INTRODUCTION

Apolipoprotein B100 (apoB)¹ is an unusually large (>500 kDa) amphipathic protein responsible for the assembly and secretion of plasma lipoproteins by the liver and intestine (reviewed in Refs. 1-3). Its concentration in plasma, as a component of LDL, is a major determinant of susceptibility to the development of atherosclerotic cardiovascular disease (4, 5). Hepatic derived apoB100-containing lipoproteins are the precursors of plasma LDL (6). Hepatic assembly and secretion of apoB-containing lipoproteins require an orchestration of many seemingly independent processes as follows: 1) the production of component lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids); 2) the synthesis of apoB, a uniquely large polypeptide containing multiple amphipathic structural domains that irreversibly associate with phospholipids (7); 3) translocation across the endoplasmic reticulum that requires an intralumenal protein complex consisting of MTP and PDI (8); and 4) the assembly of VLDL particle within the endoplasmic reticulum (1, 2, 9).

Previous studies showed a coordinate induction of the synthesis of all VLDL lipids and the assembly and secretion of apoB-containing lipoproteins in response to changes in metabolic state (e.g. during dietary carbohydrate overload (10) and fetal development (11)). In contrast, fasting caused a coordinate repression of all of these processes (12, 13). These findings led us to propose that in a coordinate manner, metabolic signals control multiple steps of the VLDL assembly/secretion pathway. Using a somatic cell genetic approach, we examined this possibility. Previous experiments showed that expression of the liver-specific gene 7-hydroxylase (EC 1.14.13.17) in non-hepatic Chinese hamster ovary cells increased the transcription of the LDL receptor gene (14), a process now known to be regulated by the SREBP family of transcription factors (15, 16). Sterol-responsive transcriptional regulation is invoked by a proteolytic cleavage of the initial translational SREBP gene product that resides in the endoplasmic reticulum membrane as a trans-membrane loop (17, 18). When the sterol content of cells is insufficient to meet metabolic demands, proteolysis of the N-terminal domain releases the DNA binding domain of SREBP(s) (mature forms), allowing them to enter the nucleus and activate transcription of genes having cognitive promoter elements. Expression of 7-hydroxylase in cells that do not normally express it increases the cellular content of mature SREBP1 (see "Results"). Moreover, since McArdle rat hepatoma cells express all of the genes required to assemble and secrete apoB-containing lipoproteins (19-21), but lack 7-hydroxylase (see "Results"), by stably expressing 7-hydroxylase via plasmid transfection in McArdle hepatoma cells, we were able to increase the cellular content of mature SREBP1 to different extents in single cell clones. These cells provide a unique opportunity to examine how SREBP1 may affect each of the steps of the VLDL assembly/secretion pathway. The results indicate that SREBP1 coordinately regulates many of the individual lipogenic and protein processing steps.

MATERIALS AND METHODS

All chemicals used for biochemical techniques were purchased from Sigma, VWR, Fisher, or Boehringer Mannheim. Cell culture medium was obtained from Life Technologies, Inc. and fetal bovine serum from Gemni. Restriction enzymes were purchased from New England Biolabs or Promega. The cDNA probes were obtained from the following: hamster MTP was a generous gift from Drs. Richard Gregg, John Wetterau, David Gordon, and their colleagues at Bristol-Myers Squibb (22); hamster HMG-CoA reductase was obtained from ATCC; rat 7-hydroxylase was a generous gift from Dr. David Russell (23); and acetyl-CoA carboxylase was a generous gift from Dr. Tim Osborne (24). A mouse hybridoma cell line producing monoclonal antibody against human SREBP-1 (a generous gift from Dr. Tim Osborne) was propagated in mice, and the resulting IgG from the ascites fluid was obtained (25). A rabbit polyclonal antibody produced from a synthetic SREBP2 peptide was a generous gift from Dr. Michael Briggs.

