apoB28. Similar results were obtained for experiments with cells expressing apoB18 (data not shown).


Figure 4: ALLN reduces the intracellular degradation of apoB28 in MME-treated cells but does not reverse the effect of MME on secretion. McArdle 7777 cells expressing apoB28 were cultured for 16 h in medium without MME (B) or with 400 µM MME (A), after which the cells were preincubated for 1 h in the same media that contained (solid symbols) or lacked (open symbols) ALLN (40 µg/ml). The proteins were pulse-labeled for 1 h with TranS-label (200 µCi/dish) in the same media, and radioactivity was chased in media that contained unlabeled methionine. After 1 h, the ALLN-containing medium was replenished with fresh ALLN. At the indicated times apoB28 was immunoprecipitated from the cells (circles) and media (triangles), and radioactivity in apoB28 was determined after separation of the proteins by polyacrylamide gel electrophoresis. Data are plotted as radioactivity in apoB as a percentage of total radioactivity in cellular apoB at the end of the 1-h pulse period (T). Radioactivity in apoB28 at the end of the pulse for MME-treated cells was 16,909 dpm/dish for cells incubated without ALLN and 16,234 dpm/dish for cells incubated with ALLN. For cells not treated with MME, radioactivity at the end of the pulse was 17,320 dpm/dish and 16,390 dpm/dish for cells incubated with and without ALLN, respectively. Similar results were obtained when the experiment was repeated.



In parallel experiments, hepatoma cells expressing apoB28 were incubated for 16 h in the presence of MME before the beginning of the pulse-chase experiment described above with ALLN. In agreement with the data shown in Fig. 2, PMME enrichment increased the intracellular degradation of apoB28 (Fig. 4A compared with Fig. 4B). By the end of the 4-h chase period the total radioactivity in apoB28 in cells plus medium had declined by approximately 60%. However, ALLN inhibited the majority of the intracellular degradation of apoB28 for the first 2 h (Fig. 4A). The increased rate of degradation of apoB28 that occurred after 2 h of the chase period may have been the result of ALLN losing some of its potency. Importantly, in spite of apoB28 being protected from degradation, the secretion of apoB28 increased only marginally, if at all, in the presence of ALLN (Fig. 4A), and did not begin to approach the level of secretion from the cells incubated without MME (Fig. 4A compared with Fig. 4B). For example, in cells incubated in the absence of MME, after 4 h of the chase period, approximately 50% of radioactive cellular apoB28 present at the end of the pulse had been secreted. In contrast, in MME-treated cells, only 20% of the radiolabeled apoB28 present at the end of the pulse had been secreted, either in the presence or absence of ALLN. Similar results were obtained for apoB18. The intracellular location of the accumulated apoproteins B in ALLN-treated cells is not known but is presently being investigated in our laboratory.

We conclude from the data presented in Fig. 4that inhibition of the intracellular degradation of apoB28 and apoB18 by ALLN did not reverse the defect in secretion caused by PMME enrichment. The increased degradation of apoproteins B induced by PMME enrichment was, therefore, secondary to the effect of PMME on translocation of the protein into the endoplasmic reticulum lumen.

Oleic Acid Treatment Does Not Reverse the Effects of MME on ApoB28 Secretion or Degradation

