© 1995 American Heart Association, Inc.
Articles |
Overexpression of Human Apolipoprotein B-100 in Transgenic Rabbits Results in Increased Levels of LDL and Decreased Levels of HDL
From the Gladstone Institute of Cardiovascular Disease (J.F., S.P.A.M., S.T., R.Q., J.M.T., S.G.Y.), San Francisco; the Cardiovascular Research Institute (J.F., S.P.A.M., J.M.T., S.G.Y.), University of California, San Francisco; the Lawrence Berkeley Laboratory (R.M.K.), University of California, Berkeley; and the Departments of Physiology (J.M.T.) and Medicine (S.G.Y.), University of California, San Francisco.
Correspondence to Stephen G. Young, MD, and John M. Taylor, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100.
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Abstract |
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Abstract In this study, an 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.
Key Words: cholesterol • apolipoprotein B • transgenic rabbits
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Introduction |
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Rabbits have been widely used for investigations of atherosclerosis and lipoprotein metabolism. On a normal rabbit-chow diet, rabbits have low total plasma cholesterol levels, very low levels of apoB and LDL, and do not develop atherosclerotic lesions. However, when the diet is supplemented with as little as 0.1% to 0.5% cholesterol and 3% fat, rabbits develop severe hypercholesterolemia, resulting in the rapid development of extensive atherosclerosis in the aorta (For review, see Reference 11 ). Because aortic atherosclerosis in rabbits can be easily induced by a simple dietary modification, cholesterol-fed rabbits have been extensively studied in atherosclerosis research.2 3 4 5
New Zealand White rabbits that are deficient in LDL receptors (Watanabe heritable hyperlipidemic rabbits) have also been studied extensively to understand the role of the LDL receptor in lipoprotein metabolism.6 7 8 9 10 11 Because of the markedly retarded clearance of the apoB-containing lipoproteins in Watanabe heritable hyperlipidemic rabbits, these animals are hypercholesterolemic on a regular chow diet (total cholesterol levels, 500 to 700 mg/dL) and develop severe atherosclerotic lesions. In these animals, most of the plasma cholesterol is found in the IDL and LDL fractions.7
In 1987, the laboratory of Barry Lewis at St. Thomas' Hospital in London characterized a genetic hyperlipidemia in chow-fed New Zealand White rabbits that was distinct from that of Watanabe heritable hyperlipidemic rabbits.12 13 These had hypercholesterolemia (total cholesterol levels ~400 mg/dL) and moderate elevations in TGs, and they developed atherosclerotic lesions. Lipoprotein turnover studies revealed that these animals had markedly increased VLDL- and LDL-apoB production rates,12 leading Lewis' group to suggest that these animals may constitute a model for human FCH, which is also characterized by the overproduction of apoB-containing lipoproteins.14 15 16 17 18 At the present time, the molecular defect in the St. Thomas' Hospital hyperlipidemic rabbits has not yet been defined. It is unclear whether the increased production of the apoB-containing lipoproteins simply results from increased synthesis of the apoB-100 protein in the liver, or is secondary to other, more complex metabolic derangements.
Over the past 3 years, we19 20 21 and Callow et al22 have developed and characterized transgenic mice that express large amounts of human apoB. These mice have elevated levels of cholesterol and TGs and represent a genetic model for increased production of apoB. However, these animals have a major drawback for examining LDL metabolism: in transgenic mice, the hepatic apoB mRNA is edited extensively, resulting in high levels of hepatic apoB-48 production.19 20 23 Because apoB-48 and apoB-100 are known to have different metabolic properties,23 24 25 it is quite conceivable that the phenotype of apoB overproduction in the human apoB transgenic mice could be different from the phenotype of apoB overproduction in animals that lack hepatic apoB mRNA editing. In addition, interpretation of the phenotype resulting from apoB overproduction in transgenic mice is complicated by the fact that human apoB-100 binds with very low affinity to the mouse LDL receptor,26 and is therefore cleared slowly from the plasma (Helen H. Hobbs, written personal communication, 1994). Furthermore, mice lack cholesteryl ester transfer protein27 28 and have hepatic lipase that circulates in the plasma.29 Therefore, the metabolism of apoB-containing lipoproteins in transgenic mice is likely to differ substantially from that in many larger mammals. In contrast to the mouse, rabbits lack apoB mRNA editing in the liver,30 31 have LDL receptors that bind human LDL with high affinity,31 have high plasma levels of cholesteryl ester transfer protein,27 and have hepatic lipase that does not circulate but is bound primarily to liver cell surfaces.32
In this study, we developed a transgenic rabbit model in which human apoB-100 (but not apoB-48) is overexpressed in the liver. To achieve our goal, we microinjected rabbit zygotes with an 80-kb fragment of human genomic DNA that spans the human apoB gene. We were successful in generating four founders expressing human apoB-100, one of which was bred to yield a line of transgenic rabbits for further study.