McArdle RH-7777 hepatoma cells, a gift from Dr. Tom Innerarity, were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose, 15% heat-inactivated fetal bovine serum, and antibiotics (penicillin, 100 units/ml; streptomycin, 100 units/ml; and fungizone, 500 µg/ml). The cells were grown at 37 °C in an atmosphere of air with 5% CO₂. A plasmid was prepared from pcDNA3 (Invitrogen) in which the coding region of rat 7-hydroxylase was placed in the EcoRI-digested linker region. This plasmid contains a cytomegalovirus promoter and encodes neomycin resistance. Stable expression of the plasmid in McArdle RH-7777 cells was achieved by calcium phosphate precipitation and selection for neomycin resistance (G418, 400 µg/ml) (26). Single cell clones of neomycin-resistant cells were obtained.

Poly⁺A RNA was isolated from cells using a modification of the guanidinium isothiocyanate method, as described (27). Two to 5 µg of the resulting mRNA was separated by 0.8% agarose gel electrophoresis, transferred to a nylon membrane, and probed with nick-translated ³²P-cDNA probes prepared from gel-purified inserts.

Cells were harvested on ice in cold phosphate-buffered saline containing a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, and 50 µg/ml leupeptin) using a rubber policeman. Nuclei and membrane fractions were obtained using the method described by Wang et al. (17). Cells were centrifuged at 1000 rpm for 10 min, and the pellets were resuspended in 10 volumes of cell homogenization buffer (10 mM HEPES-KOH at pH 7.6, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM EDTA, and proteinase inhibitors). The cells were disrupted by passage through a 22-gauge needle (15 times) and then centrifuged at 1000 rpm for 10 min. The crude nuclear pellet was extracted with an equal volume of nuclear extraction buffer (20 mM HEPES-KOH at pH 7.6, 25% glycerol, 0.5 M NaCl, 1.5 mM MgCl₂, 1 mM EDTA, and proteinase inhibitors) and centrifuged at 12,000 rpm for 30 min. The supernatant was used as nuclear extract for immunoblotting analysis. The microsomal membrane fraction was obtained by further centrifugation of the supernatant obtained from the nuclear pellet by ultracentrufugation at 45,000 rpm for 2 h using a TLA45 rotor (Beckman).

Western blotting was performed as described (28). Following SDS-PAGE (1-15% gradient), the gels were electroblotted onto nitrocellulose membranes. The nonspecific binding sites of the membranes were blocked using 10% defatted dried milk, followed by addition of the appropriate primary antibody. The relative amount of primary antibody bound to the proteins on the nitrocellulose membrane was detected with the species-specific horseradish peroxidase-conjugated IgG. Blots were developed using the enhanced chemiluminescence detection kit (Amersham Corp.).

Wild-type McArdle RH-7777 and SLW-1 cells were grown to 85% confluency on 60-mm plates, after which the cells were cultured in serum-free DMEM for 24 h. On the 2nd day, the cells were labeled for 4 h with [³⁵S]methionine (100 µCi/ml) containing serum-free DMEM in the presence of either 1 mM oleate/BSA or BSA (0.8%) alone. After labeling, cells and media were harvested as described (28), and cells were washed once with phosphate-buffered saline and solubilized in 1 ml of TETN buffer (25 mM Tris at pH 7.5, 5 mM EDTA, 250 mM NaCl, 1% Triton X-100, and proteinase inhibitors). Proteins were immunoprecipitated using specific antibodies, as described (28).

Cells were incubated in serum-containing medium with [1-¹⁴C]acetate (5 µCi, specific activity 47 mCi/mol) for 2 h, extracted with chloroform/methanol (2:1, v/v), separated on silica gel TLC plates, and the radioactivity in phospholipids, free cholesterol, triglycerides, and cholesterol esters was quantitated (29). The cellular content of free and esterified cholesterol was determined in cells cultured to ~85% confluency by gas liquid chromatography, as described (14).