Incubation of Hep G2 human hepatoma cells with oleic acid increases the secretion of apoB100 and causes a reciprocal reduction in the rate of intracellular apoB100 degradation (Furukawa et al., 1992). Addition of oleate to McArdle 7777 cells transfected with several truncated apoBs (including apoB13, apoB17, apoB23, apoB29, apoB32, and apoB36) promoted the secretion, and reduced the intracellular degradation, of only those apoBs large enough to produce buoyant lipoproteins (i.e. apoB29, apoB32, and apoB36) (White et al., 1992). The mechanisms responsible for these effects are not completely understood, but the stimulation of secretion by oleate in Hep G2 cells can be clearly dissociated from the inhibitory effect of oleate on degradation (Sakata et al., 1993). As a means of gaining further information on the mechanism which apoB secretion is inhibited by PMME enrichment, we investigated whether or not oleate would reverse the effect of MME on apoB28 secretion. McArdle 7777 cells expressing apoB28 were incubated overnight in the presence or absence of MME, after which the cells were pulse-labeled for 1 h with TranS-label, and radioactivity was chased in the presence or absence of 0.8 mM oleate. The results shown in Fig. 5B for cells incubated without MME demonstrate that oleate did not significantly enhance the secretion of apoB28 or significantly reduce the degree of its intracellular degradation. In agreement with the data presented in Fig. 1 , 2, and 4, however, MME inhibited the secretion of apoB28 (Fig. 5). The addition of oleate to both MME-treated, and untreated, cells neither stimulated the secretion, nor inhibited the degradation, of apoB28. Therefore, oleate did not reverse the effect of PMME on either secretion or degradation of apoB28.


Figure 5: Oleic acid neither increases apoB28 secretion nor reverses the defect in secretion induced by MME treatment. McArdle 7777 cells expressing apoB28 were incubated for 16 h in the presence (A) or absence (B) of MME. The cells were pulse-labeled for 1 h with TranS-label (200 µCi/dish), then radioactivity was chased in the presence (closed symbols) or absence (open symbols) of oleic acid (0.8 M) for the indicated times. ApoB28 was immunoprecipitated from cells (circles) and media (triangles), and radioactivity was measured after apoB28 had been separated by polyacrylamide gel electrophoresis. Data are presented as radioactivity in apoB28 as a percentage of total radioactivity (T) in cellular apoB28 at the end of the 1-h pulse period. Radioactivity in cellular apoB28 at the end of the pulse of MME-treated cells was 19,300 dpm/dish and 18,343 dpm/dish for cells incubated with and without oleate, respectively. For cells not treated with MME, radioactivity in apoB28 at the end of the pulse was 20,110 dpm/dish and 19,433 dpm/dish, for cells incubated with and without oleate, respectively. Similar results were obtained when the experiment was repeated.



The effect of oleate and MME on the secretion of endogenous rat apoB48 by the McArdle 7777 cells was also examined. In contrast to the effect of oleate on the secretion of truncated apoproteins B, but in agreement with the effect of oleate on apoB100 secretion from Hep G2 cells (Furukawa et al., 1992), the addition of oleate to the McArdle cells (both untreated and MME-treated) did increase the secretion of native apoB48 by approximately 2-fold (Fig. 6, A and B). However, the amount of radiolabeled apoB48 secreted from the MME-treated cells was only approximately 50% of that from untreated cells (Fig. 6, A compared with B).


Figure 6: Oleic acid increases the secretion of native apoB48 from McArdle 7777 cells. McArdle cells expressing apoB28 were incubated for 16 h with 400 µM MME (A) or without MME (B). The cells were pulse-labeled with TranS-label for 1 h, then radioactivity was chased in the presence (closed symbols) or absence (open symbols) of 0.8 M oleic acid. ApoB was immunoprecipitated from cells (circles) and media (triangles), the proteins were separated by polyacrylamide gel electrophoresis and radioactivity in apoB48 was measured. Data are presented as radioactivity in apoB48 as a percentage of total radioactivity (T) present in cellular apoB48 at the end of the pulse period. Radioactivity in cellular apoB48 at the end of the pulse for MME-treated cells was 8,302 dpm/dish and 8,520 dpm/dish in cells incubated with and without oleate, respectively. For cells not treated with MME, radioactivity in apoB48 at the end of the pulse was 7,532 dpm/dish in cells incubated without oleate and 8,007 dpm/dish for cells incubated with oleate. Similar results were obtained when the experiment was repeated.