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Methods |
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Generation of Human ApoB Transgenic Rabbits
To generate human apoB transgenic rabbits, we used an 80-kb Nru I fragment from a P1 bacteriophage clone (Dupont Merck Pharmaceutical Co, Human Foreskin Fibroblast library clone no. 1-261G, here designated p158). The Nru I fragment spanned the entire human apoB gene and contained 17.5 kb of 5' flanking sequences and 19 kb of 3' flanking sequences.19 The Nru I fragment was purified by electrophoresis on agarose pulsed-field gels.21 The Nru I fragment was dialyzed against microinjection buffer21 and adjusted to a concentration of 6 to 8 ng/µL before being microinjected into rabbit zygotes. Specific pathogen-free New Zealand White rabbits (Charles River) were superovulated and mated, and zygotes were collected.33 Nursing mothers were maintained on a breeder chow diet (Lab Rabbit Chow 5321, Purina Mills) that contains approximately 16% protein, 2.5% fat, and 18% crude fiber. After weaning at 6 weeks of age, the rabbit pups were maintained on the breeder chow diet for an additional 6 weeks. At 12 weeks of age, the rabbits were switched to a chow diet (Lab Rabbit Chow HF [high fiber], Purina Mills), which contains approximately 14% protein, 1.5% fat, and 25% crude fiber. All rabbits were given free access to food and water and were maintained on a 12-hour light/dark cycle with air temperature and humidity at 23°C and 55%, respectively. All experimental procedures were approved by the Animal Care Committee of the University of California, San Francisco.
Identification of Transgenic Rabbit Founders
To identify transgenic founder animals, we performed Southern
blot analyses of ear biopsy DNA by using a 2733-bp
HindIII probe from exon 26 of the human apoB
gene.19 To identify human apoB in the rabbit plasma, we
resolved rabbit plasma by electrophoresis on 3% to 12%
polyacrylamide gels containing 0.1% SDS.34 The
separated proteins were then electrophoretically transferred to a sheet
of nitrocellulose membrane for Western blots and probed by using a
125I-labeled monoclonal antibody, C1.4,35
which is specific for human apoB. The radioactivity within human
apoB-100 bands on the Western blots was quantified by using a gel
scanner (AMBIS, Inc).
To determine if the transgenic rabbit plasma contained human apoB-48 in
addition to human apoB-100, we analyzed rabbit VLDL samples by
Western blot analysis. For these experiments, weaned transgenic
and nontransgenic rabbits that had been maintained on a chow diet were
fasted overnight. On the next morning, the rabbits were re-fed the
rabbit chow diet. After access to rabbit chow for 6 hours, 8 mL rabbit
plasma was obtained, and VLDL particles were isolated by
ultracentrifugation.36 At the same
time, we isolated the VLDL from the plasma of a normolipidemic human
subject 90 minutes after the subject consumed 
250 mL whole milk. The
VLDL proteins were separated by electrophoresis on 3% to 6%
SDS-polyacrylamide gels. The gels were either stained with
silver or transferred to a sheet of nitrocellulose membrane for Western
blots by using the human apoB–specific monoclonal antibody
1D1.37 Antibody 1D1 binds near human apoB amino acid
residue 500 and recognizes both human apoB-48 and human
apoB-100.37 Binding of antibody 1D1 to the blots was
detected by using a horseradish peroxidase–conjugated goat
antibody to mouse IgG and a chemiluminescence kit (ECL kit,
Amersham).
Competitive Radioimmunoassay of Human ApoB-100
The concentration of human apoB-100 in transgenic rabbit plasma
was determined by using a monoclonal antibody–based competitive
radioimmunoassay performed on 96-well polyvinylchloride
plates.19 23 In this assay, the amount of human apoB in
rabbit plasma was measured by assessing the ability of rabbit plasma to
compete with 125I-labeled human LDL for binding to the
human apoB–specific monoclonal antibody 1D1.