Results are given as means ± S.D. Linear regression analysis was determined by Sigma Plot computer program, and significance of correlation constants was determined by Student's t test. Values of p < 0.05 were considered to be significant. The number of data points obtained from individual single cell clones used for linear correlations differed from experiment to experiment due to loss of a particular single cell clone. All data points were used and none were selectively deleted.

RESULTS

Expression of 7-Hydroxylase in McArdle Cells

McArdle cells were transfected with a plasmid that expresses neomycin resistance and a cytomegalovirus promoter-driven expression of the coding region of rat 7-hydroxylase. Following antibiotic selection, single cell clones were picked and grown. One single cell clone of McArdle cells stably expressing 7-hydroxylase (SLW-1 cells) was used for detailed metabolic studies, whereas single cell clones were used to establish metabolic relationships (see below). Single cell clones of transfected cells expressed 7-hydroxylase mRNA and protein of the size expected from the plasmid used (Fig. 1, A and B). While wild-type McArdle rat hepatoma cells express all of the gene products required for VLDL assembly and secretion (19-21), they show no detectable expression of 7-hydroxylase mRNA or protein (Fig. 1, A and B).

Expression of 7-hydroxylase mRNA (A) and protein (B) in McArdle cells.

Cellular Content of Mature SREBP1 Varies Linearly with 7-Hydroxylase Protein

Single cell clones of McArdle cells transfected with pcDNA-7 were subjected to Western blot analysis using repetitive immunodetection with antibodies to SREBP1, 7-hydroxylase, and albumin. Since the amount of albumin in either the cell or medium was unchanged (see below), albumin served as an internal standard for recovery and blotting. The cellular content of mature SREBP1 was increased, whereas the content of the precursor form was decreased in SLW-1 cells stably expressing 7-hydroxylase (Fig. 2A). Incubating SLW-1 cells and wild-type McArdle cells with 25-hydroxycholesterol, an established inhibitor of SREBP1 proteolytic processing (17), decreased the content of mature SREBP1 by about 90%, whereas the precursor form was slightly increased (Fig. 2B). Moreover, analysis of all of the single cell clones showed that the cellular content of mature SREBP1 varied as a linear function of 7-hydroxylase protein (Fig. 2C). Additional studies show that the cellular content of SREBP2 was not significantly affected by the expression of 7-hydroxylase (data not shown).

Effect of 7-hydroxylase on the cellular content of SREBP1.

Secretion of ApoB100 Varies as a Linear Function of 7-Hydroxylase: Increased Secretion Is Caused by a Post-transcriptional Mechanism

The secretion of apoB100, apoB48, and albumin by McArdle and SLW-1 cells was compared by Western blotting of culture medium (Fig. 3A). The blots were scanned by densitometry, and the amount of apoB100 secreted by SLW-1 cells was increased by 2-fold, compared with wild-type McArdle cells (p < 0.05). There were no significant differences in the secretion of either apoB48 or albumin by the two different cell types. Since the expression of apoB mRNA was unaffected (Fig. 3B), the increased secretion of apoB100 was the result of a post-transcriptional event. Analysis of the single cell clones showed that the secretion of apoB100 varied as a linear function of 7-hydroxylase protein (Fig. 3C).

Secretion of apoB100, apoB48, and albumin in relation to 7-hydroxylase.

Expression of MTP and 7-Hydroxylase mRNA Varies as a Linear Function

The content of MTP mRNA was significantly increased in SLW-1 cells expressing 7-hydroxylase as compared with wild-type McArdle cells. Using three individual plates of cells from each group, there was a 2.2-fold increase in the content of MTP mRNA relative to -actin in SLW-1 cells compared with wild-type McArdle cells (Fig. 4A, p < 0.05). Moreover, the relative abundance of MTP mRNA varied in proportion to the relative abundance of 7-hydroxylase mRNA (Fig. 4B). These data indicate that expression of 7-hydroxylase in McArdle cells induces the expression of MTP mRNA.

