© 1999 American Heart Association, Inc.
Original Contributions |
Overexpression of Human Hepatic Lipase and ApoE in Transgenic Rabbits Attenuates Response to Dietary Cholesterol and Alters Lipoprotein Subclass Distributions
From the Istituto di Medicina Interna e Geriatria (C.M.B.), Universitá degli Studi di Palermo, Italy; the Gladstone Institute of Cardiovascular Disease (J.F., J.M.T.), University of California, San Francisco; and the Life Sciences Division (C.M.B., P.J.B., M.R., R.M.K.), Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Berkeley.
Correspondence to Ronald M. Krauss, MD, Lawrence Berkeley National Laboratory, Donner Laboratory, Room 459, University of California, One Cyclotron Road, Berkeley, CA 94720. E-mail rmkrauss{at}lbl.gov
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Abstract |
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Abstract—The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.
Key Words: lipoproteins • apoE • hepatic lipase • rabbits • transgene
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Introduction |
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Hepatic lipase (HL) and apoE both have critical roles in the metabolism of plasma lipoproteins.1 2 Human HL is a 476–amino-acid glycoprotein that is synthesized by hepatocytes and binds to external surfaces of hepatic cells.3 4 Studies in animal models and HL-deficient humans5 6 7 8 suggested that HL is involved in the conversion of IDLs to LDLs, and in the catabolism of large lipid-rich HDLs (reviewed in Reference 11 ). HL also enhances the direct binding and uptake of cholesteryl ester–rich VLDL and chylomicron remnants by hepatocytes.9 Human apoE is a 299–amino-acid glycoprotein present mainly in triglyceride-rich particles (chylomicrons, VLDLs, and IDLs) and large HDLs. A major role of apoE in plasma lipid transport is to mediate the binding and clearance of remnant lipoproteins and large cholesterol-rich HDLs to receptors in hepatic and peripheral cells.2 10 11
To determine whether the physiological roles of HL and apoE in plasma cholesterol and lipoprotein metabolism are complementary, we generated transgenic lines of rabbits overexpressing the corresponding human proteins. Rabbits were used because they are naturally deficient in HL and they have relatively reduced levels of apoE compared with other species.12 13 We previously found that overexpression of HL in transgenic rabbits resulted in a reduction in IDLs and all subclasses of HDLs in chow-fed rabbits, as well as a 2-fold reduction in plasma cholesterol, in comparison with nontransgenic controls.14 15 Overexpression of apoE in transgenic rabbits was shown to decrease VLDL levels; this effect was due to an increased affinity of large apoE-rich remnants for the LDL receptor that was related to increased chylomicron clearance rate.16
Although VLDL+IDL and LDLs are commonly defined as lipoproteins of density <1.019 and 1.019 to 1.063 g/mL, respectively, among these intervals it is possible to recognize several heterogeneous subclasses, differing in size, density, flotation rate, electrophoretic mobility, and chemical composition.17 18 19 20 21 22 23 24 25 We previously used density-gradient ultracentrifugation (DGUC) and electrophoresis in polyacrylamide gradient gels to demonstrate the existence of multiple distinct subclasses of VLDLs and IDLs in humans.19 20 By injecting human lipoproteins into rats, we documented different metabolic pathways for distinct VLDL and IDL subclasses.21 The LDLs also contain multiple distinct subpopulations of particles.22 23 24 25 Studies have demonstrated that individual subclasses may have different metabolic properties,26 27 28 but specific effects of HL and apoE on the metabolism of these subpopulations have not been established. Plasma HDLs are also exceptionally heterogeneous particles and nondenaturing polyacrylamide gradient gel electrophoresis (GGE) can distinguish several subfractions according to size.29
In the present study, we determined whether the expression of high levels of HL or apoE has specific influences on the levels of individual lipoprotein subfractions in response to dietary cholesterol. We also tested whether the combined expression of HL and apoE has additive effects on plasma lipoprotein levels by cross-breeding rabbits overexpressing either HL or apoE. Our results demonstrate that HL and apoE have complementary and synergistic actions on lipoprotein metabolism and that both components are needed for the maximum protection against hypercholesterolemia in response to dietary cholesterol.