Cholesterol and TG Measurements
The concentrations of total cholesterol and TGs in
whole plasma and lipoprotein density fractions were determined by
enzymatic assays by using a spectrum analyzer (Abbott)
according to the manufacturer's instructions. The plasma levels of
HDL-C were determined by using the StatSpin Micro HDL-C assay kit
(StatSpin Technologies).
Isolation of Lipoprotein Density Fractions From Rabbit
Plasma
To isolate plasma from transgenic and nontransgenic rabbits, the
animals were fasted for 14 to 16 hours unless otherwise noted. Blood
was collected from the intermedial auricular artery and placed in a
tube containing aprotinin (final concentration, 50 U/mL; Miles
Laboratory) and EDTA (final concentration, 1.5 mg/mL). The plasma was
separated by centrifugation at 3000 rpm for 20 minutes
at 4°C, and plasma lipoprotein density fractions were isolated by
sequential ultracentrifugation.38
Briefly, 1 mL plasma was subjected to
ultracentrifugation in a TLA100.2 fixed-angle
rotor in a Beckman TL100 ultracentrifuge at 100 000 rpm at
4°C for 2 hours. After collecting the d<1.006 fraction,
the infranatant fluid was adjusted with KBr to d=1.020 g/mL
and respun in the ultracentrifuge. By sequentially
adjusting the density of the infranatant fluid, we isolated the
lipoproteins from the following density fractions: d<1.006,
d=1.006 to 1.020, d=1.02 to 1.04,
d=1.04 to 1.06, d=1.06 to 1.08, and
d=1.08 to 1.10 g/mL. The d=1.10 to 1.21 g/mL
fraction was then isolated by ultracentrifugation
at 100 000 rpm for 4 hours at 4° C. The lipoproteins in each density
fraction were recovered by tube slicing, dialyzed for 16 hours against
a buffer containing 10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, and 1.0
mmol/L EDTA. The final volume of each lipoprotein fraction was adjusted
to 200 µL.
Lipoprotein Analysis Procedures
We analyzed plasma and lipoprotein fractions by
electrophoresis on 1% agarose gels according to the manufacturer's
instructions (Ciba-Corning). The gels were then either stained for
neutral lipids with Fat Red 7B (Sigma) or were transferred to a sheet
of nitrocellulose membrane for Western blots. For the Western blots, we
used goat antisera specific for rabbit apolipoproteins A-I, C-III, or E
(provided by Dr Karl Weisgraber, Gladstone Institute of
Cardiovascular Disease, San Francisco, Calif) in the
primary reaction and horseradish peroxidase–conjugated antibodies
against rabbit IgG in the secondary reaction. The binding of the
apolipoprotein-specific antibodies was detected with the ECL
chemiluminescent detection kit from Amersham.
The distribution of cholesterol within the plasma lipoproteins of human apoB transgenic rabbits was also determined by size fractionation on a Superose 6 10/50 column (Pharmacia Biotech Inc).19 39 The cholesterol concentration of each fraction was determined with the Spectrum kit from Abbott Diagnostics.
Analytical ultracentrifugation, with quantification of mass as a function of flotation rate (S°f), was performed on lipoproteins of d<1.063 g/mL by using the procedures of Lindgren et al.40 Mean peak analytic ultracentrifuge flotation rate values were corrected for lipoprotein concentration and the Johnson-Ogston effect.40 LDL subfractions were isolated by equilibrium density-gradient ultracentrifugation.41 Briefly, the d=1.019 to 1.063 g/mL fraction was prepared from 4 mL plasma by sequential ultracentrifugation. The fraction was adjusted to d=1.040 g/mL in NaBr by dialysis and layered between 2.5 mL of d=1.054 g/mL and d=1.0275 g/mL NaCl solutions in a 7-mL ultracentrifuge tube. Ultracentrifugation was performed at 40 000 rpm for 40 hours at 17°C in a Beckman SW45 swinging-bucket rotor. Aliquots (0.5 mL for fractions 1 and 3 through 8; 1.0 mL for fractions 2, 9, and 10) were sequentially withdrawn by using a pipette. Salt densities were measured in a Paar densitometer by using fractions from ultracentrifuge tubes containing the salt solution without plasma.
Nondenaturing gradient gel electrophoresis of whole plasma and LDL density fractions was performed with Pharmacia 2% to 16% polyacrylamide gels.42 For electrophoresis of plasma samples, gels were stained for neutral lipid with oil red O; for LDL density fractions, gels were stained for protein with Coomassie blue R250. Gels were scanned in a Transidyne RFT densitometer. The size of lipoproteins was determined from a quadratic calibration curve based on established size markers.
