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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:889-897.)
© 1996 American Heart Association, Inc.

Articles

Selective Recruitment of ApoB-48 for the Assembly of VLDL in Rat Triacylglycerol-Enriched Hepatocytes

Peter J. Coussons; Catherine S. Bourgeois; David Wiggins; Geoffrey F. Gibbons

the Oxford Lipid Metabolism Group, Metabolic Research Laboratory, Nuffield Department of Clinical Medicine, University of Oxford, Radcliffe Infirmary, Oxford, UK.

Correspondence to Dr G.F. Gibbons, Metabolic Research Laboratory, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK.

	Abstract

Top Abstract Introduction Methods Results Discussion References

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

Key Words: apolipoprotein B • VLDL • triacylglycerol • hepatocyte culture • regulation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
It is generally agreed that in the short term, hepatic apoB is expressed constitutively.^{1 2 3 4} In vitro manipulations that give rise to changes in apoB secretion result at least partially from posttranslational regulation of apoB that involves changes in apoB degradation.^{5 6 7} Secretion of hepatic lipid may not necessarily be affected, however, since each molecule of apoB is capable of transporting a variable quantity of TAG. Thus, whole-body demands for changes in the rate of hepatic TAG secretion may be met by changing either the amount of TAG, which is "packaged" with each molecule of apoB, and/or the number of VLDL particles secreted. With respect to hormonal and nutritional regulation of hepatic VLDL secretion, therefore, an important question is whether increases in hepatic TAG production result in increases in apoB secretion. Because TAG may be synthesized de novo, from small precursors, or from extracellular fatty acids, a related question is whether apoB output is influenced by the source of hepatic TAG. Both of these questions have been previously addressed in studies of HepG2 cells^{5 8} and isolated preparations of rat liver, either primary hepatocyte cultures^{4 9} or perfused liver.¹⁰

There is little consensus concerning the response of apoB secretion to changes in the availability of extracellular fatty acid. For example, in HepG2 cells it now seems reasonably clear that extracellular oleate stimulates apoB secretion by a process that involves changes in apoB degradation.⁵ However, no such effect on the secretion of VLDL apoB has been observed following short-term exposure of isolated liver preparations from fed rats to extracellular fatty acid.^{9 10 11} In these cases increases in TAG output were observed, suggesting that this particular in vitro manipulation led to an increase in particle size only. On the other hand, in perfused livers from fasted animals, extracellular oleate stimulated the output of VLDL apoB and TAG.¹⁰ The aforementioned issues are further complicated by several observations that suggest that in vivo, nutritional manipulations in rats that change the rate of de novo hepatic lipogenesis are associated with corresponding changes in apoB secretion rates.^{9 12 13 14} Simulation of these changes either in primary rat hepatocyte culture¹⁵ or in HepG2 cells,⁸ however, had no effect on apoB output.

In longer-term cultures of hepatocytes from fed rats, the output of VLDL apoB was found to be directly dependent on the intracellular content of hepatic TAG, which itself was a function of the period over which the cells were exposed to fatty acids.¹⁶ Thus, the major objective of the present work was to determine whether VLDL apoB output could be manipulated by longer-term exposure of primary cultures of rat hepatocytes to oleate, and if so, whether this change was accompanied by changes in the net rate of apoB synthesis. A second goal was to determine whether these manipulations affected TAG content and thereby the density of the apoB-containing particles secreted by the cells. This information may be valuable for understanding the causes and mechanisms of the changes in size of hepatic VLDL particles secreted in hypertriglyceridemic subjects,^{17 18 19 20} which in turn probably influence the production rate of LDL.^{21 22} Finally, in view of the relative increase in secretion of rat hepatic apoB-48 during enhanced in vivo lipogenesis,^{13 14 23} we sought to determine the effects in vitro of increasing TAG synthesis on the output of newly synthesized apoB-48 and apoB-100 and on their relative density distribution in the cell medium.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Anti-human apoB antibody (raised in sheep) was obtained from Boehringer-Mannheim. Anti-rabbit IgG, anti-sheep IgG, and protease inhibitors were purchased from Sigma-Aldrich Co Ltd. Protein A cells (Pansorbin) were obtained from Calbiochem. [³H]Leu was obtained from Amersham International, as was the tissue solubilizer (NCS). All tissue-culture materials were obtained from Life Science Technologies (GIBCO). Amberlite MB3 was obtained from BDH Chemicals, Ltd.

Maintenance of Animals and Preparation of Hepatocyte Cultures
Animals were fed and housed as previously described.^{24 25} Hepatocytes were prepared under sterile conditions and cultured for 4 hours in Waymouth's medium with fetal calf serum and other supplements exactly as described previously.²⁴ After the 4-hour culture, the cells were transferred to supplemented, serum-free medium in either the presence or absence of oleate (0.75 mmol/L).²⁴ After 24 hours the medium was replaced with fresh medium of identical composition. After another 24 hours the medium was replaced with RPMI 1640 medium lacking Leu but containing the same supplements as those in Waymouth's medium. Oleate (0.75 mmol/L) was either present or absent as before. Oleate was added as a complex with fatty acid–free BSA.²⁵ An equivalent concentration of albumin (to give a final concentration of 0.5% wt/vol) was added to those cells that lacked oleate.