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Methods |
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Protocol
Previously described models of pathogen-free New Zealand White male rabbits transgenic for human HL,14 15 models transgenic for human apoE,16 and nontransgenic controls were used in this study. In addition, animals expressing both HL and apoE were produced by cross-breeding of the 2 transgenic strains. Seventeen animals overexpressing human HL, 7 overexpressing human apoE, 4 overexpressing both human HL and apoE, and 15 nontransgenic controls (4 to 6 months of age) were used.
The concentration of human apoE in the rabbits carrying the apoE
transgene was 14±3 mg/dL (mean±SD), similar to that previously
reported16 and 
5-fold higher than
endogenous rabbit apoE. Human HL activity level as measured
in postheparin plasma14 15 was 54±6
(mean±SD) µEq of fatty acid released ·
mL-1 · h-1 from a
triolein substrate, a level 28-fold higher than the activity found in
nontransgenic controls.
All the animals were maintained on a diet containing 17% protein, 12% crude fiber, and 5.5% fat, supplemented with 0.3% cholesterol and 3.0% soybean oil (Zeigler Bros Inc) for 10 weeks. The animals had free access to water and to the diet, and they were housed in a pathogen-free barrier facility with a 12-hour light/dark cycle at 23°C and 55% humidity. All experimental procedures with rabbits were conducted in accordance with NIH guidelines and with the approval of the Committee on Animal Research of the University of California, San Francisco.
Before analysis, rabbits were fasted overnight (14 to 16 hours); then blood was collected (between 8:30 and 9:30 AM) from the intermedial auricular artery and adjusted to a final concentration of 1.5 mg/mL EDTA and 50 U of Trasylol pancreatic protease inhibitor (Miles Laboratory). Plasma was separated by centrifugation at 3000 rpm for 20 minutes at 4°C and analyzed as described below.
Density-Gradient Ultracentrifugation
DGUC of lipoprotein fractions was performed as previously
described.20 22 In brief, lipoproteins of density (g/mL)
<1.019 (VLDL+IDL), 1.019 to 1.063 (LDLs), and 1.063 to 1.21 (HDLs)
were isolated through preparative ultracentrifugation
under standard conditions.30 Sodium bromide was added to
adjust densities and samples were centrifuged at 40 000 rpm
for 18 to 20 hours (VLDL+IDL), 24 to 26 hours (LDLs), and 24 hours
(HDLs) at 10°C in a Beckman 40.3 fixed-angle rotor. The VLDL+IDL
fraction was dialyzed against a d=1.21 g/mL sodium bromide
solution at 4°C with repeated changes over a 24- to 48-hour period. A
4.5-mL sample was placed in a 9/16x31/2-inch Ultraclear
Tube (Beckman Instruments) and sequentially overlayered with 3.0, 3.0,
and 1.5 mL, respectively, of sodium bromide solutions of
d=1.020, 1.010, and 1.000 g/mL. The latter solution
consisted of d=1.0063 g/mL solution (11.422 g/L NaCl and
0.01% EDTA) diluted 1:5 (vol/vol) with water. The tubes were
centrifuged at 17°C in a Beckman SW41 rotor in a Beckman
L5-75 ultracentrifuge. The slowest setting for acceleration was
used at the start of the run. After 6 hours at 40 000 rpm, the rotor
was allowed to coast to a stop without braking. The contents of the
tube then were withdrawn by pipetting two 0.5-mL fractions (fractions 1
and 2), three 1.0-mL fractions (fractions 3 to 5), followed by three
0.5-mL fractions (fractions 6 to 8). The bottom 6.5-mL fraction was
discarded.
The LDL fraction was dialyzed against a d=1.040 g/mL sodium bromide solution at 4°C with repeated changes during a 24- to 48-hour period. An aliquot (2.0 mL) was layered above a solution of d=1.054 g/mL (2.5 mL) in a 1/2x31/2-inch Ultraclear Tube (Beckman Instruments) and 2.5 mL of a solution of d=1.0275 g/mL was layered above the lipoprotein fraction. The tubes were centrifuged at 40 000 rpm for 40 hours at 17°C in a Beckman SW45 rotor in a Beckman L5-75 ultracentrifuge under the same conditions as above. The contents of the tube then were withdrawn by pipetting one 0.5-mL fraction (fraction 1), one 1.0-mL fraction (fraction 2), followed by six 0.5-mL fractions (fractions 3 to 8) and two 1.0-mL fractions (fractions 9 and 10). The bottom 0.5-mL fraction was discarded.