Sizing of the lipoproteins within several lipoprotein fractions (d <1.006, d=1.006 to 1.02, d=1.02 to 1.04, and d=1.04 to 1.06 g/mL) was also performed by using negative-staining electron microscopy.33 43
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Results |
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Generation and Identification of Human ApoB Transgenic Rabbits
A total of 190 New Zealand White rabbit zygotes were microinjected with an 80-kb Nru I fragment spanning the human apoB gene and implanted into the oviducts of eight foster mothers. A total of 24 live pups were born. Two female pups (rabbits 488 and 865) and two male pups (rabbits 844 and 845) were transgenic as determined by Southern blot analysis of ear biopsy DNA using a 32P-labeled probe from exon 26 of the human apoB gene19 (data not shown). Founder rabbit 488 was mated with a male nontransgenic rabbit. One of four surviving pups, a female, was transgenic; this animal (rabbit 818) was also analyzed.
To examine the expression of human apoB in the transgenic rabbits,
rabbit plasma samples were resolved by SDS–polyacrylamide gel
electrophoresis followed by Western blotting with the human
apoB–specific monoclonal antibody C1.4 (Fig 1
). Antibody C1.4 binds near apoB-100
amino acid residue 500,35 and therefore it binds to both
human apoB-48 and apoB-100. The plasma of fasting transgenic rabbits
contained human apoB-100 but no human apoB-48. As a positive control,
we analyzed the plasma of a human apoB transgenic mouse that
had a human apoB-100 level of 60 mg/dL23 ; the plasma of
this mouse contained both apoB-48 and apoB-100, as expected (Fig 1). (In transgenic mice the human apoB transgene is
expressed in the liver and not in the intestine. Both human apoB-48 and
human apoB-100 in transgenic mouse plasma are produced in the
transgenic mouse liver.) Quantitation of the Western blots revealed
that the concentration of human apoB-100 in the plasma of founder
rabbit 488 was approximately the same as that in a high-expression
transgenic mouse. Rabbits 845 and 818 had significantly higher
concentrations of human apoB-100 in their plasma than the transgenic
mouse (see Fig 1 legend).
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To confirm the expression of human apoB in transgenic rabbit plasma and
to examine the possibility that low levels of human apoB-48 might be
present in rabbit plasma, we isolated the VLDL from postprandial
rabbit and human plasma and examined the VLDL proteins by using
SDS–polyacrylamide gel electrophoresis and Western blotting.
On a silver-stained gel both apoB-100 and apoB-48 could be detected
in transgenic rabbit VLDL, nontransgenic rabbit VLDL, and human VLDL
(Fig 2A). Fig 2B shows a
Western blot using the human apoB–specific monoclonal antibody 1D1,
which binds near apoB-100 amino acid 500 and therefore recognizes both
apoB-48 and apoB-100. As expected, antibody 1D1 detected both apoB-48
and apoB-100 in human VLDL. However, only human apoB-100 (and no human
apoB-48) was present in the VLDL of the transgenic rabbits. These
results were consistent with the previously determined
liver-specific expression of the 80-kb human apoB
transgene19 23 and the absence of apoB mRNA editing in the
rabbit liver.30
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Human ApoB-100 Concentrations in Transgenic Rabbits
To measure human apoB-100 levels in the plasma of the transgenic
rabbits, we used a competitive radioimmunoassay (Table).
On the chow diet at 6 months of age, transgenic founder 488 had a human
apoB-100 level of 44 mg/dL. On the breeder chow diet at 10 weeks of
age, the F1 transgenic rabbit 818 had human apoB-100 levels of 100
mg/dL. On a chow diet at 3 months of age, the apoB-100 level in animal
818 remained high: 98 mg/dL. Founder 845 had human apoB-100 levels of
53 mg/dL on the breeder chow diet at 10 weeks of age and 33 mg/dL at 3
months of age on the chow diet. Two other founder animals (844 and 865)
had low human apoB-100 levels (<12 mg/dL) on both the breeder chow and
regular chow diet.