Preparation of Rabbit Anti-Rat ApoB Antisera
Rat plasma was enriched in VLDL by injecting the rats (250 g) with Triton WR-1339 (1.0 mL of a 10% solution in 0.9% saline) into a tail vein, which prevented VLDL catabolism²⁶ and removed most of the peripheral apoproteins.²⁷ VLDL was isolated as described²⁴ in a Beckman L8-70M ultracentrifuge with a 50.4 rotor at 40 000 rpm for 16 hours at 4°C. VLDL (5.0 mg protein per milliliter) was delipidated, and the residual small peptides were removed with isopropanol by the method of Egusa et al.²⁸ The pellet of insoluble apoB was collected by centrifugation and washed twice more, each with 5 mL isopropanol. The residual material was solubilized by heating at 80°C for 20 minutes in a buffer composed of 3% SDS, 20% (vol/vol) 2-mercaptoethanol, and 20% (vol/vol) glycerol in 0.05 mol/L Tris HCl (pH 6.8). The SDS was removed by dialysis at room temperature for 24 hours against two 4.0-L changes of 0.05 mol/L Tris buffer and 0.1% Triton X-100 (pH 7.5) with a 50 000-cutoff membrane and 25 g of Amberlite MB3 in the dialysate. Finally, the material was dialyzed overnight against 0.01% ammonium formate at 4°C and then emulsified with Freund's adjuvant.

New Zealand White rabbits were immunized by multiple intramuscular injections of the antigen and boosters at 6-week intervals. Sera were obtained 2 weeks after each boost. The specificity of the sera was tested against rat VLDL that had been electroblotted onto Immobilon membranes. The antibody cross-reacted exclusively with apoB-100 and apoB-48. No cross-reaction was observed between apoB antiserum and apoE, C, or A. Furthermore, in some experiments preimmune serum was used to check the specificity of the anti-apoB antiserum. In these cases between 200 and 1000 disintegrations per minute of label was immunoprecipitated from the cell medium. This amount represented <5% of the label immunoprecipitated by the anti-apoB antiserum after 24-hour culture.

Immunoprecipitation of ApoB
One hour after addition of RPMI 1640 to the cultures, [³H]Leu was added (50 µCi, 167 Ci/mmol). The cells were cultured for as long as 24 hours in the presence or absence of oleate (0.75 mmol/L). After 0, 1, 2, 8, or 24 hours of culture the medium was removed and the cells were washed three times, each with 3.0 mL PBS. The cells were then lysed at 80°C in 1.5 mL lysis buffer (0.15 mol/L NaCl, 1.0% Triton X-100, and 0.5% SDS in 0.05 mol/L Tris, pH 7.4) containing a mixture of proteinase inhibitors.^{29 30} Aliquots of the total cell lysate and medium were taken for TCA precipitation and analysis of [³H]Leu incorporation into total labeled protein. Cellular protein content was measured by the method of Lowry et al.³¹ In some cases the cell medium was separated into a VLDL-containing fraction (d<1.006 g/mL) and a VLDL infranatant (d>1.006 g/mL; see below).

Cell lysates were heated to 85°C for 30 minutes and allowed to cool to room temperature before centrifugation at 1500 rpm in an MSE Mistral centrifuge for 20 minutes. An aliquot of supernatant (0.75 mL) was removed and diluted to 3.0 mL with a solution of 0.01 mol/L Tris HCl (pH 7.4), 0.15 mol/L NaCl, and 1.0% (wt/vol) BSA. Ten microliters of the rabbit anti-rat apoB antiserum (diluted to 100 µL with the aforementioned diluent) was then added. This dilution of antiserum had been previously shown to quantitatively precipitate 5.0 µg of ¹²⁵I-labeled apoB from rat VLDL after it had been treated in the same way as described above for the cell lysates. Immune complexes were collected by adding 150 µL of staphylococcal protein A cells (0.25 g/mL) followed by incubation at room temperature for 45 minutes with gentle rocking. Protein A cells were pretreated by boiling in Tris HCl buffer (0.05 mol/L) with 0.15 mol/L NaCl, 10% (vol/vol) 2-mercaptoethanol, and 3% (wt/vol) SDS.²⁹ The immune complexes were sedimented by centrifugation and washed five times with the aforementioned diluent. Finally, the immune complexes were suspended in 1.0 mL of 0.25 mol/L sucrose, and 330 µL was taken for scintillation counting. ApoB was immunoprecipitated from the whole-cell medium (1.0 mL), VLDL (1.0 mL), and the d=1.006 g/mL infranatant (1.0 mL) in essentially the same way; in these instances, however, 1.0 mL of lysate buffer was added before heating and then diluted to 4.0 mL with the same diluent. To this solution was finally added 10 µL antiserum that had been diluted to 100 µL with the diluent.

SDS-PAGE
The remainder of the suspended immunoprecipitate (after 330 µL was removed for radioassay) was transferred to microcentrifuge tubes (Eppendorf). Immune complexes were harvested by centrifugation at 800g for 5 minutes at 4°C. The pellets were resuspended in 120 µL of sample buffer composed of 0.05 mol/L Tris (pH 6.8) containing 3% (wt/vol) SDS, 20% (vol/vol) 2-mercaptoethanol, 20% (vol/vol) glycerol, 0.05% (wt/vol) bromophenol blue, and Triton-treated rat plasma VLDL apoB (final concentration, 0.2 mg/mL). Samples were heated at 90°C for 15 minutes and allowed to cool to room temperature. The protein A cells were then pelleted by centrifugation at 8000 rpm for 5 minutes at 4°C. The supernatant was loaded onto a 4% to 20% gradient polyacrylamide gel (20x20 cm) and run for 18 hours at 15 mA. The residual material associated with the protein A pellet was resuspended in 1.0 mL of water, mixed with 10 mL of Optiphase R scintillation fluid, and counted. Typically <10% of the original immunoprecipitate label remained in the residue. After electrophoresis the proteins were visualized by staining the gels overnight with Coomassie Brilliant Blue R (0.1% wt/vol dissolved in destaining solution and filtered) and destained in a solution of acetic acid (7.5% vol/vol), methanol (10% vol/vol), and water (82.5% vol/vol). Areas of the gel corresponding to apoB-48 and apoB-100 markers were excised with a razor blade. Samples were placed in plastic scintillation vials and 1.0 mL of 30% (vol/vol) H₂O₂ was added. The vials were capped and incubated overnight at 25°C. One milliliter of tissue solubilizer (NCS) was added to each sample and incubation continued for 24 hours at room temperature. The solutions were then neutralized with 150 µL acetic acid, mixed, and incubated for another 24 hours. Finally, 10 mL of scintillation fluid (Optiphase R) was added to each vial and the samples were mixed. The samples were then stored in the dark for 24 hours to reduce chemiluminescence before the radioactivity was counted. The total yield of radioactivity recovered from each gel was typically >90% of that originally loaded onto the gel.