Nondenaturing Polyacrylamide GGE
Nondenaturing polyacrylamide GGE of d<1.019
and d=1.019 to 1.063 g/mL lipoprotein fractions was
performed at 10°C in 2% to 14% polyacrylamide gradient gels
for 24 hours at 125 V in Tris (0.09 mol/L)/boric acid (0.08
mol/L)/Na2EDTA (0.003 mol/L) buffer (pH 8.3) as
described elsewhere,19 29 except that the gradient
gels were prepared in our laboratory essentially as described by
Rainwater et al.31 Gels were fixed and stained for lipids
in a solution containing oil red O in 60% ethanol at 55°C or for
proteins in a solution containing 0.1% Coomassie Brilliant Blue R-250,
50% ethanol, and 9% acetic acid (vol/vol). Gels were scanned at 530
nm (for oil red O staining) or at 555 nm (for Coomassie
Brilliant Blue staining) with a Transidyne densitometer.
Migration distance for each absorbance peak was determined, and the
molecular diameter corresponding to each peak was calculated from a
calibration curve generated from the migration distance of size
standards of known diameter, which included carboxylated latex beads
(Duke Scientific), thyroglobulin, and apoferritin (HMW Std;
Pharmacia) with molecular diameters of 380, 170, and 122 Å,
respectively, and lipoprotein calibrators of previously determined
particle size.
The HDL fractions were analyzed with GGE as described by Nichols et al,29 using 3% to 31% gradient gels prepared in our laboratory according to the method of Rainwater et al.31 Gels were fixed and protein bands stained with Coomassie Brilliant Blue R-250 as described above. Densitometry was used to measure areas in 5 particle size ranges corresponding to human HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c.29 Estimates of the plasma concentrations of these HDL subclasses were obtained by multiplying the percentage of total area for each band by the total plasma HDL protein concentration as described below. Fractions with a diameter of >120 Å were considered HDL1.
Analytical Ultracentrifugation
Analytical ultracentrifugation (AnUC) of VLDLs,
IDLs, and LDLs was performed in 3 rabbits overexpressing HL, 3 rabbits
overexpressing apoE, 4 rabbits overexpressing both HL and apoE, and 3
nontransgenic controls as previously described.30
Centrifugation was performed at 26°C and 52 640 rpm
at a salt density of 1.063 g/mL in a Spinco Model E instrument equipped
with Schlieren optics. Total lipoprotein mass was estimated as a
function of Svedberg flotation rate (Sf°), using computer-assisted
analytical techniques.
Chemical Composition Determinations
Triglyceride total and unesterified cholesterol levels were
measured by using enzymatic methods and a Gilford Impact 400E
analyzer (Gilford Instruments).32 33 HDL
cholesterol was measured after heparin–manganese
precipitation of the apoB containing lipoproteins.34
Protein concentrations were determined according to the method
of Markwell et al35 and phospholipids quantification was
determined according to the method of Bartlett.36
The total mass of lipoproteins was estimated by adding the mass of all
components in each fraction.
Statistical Analysis
Statistical analysis was performed by using StatView II
software. The differences between all transgenic rabbit groups and
controls were evaluated by the 2-tailed Student's t test
for unpaired data or by the Mann–Whitney U test for
variables found not normally distributed when tested with the
Kolgomorov–Smirnov test. Statistical significance between groups had a
probability value of <0.05.
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Results |
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Plasma Lipid and Lipoprotein Concentrations
Figure 1
shows the plasma lipids of
nontransgenic and transgenic animals on the chow diet and after 10
weeks on the 0.3% cholesterol–enriched diet. In
nontransgenic rabbits on chow, HDL cholesterol comprised
75% of circulating plasma cholesterol, whereas in both
HL and apoE transgenic animals, there was a pronounced shift in
lipoprotein cholesterol distribution with HDL
cholesterol comprising 30% and 50% of the total.
Combined expression of HL and apoE resulted in lower levels of HDL
cholesterol and triglyceride than were found in
the HL transgenic rabbits (Figure 1). In all experimental
groups, the increased plasma cholesterol levels on the
high-cholesterol diet were almost entirely (>95%) in the
non-HDL fraction (Figure 1). Compared with nontransgenic
controls, both the HL and the apoE transgenic rabbits had dramatically
lower concentrations of total and HDL cholesterol; levels
of triglyceride were also significantly lower in the HL
rabbits. Rabbits overexpressing both apoE and HL had remarkably low
concentrations of plasma lipids compared with the other experimental
groups.