DISCUSSION

A prime candidate for regulation of very low density lipoprotein secretion from the liver is the movement of apoB across the endoplasmic reticulum membrane into the lumen (Thrift et al., 1992). We have investigated whether or not the translocation process is linked to the ability of apoB to associate with neutral lipids during formation of a floating lipoprotein particle. We have used the rat hepatoma cell line, McArdle 7777, stably transfected with truncated apoB variants, some of which do not require assembly with core lipids (triacylglycerols and cholesteryl esters) for secretion. A direct linear correlation has been reported previously between the size of the apoB molecule and the extent of its association with lipids. In cells expressing various truncated variants of human apoB100, as the length of the apoB variant increased the density of the secreted apoB-containing particles decreased and the size of the particles increased. For example, apoproteins B15, B18, and B23 were isolated from the bottom of a salt gradient, at a density of 1.23 g/ml, whereas apoproteins B28, B31, B37, B48, and B53 (Yao et al., 1991), as well as apoproteins B60, B72, B80, B88, B94, and B100 (McLeod et al., 1994) exhibited a progressive decrease in density from 1.17 g/ml for apoB28 to 1.02 g/ml for apoB100. These studies implied that apoB15, apoB18 and apoB23 are secreted essentially devoid of significant amounts of associated lipid, whereas apoB variants larger than apoB23 are associated with progressively more lipid as the size of the apoprotein B increases.

In our studies, when rat hepatoma cells expressing apoB15, apoB18, apoB23, and apoB28 were enriched in PMME, the secretion of all four truncated apoproteins B was inhibited by 42-57%, compared with that in the same cells not enriched in PMME. Secretion of native apoB100 and apoB48 by the transfected McArdle 7777 cell lines was also inhibited by PMME enrichment, as has been observed previously in primary rat hepatocytes (Vance, 1991). Therefore, the PMME effect was clearly independent of the assembly of apoB with significant amounts of neutral lipids or formation of a floating lipoprotein particle. PMME enrichment of endoplasmic reticulum membranes specifically disrupted the translocation process per se and appeared to be specific for apoB.

A second significant observation from the present study was that inhibition of intracellular apoB degradation did not reverse the inhibitory effect of PMME on secretion. PMME enrichment of cells expressing apoB18 and apoB28 caused an increased intracellular degradation of the apoproteins B. In addition, in PMME-enriched microsomes, apoB28 and apoB18 were more susceptible to degradation by exogenously added trypsin, than in control microsomes, suggesting that PMME enrichment impaired the translocation of these apoproteins. When the protease inhibitor ALLN, which inhibits apoB degradation, was added, the PMME-induced increase in degradation was prevented and the apoproteins B accumulated intracellularly. ALLN did not, however, restore the rate of secretion of apoB28 or apoB18 to the level of secretion from cells not treated with MME. These experiments suggest that the increased degradation of apoB18 and apoB28 was the consequence, not the cause, of the decreased rate of movement of these proteins through the secretory pathway. Our observation complements the report of Sakata et al.(1993) that in Hep G2 cells apoB100 secretion is regulated independently by proteolysis and by lipid supply. Parallel experiments in primary rat hepatocytes were not possible, because incubation of these cells with ALLN, even at concentrations as low as 10 µg/ml, inhibited the synthesis and secretion of proteins in general and induced cell death.

Oleic acid has been reported to increase the secretion of apoB100 and to decrease its intracellular degradation in the human hepatoma cell line Hep G2 (Dixon et al., 1991; Sakata et al., 1993). Oleate presumably increases the secretion of apoB-containing lipoproteins from these cells because the increased supply of fatty acids stimulates lipid synthesis. In contrast, in cultured rat hepatocytes, oleate does not increase apoB secretion,() even though the synthesis of phospholipids and triacylglycerols is increased (Pelech et al., 1983). In the present study, we examined whether or not oleate was capable of restoring the level of secretion of the truncated apoproteins B from McArdle cells enriched in PMME to the levels of secretion found for cells not enriched in PMME. Oleate did not stimulate the secretion of apoB28 from either control or PMME-enriched McArdle 7777 cells, nor did oleate reverse the inhibition of secretion of apoB28 or its accelerated degradation, caused by the increased cellular content of PMME. The secretion of native rat apoB100 and apoB48 from cell lines expressing the truncated apoproteins B was, however, stimulated approximately 2-fold by incubation of the cells with oleate. These data might reflect the finding that, unlike apoB100 and apoB48, the truncated apoproteins B used in our study do not require the association with large amounts of neutral lipid for secretion (Yao et al., 1991). Interestingly, we have recently shown that in vitro translated apoB15 isolated from the lumen of rat liver microsomes is associated with some phosphatidylcholine.