Determination of ApoB and TAG Associated With the Cell Medium, VLDL, and the d>1.006 g/mL Infranatant
In some experiments we performed assays on the total-cell medium without prior fractionation into VLDL and the d>1.006 g/mL infranatant. In other experiments when these density fractions were required, the cell medium was removed and added to a mixture of antibiotics, antioxidants, and protease inhibitors,³² and the mixture was centrifuged for 16 hours at 40 000 rpm (154 000g) in a Beckman L8-70 ultracentrifuge with a 50.4 rotor. The floating VLDL fraction (d<1.006 g/mL) was separated from the infranatant (d>1.006 g/mL) by slicing the tube. Lipids (including TAG) were extracted from all fractions (including the cells) by the method of Folch et al.³³ TAG mass was determined by the method of Trinder³⁴ using a kit supplied by Boehringer-Mannheim. The mass of apoB in the medium, VLDL, and the d>1.006 g/mL infranatant was determined with a direct, noncompetitive ELISA with rat plasma VLDL apoB as the standard.³⁵

	Results

Top Abstract Introduction Methods Results Discussion References

 
To assess whether changes in intracellular TAG content were required to trigger changes in apoB output, the TAG concentration was increased substantially by culturing the cells for 3 days with extracellular oleate (0.75 mmol/L). This treatment increased the intracellular TAG content to 575±62 compared with 38±6 µg/mg protein (P<.001) in cells that were cultured without oleate. These TAG-enriched cells secreted much more TAG (225±30 µg/mg protein) into the medium during the final 24-hour culture period than did hepatocytes that had not been cultured with oleate (40±10 µg/mg protein, P<.01). By contrast there was only a small increase in apoB secretion (measured as total mass) into the whole medium of the cells (4.22±0.94 versus 3.72±0.75 µg/mg cell protein).

In the radiolabeling experiments secretion of newly synthesized, ³H-labeled, immunoprecipitable apoB into the medium from TAG-enriched cells was only slightly higher than that secreted by the control cells (Fig 1). Thus, compared with the control cells, the TAG-enriched hepatocytes had increases in TAG secretion that were much greater than the increases in apoB secretion, suggesting an increase in particle size. This might be expected to decrease the density of the secreted particles in the TAG-enriched hepatocytes. To verify or refute this hypothesis, in subsequent similar experiments the secreted apoB-containing lipoproteins were separated by ultracentrifugation into d<1.006 and d>1.006 g/mL fractions. In these experiments the d<1.006 g/mL fraction (which represented secreted VLDL) from TAG-enriched cells contained substantial quantities of TAG and apoB (measured as total mass), both of which appeared in the VLDL fraction at a linear rate (Fig 2). In control cells the VLDL fraction contained virtually no TAG and very little apoB (Fig 2). However, the d>1.006 g/mL fraction (containing the heavier particles) from control cells contained a larger proportion of the total secreted apoB than did that from TAG-enriched cells (Table 1). In control and TAG-enriched cells, the apoB in the d>1.006 g/mL infranatant was associated with very little TAG (Table 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Secretion of newly synthesized apoB into the medium. Cells were cultured for 48 hours in supplemented Waymouth's medium with (TAG-enriched cells, ) or without (control cells, ) oleate. The medium was changed to Leu-free RPMI 1640 medium containing the same supplements and with or without oleate as appropriate. Cells were cultured for another 1, 2, or 8 hours with 50 µCi [3H]Leu, after which the radiolabeled apoB was immunoprecipitated from the cell medium. Each point represents the mean±SEM of 6 independent cell preparations.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of cellular TAG enrichment on TAG and apoB secretion in rat hepatocytes. Hepatocytes were cultured for 48 hours with (TAG-enriched cells, ) or without (control cells, ) oleate as described in the legend to Fig 1 and "Methods." After transfer to RPMI 1640 medium, the cells were cultured for another 8 or 24 hours with or without oleate, as during the previous 48 hours. After 8 or 24 hours aliquots of the cell medium were removed for preparation of VLDL fraction. TAG content was measured enzymatically and total apoB was measured with a noncompetitive ELISA. Each point is the mean±SEM of 4-9 independent cell preparations. *P<.05, **P<.01, and ***P<.001 are significantly different from corresponding values in control cells.