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P<0.001, §P<0.0001 versus controls (same
diet). For abbreviations, see legend to Table 1
Measurements of total mass concentrations of the major plasma
lipoprotein fractions (Table) showed
effects of the high-cholesterol diets consistent
with those found for plasma cholesterol and
triglyceride. On the high-cholesterol diet,
compared with controls, levels of VLDL+IDL were significantly lower in
both HL and apoE transgenic rabbits and were further reduced in the
combined transgenic animals. LDL mass concentrations were significantly
lower in the HL transgenics than in the controls; mean values
for the other 2 groups were similar, but the differences from the
controls did not reach statistical significance. Differences in HDL
mass concentrations among the groups paralleled those for HDL
cholesterol (Figure 1
), although levels in the
combined transgenic animals, and in the HL animals on chow, were too
low to permit measurement.
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Lipoprotein Subfraction Analyses
Density and Size Distributions of VLDLs, IDLs, and LDLs
In subsets of each experimental group on the
high-cholesterol diet, AnUC measurements were performed to
evaluate the distribution of lipoprotein mass within the spectrum of
VLDL, IDL, and LDL subfractions. In addition, nondenaturing GGE was
performed on plasma and the VLDL+IDL (d<1.019 g/mL) and LDL
(d=1.019 to 1.063 g/mL) fractions from all animals to assess
the diameter of the major species in each fraction. Mean AnUC profiles
for each subgroup are shown in Figure 2.
The Sf° ranges designating VLDLs, IDLs, and LDLs are similar to those
for humans,30 except that rabbit VLDLs extend to a
lower value (17 versus 20), which corresponds to a buoyant density of
1.010 g/mL (Rizzo M, Barbagallo CM, and Krauss RM, unpublished
observations). Several components were observed within the VLDL Sf°
range in the nontransgenic rabbits. These were grouped into intervals
designated large, medium, and small VLDLs. In addition, there was a
peak of very large VLDLs extending to Sf° >400, a region that
includes chylomicron-sized particles.30
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In the HL transgenic rabbits, AnUC revealed a predominance of large and
very large VLDLs, with a marked depletion of medium and small VLDLs and
IDLs in comparison with nontransgenic controls. The diameter of the
major VLDL peak, as assessed by GGE, was not different between HL
transgenic and control rabbits (ie, 448±4 versus 450±3 Å,
respectively). There was, however, a shift to smaller, denser LDL
particles (Sf° 4 to 6, Figure 2) in the HL transgenic rabbits.
Mean diameter of the major LDL GGE band (263±2 Å) was significantly
smaller than that in controls (290±3 Å, P<0.001).
In sharp contrast to the results for the HL transgenic rabbits, the
apoE transgenic animals exhibited near absence of large and medium
VLDLs with a selective accumulation of small VLDLs and IDLs (Figure 2). This finding was corroborated by GGE (results not shown),
which revealed a significantly smaller diameter of the major VLDL
species in the HL rabbits (334±4 Å) than in the controls
(P<0.0001). AnUC also indicated a shift toward LDLs of
higher Sf° in apoE transgenic rabbits than controls (Figure 3), but mean peak LDL particle diameter
as measured by GGE was not significantly larger than that in controls
(298±3 versus 290±4 Å, respectively).
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In the combined HL+apoE transgenic rabbits, amounts of all VLDL species were much lower than in the other groups, with little detectable large and medium VLDLs of Sf° >80 and with reduced IDLs and LDLs compared with controls. Peak particle diameters of the VLDL+IDL (320±4 Å) and LDL (272±3 Å) fractions were significantly smaller than those in controls (P<0.01 and 0.05, respectively).
