Disruption of LDL receptor gene in transgenic
SREBP-1a mice unmasks hyperlipidemia
resulting from production of lipid-rich VLDLJay D. Horton,1 Hitoshi Shimano,1 Robert L. Hamilton,2 Michael S. Brown,1and Joseph L. Goldstein1
1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
2Cardiovascular Research Institute and Department of Anatomy, University of California–San Francisco, San Francisco,California 94143, USA
Address correspondence to: Joseph L. Goldstein and Michael S. Brown, Department of Molecular Genetics, University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard, Room L5-238, Dallas, Texas 75235, USA. Phone: (214) 648-2141;
Fax: (214) 648-8804; E-mail: jgolds@mednet.swmed.edu (Dr. Goldstein); mbrow1@mednet.swmed.edu (Dr. Brown).
J.D. Horton and H. Shimano contributed equally to this work.
Received for publication January 9, 1999, and accepted in revised form February 23, 1999.

Transgenic mice that overexpress the nuclear form of sterol regulatory element binding protein-1a(SREBP-1a) in liver (TgBP-1a mice) were shown previously to overproduce cholesterol and fatty acids
and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol (∼45 mg/dl) and triglycerides (∼55 mg/dl)
were not elevated, perhaps owing to degradation of lipid-enriched particles by low-density lipoprotein(LDL) receptors. To test this hypothesis, in the current studies we bred TgBP-1a mice with LDL receptor knockout mice. As reported previously, LDLR–/– mice manifested a moderate elevation in plasmacholesterol (∼215 mg/dl) and triglycerides (∼155 mg/dl). In contrast, the doubly mutant TgBP1a;LDLR–/– mice exhibited marked increases in plasma cholesterol (∼1,050 mg/dl) and triglycerides(∼900 mg/dl). These lipids were contained predominantly within large very-low-density lipoprotein(VLDL) particles that were relatively enriched in cholesterol and apolipoprotein E. Freshly isolated
hepatocytes from TgBP-1a and TgBP-1a;LDLR–/– mice overproduced cholesterol and fatty acids andsecreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP1a mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude
that the TgBP-1a mice produce large lipid-rich lipoproteins, but these particles do not accumulate inplasma because they are degraded through the action of LDL receptors.
J. Clin. Invest. 103:1067–1076 (1999).

Introduction
The synthesis of cholesterol and unsaturated fatty acids
is regulated by a family of membrane-bound transcription factors called sterol regulatory element binding proteins (SREBPs) (1). The tripartite SREBPs consist of an
NH2-terminal domain of ∼480 amino acids that is a transcription factor of the basic-helix-loop-helix-leucine zipper family, a central membrane attachment domain of∼80 amino acids containing two membrane-spanningsegments and a COOH-terminal regulatory domain of∼590 amino acids. The SREBPs are bound to membranesof the endoplasmic reticulum (ER) and outer nuclear
envelope in a hairpin fashion, with the NH2- and COOHterminal domains projecting into the cytosol (2). In
sterol-deprived cells, a two-step proteolytic process
releases the NH2-terminal domains, allowing them toenter the nucleus where they enhance transcription ofmultiple genes involved in the synthesis of cholesteroland fatty acids and in their receptor-mediated uptake
from plasma lipoproteins. When sterols build up in cells,
the proteolytic release process is inhibited, the NH2-terminal domains of the SREBPs remain membrane bound,and transcription of the target genes declines (1).
The Journal of Clinical Investigation|

The three known isoforms of SREBP are produced by
two genes (1). SREBP-1a and SREBP-1c (also known as
ADD1) (3) are encoded by a single gene by using alternate promoters that give rise to alternate first exons (4,5). The proteins differ in the length of an acidic sequenceat the extreme NH2-terminus that mediates the interaction with the coactivator CBP (6). SREBP-1a has a long
acidic sequence and is a potent activator; SREBP-1c has
a shorter acidic sequence and is only weakly active (7, 8).
The third isoform, SREBP-2, has a long acidic sequence
like that of SREBP-1a, and it too is a potent activator oftranscription (8). SREBP-1a and -2 can each activatetranscription of the gene encoding the low-density
lipoprotein (LDL) receptor as well as genes in the cholesterol and fatty acid biosynthetic pathways, but they
have different relative efficiencies. SREBP-1 isoforms
tend to favor activation of genes of fatty acid biosynthesis, whereas SREBP-2 is more potent in activating genesof cholesterol biosynthesis (7–9).
The biochemistry of the SREBPs was elucidated
through experiments in cultured cells. The relevance tohepatic physiology was established by two types of experiments: regulatory and genetic. The regulatory experiApril 1999|

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Figure 1
Amounts of various mRNAs in livers of (A) wild-type, (B) TgBP-1a, (C) LDLR–/–, and (D) TgBP-1a;LDLR–/– mice, as measured by blot hybridization.
Total RNA isolated from livers of mice described in experiment A of Table 1 was pooled, and 15-µg aliquots were subjected to electrophoresis andblot hybridization with the indicated 32P-labeled probe. The amount of radioactivity in each band was quantified as described in Methods. The probe
used for SCD was a mouse SCD-1 cDNA fragment that detects both SCD-1 and SCD-2 mRNAs (see ref. 35). The increase for each mRNA relative tothat of wild-type mice was calculated after correction for loading differences with GAPDH as described previously (12). These values are shown below
each blot. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTP, microsomal triglyceride transfer protein; SCD, stearoyl CoA desaturase.