View this table: [in this window] [in a new window]   Table 1. Distribution of Secreted ApoB and TAG Between VLDL and d>1.006 g/mL Fractions

It might be argued that the increased secretion of VLDL TAG and apoB by hepatocytes exposed to oleate for 3 days compared with that of non–oleate treated controls resulted from the presence of oleate itself and not prior cellular TAG enrichment. To investigate this possibility we measured VLDL secretion in the presence of oleate in cells previously exposed to oleate for 48 hours and compared it with that of cells cultured in the absence of oleate for the same period. The latter cells were depleted in cellular TAG compared with the former (Table 2). If exposure to oleate during the VLDL secretion period were the only prerequisite for an increase in VLDL secretion, then the output of VLDL TAG and apoB should be identical in both types of cell. This was not the case. Table 2 shows that despite identical final culture conditions, TAG-enriched cells secreted significantly more VLDL apoB than did those cells that were TAG depleted. Furthermore, exposure of freshly isolated cell cultures to oleate for 24 hours had no effect on VLDL apoB secretion compared with those cultures in which oleate was absent (4.98±1.08 versus 6.58±0.85 µg/mg per 24 hours, respectively). These "younger" cell cultures had insufficient time to accumulate the amount of intracellular TAG required to stimulate VLDL apoB output. Nevertheless, under these conditions oleate resulted in a small but significant increase in VLDL TAG output (112±1.4 versus 98.3±1.7 µg/mg per 24 hours, P<.01).

View this table: [in this window] [in a new window]   Table 2. Effect of Preloading Cells With TAG on the Secretion of VLDL TAG and ApoB Without Oleate

The effects of cellular TAG enrichment on the distribution of newly synthesized, immunoprecipitable apoB between VLDL and the d>1.006 g/mL infranatant (Fig 3) were similar to those observed for the distribution of apoB mass. TAG enrichment had no significant effect on the secretion of labeled apoB into the d>1.006 g/mL infranatant but increased apoB secretion into VLDL by a factor of 3. The sum of labeled apoB (VLDL plus infranatant) secreted from the TAG-enriched cells was 219 000±42 800 dpm/mg protein per 8 hours. The corresponding value for control cultures was 161 000±28 000 dpm/mg per 8 hours. These values are similar to those for whole, unfractionated medium when it was analyzed for apoB (Fig 1). The small difference in total output between TAG-enriched and control cells can be almost entirely accounted for by a large, relative increase in apoB output that is specifically associated with VLDL. Thus, an important consequence of cellular TAG enrichment was an increase in the proportion of total secreted apoB that was associated with VLDL, from 9.5±1.9% to 20.8±4.1% (P<.05) after 8 hours and from 16.2±4.3% to 37.1±1.9% (P<.01) after 24 hours.

View larger version (13K): [in this window] [in a new window]   Figure 3. Appearance of newly synthesized apoB in d>1.006 (A) and d<1.006 (B) g/mL fractions of cell medium: effects of cellular TAG enrichment. Rat hepatocytes were cultured as described in the legend to Fig 1, transferred to RPMI 1640 medium, and cultured with 50 µCi [3H]Leu for 1, 2, or 8 hours with () or without () 0.75 mmol/L oleate. After 1, 2, or 8 hours the cell medium was centrifuged (see "Methods") and separated into d<1.006 and d>1.006 g/mL fractions. Radiolabeled apoB was immunoprecipitated from each fraction. Each point is the mean±SEM of 4 independent cell preparations. *Significantly different from corresponding values in control cells.

Although most of the extracellular apoB was found in the VLDL infranatant, we are confident that this occurrence resulted from direct secretion rather than cell damage. First, if the latter explanation were correct, cell damage would be expected to increase with increasing incubation time. Thus, the proportion of labeled apoB in the infranatant would increase with time. However, this was not the case. In TAG-rich cells at 8 hours, 79.2±4.1% of the total apoB appeared in the infranatant; at 24 hours this value was 62.9±1.9%. A similar pattern was observed for TAG-depleted cells. Second, it has been our experience over a number of years that if cells are damaged in any way (eg, low initial viability, toxic concentrations of drugs, etc), then the amounts of apoB recovered from the medium (from both d>1.006 and d<1.006 g/mL fractions) are very low. Finally, there were no significant differences in the amounts of total TCA-precipitable label in the medium during 8-hour culture of freshly isolated cells compared with older cells that had been precultured for 48 hours. Thus, older cells did not "leak" labeled proteins into the medium. Neither was there any effect of oleate treatment on total TCA-precipitable, labeled protein secretion into the medium. For freshly isolated cells these values were (in dpmx10^-6 per milligram cell protein) 0.80±0.37, 1.46±0.39, and 6.33±1.17 for 1, 2, and 8 hours, respectively, without oleate. With oleate the corresponding values were 0.39±0.07, 1.43±0.09, and 5.72±0.64. For older cell cultures the corresponding values were 0.43±0.13, 1.56±0.71, and 4.45±1.31 (oleate absent) and 0.46±0.20, 1.37±0.36, and 6.09±1.30 (oleate present). Nor did TAG enrichment have any effect on the incorporation of label into TCA-precipitable material within the cell. These values were (in dpmx10^-7 per milligram protein) 1.67±0.24/8 h (TAG enriched) and 1.51±0.24/8 h (TAG depleted). It is unlikely that the apoB in the d>1.006 g/mL infranatant resulted from extracellular metabolism of secreted VLDL (eg, lipolysis of VLDL TAG). If this had occurred then the proportion of total secreted apoB in the infranatant would have increased. This was not observed (see above).

