Hepatic lipase affects both HDL and ApoB-containing lipoprotein levels in the mouse

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Hepatic lipase a¡ects both HDL and ApoB-containing lipoprotein levels in the mouse Sylvie Braschi a , Nicole Couture a , Adriana Gambarotta a , Benoit R. Gauthier a , Cynthia R. Co¤ll a , Daniel L. Sparks a , Nobuyo Maeda b , Joshua R. Schultz a; * a

Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Departments of Pathology and Laboratory Medicine, and Biochemistry, University of Ottawa, H445A, 1053 Carling Avenue, Ottawa, Ont. K1Y 4E9, Canada b Department of Pathology, University of North Carolina, Chapel Hill, NC 27599-7525, USA Received 12 February 1998; accepted 17 March 1998

Abstract Transgenic mice were created overproducing a range of human HL (hHL) activities (4^23-fold increase) to further examine the role of hepatic lipase (HL) in lipoprotein metabolism. A 5-fold increase in heparin releasable HL activity was accompanied by moderate (approx. 20%) decreases in plasma total and high density lipoprotein (HDL) cholesterol and phospholipid (PL) but no significant change in triglyceride (TG). A 23-fold increase in HL activity caused a more significant decrease in plasma total and HDL cholesterol, PL and TG (77%, 64%, 60%, and 24% respectively), and a substantial decrease in lipoprotein lipids amongst IDL, LDL and HDL fractions. High levels of HL activity diminished the plasma concentration of apoA-I, A-II and apoE (76%, 48% and 75%, respectively). In contrast, the levels of apoA-IV-containing lipoproteins appear relatively resistant to increased titers of hHL activity. Increased hHL activity was associated with a progressive decrease in the levels and an increase in the density of LpAI and LpB48 particles. The increased rate of disappearance of 125 I-labeled human HDL from the plasma of hHL transgenic mice suggests increased clearance of HDL apoproteins in the transgenic mice. The effect of increased HL activity on apoB100-containing lipoproteins was more complex. HL-deficient mice have substantially decreased apoB100-containing low density lipoproteins (LDL) compared to controls. Increased HL activity is associated with a transformation of the lipoprotein density profile from predominantly buoyant (VLDL/IDL) lipoproteins to more dense (LDL) fractions. Increased HL activity from moderate (4-fold) to higher (5-fold) levels decreased the levels of apoB100-containing particles. Thus, at normal to moderately high levels in the mouse, HL promotes the metabolism of both HDL and apoB-containing lipoproteins and thereby acts as a key determinant of plasma levels of both HDL and LDL. z 1998 Elsevier Science B.V. Keywords: HL transgenic and de¢cient mice; Apolipoproteins A-I, A-II, A-IV, B100/48 and E

1. Introduction Human hepatic lipase (hHL) is a 476 amino acid

* Corresponding author. Fax: +1 (613) 761-5281; E-mail: [email protected]

glycoprotein with an apparent molecular mass of approx. 65 kDa. It is synthesized and secreted by liver parenchymal cells into hepatic sinusoids, where it binds to heparan sulfate proteoglycans (HSPGs) anchored on hepatocytes and endothelial cells [1,2]. HL has two known catalytic activities, glyceride hydrolase and phospholipase, that catalyze the hydrol-

0005-2760 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 4 6 - 0

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ysis of glycerolipids of plasma lipoproteins speci¢cally mono-, di-, and triacylglycerol and phospholipid (PL) in intermediate density lipoprotein (IDL), VLDL, chylomicron remnants, and the high density lipoproteins (HDL) [3^7]. HL hydrolyzes TG and PL in HDL2 and apoE-rich HDL1 and is important in HDL remodeling and hepatic HDL cholesterol uptake after cholesteryl ester transfer protein (CETP)mediated transfer of TG into HDL in exchange for cholesteryl ester (CE) [8,9]. The variation in HL and apoA-I genes is reported to be a major cause of genetically determined variation in plasma HDL cholesterol levels in humans [10,11]. However, our understanding of the role of HL in human lipoprotein metabolism and atherosclerosis is complex and certain aspects remain unclear. Common features among subjects with complete HL de¢ciency include elevated fasting serum TG levels, elevated plasma cholesterol, an accumulation of TG-rich remnant particles in the density ranges of IDL and LDL and premature atherosclerosis [5,12^15]. Patients de¢cient for HL have low LDL but accumulate cholesterol-rich, L very low density lipoproteins (L-VLDL). In addition, HL de¢ciency in humans has been associated with the accumulation of IDL resulting from a lack of VLDL to IDL conversion. The increase in plasma concentrations of L-VLDL suggests that HL also plays an important role in the hepatic clearance of VLDL remnants. In support of this, injection of speci¢c HL antisera into fasted and lipid-fed animals (cats, rats and monkeys) results in the accumulation of both VLDL and chylomicron remnants in plasma [6,16^18]. HL may contribute to remnant clearance via a hepatic receptor-mediated uptake pathway through the low density lipoprotein (LDL) receptor-related protein (LRP), which also appears to have a¤nity for HL [19^21]. Although HL has no speci¢c apoprotein dependence, such as the dependence of LpL on apoC-II, one study has suggested that the 12 kDa C-terminal fragment of apoE (residues 192^299) activates its phospholipase activity [22]. The common apoE isoforms di¡er signi¢cantly in their ability to stimulate hydrolysis of HDL-associated PL (apoE3 s apoE2 s apoE4). Evidence also appears to suggest that the apoA-IV [23] and apoA-II [24^28] composition of plasma lipoproteins may modulate HL activity. The in£uence of apoA-II is currently unclear, with some