ments demonstrated that the NH2-terminal fragment of
SREBP-2 increases in hepatic nuclei of hamsters andmice that are rendered cholesterol-deficient by treatment
with cholesterol synthesis inhibitors and bile acid–binding resins (4, 10, 11). Conversely, the amounts of nuclear
SREBP-1 and SREBP-2 are reduced in hepatic nuclei of
hamsters that are fed a high-cholesterol diet (11). Thesechanges correlate with changes in the amounts of mRNAfor the LDL receptor and enzymes of the cholesterol
biosynthetic pathway.
The genetic experiments involved the production oftransgenic mice that express truncated nuclear forms of
SREBP-1a, -1c, or -2 in the liver (7, 9, 12). The transgeneencoded SREBPs terminate after the basic-helix-loophelix-leucine zipper domain, but before the membrane
attachment domain. These proteins enter the nucleus
without proteolysis, and hence they are not susceptible
to feedback regulation. The transgenic animals expressing truncated SREBP-1a and -2 had enlarged livers,
owing to the accumulation of large amounts of triglycerides and cholesteryl esters. Despite the accumulationof fat, the rate of synthesis of cholesterol and fatty acids
remained high, owing to large increases in hepatic
mRNAs for multiple enzymes in the cholesterol andfatty acid biosynthetic pathways (9, 12). These experiments demonstrated that forced overexpression of
nuclear SREBPs can overcome the normal system of
feedback regulation and lead to massive overproductionof lipids in the liver.
The aforementioned studies with the transgenic mice
produced one puzzling result. Despite the massive accumulation of cholesteryl esters and fatty acids in the liver,the plasma levels of these lipids were not elevated; in fact,
plasma triglycerides were significantly reduced (9, 12).
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We hypothesized that the livers of these transgenic mice
were producing lipid-rich lipoproteins, but the plasma
level did not rise because of the action of hepatic LDL
receptors, whose mRNA increased approximately sixfold
under the influence of the SREBP transgenes (9, 12). The
current experiments were designed to test this hypothesis. For this purpose, we mated SREBP-1a transgenic
mice with a previously described line of mice in which
the LDL receptor gene was disrupted by gene targeting(13). The LDLR–/– mice were shown previously to havemoderate elevations in plasma LDL (total plasma cholesterol levels ∼240 mg/dl in LDLR–/– mice versus ∼110
mg/dl in wild-type littermates). Plasma cholesterol rosemarkedly only when the LDLR–/– mice were placed on a
1.25% cholesterol diet (plasma cholesterol ∼2,000 mg/dl
in LDLR–/– mice versus ∼250 mg/dl in wild-type mice on
the same high-cholesterol diet) (14). If low plasma lipids
in the SREBP-1a transgenic mice are attributable toclearance by LDL receptors, we predicted that the plasma lipid levels would rise markedly when the SREBP-1atransgenic mice were crossed with LDLR–/– mice, andindeed this is what we observed.

Methods
Animals. Transgenic mice overexpressing amino acids 1–460 ofhuman SREBP-1a under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter were described previously (12). Males hemizygous for the SREBP-1a transgene(TgBP-1a) were bred to SJL × C57BL6/J F1 females to produce
TgBP-1a and wild-type controls. TgBP-1a;LDLR–/– mice weregenerated by crossing TgBP-1a hemizygous males with LDLR–/–
females (13). The TgBP-1a;LDLR+/- mice were bred to produce
TgBP-1a;LDLR–/– offspring. The current studies were performed
with offspring from TgBP-1a;LDLR–/– males bred to LDLR–/–
females. All mice were housed in colony cages with a 14-h
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Table 1
Phenotypic comparison of wild-type, TgBP-1a, LDLR–/–, and TgBP-1a;LDLR–/– mice
Genotype of miceParameters

Wild-type

TgBP-1aLDLR–/–

TgBP-1a;LDLR–/–

6 females
20 ± 0.5
1.0 ± 0.05
2.7 ± 0.1
20 ± 4.0
58 ± 3.867 ± 7.2

6 females
23 ± 0.5
1.6 ± 0.06A
4.4 ± 0.3A
58 ± 2.2A
35 ± 3.4A
53 ± 6.7

6 females
21 ± 0.7
0.88 ± 0.03
4.4 ± 0.2A
36 ± 6.6
205 ± 25A
144 ± 15A

6 females
23 ± 0.8
1.7 ± 0.05A
12.7 ± 0.9A
63 ± 6.2A
812 ± 92A
841 ± 131A

6 males
25 ± 0.9
1.2 ± 0.08
2.4 ± 0.04
12 ± 2.3
82 ± 3.3
133 ± 11

6 males
27 ± 0.8
2.4 ± 0.2A
7.8 ± 0.9A
95 ± 11A
50 ± 3.8A
63 ± 5.1A

6 males
23 ± 0.5
1.0 ± 0.03
3.3 ± 0.3A
14 ± 2.4
233 ± 14A
173 ± 9.9

6 males
25 ± 0.8
2.1 ± 0.1A
18 ± 1.7A
74 ± 9.1A
1,308 ± 164A
949 ± 155A

Experiment A
No. and sex
Body weight (g)Liver weight (g)Liver cholesterol content (mg/g)
Liver triglyceride content (mg/g)
Total plasma cholesterol (mg/dl)
Total plasma triglycerides (mg/dl)
Experiment B
No. and sex
Body weight (g)Liver weight (g)Liver cholesterol content (mg/g)
Liver triglyceride content (mg/g)
Total plasma cholesterol (mg/dl)
Total plasma triglycerides (mg/dl)