The distribution of label between apoB-48 and apoB-100 in the VLDL and d>1.006 g/mL fractions is shown in Fig 4. In the VLDL fraction, cellular TAG enrichment significantly increased the amount of label associated with apoB-48 but had little or no effect on that associated with apoB-100. No significant changes occurred in the d>1.006 g/mL fraction. This differential effect on apoB-48 in VLDL was more evident when the relative changes in apoB-48 and apoB-100 were calculated as a function of incubation time. In this case TAG enrichment increased the appearance of labeled apoB-48 in the VLDL fraction by 56±12.1% (P<.05), 122±52.6% (P<.05), and 248±98% (P<.05) after 1, 2, and 8 hours, respectively. There were no significant corresponding effects on the appearance of apoB-100 in the VLDL fraction.

View larger version (17K): [in this window] [in a new window]   Figure 4. Secretion of apoB-48 and apoB-100 into VLDL (A) and d>1.006 g/mL (B) fractions. Rat hepatocytes were cultured for 48 hours as described in the legend to Fig 1 before transfer to RPMI 1640 medium and addition of 50 µCi [3H]Leu. After culture for another 1, 2, or 8 hours with (TAG-enriched cells; , ) or without (control cells; , ) 0.75 mmol/L oleate, the medium was removed and separated into d<1.006 (VLDL) and d>1.006 g/mL fractions by ultracentrifugation. Total labeled apoB was immunoprecipitated from each fraction and apoB-48 was separated from apoB-100 by SDS-PAGE. Each point represents the mean±SEM of 4 separate experiments. *Significantly different from corresponding values in control cells. Circles represent apoB-48; triangles, apoB-100.

In TAG-enriched cells the large increase in the secretion of apoB-48 specifically into the VLDL fraction of the medium suggests that the relatively small increase in total secretion of apoB into the unfractionated medium (Fig 1) may have exclusively been due to an increase in apoB-48 secretion. To test this possibility the unfractionated medium of TAG-enriched and control cells was separated into d>1.006 and d<1.006 g/mL fractions, and radiolabeled apoB-48 and apoB-100 were isolated from each fraction. Table 3 shows that the total amount of secreted apoB-48 in the two density fractions was significantly higher in TAG-enriched than control cells. This was accompanied by an increase in the net incorporation of radiolabel into cellular apoB-48 in TAG-enriched cells after 1 and 2 hours of culture (Fig 5). Cellular TAG enrichment had no effect on either the secretion of labeled apoB-100 into the medium (Table 3) or the net synthesis of apoB-100 by the cells (Fig 5).

View this table: [in this window] [in a new window]   Table 3. Effect of Cellular TAG Enrichment on Total (VLDL+Infranatant) Secretion of ApoB-48 and ApoB-100

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of TAG enrichment on net cellular synthesis of apoB-48 and apoB-100. Cells were cultured for 48 hours as described in the legend to Fig 1 before transfer to RPMI 1640 medium and addition of 50 µCi [3H]Leu. After culture for another 1, 2, or 8 hours with (, ; TAG-enriched cells) or without (, ; control cells) oleate, radiolabeled apoB was immunoprecipitated from the cell lysate. Labeled apoB-48 and apoB-100 were separated by SDS-PAGE and their bands excised from the gel and assayed for 3H radioactivity by scintillation counting. Values are expressed as radioactivity incorporated per milligram of cell protein. Each point represents the mean±SEM of 4 independent cell preparations.

Fig 1 shows that the total labeled apoB immunoprecipitated from the unfractionated medium exceeded the sum of apoB-48 and apoB-100 isolated by electrophoresis after separate immunoprecipitation of apoB from VLDL and infranatant fractions (Table 3). Some of this discrepancy may be accounted for by manipulative losses inherent to the centrifugation procedure. A contribution to the shortfall, however, was the presence of an immunoprecipitable, labeled peptide with an apparent M_r of 70 kD after electrophoresis in the infranatant fraction only. This peptide was neither precipitated by preimmune rabbit serum nor removed by pretreatment with an anti-rat albumin antibody. We are currently investigating the origin and regulation of this labeled peptide.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
An important question regarding the synthesis of VLDL is whether an increase in fatty acid flux to the hepatocyte increases the availability and secretion of apoB. This question has not yet been answered conclusively and may depend on the particular in vitro model,^{4 5 8 9 11} the metabolic source of fatty acids,^{5 13 36} and the nutritional status of donor animals.¹⁰ Since our previous results¹⁶ had suggested that the availability of intracellular TAG rather than the extracellular concentration of oleate was an important factor in the control of VLDL apoB secretion, we studied apoB secretion in hepatocytes that had been cultured for 3 days with or without oleate. This resulted in the accumulation or depletion, respectively, of intracellular TAG stores. During the final 24 hours of culture, TAG-enriched cells secreted much more TAG into the total medium. This change, however, was accompanied by only a small increase in secretion of total apoB mass (Table 1) and newly synthesized apoB (Fig 1). Fractionation of the whole medium into VLDL (d<1.006 g/mL) and the d>1.006 g/mL infranatant showed that TAG-rich cells secreted a much higher proportion of newly synthesized apoB into VLDL than did control cells (Fig 3). A similar pattern was also observed for the distribution of the total mass of apoB (Table 1). The secretion rate of apoB into VLDL paralleled that of TAG into this fraction in both types of cell (Table 1 and Fig 2).