Expression of MTP mRNA correlated with 7-hydroxylase mRNA.

Expression of 7-Hydroxylase in McArdle Cells Coordinately Induces the Synthesis and Secretion of All VLDL Lipids and Decreases Cellular Cholesterol Content

Compared with wild-type cells, in SLW-1 cells the synthesis of all VLDL lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids) was significantly increased as determined by the incorporation of [¹⁴C]acetate (Fig. 5A). Since the cellular content of mature SREBP is sensitive to the sterol content of cells (15-18), we determined the cellular content of free and esterified cholesterol in McArdle and SLW-1 cells (Fig. 5B). In SLW-1 cells, expressing 7-hydroxylase, the cellular content of both free and esterified cholesterol was 80 and 50% of the levels in McArdle cells (Fig. 5B). This experiment was repeated two times and similar results were obtained.

Expression of HMG-CoA Reductase and Acetyl-CoA Carboxylase Varies as a Linear Function of 7-Hydroxylase mRNA

Recent reports indicate that several genes, whose products control both sterol and fatty acid synthesis, are targets for SREBP-induced transcription (15, 24, 30-33). The expression of the mRNA's encoding HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, and acetyl-CoA carboxylase, the rate-limiting enzyme in fatty acid synthesis, was increased in SLW-1 cells compared with wild-type McArdle cells (Fig. 6A). In contrast, the relative abundance of SREBP1 mRNA was similar in both cell types. Moreover, the expression of HMG-CoA reductase (Fig. 6B) and acetyl-CoA carboxylase mRNA (Fig. 6C) varied as a linear relationship with the expression of 7-hydroxylase mRNA. These data suggest that the expression of genes responsible for the biosynthesis of cholesterol and the fatty acid components in VLDL is coordinately influenced by the expression of 7-hydroxylase in McArdle cells.

Relationship between the mRNA expression of HMG-CoA reductase, acetyl-CoA carboxylase, and SREBP1 to 7-hydroxylase mRNA.

Expression of 7-Hydroxylase in McArdle Cells Blocks Oleic Acid-stimulated ApoB Secretion

Oleic acid stimulates the synthesis of glycerolipids and increases the secretion of apoB100 when added to the culture medium of McArdle hepatoma cells (2). To examine if 7-hydroxylase-mediated induction of glycerolipid synthesis (Fig. 5) "saturates" the lipid requirement for maximal secretion of apoB100, we examined the effect of oleic acid on the cellular content and secretion of apoB100 in wild-type and SLW-1 cells (Fig. 7). In McArdle and SLW-1 cells, oleic acid increased the cellular content of apoB100 by 2.5-fold (p < 0.01) and 1.5-fold (p < 0.05), respectively (Fig. 7A). Adding oleic acid to the serum-free BSA-containing medium increased the secretion of apoB100 by McArdle cells (Fig. 7B). The oleic acid stimulation of apoB100 secretion by McArdle cells was specific since it did not significantly affect the secretion of apoB48 or total protein (data not shown). These data confirm those of others showing that in McArdle hepatoma cells, oleic acid stimulates apoB100 secretion but not apoB48 secretion (34). Moreover, although oleic acid increased the secretion of apoB100 by wild-type McArdle cells, it had no affect on the secretion of apoB100 by SLW-1 cells expressing 7-hydroxylase (Fig. 7B). Since oleic acid did significantly increase the cellular content of apoB100 in SLW-1 cells, albeit less than the increase observed in wild-type McArdle cells (Fig. 7A), the unaltered apoB100 secretion in SLW-1 cells cannot be due to impaired apoB100 synthesis. It appears that SLW-1 cells respond similarly to oleic acid as do primary cultured hepatocytes: increased content of cellular apoB but no change in secretion (35, 36).