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Effect of Human ApoB Expression on Plasma Lipid
Levels
Total plasma TG and cholesterol levels in the human
apoB transgenic rabbits were consistently higher than those of
all age-, sex-, and diet-matched nontransgenic rabbits
(Table
). In all the transgenic rabbits TG levels were
two- to threefold higher than in control nontransgenic rabbits
(Table). The total cholesterol level in the
plasma of transgenic founder 488 was 136 mg/dL on the chow diet, which
was 2.8-fold higher than that of nontransgenic littermate controls. F1
transgenic rabbit 818 had a total plasma cholesterol level
of 308 mg/dL, which was 2.7-fold higher than the corresponding levels
in nontransgenic littermate controls. The cholesterol level
was also elevated, but to a lesser degree, in human apoB transgenic
founder rabbit 845. In each of the three animals that expressed high
levels of human apoB-100 (animals 488, 818, and 845), levels of HDL-C
were markedly reduced compared with the nontransgenic control animals
(Table). The two other founder rabbits expressing lower
levels of human apoB-100 in their plasma (844 and 865) did not have
elevated plasma cholesterol or TG levels compared with
nontransgenic rabbits, suggesting that the plasma lipid changes that we
observed in the high-expressor animals were a direct consequence of
the level of human apoB expression.
Analysis of the Lipoprotein Phenotype of Human ApoB
Transgenic Rabbits
To determine the effect of human apoB expression on the plasma
lipoproteins, we analyzed rabbit plasma by agarose gel
electrophoresis. Staining for neutral lipids with fat red 7B showed
that the plasma of the transgenic rabbits had increased levels of
ß-migrating lipoproteins and decreased levels of 
-migrating
lipoproteins (Fig 3A). Western blot
analysis indicated that virtually all the human apoB-100 was in
the ß-migrating lipoproteins, as expected (Fig 3A).
To analyze the distribution of human apoB in different
lipoprotein fractions, seven density fractions were isolated from the
plasma of transgenic rabbit 818 and analyzed by agarose gel
electrophoresis and Western blotting (Fig 3B). Human
apoB-100 was found in the VLDL (d<1.006 g/mL) and IDL
(d=1.006 to 1.020 g/mL) fractions, but most was in the LDL
fractions (d=1.02 to 1.04 and d=1.04 to 1.06
g/mL). In nontransgenic rabbits, rabbit apoB also was found in the
VLDL, IDL, and LDL fractions with the same distribution as reported
previously.33 In early studies the distribution of rabbit
apoB in transgenic rabbit plasma appeared to be similar to that in the
nontransgenic animals (data not shown). We would expect the density
distribution of rabbit and human apoB-100 in transgenic rabbit plasma
to be similar; in transgenic mice, the distribution of human apoB-100
and apoB-48 was identical to the distribution of mouse apoB-100 and
apoB-48.23
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Analysis of the distribution of cholesterol and TG
in the lipoprotein fractions of the human apoB transgenic rabbits
revealed a striking increase in the amount of LDL-C (d=1.02
to 1.06 g/mL) and a substantial decrease in the amount of HDL-C in the
HDL (Fig 4A). Of note, the transgenic
rabbit LDL was enriched in TGs: the TG/cholesterol ratio in
the d=1.019 to 1.063 g/mL fraction was 1.02 (versus 0.38 in
a nontransgenic control). Indeed, the increased plasma TG
concentrations in the transgenic animals were completely accounted for
by the high TG levels in the LDL fractions (Fig 4B). We
also assessed the distribution of cholesterol within the
plasma lipoprotein fractions by Superose 6 10/50
chromatography to confirm the results obtained from the
ultracentrifugation studies (Fig 5). On both the normal chow diet (Fig 5A) and the breeder chow diet (Fig 5B)
there were striking increases in LDL-C levels accompanied by dramatic
decreases in HDL-C in the transgenic rabbits compared with age- and
sex-matched nontransgenic rabbits.
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We further analyzed the plasma LDL of the transgenic and
nontransgenic rabbits by analytical
ultracentrifugation. Fig 6 shows the
analytic ultracentrifuge schlieren profiles of
S°f=0 to 20 lipoproteins from the plasma of a
nontransgenic rabbit, a transgenic rabbit, and a human plasma, with
measurements of total mass for the S°f=0 to 12 (LDL) and
S°f=12 to 20 (IDL) fractions. The nontransgenic rabbit
had only small amounts of LDL, and a significant fraction of the
S°f=0 to 20 lipoproteins was actually in the IDL range
(S°f=12 to 20). In contrast, most of the lipoprotein mass
in transgenic rabbit plasma was within the LDL range
(S°f=0 to 12). The distribution for the transgenic rabbit
plasma was remarkably similar to that observed in the plasma from a
moderately hyperlipidemic human subject (total
cholesterol, 261 mg/dL; TGs, 104 mg/dL). In addition to the
analytical ultracentrifugation studies, the density
distribution of LDL protein was examined in detail by equilibrium
density-gradient ultracentrifugation (Fig 7). In the nontransgenic rabbits, most of
the LDL protein was located in fractions with densities <1.030 g/mL
(Fig 7A). In contrast, most of the LDL protein in the
transgenic rabbit plasma was located in subfractions having densities
between d=1.030 g/mL and d=1.046 g/mL (Fig 7B), similar to the density of LDL in human plasma (Fig 7C).