The results suggest that the large increase in TAG transport as VLDL in TAG-enriched cells (Fig 2) is achieved at the expense of only a small increase in total secretion of immunoprecipitable apoB into unfractionated medium (Fig 1). The major contributor to the increased VLDL secretion by these cell cultures was greater recruitment of available apoB for the assembly of buoyant particles (Fig 3). In this respect it is noteworthy that even when TAG transport from the cell was greatly diminished (as in TAG-depleted cells), the total output of immunoreactive apoB remained relatively high (Table 1 and Fig 1). In this case, most of the apoB was secreted into a higher-density fraction of the cell medium and was associated with only very small amounts of TAG (Table 1). In this respect TAG-depleted cells resemble HepG2 cells, which also secrete TAG with mainly small, dense particles.⁵

The present work also provides evidence that the higher buoyancy of apoB-containing particles secreted by TAG-enriched cells is associated with greater recruitment of apoB-48 rather than apoB-100 into VLDL (Fig 4). This was due not only to greater utilization of apoB-48 (which otherwise would have been secreted as denser particles) but also to overall stimulation of total apoB-48 secretion by TAG-enriched cells (Table 3). In this case there was an increase in the net incorporation of label into newly synthesized apoB-48 after 1 and 2 hours of culture. This was not observed for apoB-100 (Fig 5). It may be argued that the different net synthesis rates of apoB-48 in control (TAG depleted) versus TAG-enriched (Fig 5) cells may result from a decreased basal level of apoB-48 synthesis in control cells rather than a relative increase in apoB-48 synthesis in TAG-enriched cells. To address this issue we measured net rates of apoB-48 synthesis in freshly isolated hepatocytes with "normal" levels of intracellular TAG. In this case apoB-48 synthesis after 1 and 2 hours of culture corresponded to incorporation of 3117±882 and 3427±1107 dpm/mg cell protein, respectively. These values are similar to those for apoB-48 synthesis after 1 and 2 hours in TAG-depleted (control) cells (Fig 5). Similarly, in freshly isolated cells the corresponding values for apoB-48 in VLDL plus infranatant were 1131±440 and 3073±778 dpm/mg protein after 1 and 2 hours, respectively. Corresponding values in TAG-depleted (control) cells were not lower than these (Table 3). Thus, we conclude that "basal" levels of apoB-48 synthesis and secretion did not decrease in the TAG-poor cells and that the differences reported in Table 3 and Fig 5 resulted from a stimulatory effect of TAG enrichment on apoB-48 synthesis and secretion. Although lesser degradation of apoB-48 may be a contributing factor, increased synthesis may also play a role, since the observed effects occurred very rapidly. It is possible that this latter effect results from control of the apoB-100 mRNA editing mechanism,^{37 38} as has recently been proposed for the increase in apoB-48 that occurs after long-term exposure of rat hepatocytes to insulin.³⁹ The present findings are consistent with those in which physiological and developmental alterations that result in a higher VLDL TAG output in the rat are accompanied by a selective increase in apoB-48 rather than apoB-100 secretion.^{14 23 37}

We cannot of course exclude the possibility that at least some of the increase in the quantity of secreted VLDL TAG in TAG-enriched cells is due to increased TAG loading onto apoB-100–containing VLDL particles. Any resulting increase in size (and decrease in density) of these particles would not have been detected by the method used for VLDL isolation. Any such change must have been limited to VLDL itself, since we were unable to detect any change in buoyancy of apoB-100–containing particles across the d=1.006 g/mL boundary. A reasonable assumption is that the twofold to threefold increase in the apoB-48 content of VLDL resulted from increased buoyancy of the apoB-48–containing particles because of an increase in TAG content. This increase was probably a major contribution to the increased TAG content of VLDL secreted by TAG-enriched cells.

At first sight the aforementioned observations appear to be at odds with several reports on the ability of apoB to bind lipid and transport it from the cell and how this ability varies in proportion to the molecular mass of the apoB fragment.^{40 41 42 43} Thus, the capacity of apoB-48 to transport lipid from the cell might be expected to be lower than that of apoB-100. The aforementioned reports, however, considered only the amount of TAG that became associated with the apoB fragment during its translation and translocation into the lumen of the secretory apparatus. A recent report⁴⁴ has provided other evidence that in the rat hepatoma cell line McA-RM7777, apoB-48 is preferentially recruited for assembly of VLDL particles in cells cultured with oleate. The authors concluded that without oleate, transfer of cellular TAG to apoB-100 and apoB-48 occurred only during translocation. Although this was sufficient to give the nascent apoB-100–containing particles the buoyancy of VLDL, the smaller apoB-48 became relatively lipid depleted in the secretory lumen. With oleate, however, the increase in cellular TAG content effected a selective, posttranslational, second-step TAG transfer to the nascent, small apoB-48–containing particles, which greatly increased production of particles with the buoyancy of VLDL. Differences in kinetics between newly synthesized apoB-48 and apoB-100 in the rat support the notion of differential hepatic metabolism prior to secretion.⁴⁵ Our results suggest that a similar mechanism may be operating in primary rat hepatocyte cultures under circumstances that favor increased transport of TAG from the cell. This would cause preferential utilization of apoB-48 and might provide a mechanistic explanation for the selective response of apoB-48 metabolism to different physiological circumstances.^{6 14 23} The differential processing of apoB-48 and apoB-100 with respect to lipoprotein assembly suggests a possible mechanism for the lack of secretion of intestinal apoB-48 in the rare autosomal recessive disorder, chylomicron retention disease.⁴⁶ This disorder results from neither defective synthesis nor editing of apoB mRNA in the gut⁴⁷ but may arise from a defect in the gene that is normally responsible for lipid transfer to the nascent apoB-48–containing particle.^{44 47}

We conclude that the increased transport of TAG as VLDL from TAG-enriched rat hepatocytes is achieved by a combination of at least two mechanisms: (1) a relatively minor increase in apoB net synthesis and secretion, most or all of which can be accounted for by apoB-48, and (2) a major change in apoB distribution from heavy to light particles, mainly due to selective recruitment of apoB-48 into the d<1.006 g/mL fraction.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay PAGE = polyacrylamide gel electrophoresis TAG = triacylglycerol TCA = trichloroacetic acid