Each value represents the mean ± SEM. Wild-type and TgBP-1a mice were littermate offspring of TgBP-1a male × (C57BL6/J × SJL) F1 female matings. LDLR–/– and TgBP1a;LDLR–/– mice were littermate offspring of TgBP-1a;LDLR–/– male × LDLR–/– female matings. Mice in experiment A were 11–14 weeks old, and mice in experiment B were 9
weeks old at the time of study. All mice were fasted 4 h and sacrificed during the early phase of the light cycle. AP < 0.01 (Student’s t test) compared with the wild-type values.

light/10-h dark cycle and were maintained on Teklad 6%(wt/wt) Mouse/Rat Diet 7002 from Harlan Teklad Premier Laboratory Diets (Madison, Wisconsin, USA).
Blot hybridization of RNA. Total RNA was prepared from the livers of mice described in experiment A of Table 1 using the
RNeasy Total RNA kit (QIAGEN Inc., Valencia, California, USA).
Equal aliquots of total RNA from mice of each genotype were

Figure 2
FPLC profiles of plasma lipoproteins from wild-type (open circles), TgBP1a (filled circles), LDLR–/– (open triangles), and TgBP-1a;LDLR–/– (filled triangles) mice. Plasma from mice described in experiment B of Table 1 waspooled (2.5 ml for each genotype) and subjected to ultracentrifugation
at d = 1.215 g/ml. The lipoprotein fractions (d < 1.215 g/ml) were subjected to gel filtration by FPLC, and the cholesterol content of each fraction was measured as described in Methods. FPLC, fast performance liquid chromatography.

The Journal of Clinical Investigation|

pooled (total of 15 µg) and subjected to Northern blot analysis
with the indicated cDNA probes as described previously (12).
cDNA probes for mouse apo AII, apo CI, apo CII, and apo CIII
were prepared with reverse transcriptase–PCR using mouse liverpoly(A)+ RNA as a template (12). The PCR primers usedwere as follows: apo AII: 5′ primer, 5′-GAATTCCATAGAATCGCAGCACTGTT-3′, and 3′ primer, 5′-GAATTCAACTCCTTCCGCATTTATT-3′ (15); apo CI: 5′ primer,
5′-ATGGGCCTCCTGAGAGATCCTTAGATCCAG-3′,
and3′ primer, 5′-TCTGTGATGAAGAGGGACCTGGCACATTAC-3′(16); apo CII: 5′ primer, 5′-TGGAGTGAGCCAGGATAGTCCTTCCTGCCA-3′, and 3′ primer, 5′-GAACCTGGACTCATTTATTCTTTCACTGAA-3′ (17); apo CIII: 5′ primer, 5′-CCTGCTCAGTTTTATCCCTAGAAGCAGCTA-3′, and 3′ primer,
5′-CATACTTTCCCCTTAAAGCAACCTTCAGG-3′ (18). Northern blot filters were exposed to a Fuji PhosphorImager and quantified using a Bio-Imaging Analyzer with BAS1000 MacBAS software (Fuji Medical Systems, Stamford, Conneticut, USA).
Fast performance liquid chromatography analysis. Pooled plasma (3ml) from six male mice of each genotype (experiment B in Table1) was subjected to ultracentrifugation at a density (d) of 1.215
g/ml. The resulting lipoprotein fractions (d < 1.215) were adjusted to a final volume of 3 ml with a buffer containing 0.15 M
sodium chloride, 0.01% (wt/vol) sodium EDTA, and 0.02%(wt/vol) sodium azide at pH 7.2, after which 2 ml was subjected
to gel filtration with fast performance liquid chromatography(FPLC) (19). Fractions (2 ml) were collected, and 0.1 ml fromeach fraction was used to determine the content of total cholesterol and triglycerides (12, 19).
For apolipoprotein analysis, equal aliquots (0.75 ml) from eachof three consecutive FPLC fractions were pooled (total of 1.5 ml)
and delipidated with 15 ml of ethanol/ether (1:1), and the precipitates were collected by centrifugation at 1,000 g for 15 min.
The precipitated apolipoproteins were dissolved in SDS loading
buffer containing 50 mM Tris-chloride (pH 6.8), 5.5% (vol/vol) βmercaptoethanol, 7% (wt/vol) SDS, 2.5% (wt/vol) sodium myristyl
sulfate, 0.5% (wt/vol) sodium cetyl sulfate, 0.1% (vol/vol) bromophenol blue, and 10% (vol/vol) glycerol (20). The samples wereboiled for 10 min and subjected to electrophoresis on 3–15% SDS

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Figure 3
SDS-PAGE of plasma apolipoproteins from (a)wild-type, (b) TgBP-1a, (c) LDLR–/–, and (d)TgBP-1a;LDLR–/– mice. Equal aliquots (0.75 ml)from three consecutive FPLC fractions shown in
Figure 2 were pooled (total of 1.5 ml) and delipidated, and the apoproteins were precipitatedas described in Methods. The apoproteins were
subjected to 3–15% gradient SDS-PAGE andwere stained with Coomassie blue. The positions of migration of apo B-100, apo B-48, apo
E, and apo AI are indicated.