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Lipoprotein sizes were estimated by gradient polyacrylamide gel
electrophoresis. In nontransgenic rabbit plasma (Fig 8A), the major electrophoretic peak was
in particles within the LDL size range; the calculated diameter for
these particles was 277Å. In contrast, the major peak in transgenic
rabbit plasma was in smaller particles (Fig 8B); the
diameter of these particles was 242Å. Similar findings were made
when the ultracentrifuge-isolated LDL fractions were
analyzed by gradient polyacrylamide gels (Fig 8C and 8D). The diameter of particles in the main
LDL peak in the nontransgenic rabbit was 272Å, versus 240Å in the
transgenic animal.
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We also used negative-stain electron microscopy to assess the sizes
of the LDL fractions (d=1.02 to 1.04 g/mL and 1.04 to 1.06
g/mL) from transgenic and nontransgenic rabbits. Although the particle
diameters that we observed by this technique were less than those
calculated from the nondenaturing gradient polyacrylamide gels,
the conclusions were the same: the LDL particles in the transgenic
animals were significantly smaller than those in the nontransgenic
animals. The LDL fractions isolated from a transgenic rabbit contained
a homogeneous collection of spherical particles; the particle diameters
(mean±SD) for lipoproteins within the d=1.02 to 1.04 and
1.04 to 1.06 g/mL fractions were 199.6±17.9 and 187.3±14.2 Å,
respectively. The mean particle diameter for the nontransgenic
d=1.02 to 1.04 g/mL fraction was larger: 214±33Å. The
nontransgenic d=1.04 to 1.06 g/mL fraction appeared to
contain a mixture of HDL-sized particles (100 Å) and LDL-sized
particles with diameters ranging from 180 to 220 Å.
The increased content of TG-enriched LDL and the reduced amounts of HDL
in the plasma of transgenic rabbits suggested that there might be
significant changes in the metabolism of other
apolipoproteins. Therefore, the distributions of apoE, apoC-III, and
apoA-I in the lipoprotein density fractions were examined by using
Western blots of 1% agarose gels (Fig 9). In an agarose gel stained for neutral
lipid with fat red 7B (Fig 9A), which is shown for
reference, there was an increased number of ß-migrating lipoproteins
and a decreased number of -migrating lipoproteins in the transgenic
rabbits, consistent with the results shown in Figs 3, 4, and 5.
The transgenic rabbits had markedly decreased amounts of apoA-I in
their HDL fraction (Fig 9B), consistent with
the observation that these animals had reduced amounts of HDL-C in
their plasma. In transgenic rabbits about one-half the apoE was
located within the TG-rich, ß-migrating large LDL (d=1.02
to 1.04 g/mL). In contrast, only minor amounts of apoE were found in
the d=1.02 to 1.04 g/mL fraction of nontransgenic rabbits;
most of the apoE was found in the VLDL and the large HDL (Fig 9C). Similarly, in the transgenic rabbits, virtually
all the apoC-III was located in the ß-migrating LDL rather than in
VLDL and HDL, which was typical of the nontransgenic rabbits (Fig 9D).