	Acknowledgments

 
This work was supported by a Programme Grant from the Medical Research Council (United Kingdom). Geoffrey F. Gibbons is a member of the External Scientific Staff of the Medical Research Council and University Research Lecturer at the University of Oxford. We thank Madge Barber for typing the manuscript and the staff of the Medical Illustration Department, John Radcliffe Hospital, Oxford, for preparing the diagrams. Peter J. Coussons is currently at the Department of Pathology, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QP, UK. Catherine S. Bourgeois is currently at Faulding Pharmaceuticals, Greenhill Rd, Adelaide, South Australia 5000, Australia.

Received June 25, 1995; revision received December 14, 1995;

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Pullinger CR, North JD, Teng B-B, Rifici VA, Ronhild de Brito AE, Scott J. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life. J Lipid Res. 1989;30:1065-1077.[Abstract]
2. Dashti N, Williams DL, Alaupovic P. Effects of oleate and insulin on the production rates and cellular mRNA concentrations of apolipoproteins in HepG2 cells. J Lipid Res. 1989;30:1365-1373.[Abstract]
3. Boren J, Wettesten M, Sjoberg A, Thorlin T, Bondjers G, Wiklund O, Olofsson S-O. The assembly and secretion of apoB 100 containing lipoproteins in HepG2 cells: evidence for different sites for protein synthesis and lipoprotein assembly. J Biol Chem. 1990;265:10556-10564.[Abstract/Free Full Text]
4. Wang H, Chen X, Fisher EA. N-3 fatty acids stimulate intracellular degradation of apoprotein B in rat hepatocytes. J Clin Invest. 1993;91:1380-1389.
5. Dixon JL, Ginsberg HN. Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells. J Lipid Res. 1993;34:167-179.[Abstract]
6. Sparks JD, Sparks CE. Hormonal regulation of lipoprotein assembly and secretion. Curr Opin Lipidol. 1993;4:177-186.
7. Sparks JD, Sparks CE. Insulin regulation of triacylglycerol-rich lipoprotein synthesis and secretion. Biochim Biophys Acta. 1994;1215:9-32.[Medline] [Order article via Infotrieve]
8. Cianflone K, Dahan S, Monge JC, Sniderman AD. Pathogenesis of carbohydrate-induced hypertriglyceridemia using HepG2 cells as a model system. Arterioscler Thromb. 1992;12:271-277.[Abstract/Free Full Text]
9. Davis RA, Boogaerts JR. Intrahepatic assembly of very low density lipoproteins: effect of fatty acids on triacylglycerol and apolipoprotein synthesis. J Biol Chem. 1982;257:10908-10913.[Abstract/Free Full Text]
10. Salam WH, Wilcox HG, Heimberg M. Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver. Biochem J. 1988;251:809-816.[Medline] [Order article via Infotrieve]
11. Patsch W, Tamai T, Schonfeld G. Effect of fatty acids on lipid and apoprotein secretion and association in hepatocyte cultures. J Clin Invest. 1983;72:371-378.
12. Davis RA, Clinton GM, Borchardt RA, Malone-McNeal M, Tan T, Lattier GR. Intrahepatic assembly of very low density lipoproteins: phosphorylation of small molecular weight apolipoprotein B. J Biol Chem. 1984;259:3383-3386.[Abstract/Free Full Text]
13. Gibbons GF. Assembly and secretion of hepatic very-low-density lipoprotein. Biochem J. 1990;268:1-13.[Medline] [Order article via Infotrieve]
14. Coleman RA, Haynes EB, Sand TM, Davis RA. Developmental coordinate expression of triacylglycerol and small molecular weight apoB synthesis and secretion by rat hepatocytes. J Lipid Res. 1988;29:33-42.[Abstract]
15. Boogaerts JR, Malone-McNeal M, Archambault-Schexnayder J, Davis RA. Dietary carbohydrate induces lipogenesis and very-low-density lipoprotein synthesis. Am J Physiol. 1984;246:E77-E83.[Abstract/Free Full Text]
16. Gibbons GF, Bartlett SM, Sparks CE, Sparks JD. Extracellular fatty acids are not utilized directly for the synthesis of very-low-density lipoprotein in primary cultures of rat hepatocytes. Biochem J. 1992;287:749-753.
17. Taskinen M-R, Beltz WF, Harper I, Fields RM, Schonfeld G, Grundy SM, Howard BV. Effects of NIDDM on very-low-density lipoprotein triglyceride and apolipoprotein B metabolism: studies before and after sulfonylurea therapy. Diabetes. 1986;35:1268-1277.[Abstract]
18. Howard BV. Lipoprotein metabolism in diabetes mellitus. J Lipid Res. 1987;28:613-628.[Medline] [Order article via Infotrieve]
19. Howard BV, Abbott WGH, Egusa G, Taskinen MR. Coordination of very low-density lipoprotein triglyceride and apolipoprotein B metabolism in humans: effects of obesity and non-insulin-dependent diabetes. Am Heart J. 1987;113:522-526.[Medline] [Order article via Infotrieve]
20. Taskinen M-R, Packard CJ, Shepherd J. Effect of insulin therapy on metabolic fate of apolipoprotein B-containing lipoproteins in NIDDM. Diabetes. 1990;39:1017-1027.[Abstract]
21. Packard CJ, Munro A, Lorimer AR, Gotto AM, Shepherd J. Metabolism of apolipoprotein B in large triglyceride-rich very low density lipoproteins of normal and hypertriglyceridemic subjects. J Clin Invest. 1984;74:2178-2192.