gradient gels. The gels were stained with Coomassie blue and calibrated with Rainbow high-molecular-weight markers (Amersham Corp., Burlington, Massachusetts, USA).
Plasma lipoprotein isolation. For the experiments in Table 2 andFigure 4, mice 10–14 weeks old were fasted 4 h, and plasma wascollected in sodium EDTA (1 mg/ml) at pH 8 and aprotinin (2mg/ml). Equal aliquots of plasma (0.4 ml) from 10 mice of eachgenotype (five males and five females) were pooled (total of 4ml), and lipoproteins were separated by sequential ultracentrifugation at densities of 1.006, 1.019, 1.063, and 1.215 toobtain very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL, and high-density lipoprotein(HDL), respectively (21). Each lipoprotein fraction was dialyzed
against a buffer containing 150 mM NaCl, 10 mM sodium
phosphate (pH 7.4), and 0.01% sodium EDTA. The triglyceride
content in each lipoprotein fraction was determined as
described above for plasma samples. Phosphatidylcholine wasmeasured with the enzyme phospholipase D according to the
manufacturer’s instructions (Phospholipids B; Wako Chemicals
USA Inc., Richmond, Virginia, USA). Total and unesterified cholesterol were measured fluorometrically as described (22), andesterified cholesterol was calculated by subtraction.
Electron microscopy of plasma VLDL. Sixteen-week-old malemice were fasted for 4 h, and plasma was collected in sodium
EDTA (1 mg/ml) at pH 8 and aprotinin (2 mg/ml) from theinferior vena cava of anesthetized mice. The plasma from four
mice of each genotype was pooled, and VLDL (d < 1.006) was
isolated by ultracentrifugation (21). Negatively stained preparations of VLDL particles were prepared as described (23). The
VLDL particles were photographed at 60,000 diameters and 80
kV in a Seimens 101 electron microscope (Seimens Corp., Iseline, New Jersey, USA).
Clearance of 125I-labeled VLDL from plasma. 125I-labeled VLDL from
TgBP-1a;LDLR–/– mice was prepared as follows. Plasma was collected from five male TgBP-1a;LDLR–/– mice after a 4-h fast,pooled, and ultracentrifuged at d = 1.006. The VLDL fraction (1.3mg protein) was radiolabeled with sodium 125I by the iodine
monochloride method as described previously (21). The specificactivity of the 125I-labeled VLDL was 860 cpm/ng protein. Of the1070

The Journal of Clinical Investigation|

total 125I-radioactivity in VLDL, ∼30% was contained in apo B, as
estimated by isopropanol precipitation (24).
All recipient mice were fasted 1 h and anesthetized withsodium pentobarbital (60 mg/kg). Each mouse received an
intravenous bolus of 0.1 ml of 0.15 M NaCl containing 15 µgof 125I-labeled VLDL (860 cpm/ng apo B protein) via the rightexternal jugular vein. Blood was collected from the left jugular vein 1 min after the 125I-labeled VLDL injection and at theindicated times thereafter. Two methods were used to measure
the content of 125I-labeled apo B in plasma. Method 1 measured total 125I-labeled apo B (apo B-48 plus apo B-100) by isopropanol precipitation (24). An aliquot (10 µl) of each plasma
sample was adjusted to 250 µl with 0.15 M NaCl containing
human LDL (100 µg) as a carrier. An equal volume of isopropanol (250 µl) was added, and the samples were placed on
ice for 30 min. The samples were centrifuged at 15,000 g for 10
min to precipitate apo B. The content of 125I was determinedby γ-scintillation spectrometry of samples from the originalplasma and from the supernatant portion of the isopropanoltreated plasma. The amount of radiolabeled apo B was determined by subtracting the radioactivity in the supernatantfrom that in the original plasma. Method 2 measured 125Ilabeled apo B-48 directly by subjecting 2 µl of each plasma
sample to 3–15% gradient SDS-PAGE, after which the gel wasdried, and the signals generated by apo B-48 were quantified
by a Fuji PhosphorImager as described above.
Lipid synthesis and secretion in mouse primary hepatocytes. Mice
(14–16 weeks old) of the indicated genotype were anesthetized,
and their livers were perfused via the portal vein with 30 ml ofHanks’ buffered saline, without calcium or magnesium, supplemented with 10 mM HEPES (pH 7.4), 0.5 mM sodium
EGTA, and gentamycin (0.1 mg/ml). The buffer was prewarmed
to 37°C and infused at a rate of ∼3 ml/min. The livers were thenperfused with Liver Digest Medium containing collagenase (catalogue no. 17703-034; GIBCO-BRL, Grand Island, New York,
USA) for ∼10 min at a flow rate of ∼3 ml/min. The livers were
removed from the animals, the hepatic capsule was stripped,
and the dissociated cells were dispersed by shaking, followed byfiltration at 4°C through gauze into an equal volume of ice-coldApril 1999|

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Table 2
Lipid comparison of plasma lipoprotein fractions from wild-type,
TgBP-1a, LDLR–/–, and TgBP-1a;LDLR–/– mice
Lipoprotein
fraction

Composition of lipoprotein fraction
Wild-type

TgBP-1aLDLR–/–
mg/dl

TgBP-1a;LDLR–/–

Ultrastructure. The livers of one male and one female 16-weekold TgBP-1a mice, fed ad libitum, were perfused and fixed underpentobarbital anesthesia and prepared for electron microscopy
as described (26). Liver lipids were stained by a modification of
the Angermüller and Fahimi (27) imidazole-buffered osmium
tetroxide technique as described (28). Two blocks of liver fromeach mouse were thin-sectioned, and ∼100 electron photomicrographs were examined in this study.