To examine the possibility that 7-hydroxylase increases apoB100 secretion through a SREBP1-mediated process, McArdle and SLW-1 cells were cultured with and without 25-hydroxycholesterol (2 µg/ml) plus cholesterol (10 µg/ml) for 24 h. The proteolytic processing of transmembrane SREBP precursors to the mature transcriptionally active forms is inhibited by 25-hydroxycholesterol (17). Similar to its ability to decrease the cellular content of mature SREBP1 in SLW-1 cells (Fig. 2B), 25-hydroxycholesterol also decreased the secretion of apoB100 to levels that were similar to those of wild-type McArdle cells (Fig. 8). In the absence of 25-hydroxycholesterol, SLW-1 cells secreted 3.1-fold more apoB100 (p < 0.05) than McArdle cells. In the presence of 25-hydroxycholesterol, both cell types secreted similar amounts of apoB100. Although 25-hydroxycholesterol did not significantly affect the secretion of apoB100 in wild-type McArdle cells, it decreased the secretion of apoB100 in SLW-1 cells by 71% (p < 0.02). The secretion of apoB48 and albumin by both groups of cells was similar and unaffected by 25-hydroxycholesterol. This experiment was repeated twice and similar results were obtained. The combined data suggest that the augmented secretion of apoB100 displayed by McArdle cells expressing 7-hydroxylase can be reversed to levels detected in wild-type McArdle cells by 25-hydroxycholesterol. The additional finding that 25-hydroxycholesterol also restores the level of mature SREBP1 in SLW-1 cells to those of McArdle cells suggests that SREBP1 mediates the augmented secretion of apoB100 caused by expression of 7-hydroxylase.

DISCUSSION

SREBP was first identified as the sterol-responsive transcription factor that mediates the regulation of human LDL receptor gene (15). Its rat homologue ADD1 was identified as a transcription factor involved in adipocyte differentiation (37), suggesting that it may play a diverse role in regulating the expression of genes involved in the biosynthesis and metabolism of several classes of lipids. Subsequent studies show that many of the genes encoding isoprenoid/cholesterol biosynthetic enzymes are SREBP-responsive: HMG-CoA synthase (17), HMG-CoA reductase (31), isopentyfarnasyldiphosphate synthase (30), and squalene synthase (32). More recently, it has been reported that the genes for fatty acid synthase (33) and acetyl-CoA carboxylase (24) are both SREBP targets. The data derived from our studies now extend the metabolic regulatory importance of SREBP(s) to having a significant influence on controlling several steps of the VLDL assembly/secretion pathway.

Through the expression of 7-hydroxylase in McArdle hepatoma cells, we were able to obtain single cell clones having a fairly large variation in the cellular content of mature SREBP1 that varied as a linear function of 7-hydroxylase protein content (Fig. 2C). It is interesting to note that in vivo the cellular content of mature SREBP2 is increased in the livers of hamsters treated with bile acid sequestrants, which induce the expression of 7-hydroxylase (38). The mechanism through which 7-hydroxylase expression increases the cellular content of mature SREBP1 (this study) or SREBP2 in hamsters is not firmly established but may involve altering the signaling of SREBP cleavage by regulatory sterols (39).