DGUC of ApoB-Containing Lipoproteins
VLDL+IDL
Figure 3 displays mean protein concentrations for
d<1.019 g/mL lipoprotein subfractions separated by DGUC in
all groups of animals. Also shown are mean particle diameters of the
predominant peaks within these fractions, which permit their grouping
into large, medium, and small VLDLs and IDLs. Consistent with
the AnUC results, control rabbits had an accumulation of particles in
all VLDL fractions, with lower levels of IDLs. In the HL transgenic
rabbits, levels of all VLDL and IDL particles were reduced
significantly compared with controls, with the exception of fraction 1,
the largest fraction (diameter >400 Å), and of fraction 9, the
smallest fraction. For rabbits overexpressing apoE, levels of the 3
fractions having the largest particle diameter were significantly lower
than controls. In rabbits expressing both HL and apoE, DGUC
analyses again indicated very low concentrations of VLDL along
the entire density and size spectrum. GGE analysis of
individual DGUC fractions (data not shown) revealed that the higher
density VLDL fractions contained both small VLDLs and abnormally large
particles corresponding to the large VLDL peak (Sf° >400) observed
in whole plasma (Figure 2). The failure to detect such particles
as lipoproteins of Sf° >80 by analytic
ultracentrifugation (Figure 2) suggests abnormal
physical properties of these larger sized particles.
Mean concentrations of LDL-density subfractions are shown in Figure 4. Control rabbits accumulated
predominantly large LDLs, whereas HL rabbits had significantly lower
mass levels of large LDLs in fractions 1 through 3 and significantly
increased levels of smaller, denser LDLs in fractions 6 and 7 than
nontransgenic animals. Rabbits overexpressing apoE showed significant
reductions of mass of all LDL fractions except the largest and the
combined HL+apoE transgenic rabbits had significantly lower levels of
the large LDL subclasses compared with controls.
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HDL Subfraction Analyses
Distributions of protein mass within HDL subspecies as assessed by
GGE are shown in Figures 5 and 6. In nontransgenic controls, particles
corresponding to human HDL2b and
HDL2a were most abundant with lesser amounts of
larger sized particles, as well as a band corresponding in size to
human HDL3a. In HL transgenic rabbits, we found
significantly reduced concentrations in all the major subclasses, with
a marked narrowing of the particle size distributions corresponding to
HDL2b and HDL2a. ApoE
transgenic rabbits had a selective reduction in
HDL1 and HDL2b, with
nonsignificant changes in levels of smaller sized HDL particles and
combined HL+apoE transgenic rabbits showed a marked depletion of
particles across the spectrum of subclasses.
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Discussion |
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The cholesterol-fed rabbit has been used for many years as an experimental model for the study of diet-induced hypercholesterolemia and atherosclerosis.38 In previous studies in HL transgenic rabbits, we reported a marked reduction of IDLs and HDLs on a chow diet, as well as a reduced plasma cholesterol response to a high-cholesterol diet.14 15 Here, we have used these HL transgenic rabbits, as well as rabbits expressing high levels of human apoE,16 to determine the influence of these proteins singly and in combination on the plasma lipid and lipoprotein profiles induced by high-cholesterol feeding. We found that increased expression of either gene resulted in a substantial attenuation of the plasma cholesterol response to the diet and that there was a further dramatic reduction in this response in the HL+apoE double transgenic animals. This appeared to result from complementary and synergistic effects of HL and apoE on the metabolism of apoB-containing lipoproteins, as well as HDLs.
We demonstrated by three different methods (GGE, AnUC, and DGUC) that the diet-induced hypercholesterolemia in the nontransgenic rabbits was characterized by high levels of multiple distinct lipoprotein subpopulations: large, buoyant VLDLs; medium VLDLs; small, dense VLDLs; IDLs; and large, buoyant LDLs. Our results indicate that HL and apoE affect the metabolism of these subspecies differently. Increased expression of HL caused a reduction of all species except large VLDLs. In contrast, apoE transgenic rabbits had a marked reduction of these large VLDL particles, with selective accumulation of IDLs and large LDLs.
HL has both triglyceride hydrolase and phospholipase activities, allowing it to participate in the final steps of lipolysis initiated by lipoprotein lipase, mediating the conversion of IDLs to LDLs.1 39 40 41 In addition, it enhances cell association of triglyceride-rich lipoprotein remnants by initiating their binding to cell surface heparin sulfate proteoglycans.9 42 Our results showing significantly reduced levels of small VLDLs and IDLs in HL transgenic rabbits are consistent with these functions of HL and with earlier suggestions that endogenous HL deficiency in rabbits may contribute to their hyperlipidemic response to cholesterol feeding.43 44 The accumulation of very large lipoproteins in these animals suggests that HL does not interact significantly with these particles. It has been reported previously that chylomicrons are poor substrates for HL in comparison with smaller lipoproteins.45 In our study, samples were collected after an overnight fast, making it unlikely that our results are caused by the presence of significant amounts of chylomicrons in the VLDL fraction.46 47
The reduced levels of IDLs, and a shift toward smaller, denser LDL particles in the HL transgenic animals, are consistent with evidence that HL is involved in the production of smaller LDLs from IDLs and large LDL precursors.24 48 However, we found that plasma levels of LDLs were reduced in these animals. Therefore, either LDLs were cleared more rapidly or there was increased clearance of LDL precursors.