VLDL
Total cholesterol
Free cholesterol
Cholesteryl ester
Triglycerides
Phospholipids
Protein

2.3
1.3
0.9
30
5.9
11

8.3
2.4
5.9
27
6.4
7.3

22
11
10
58
18
32

440
110
330
550
220
160

0.15
0.06
0.09
–
–
2.5

0.26
0.10
0.16
–
–
6.8

8.4
3.0
5.4
4.1
3.7
14

59
9.2
50
8.0
27
32

4.7
2.1
2.6
0.93
4.4
14

3.8
1.0
2.8
–
1.5
9.3

67
22
45
13
41
80

81
17
64
5.6
60
81

30
9.8
20
–
49
93

12
2.2
9.5
1.5
15
48

58
17
41
1.0
80
150

22
5.4
17
1.5
25
54

Results

IDL
Total cholesterol
Free cholesterol
Cholesteryl ester
Triglycerides
Phospholipids
Protein
LDL
Total cholesterol
Free cholesterol
Cholesteryl ester
Triglycerides
Phospholipids
Protein
HDL
Total cholesterol
Free cholesterol
Cholesteryl ester
Triglycerides
Phospholipids
ProteinFasting plasma samples were collected from 10 (five male and five female) mice ofeach genotype, pooled, and subjected to sequential ultracentrifugation for measurement of lipid and protein concentrations as described in Methods. (–) denotes
values that were too low for accurate measurement.

DMEM (catalog no. 11885-084; GIBCO BRL) containing 5%(vol/vol) FCS, 10 mM HEPES (pH 7.4), and gentamycin (0.1
mg/ml). The cells were pelleted and washed twice at 4°C with thesame medium. The cells were counted with an hemacytometer,and 1.5 × 106 cells were plated onto 60-mm mouse collagen
IV–coated dishes (Fisher Scientific Co., Pittsburgh, Pennsylvania, USA) in the above medium and incubated at 37°C in 81%
air/9% CO2 for 3 h. The cells were then washed with DMEM containing 5% human lipoprotein–deficient serum (21). They werethen incubated with DMEM containing 5% human lipoprotein–deficient serum, gentamycin (0.1 mg/ml), and 0.5 mM
sodium [14C]acetate (18 dpm/pmol). After incubation for thetimes indicated, the medium was removed and centrifuged as
described above for preparation of mouse plasma VLDL. The
cells were harvested by scraping into 2 ml of 0.1 N sodiumhydroxide, followed by 2 ml water (25). The content of 14Clabeled cholesterol and 14C-labeled fatty acids in the cells and in
the VLDL fraction of medium was quantified by thin-layer chromatography and scintillation counting as described previously(8). The cellular protein content was measured by the BCA Protein Assay Kit (Pierce Chemical Co., Rockford, Illinois, USA).
The data are expressed as nanomoles of [14C]acetate incorporated into cholesterol or fatty acids per milligram of cell protein.

The Journal of Clinical Investigation|

Figure 1 compares the levels of various mRNAs, as determined by Northern blotting, in pooled samples from livers of wild-type mice, transgenic SREBP-1a mice (TgBP1a), LDLR–/– mice, and TgBP-1a;LDLR–/– mice, all studied
in the same experiment. As expected, the TgBP-1a mice
showed an elevation in the mRNA for the LDL receptor(3.5-fold). This mRNA was missing in the LDLR–/– and
TgBP-1a;LDLR–/– mice. The level of mRNA expression for
the SREBP-1a transgene in the TgBP-1a;LDLR–/– mice was∼80 percent of that in the TgBP-1a mice. The elevations
in mRNAs for the cholesterologenic enzymes (HMG CoA
synthase, HMG CoA reductase, and squalene synthase)were nearly identical in the TgBP-1a and TgBP-1a;LDLR–/–
mice. Similar findings pertained to the enzymes of fatty
acid synthesis and esterification (acetyl CoA carboxylase,fatty acid synthase, stearoyl CoA desaturase, and glycerol3-phosphate acyltransferase). The mRNAs for apo AI, AII,
B, CI, CII, CIII, and E were not significantly or consistently altered in the gene-manipulated mice.
Consistent with the elevations in mRNAs for lipid-synthesizing enzymes, the livers of the TgBP-1a and TgBP1a;LDLR–/– mice were enlarged, pale in color, and enrichedwith lipid. Table 1 summarizes two experiments in whichquantitative values were obtained on livers and plasma ofmatched female and male mice of the four genotypes. The
liver weights of the TgBP-1a and TgBP-1a;LDLR–/– mice
were elevated equally when compared with the nontransgenic mice. The degree of increase was not as profound as
in our previous study of TgBP-1a mice because the current
animals were not placed on a low-carbohydrate/high-protein diet to induce high-level expression of the transgene,which was driven by the promoter of the gluconeogenic
enzyme PEPCK (12). To avoid the possible influence ofsuch a diet on the measured metabolic parameters, wedeliberately maintained the animals on a normal chow
diet, which allows a moderate, but not superabundant,
expression of the transgene. The hepatic content oftriglyceride was elevated equally in the TgBP-1a and TgBP1a;LDLR–/– mice. The hepatic cholesterol content was elevated significantly in the LDLR–/– mice when comparedwith wild-type mice. The LDL receptor deficiency and the
SREBP-1a overexpression combined to produce a higher
hepatic cholesterol content in the TgBP-1a;LDLR–/– micethan in the TgBP-1a animals (Table 1).
As reported previously, total plasma cholesterol andtriglyceride levels were either normal or slightly reducedin TgBP-1a mice (12). Plasma cholesterol was elevated to
the range of 205–233 mg/dl in the LDLR–/– mice (Table 1).
When the two genetic alterations were combined, the plasma levels of cholesterol and triglycerides rose markedly(total plasma cholesterol of 812–1308 mg/dl and triglycerides of 841–949 mg/dl in the TgBP-1a;LDLR–/– mice).
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these apolipoproteins were shifted toward larger particles. The amount of apo AI was significantly reduced.
To study the composition of the plasma lipoproteins
more quantitatively, we pooled plasma from 10 mice ofeach genotype and separated the lipoproteins by sequential ultracentrifugation at increasing densities as describedin Methods (Table 2). The VLDL fractions from the TgBP1a;LDLR–/– mice contained a major increase in the number of particles, as revealed by an increased content of proteins and lipids. These particles were relatively enriched incholesterol, as determined by the marked increase in thecholesterol/protein ratio when compared with VLDL
from wild-type mice (Figure 4a and Table 2). The total
triglyceride content of the VLDL particles was also
increased, but the triglyceride/protein ratio remained relatively normal (Figure 4b). As a result of these combinedchanges, the cholesterol/triglyceride ratio was elevatedeightfold in the VLDL of TgBP-1a;LDLR–/– mice comparedwith wild-type mice (Figure 4c). A similar enrichment incholesterol was found in the IDL fraction of TgBP1a;LDLR–/– mice (Table 2). The content of cholesterol in
the LDL fraction of the TgBP-1a;LDLR–/– mice was elevated to the same degree as in the LDLR–/– mice (Table 2). The
content of both cholesterol and protein in the HDL fraction was reduced in the TgBP-1a;LDLR–/– mice (Table 2).
Figure 5 shows electron micrographs of negatively
stained VLDL fractions from fasted animals of the four
genotypes. These images confirm that the VLDL from
TgBP-1a mice and TgBP-1a;LDLR–/– mice contain an
Figure 4
Composition of plasma VLDL (d < 1.006 g/ml) from wild-type (WT), TgBP1a, LDLR–/–, and TgBP-1a;LDLR–/– mice. The ratios of cholesterol/protein,
triglycerides/protein, and cholesterol/triglycerides were calculated from thevalues shown in Table 2. VLDL, very-low-density lipoprotein.