The following data and interpretations support the conclusion that SREBP1 coordinately regulates the assembly and secretion of apoB-containing lipoproteins. Since 7-hydroxylase expression via plasmid transfection is the manipulation we employed to obtain SLW-1 cells, changes in lipoprotein assembly and secretion are the resulting consequence of this liver-specific gene (23). These consequences include linear relationships between 7-hydroxylase mRNA and the expression of mRNAs encoding the enzymes regulating fatty acid and sterol synthesis (acetyl-CoA carboxylase and HMG-CoA reductase) and MTP. As a result of the increased expression of these mRNAs and perhaps others (not studied), SLW-1 cells display increased synthesis of all lipoprotein lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids). These changes in lipid biosynthesis and MTP expression may be responsible for the increased secretion of apoB100. Moreover, as shown by the direct linear relationship between the cellular content of 7-hydroxylase and mature SREBP1 (Fig. 3C), it is likely that the changes in the expression of genes regulating sterol and fatty acid biosynthesis and the assembly of apoB-containing lipoproteins (i.e. MTP) caused by 7-hydroxylase expression are mediated through the variation of the cellular content of mature SREBP1. The additional findings that 1) expression of 7-hydroxylase (SLW-1 cells) decreased the cellular content of both free and esterified cholesterol (Fig. 5B), and 2) 25-hydroxycholesterol concomitantly decreases the cellular content of mature SREBP1 in SLW-1 cells and decreases the secretion of apoB100 to the same levels exhibited by wild-type McArdle cells provide compelling evidence that the cellular content of mature SREBP1 is responsible for these changes. Based on this rationale, we propose that SREBP1 is a common regulatory mediator responsible for coordinate regulation of the lipoprotein assembly and secretion pathway in McArdle hepatoma cells. It is important to point out that in other cell types (e.g. liver in vivo (38)), SREBP2 may mediate changes in lipoprotein assembly and secretion. Recent studies in our lab show that in a differentiated hepatoma cell line found to express liver-specific genes not expressed by McArdle cells, SREBP2 induces the expression of the MTP gene.² Thus, depending upon the cell type and physiologic conditions, SREBP1 or SREBP2 may act to mediate changes in lipoprotein assembly and secretion. This coordinate regulation provides a means for the efficient utilization of both lipids and apoB100 for transport from the liver to peripheral tissues.

It is now established that under most conditions, hepatic secretion of apoB is regulated post-transcriptionally by a process that determines the portion of de novo synthesized apoB that either enters the lipoprotein assembly/secretion pathway or is degraded in the endoplasmic reticulum (1, 2, 9). Studies using cultured hepatocytes and perfused livers of rats showed that the translocation of apoB across the endoplasmic reticulum is inefficient (40). One pool consisting of incompletely translocated apoB appeared to be diverted into the degradation pathway, whereas the pool of apoB that is completely translocated into the lumen can ultimately be secreted as a lipoprotein particle (40). Additional studies using human hepatoma HepG2 cells (41) and McArdle rat hepatoma cells (42) showed that the availability of glycerolipids (triglycerides and phospholipids) affects the efficiency of apoB translocation across the endoplasmic reticulum. It appears that when the availability of lipids is insufficient, translocation of de novo synthesized apoB is disrupted causing it to be degraded. The majority of the degradation of translocation-arrested apoB occurs co-translationally in HepG2 cells (43). While the availability of lipid is required for efficient translocation and utilization of apoB (9), the presence and activity of MTP is essential. In Chinese hamster ovary cells, which do not normally express MTP, essentially all of the apoB synthesized from a stably expressed plasmid is translocation arrested and rapidly degraded by a ALLN-inhibitable process (26, 28). In addition, blocking the functional activity of MTP also blocks the secretion of apoB by hepatoma cells, which do express MTP (44). Our results showing that the increased secretion of apoB100 correlated with MTP and mature SREBP1, but was not associated with any detectable change in apoB mRNA (Fig. 3B), suggest that a post-transcriptional mechanism is responsible. The additional finding that oleic acid increases the secretion of apoB100 by wild-type McArdle hepatoma cells, but has no effect in McArdle cells expressing 7-hydroxylase (Fig. 7), supports the proposal that SREBP1 induction of genes regulating lipogenesis saturates the requirement for lipids in the lipoprotein assembly pathway of SLW-1 cells. Our findings that in SLW-1 cells oleic acid increased the cellular content of apoB100, but not its secretion, suggest that the intracellular degradation was blocked. The inability of oleic acid to increase the secretion of apoB100 in SLW-1 cells suggests that one or more of the processes required to assemble and secrete apoB100 lipoproteins other than lipid availability is limiting. Based on these data it is tempting to speculate that in McArdle cells, when the availability of lipids is in excess of what is required for lipoprotein assembly, MTP becomes rate-limiting for apoB100 translocation and the subsequent assembly and secretion of apoB100-containing lipoproteins.