In humans, a strong relation has been shown between plasma triglyceride levels and LDL density, a phenomenon thought to reflect in part lipolysis of larger LDLs that have become enriched in triglyceride as a result of cholesteryl ester transfer protein activity.49 In this regard, it is noteworthy that reduced LDL size and density occurred with HL overexpression despite reduced plasma levels of triglyceride. This suggests that hepatic lipase may not require cholesteryl ester transfer protein–mediated triglyceride enrichment of LDLs to generate smaller, denser LDL particles.
The reduction of HDLs in the HL transgenic rabbits occurred in all size fractions, but there was a marked narrowing of the particle distribution within HDL2b and HDL2a. Preliminary data on HDL composition showed a low cholesteryl ester content in HL transgenic rabbits (data not shown), and it is possible that this contributed to reduced polydispersity in the HDL2b and HDL2a particle distributions. The basis for this is not evident, although it has been reported that HL may promote selective hepatic removal of HDL cholesteryl esters as a result of its phospholipase activity.50 It may also be that a subset of HDL2 particles are resistant to HL.
In contrast with HL transgenic rabbits, lower levels of total VLDL+IDL found in cholesterol-fed animals overexpressing apoE in this study were accounted for by a marked reduction in levels of large VLDL, presumably as a result of increased clearance. This mechanism is supported by the previous finding of an increased receptor affinity of VLDLs from apoE transgenic rabbits.16 Previous studies have indicated that apoE is more effective in promoting the clearance of larger than smaller VLDL particles.51 52 53 54 55 An enrichment of apoE on the lipoprotein surface relative to apoB or to apoCIII56 or, alternatively, differences in steric accessibility or domain availability may mediate apoE function on distinct VLDL subclasses. It also has been reported that lipoprotein lipase–mediated hydrolysis of large VLDLs may be influenced by VLDL apoE content.57
The increased removal of larger VLDLs in apoE transgenic rabbits may saturate or compete with mechanisms responsible for clearance of smaller remnant particles.58 This may explain the accumulation of triglyceride-enriched particles in the IDL/large LDL particle spectrum in the apoE transgenic animals.
Analyses of HDLs in apoE transgenic rabbits revealed a selective reduction in larger particles, including those corresponding to human HDL2b. It is known that apoE is preferentially transported on larger HDLs, including HDL1.59 Therefore, it is possible that overexpression of apoE increases the amount of apoE on these particles, and thereby enhances their plasma clearance by receptor-dependent pathways.
The complementary functions of HL and apoE in lipoprotein metabolism were further demonstrated by simultaneous expression of both proteins in transgenic rabbits, which led to marked reductions of lipoproteins across the entire size and density spectrum. However, the extent of the reductions in plasma lipoprotein concentrations suggests that the interaction of HL and apoE is not merely additive, but synergistic. ApoE and HL may cooperate in the VLDL clearance process by facilitating the association of VLDLs with heparin sulfate proteoglycans on cell surfaces. In a similar manner, it has been reported that chylomicron remnants that are partially depleted of phospholipid and triglyceride content by HL treatment may have an accelerated clearance due to an enhanced exposure of apoE.60 61 62 In the case of HDLs, the synergistic effects of HL and apoE resulted in extremely low plasma levels of all the major HDL subspecies. The basis for this effect, and its potential impact on the development of atherosclerosis in this model, remains to be clarified.
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Acknowledgments |
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This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Division of the US Department of Energy under contract no. DE-AC03-76SF00098; by NIH grants HL 18574 (R.M.K.) and HL 51588 (J.M.T.); and by a grant from the National Dairy Promotion and Research Board, administered in cooperation with the National Dairy Council. Dr Barbagallo was the recipient of grants from Consiglio Nazionale delle Ricerche, Rome, Italy, and from the International Atherosclerosis Society, Houston, Tex. The authors thank Laura Holl and Joseph Orr for laboratory analyses, Ricky Quan for rabbit management, and Linda Abe for preparation of figures.
Received January 16, 1998; accepted August 19, 1998.
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