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Discussion |
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In this study we report the development and characterization of transgenic rabbits expressing human apoB-100. The rabbits were generated by microinjecting rabbit zygotes with an 80-kb fragment of human genomic DNA spanning the complete human apoB gene. The expression of high levels of human apoB-100 produced dramatic changes in the lipoprotein phenotype of the transgenic rabbits compared with that of nontransgenic control rabbits. In nontransgenic rabbits on a chow diet, the total plasma cholesterol levels were relatively low (typically <50 to 60 mg/dL), and most of the cholesterol was located in
-migrating lipoproteins in
the HDL fraction. The levels of LDL-C in these animals were quite low,
and the LDL consisted mainly of large particles (270 to 275Å as
assessed by gradient gel electrophoresis). In contrast, transgenic
rabbits that expressed high levels of human apoB had two- to threefold
elevations in total plasma cholesterol levels, and most of
it was within LDL. The size of the LDL particles in the transgenic
rabbits (240Å as assessed by gradient gel electrophoresis) was
smaller than in the nontransgenic rabbits and in the size range of LDL
particles in human plasma.42 Moreover, the flotation
characteristics and density of the transgenic rabbit LDL resembled
human LDL as assessed by analytical
ultracentrifugation or by equilibrium
density-gradient ultracentrifugation. Of note,
high levels of human apoB expression in the rabbit resulted in reduced
levels of apoA-I and HDL-C. In the chow-fed founder transgenic
rabbit 488, the combination of higher LDL-C levels and lower HDL-C
levels resulted in a plasma cholesterol distribution
profile (Fig 5A) that was similar to that observed in
normolipidemic humans, with approximately two thirds of the
cholesterol in the LDL fraction and most of the remainder
in the HDL fraction. One of the most intriguing findings in this study was that the LDL in the transgenic rabbits was markedly enriched in TGs and contained large amounts of apoE and apoC-III. In the human apoB transgenic mice we also noted TG-enriched LDL,19 20 so it seems possible that TG-rich LDL is a hallmark feature of overproduction of apoB protein by the liver. In the transgenic mice we initially hypothesized that the TG enrichment of the LDL might be due to the absence of plasma cholesteryl ester transfer protein activity in that species,27 28 which might interfere with the transfer of TGs from the apoB-containing lipoproteins to the HDL. However, the finding of TG-rich LDL in the transgenic rabbits argues for an alternative explanation, since rabbits have high levels of cholesteryl ester transfer protein in their plasma.27 28 An attractive explanation for the production of TG-rich LDL is that hepatic overproduction of apoB leads to the assembly of nascent TG-rich particles that have the size and density of LDL rather than VLDL. Assuming that the increased synthesis of apoB-100 in the liver does not, in some indirect manner, lead to increased production of neutral lipids, then it is reasonable to postulate that a smaller amount of neutral lipids would be available for each newly synthesized apoB molecule. According to this reasoning, nascent hepatic lipoproteins in the transgenic rabbits might closely resemble the TG-rich LDL particles in the plasma, whereas the nascent hepatic lipoproteins in nontransgenic animals would be more buoyant and significantly larger. It is noteworthy that the LDL of the transgenic rabbits contain substantial amounts of apoC-III and apoE, two apolipoproteins that are normally abundant in TG-rich VLDL.
The hypothesis that hepatic overexpression of apoB results in smaller, denser nascent lipoproteins remains to be tested. However, even if our future studies confirm this hypothesis, we would still be left with the question of why these animals have increased levels of TGs in the plasma. Both human apoB transgenic rabbits and human apoB transgenic mice were hypertriglyceridemic on chow diets, and most of the TGs were in the LDL fraction of both species. It is possible that the TGs within the LDL-sized particles are not as accessible to lipoprotein lipase as those in VLDL-sized particles44 or that the relatively high levels of cholesteryl ester in the core of these particles interfere with lipolysis.45 In addition, the naturally low levels of hepatic lipase activity that are characteristic of the rabbit46 may contribute to the high TG levels found in the LDL. Finally, it is conceivable that the human apoB-containing lipoproteins may not be an ideal substrate for the rabbit lipoprotein lipase.
Humans with FCH have moderate elevations in plasma cholesterol, TGs, and apoB-100, and kinetic studies have indicated that these patients have increased production of apoB-containing lipoproteins.14 15 16 17 18 The LDL of these patients tends to be smaller and denser than that of normal control human subjects, a finding that is largely explained by the fact that the LDL of these patients is depleted in cholesteryl esters.47 The existence of small, dense LDL in FCH patients is associated, to an extent, with hypertriglyceridemia.47 48 However, most of the TGs in the plasma of FCH patients are found in the VLDL and IDL rather than the LDL fraction.47 This is completely unlike the distribution of TGs in the human apoB transgenic rabbits and mice. Of note, many FCH patients have a slight reduction in HDL-C levels,47 48 a finding that is also observed in transgenic animals that overexpress apoB. At the present time, the mechanism whereby overproduction of the apoB protein lowers HDL-C levels is not understood, and it is not clear if the low levels of HDL in human patients and transgenic animals share a common mechanism.