To determine the lipoprotein distribution of the excess
plasma cholesterol, we fractionated the plasma lipoproteins by FPLC, which separates particles on the basis ofsize (Figure 2). These data repeat the earlier observationthat the elevated cholesterol in LDLR–/– mice is contained
within particles of the LDL size range (13). In contrast,
the TgBP-1a;LDLR–/– animals exhibited a marked increasein large lipoproteins that extended from super-large
VLDL to IDL and LDL.
Figure 3 shows the protein composition of various
lipoproteins isolated from the FPLC column. The
apolipoproteins were stained with Coomassie blue andidentified on the basis of size. In the plasma of wildtype mice, the major apolipoprotein seen in the
VLDL/IDL/LDL fractions was apo E (Figure 3a). Apo
AI was abundant in the HDL fraction. In the TgBP-1a
plasma, apo E was no longer visible (Figure 3b). Apo AI
was present but reduced. The LDLR–/– mice showed
obvious elevations in apo B-48 and apo B-100, whichwere enriched in the LDL fraction (Figure 3c). The
amounts of apo E and apo AI remained relatively normal. The TgBP-1a;LDLR–/– mice exhibited a major
increase in apo E, most of which was found in the
VLDL fraction (Figure 3d). The total amounts of apo B48 and apo B-100 were also increased, even when compared with the elevated levels in LDLR–/– mice, and1072

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Figure 5
Electron microscopy of negatively stained plasma VLDL (d < 1.006 g/ml)
from (a) wild-type, (b) TgBP-1a, (c) LDLR–/–, and (d) TgBP-1a;LDLR–/–mice.
After a 4-h fast, plasma samples from four male mice of each genotype werepooled, and VLDL was isolated for viewing by electron microscopy as
described in Methods. ×108,000

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Figure 6
Plasma clearance of 125I-labeled VLDL in wild-type (WT), TgBP-1a,
LDLR–/–, and TgBP- 1a;LDLR–/– mice. VLDL was isolated from four fasted male TgBP-1a;LDLR–/– mice and labeled with 125I as described in Methods. 125I-labeled VLDL (15 µg protein, 860 cpm/ng apo B protein) was
injected intravenously into each of five 12-week-old male mice with theindicated genotype. Blood was drawn at the indicated times to quantify
the content of 125I-labeled apo B in plasma. (a and b) Plasma content of
125I-labeled total apo B was measured by isopropanol precipitation followed by scintillation counting (as described in Methods). (c and d) Plasma samples were subjected to SDS-PAGE, and the signal generated by
apo B-48 was quantified using a Fuji PhosphorImager as described in
Methods. The data are plotted as the percent of the zero time value. *P< 0.05 (Student’s t test) compared with wild-type values. Each value represents the mean ± SEM of data from five mice.

increased number of extremely large particles (>80 nm indiameter) that are in the size range normally associatedwith chylomicrons (28). These very large particles arelikely to account for the cholesterol-rich nature of the
VLDL fraction in the transgenic mice, as indicated by thecompositional data in Table 2.
The data presented so far are consistent with the
hypothesis that the production of LDL receptors in the
TgBP-1a mice compensates for the oversynthesis of lipidrich lipoproteins and that this compensation is prevented in the TgBP-1a;LDLR–/– mice, thereby unmasking a
massive elevation in plasma VLDL. To test this hypothesis more directly, we measured the ability of mice fromboth genotypes to clear 125I-labeled VLDL from plasma.
For this purpose, we isolated VLDL from fasting TgBP1a;LDLR–/– mice and labeled the protein component with
125I. As shown in Figure 6a, the total apo B componentof this 125I-labeled VLDL was cleared more rapidly fromthe circulation in the TgBP-1a mice compared with wildtype mice. In contrast, the LDLR–/– mice exhibited amarked delay in this clearance (Figure 6b). Similar results
were obtained when we specifically followed the 125Ilabeled apo B-48 component, as determined by SDSPAGE followed by autoradiography (Figure 6, c and d). In
experiments not shown, we found that the clearance of
the apo B-100 and apo B-48 components of 125I-labeled
The Journal of Clinical Investigation|