A novel mechanism for regulation of apoB translocation across the endoplasmic reticulum membrane by the process of translocational pausing has been described by Chuck et al.(1990). The existence of translocational pausing has, however, been challenged by others (Pease et al., 1991; Shelness et al., 1994). Although the process is unusual, translocational pausing has also been observed for the prion protein and for GRP94, a luminal endoplasmic reticulum protein widely distributed in eukaryotic cells (Nakahara et al., 1994). ApoB100 has been proposed to contain throughout its sequence multiple (perhaps 40) topological domains designated as pause-transfer sequences which are degenerate sequences that act independently of one another (Chuck and Lingappa, 1993). Six putative pause-transfer sequences have been identified in apoB15 (Chuck and Lingappa, 1993). The presence of a pause-transfer sequence in a protein has been reported to cause the temporary arrest of the protein within the aqueous, proteinaceous translocation channel in the membrane during co-translational translocation; after pausing has occurred, translocation resumes. The pausing process was inferred, because discrete fragments of apoB15 were detected in the microsomal lumen from in vitro translation experiments in which the microsomes were treated post-translationally with an exogenous protease (Chuck et al., 1990; Chuck and Lingappa, 1992). When a pause-transfer sequence of apoB was engineered into a chimeric secretory protein, translocational pausing of the protein was induced, whereas the same chimera lacking the pause-transfer sequence did not pause during translocation (Chuck and Lingappa, 1992). A possible function of the multiple pause-transfer sequences in apoB might be to allow time for the protein either to become ``painted'' with (phospho)lipid or to become correctly folded so that the large hydrophobic protein assumes a stable conformation and can enter the aqueous lumen. An attractive hypothesis related to our present study is that PMME enrichment of membranes might specifically affect translocational pausing so that apoB would remain paused in the translocation channel and that restarting of translocation after the pausing might be delayed. As a result, the apoB would be exposed to the protease, which is present either in the cytosol or in the endoplasmic reticulum membrane, for an increased length of time and the apoB would be degraded.

We conclude that the defect in apoB translocation induced by PMME enrichment is dependent on neither the length of the apoB variant nor on the acquisition of neutral core lipids by the apoB. Moreover, the increase in the intracellular degradation of apoB is the result, rather than the cause, of the decreased translocation of apoB into the microsomal lumen.


FOOTNOTES

*
This work was supported by a grant from the Heart and Stroke Foundation of Alberta. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral fellow of the Alberta Heritage Foundation for Medical Research.

To whom correspondence should be addressed: Lipid and Lipoprotein Research Group, 315 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, T6G 2S2 Canada. Tel.: 403-492-7250; Fax: 403-492-338; E-mail: jvance@gpu.srv.ualberta.ca.

The abbreviations used are: apo, apolipoprotein; ALLN, N-acetyl-leucyl-leucyl-norleucinal; DMEM, Dulbecco's modified Eagle's medium; MME, monomethylethanolamine; PMME, phosphatidylmonomethylethanolamine.

Z. Yao, S. Selby, and S. Lingrell, unpublished data.

A. E. Rusiol, S. L. Chuck, V. R. Lingappa, and J. E. Vance, submitted for publication.

J. E. Vance, unpublished data.


ACKNOWLEDGEMENTS

We thank Zemin Yao (University of Alberta) for the gift of the McArdle 7777 cell lines and Penney Bandura for excellent technical assistance.


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