The St. Thomas' Hospital hyperlipidemic rabbits12 13 have elevated cholesterol and TG levels, normal LDL receptor activity, and increased VLDL- and LDL-apoB production rates. The molecular mechanisms underlying the hyperlipidemia in these animals have not been defined. Although we have not performed side-by-side comparisons of the St. Thomas' Hospital rabbit and the human apoB transgenic rabbit, the phenotypes of the two animals appear to differ. The St. Thomas' Hospital rabbit did not manifest a marked TG enrichment of the LDL fraction.12 In addition, the St. Thomas' Hospital rabbit had significantly increased levels of VLDL and IDL cholesterol, which we did not encounter in the human apoB transgenic rabbits. Based on these phenotypic differences, it seems unlikely that overproduction of the apoB protein is the sole metabolic defect in the St. Thomas' Hospital rabbit. This conclusion, of course, rests on the assumption that the basic physiological properties of human apoB (in the transgenic rabbit) and rabbit apoB (in the St. Thomas' Hospital rabbit) are similar.
In the present study, we generated transgenic rabbits with an 80-kb fragment of genomic DNA spanning the human apoB gene, including 17.5 kb of the 5' flanking sequences and 19 kb of 3' flanking sequences.19 20 21 In multiple lines of transgenic mice generated with the same DNA fragment, high levels of human apoB expression were observed in the liver with no apoB expression in the intestine. The absence of transgene expression in the intestine was surprising since prior reporter-gene expression studies have suggested that the apoB intestinal control element might be located within several hundred bases of the transcriptional start site.49 The results of our current study indicate that the pattern of expression of the 80-kb transgene is almost certainly the same in the rabbit and the mouse. On a silver-stained SDS–polyacrylamide gel there was ample apoB-48 in the postprandial VLDL of the transgenic rabbit, but none of the apoB-48 was human apoB-48. Rabbit intestinal apoB mRNA editing factor edits the human apoB mRNA efficiently (T. Innerarity and S. Yamanaka, unpublished data, 1995). Consequently, we would expect to find human apoB-48 in the transgenic rabbit VLDL if the 80-kb human apoB transgene were expressed in the intestine. The absence of human apoB-48 in the transgenic rabbit VLDL fraction, together with the absence of human apoB expression in the intestines of transgenic mice, suggests that the intestinal tissue-specific DNA sequence element is not contained within the 80-kb fragment of human genomic DNA.
The fact that transgenic rabbits synthesize exclusively apoB-100 in the liver will make these animals especially useful for studies of lipoprotein metabolism and assembly. In the human apoB transgenic mice,19 the synthesis of both apoB-48 and apoB-100 in the mouse liver produces unwanted complexities for certain studies of lipoprotein metabolism. Transgenic mouse studies have led us to conclude that apoB-48 and apoB-100 probably have markedly different metabolic properties.23 For example, in chow-fed transgenic mice, approximately 70% of the human apoB mRNA in the mouse liver is edited, but there is a huge preponderance of apoB-100 in the plasma.23 In transgenic mice that are fed a high-fat diet, human apoB-48 levels in the LDL fraction increase dramatically, but human apoB-100 levels are unchanged.23 The study of apoB metabolism in transgenic rabbits should avoid these complications, and studies of dietary manipulation and drug intervention in these animals should be more relevant to humans. Similarly, because the size and assembly process for apoB-48– and apoB-100–containing lipoproteins appear to differ,50 51 the use of transgenic mice to study the effects of overexpression of apoB on lipoprotein assembly would be quite difficult. In contrast, the transgenic rabbits present an excellent model for assessment of the effects of apoB overproduction on the size, density, and composition of nascent hepatic lipoproteins.
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Acknowledgments |
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This work was funded in part by National Institutes of Health grants HL 51588, HL 41633, and HL 18754, a grant from the National Dairy Promotion and Research Board, and a contract from the US Department of Energy (DEAC0376SF00098). J. Fan and S. McCormick were recipients of fellowship awards from the American Heart Association, California Affiliate. We thank V. Pierotti for preparing the p158 DNA for microinjection, K. MacLeod for help with rabbit surgery, K. Weisgraber for providing rabbit antisera, G. Schonfeld for antibody C1.4, R. Milne and Y. Marcel for antibody 1D1, J. McGuire, J. Wang, and P. Blanche for the lipid and lipoprotein analysis, D. Sanan and D. Newland for electron microscopy staining of negatively stained lipoproteins, A. Corder for graphic arts, G. Howard for editorial assistance, and L. Hymowitz for manuscript preparation.
Received June 23, 1995; accepted August 16, 1995.
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