VLDL were just as defective in the TgBP-1a;LDLR–/– miceas in the LDLR–/– littermates, indicating that the delayed
clearance in the doubly mutant mice was due toimpaired LDL receptor function.
To compare the rates of lipid synthesis and secretionin the mice of the four genotypes, we turned to the useof freshly isolated hepatocytes (Figure 7). To mimic invivo conditions as closely as possible, these cells werestudied three hours after plating. The time required toprepare the hepatocytes before plating was ∼40 minutes. The cells were incubated for various intervals with
[14C]acetate, after which the cells and medium were
harvested separately and the content of total 14Clabeled cholesterol and 14C-labeled fatty acids (unesterified plus esterified) was measured. As expected,
hepatocytes from the TgBP-1a and TgBP-1a;LDLR–/–mice synthesized much more 14C-labeled cholesterol
and 14C-labeled fatty acids than did the wild-type and
LDLR–/– mice (filled symbols in Figure 7, a and d). They
also secreted more of these lipids into the culture medium (filled symbols in Figure 7, b and e). An interesting
phenomenon emerged when we calculated the efficiency of secretion, i.e., the ratio between the amount ofradioactive lipids in the medium compared with thecells. Relative hypersecretion was seen in those animals
lacking LDL receptors, i.e., the LDLR–/– and TgBP1a;LDLR–/– mice (triangles in Figure 7, c and f). Similar
results were obtained when this experiment was repeated on three other occasions.
To further test the hypothesis that the livers of TgBP-1a
mice are producing increased amounts of lipid-enriched
lipoproteins, we studied the liver by electron microscopy.
At low magnification (approximately ×12,000), the excess
nonpolar lipids in the liver were apparently partitioned
largely within two distinct compartments of the hepatocytes: large cytosolic lipid droplets (∼1–3 µm in diameter)and smaller lipid-staining particles (∼100–250 nm) within the membrane-bound compartments of the VLDL
secretory pathway. Remarkably, there were very few lipidstaining droplets between these two size ranges (images
not shown). The hepatocyte VLDL secretory compartments were better resolved at higher magnification(×27,000), as shown in Figure 8, a–d.
The specialized compartment of ER that has beenstrongly implicated in nascent VLDL assembly — namely, the ribosome-free regions of the terminal ends of therough ER (RER) (Figure 8a) – was far more evident in thehepatocytes of the TgBP-1a mice than in those of mice orof other species studied previously (29). In many hepatocytes of the TgBP-1a mice, virtually all of these putative
VLDL assembly compartments appeared to contain achylomicron-sized nascent VLDL precursor particle. Thismorphologic observation alone strongly suggests thatVLDL core-lipid assembly in the fatty livers of TgBP-1a
mice may be greatly stimulated, even though the level of
apo B mRNA remains unchanged. Similarly sized lipidstaining particles were commonly found associated withthe smooth ER (SER), mostly around the periphery of thelarge patches of tightly packed membranes of SER proper (Figure 8b). These putative nascent VLDL particles ofchylomicron diameter were often observed singly within
forming secretory vesicles of the Golgi apparatus (Figure
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Figure 7
Rates of lipid synthesis and secretion by primary hepatocytes from wildtype (open circles), TgBP-1a (filled circles), LDLR–/– (open triangles), and TgBP1a;LDLR–/– (filled triangles) mice. Primary hepatocytes were prepared fromone mouse of each genotype as described in Methods. The hepatocytes
were incubated with DMEM containing 5% FCS for 3 h at 37°C, after which
the cells were incubated with 0.5 mM sodium [14C]acetate (18 dpm/pmol)in DMEM supplemented with 5% human lipoprotein–deficient serum. At
the indicated time, the medium was removed for centrifugation at d < 1.006
g/ml, the monolayers were washed, and the cells were harvested. The content of 14C-labeled cholesterol and fatty acids in cells and medium wasquantified as described in Methods. Each value is the average of duplicate
incubations. Similar results were obtained in three other experiments inwhich different groups of mice were studied at a single time point.

8c) or as clusters of a few particles (Figure 8d). This finding differs from hepatocytes of many species, including
those of wild-type mice, in which nascent VLDL arealmost always clustered in large numbers within forming
Golgi secretory vesicles (29).
Interestingly, late hepatocytic endosomal compartments, which have been shown in rat livers to be prelysosomal multivesicular bodies (MVB) filled with remnantsof apo B–containing VLDL and chylomicrons (30), contained lipid-staining particles that were not distinguishable from those within the secretory organelles (Figure8c). In some cases, the large cytosolic lipid droplets (LD)appeared to be closely associated with the surface oftightly packed SER membranes (Figure 8b).