22. Eisenberg S. Lipoprotein abnormalities in hypertriglyceridemia: significance in atherosclerosis. Am Heart J. 1987;113:555-561.[Medline] [Order article via Infotrieve]
23. Windmueller HG, Spaeth AE. Regulated biosynthesis and divergent metabolism of three forms of hepatic apolipoprotein B in the rat. J Lipid Res. 1985;26:70-81.[Abstract]
24. Bartlett SM, Gibbons GF. Short- and longer-term regulation of very-low-density lipoprotein secretion by insulin, dexamethasone and lipogenic substrates in cultured hepatocytes: a biphasic effect of insulin. Biochem J. 1988;249:37-43.[Medline] [Order article via Infotrieve]
25. Van Harken DR, Dixon CW, Heimberg M. Hepatic lipid metabolism in experimental diabetes, V: the effect of concentration of oleate on metabolism of triglycerides and on ketogenesis. J Biol Chem. 1969;244:2278-2285.[Abstract/Free Full Text]
26. Otway S, Robinson DS. The use of a non-ionic detergent (Triton WR-1339) to determine rates of triglyceride entry into the circulation of the rat under different physiological conditions. J Physiol (Lond). 1967;190:321-332.[Abstract/Free Full Text]
27. Fisher WR, Schumaker VN. Isolation and characterization of apolipoprotein B-100. Methods Enzymol. 1986;128A:247-262.
28. Egusa G, Brady DW, Grundy SM, Howard BV. Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B. J Lipid Res. 1983;24:1261-1267.[Abstract]
29. Sparks JD, Sparks CE. Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes. J Biol Chem. 1990;265:8854-8862.[Abstract/Free Full Text]
30. Sparks JD, Zolfaghari R, Sparks CE, Smith HC, Fisher EA. Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats. J Clin Invest. 1992;89:1418-1430.
31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]
32. Edelstein C, Scanu AM. Precautionary measures for collecting blood destined for lipoprotein isolation. Methods Enzymol. 1986;128:151-155.[Medline] [Order article via Infotrieve]
33. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497-509.[Free Full Text]
34. Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem. 1969;6:24-30.
35. Duerden JM, Gibbons GF. Restoration in vitro of normal rates of very-low-density lipoprotein triacylglycerol and apoprotein B secretion in hepatocyte cultures from diabetic rats. Biochem J. 1993;294:167-171.
36. Wang H, Yao Z, Fisher EA. The effects of n-3 fatty acids on the secretion of carboxyl-terminally truncated forms of human apoprotein B. J Biol Chem. 1994;269:18514-18520.[Abstract/Free Full Text]
37. Leighton JK, Joyner J, Zamarripa J, Deines M, Davis RA. Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: evidence that the total amount of apoB secreted is regulated post-transcriptionally. J Lipid Res. 1990;31:1663-1668.[Abstract]
38. Inui Y, Giannoni F, Funahashi T, Davidson NO. REPR and complementation factor(s) interact to modulate rat apolipoprotein B mRNA editing in response to alterations in cellular cholesterol flux. J Lipid Res. 1994;35:1477-1489.[Abstract]
39. Thorngate FE, Raghow R, Wilcox HG, Werner CS, Heimberg M, Elam MB. Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by altering apolipoprotein B mRNA editing. Proc Natl Acad Sci U S A. 1994;91:5392-5396.[Abstract/Free Full Text]
40. Yao ZM, Blackhart BD, Linton MF, Taylor SM, Young SG, McCarthy BJ. Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells: evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins. J Biol Chem. 1991;266:3300-3308.[Abstract/Free Full Text]
41. Boren J, Graham L, Wettesten M, Scott J, White A, Olofsson S-O. The assembly and secretion of apoB 100-containing lipoproteins in HepG2 cells: apoB 100 is cotranslationally integrated into lipoproteins. J Biol Chem. 1992;267:9858-9867.[Abstract/Free Full Text]
42. Graham DL, Knott TJ, Jones TC, Pease RJ, Pullinger CR, Scott J. Carboxyl-terminal truncation of apolipoprotein B results in gradual loss of the ability to form buoyant lipoproteins in cultured human and rat liver cell lines. Biochemistry. 1991;30:5616-5621.[Medline] [Order article via Infotrieve]
43. Spring DJ, Lee SM, Puppione DL, Phillips M, Elovson J, Schumaker VN. Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apoB-48-like apolipoprotein. J Lipid Res. 1992;33:233-240.[Abstract]
44. Boren J, Rustaeus S, Olofsson SO. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells. J Biol Chem. 1994;269:25879-25888.[Abstract/Free Full Text]
45. Swift LL. Assembly of very low density lipoproteins in rat liver: a study of nascent particles recovered from the rough endoplasmic reticulum. J Lipid Res. 1995;36:395-406.[Abstract]
46. Levy E, Marcel Y, Deckelbaum RJ, Milne R, Lepage G, Seidman E, Bendayan M, Roy CC. Intestinal apoB synthesis, lipids, and lipoproteins in chylomicron retention disease. J Lipid Res. 1987;28:1263-1274.[Abstract]
47. Patel S, Pessah M, Beucler I, Navarro J, Infante R. Chylomicron retention disease: exclusion of apolipoprotein B gene defects and detection of mRNA editing in an affected family. Atherosclerosis. 1994;108:201-207.[Medline] [Order article via Infotrieve]

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