Our results may explain why oleic acid stimulation of lipogenesis is not associated with increased apoB secretion in primary rat hepatocytes (35, 36), whereas oleic acid increases the secretion of apoB by hepatoma cells (HepG2 (45-47) and McArdle (34, 48, 49)). In hepatoma cells, glycerolipid biosynthesis may be limiting for apoB secretion, whereas in primary hepatocytes other processes may be limiting.

There are other examples in which limitations in the availability of lipids in addition to triglycerides restrict the ability of the liver cell to assemble and secrete apoB-containing lipoproteins. Decreased production of cholesterol and cholesterol esters caused by drugs that competitively inhibit HMG-CoA reductase and/or ACAT also decrease the assembly and secretion of apoB-containing lipoproteins in some (50, 51) but not all (52) studies. Decreased production of phosphatidylcholine (via choline deficiency (53) and altered head-group precursors (54)) also blocks the secretion of apoB-containing lipoproteins. It seems reasonable to propose that a deficiency in the availability of one or more of the lipid components required for the intracellular processing of apoB and assembly of the lipoprotein particle can impair the overall pathway. Optimal utilization of lipids and apoB for lipoprotein assembly requires that each essential protein and lipid component be in sufficient supply relative to the others to assemble the lipoprotein particle.

Coordinate changes of the individual processes required for VLDL assembly and secretion are invoked in response to physiologic state (reviewed in Ref. 55). For example, carbohydrate "overload" leads to a coordinate increase in the synthesis of all lipoprotein lipids and an amplification of all of the processes required to increase the secretion of apoB-containing lipoproteins (10). Conversely, fasting leads to the opposite coordinated responses: decreased apoB translocation, increased apoB degradation, decreased lipid biosynthesis, and decreased lipoprotein secretion (12, 13). Fetal nutritional development also shows a coordinated change in apoB processing, lipogenesis, and lipoprotein secretion (11). These findings have led us to propose that one or more metabolic signals is responsible for the coordinate regulation of lipid biosynthesis and the intracellular processing of apoB.

Our present data show that expression of 7-hydroxylase in McArdle cells alters the cellular content of mature SREBP1 in a manner that coordinately changes the availability of lipids and the processes required to efficiently assemble apoB-containing lipoproteins. We also observed that in McArdle cells expressing 7-hydroxylase, 25-hydroxycholesterol decreases both the cellular content of mature SREBP1 and the secretion of apoB100. Our findings may explain the well-established observation that in patients treated with bile acid sequestrants, known to increase the expression of 7-hydroxylase, there is a concomitant increase in the secretion of VLDL triglycerides (56, 57). Furthermore, in some patients, there appears to be a direct correlation between the rate of bile acid synthesis, a parameter linked to 7-hydroxylase activity, and VLDL-triglyceride secretion (56, 57). The combined data suggest that changes in cholesterol metabolism may alter VLDL assembly and secretion by 1) affecting the amount of cholesterol and cholesterol esters that are available for VLDL assembly and 2) by altering the cellular content of mature SREBP. It is through the second parameter (cellular content of mature SREBP) that coordinate response of the gene products controlling the availability of glycerol lipids (i.e. triglycerides and phospholipids) and MTP is linked to the first parameter (cholesterol metabolism). Optimal production of VLDL in mammals may require coordinate induction of all of the gene products necessary for the synthesis of individual lipids and for their assembly with apoB (e.g. MTP). This may be comparable to other species (e.g. birds) in which VLDL secretion by the liver is optimized by a coordinate regulation of gene expression by estrogen (58-61). However, it is important to emphasize that mammalian VLDL production may not be optimal under all conditions. Variation in physiologic conditions may determine which of the many processes are rate-limiting for VLDL assembly and/or secretion.