Discussion
The current data provide an explanation for the previous
paradoxical observation that plasma levels of cholesterol
and triglycerides are normal or slightly reduced in SREBP1074

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1a transgenic mice despite massive overproduction andaccumulation of these lipids in the liver (9, 12). The datasuggest that the livers of these transgenic mice produce
extremely enlarged, cholesterol-enriched VLDL but thatthese particles do not accumulate in plasma because they
are degraded through the action of LDL receptors. WhenLDL receptors were eliminated by producing TgBP1a;LDLR–/– doubly mutant mice, this degradation was prevented and massive hyperlipidemia was unmasked.
In the SREBP-1a transgenic mice, the clearance of 125Ilabeled VLDL was faster than normal despite massive
hepatic cholesterol accumulation, which ordinarily
would have downregulated LDL receptors. Thisenhanced clearance was mediated by LDL receptors
because it was abolished in the TgBP-1a;LDLR–/– mice.
The notion that the TgBP-1a animals overproduce largecholesterol-rich VLDL particles is supported by two
observations. First, when freshly isolated hepatocytes
from these animals were incubated with [14C]acetate, they
secreted increased amounts of 14C-labeled cholesterol and
14C-labeled fatty acids. Second, electron micrographs oflivers from TgBP-1a mice showed large lipoproteins thatwere formed in the ER and carried to the Golgi where
they were observed within budding secretory vesicles.
Receptor-mediated degradation of the cholesterolenriched VLDL particles could have occurred at one or
all of three levels: (a) before secretion from the hepatocytes; (b) after secretion but before escape from the
Dissé’s space into the hepatic sinusoids; and (c) after theparticles had circulated in plasma. We believe it likelythat some, but not all, of the nascent VLDL wasdestroyed before secretion from the cell. This is based on
findings from the studies in which hepatocytes wereincubated with [14C]acetate. These studies showed thatthe relative efficiency of secretion, as determined by theratio between secreted lipid compared with the amountretained within the cell, was higher in LDL receptor–deficient mice. This relative hypersecretion was observed in
the LDLR–/– mice as well as in the TgBP-1a;LDLR–/– mice,even though the absolute rates of lipid synthesis andsecretion differed markedly between these two genotypes(Figure 7, c and f). This finding would be explained if
LDL receptors, functioning within hepatocytes, mediate
the degradation of some VLDL particles before theirsecretion. These findings would also be consistent withnumerous studies of lipoprotein turnover in humans
that show an apparent oversecretion of VLDL (and perhaps LDL) in homozygotes with familial hypercholesterolemia who lack LDL receptors (31). This finding is
also consistent with recent data from Twisk and coworkers (32), who showed a decrease in intrahepatic degradation of apo B–containing lipoproteins in LDLR–/– mice,
which also lack LDL receptors .
The electron microscopic images in Figure 8c suggestthat some of the large lipoprotein particles are secreted andthen taken back into the cells. This conclusion is based on
the finding of large lipoprotein-like particles within multivesicular bodies that ordinarily contain lipoproteinstaken up from the surface by receptor-mediated endocytosis (30). The chylomicron-sized VLDL particles are solarge (many >150 nm) that they might be hindered in penetrating through the sinusoidal endothelial pores, whose
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Figure 8
Electron micrographs of thin sections depicting lipoprotein assembly and degradation in fatty hepatocytes of TgBP-1a mouse liver. These hepatocytes
contained numerous very large (~100–250 nm) nascent VLDL particles (open arrows) in the following areas: (a) within the lumen of the unique smoothsurfaced (ribosome-free) terminal ends of rough ER (RER); (b) within the smooth ER (SER) proper; (c and d) within forming secretory vesicles of the Golgi
apparatus (G), singly (c) or clustered (d). Multivesicular bodies (MVB) that contained partially degraded lipoproteins the same size as nascent VLDL were
often seen (c). Larger than normal patches of tightly packed membranes of SER were common in these fatty hepatocytes and sometimes appeared to be
associated with cytosolic lipid droplets (LD) (b). Differences in the intensity of lipid staining between nascent VLDL particles of different diameters may
reflect differential absorbency of the imidazole mordant and/or differences in unsaturated fatty acid content of the core lipids (27). ×27,000.

diameters are limited to about 120 nm (33). They would
then remain retarded in the Dissé’s space, from which theywould be cleared by cell-surface hepatocyte LDL receptors
and delivered to multivesicular bodies. In the absence of
LDL receptors, some of these particles may more readily
pass into the sinusoids from the Dissé’s space.
The large size of the secreted VLDL particles is likely
attributable to an imbalance between the large amountof triglyceride and cholesteryl esters and the limiting
amount of apo B, whose mRNA levels do not increase in
the TgBP-1a mice. A similar phenomenon has been
observed in enterocytes of apo B–deficient mice (28).
The observation of marked hyperlipidemia in the TgBP1a;LDLR–/– mice, but not in TgBP-1a mice, provides further support for the emerging concept that LDL receptors
are able to compensate for large increases in cholesterol
input from either dietary sources or endogenous synthesis and that significant hypercholesterolemia occurs only
when the function of the LDL receptor is impaired
through one of several mechanisms — namely, feedback
suppression of the SREBP pathway, mutation in the LDL
receptor gene, or mutation in the genes encoding either of
its ligands, apo B or apo E (14, 31, 34).

Acknowledgments
We thank Scott Clark, Robin Craddock, and Debra Morgan for
excellent technical assistance; Richard Gibson for invaluable help
The Journal of Clinical Investigation|

with animal care; Jinny Wong for help with outstanding electron
microscopy; and our colleague Shinji Ikemoto for help with primary hepatocytes. This study was supported by grants from the
National Institutes of Health (HL-20948 and HL-60844), theMoss Heart Foundation, and the Perot Family Foundation. J.D.
Horton was the recipient of a Postdoctoral Fellowship for Physicians from the Howard Hughes Medical Institute.
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