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studies suggesting an activating [24^26] and others supporting an inhibiting role [27,28]. The current study was carried out utilizing newly produced mice overproducing a range of hHL activities and previously derived HL-de¢cient (HL3/3) mice [29] to further characterize the in£uence of HL on lipoprotein metabolism and to determine whether apoprotein-speci¢c lipoprotein populations could be identi¢ed as preferred substrates of HL in vivo. The results indicate that HL functions in a somewhat indiscriminate manner with regard to HDL apoprotein composition and appears to e¡ectively enhance HDL clearance rates. We also provide evidence from HL-de¢cient compared to control and the hHL transgenic mice which supports the important role that HL plays in a¡ecting the plasma levels and densities of apoB-containing VLDL/LDL. 2. Methods 2.1. Production of transgenic mice Fertilized eggs were collected from 6-week-old superovulated C57BL/6 females after mating overnight with C57BL/6 males (all production animals were obtained from Charles River Breeding Laboratory, Wilmington, MA). A 9.5 kb SalI-SpeI fragment containing approx. 3 kb of 5P-£anking sequence, the ¢rst exon, ¢rst intron, the ¢rst 6 nucleotides of the second exon, a full-length 1.6 kb human HL cDNA, a 254 bp polyadenylation signal from apoE and the 3.8 kb hepatic control region (LE1) was isolated away from the pLIV10.hHL parent vector (pBSSK plasmid, Stratagene, La Jolla, CA) (kindly provided by J. Taylor, Gladstone Institute, San Francisco, CA [30] and H. Will, University of Hamburg, Germany). DNA used for embryo microinjection was puri¢ed from agarose gels using GeneCapsule (Geno Technology, St. Louis, MO), microdialyzed, and resuspended at 3 Wg/ml in 10 mM Tris (pH 7.4), 0.10 mM EDTA. The puri¢ed heterologous DNA construct was microinjected into the male pronucleus of fertilized eggs and surviving eggs were reimplanted by oviduct transfer into pseudo-pregnant CD1 females. Integration of the human DNA into the murine genome was determined by the method of Southern [31] using 10 Wg of mouse tail DNA, digested

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with SacII, electrophoresed on 0.8% agarose gels, and transferred to nylon membranes (HyBond-N, Amersham Life Sciences). Membranes were hybridized overnight (in 6USSC, 5UDenhardt's and 100 mg/ml salmon sperm DNA at 42³C) to a 410 bp random primed 32 P-labeled SacII fragment prepared from the human HL cDNA, and washed, dried and exposed for 24^36 h. Transgene copy number was estimated by densitometric scanning of representative Southern blots. Increased transgene copy number, as determined by this semi-quantitative determination, roughly correlates with increased post-heparin plasma HL activity. Subsequent analysis focused primarily on transgenic progeny of the male founder lines hHL6784+ (hemizygote with moderate expression; 4^5 copies; designated hHLmed‡ ), hHL6784+/+ (homozygote of hHL6784+ with moderately high expression; 8^10 copies; hHLmed‡=‡ ) and 6881 (highest expression; 30^35 copies; hHLhi‡ ), as well as HL3/3 mice [29]. Mice were maintained on 12 h light/dark cycles (7.00^19.00 h) with free access to food (standard chow diet) and water. All animal care and treatment protocols were approved by the University of Ottawa Animal Care Committee in accordance with guidelines established by the Canadian Council on Animal Care. 2.2. RNA extraction, puri¢cation and Northern blot analysis Total RNA was isolated from age- and sexmatched control and transgenic mice tissues (e.g., adrenal, brain, heart, intestine, liver, lung, kidney, testes etc.) using the TRI reagent (Molecular Research Center, Cincinnati, OH) [32,33]. Polyadenylated mRNA [poly(A)‡ mRNA] was isolated using the Poly(A)‡ Track kit (Promega, Madison, WI). Mouse and human HL and mouse GAPDH gene expression were assessed by Northern blot analysis using random primed, 32 P-labeled mHL (669 bp PstI fragment), hHL (410 bp SacII fragment) and GAPDH (1.2 kb PstI-XbaI fragment) cDNA fragments. Based on our experience using the speci¢ed hHL cDNA probe hybridized to nontransgenic control mouse RNA, we estimate that the degree of cross-hybridization is less than 3% and therefore we consider these probes to be essentially species-specific.

2.3. Plasma hepatic lipase activity Total HL activity was assayed in post-heparin plasma by incubation with a radiolabeled triolein substrate in the presence of 2 M NaCl as previously described [34^36]. To determine total plasma HL activity, mice were fasted for 6 h and injected intraperitoneally with heparin (500 IU/kg). Results are expressed as nanomoles of free fatty acids (FFA) released per minute per milliliter of plasma (nmol FFA/min/ml). 2.4. Lipid and lipoprotein analyses Plasma lipid and lipoprotein analyses were determined after a 6 h fast from blood obtained via tail vein bleeding. Lipoprotein lipid analysis was run with a group of six individual mice (for transgenics and controls). The exception to this was the highest expressing hHLhi‡ founder line, a male that did not produce progeny. Total cholesterol (TC), phospholipid (PL) and triglyceride (TG) levels in plasma were determined enzymatically with reagents from Boehringer (Indianapolis, IN), Wako (Richmond, VA) and Sigma (St. Louis, MO), respectively. Total TG levels in plasma were corrected for the plasma free glycerol levels, which was measured with enzymatic reagents from Sigma (St. Louis, MO). All plasma lipid determinations were done in triplicate for each mouse group and the results are expressed as mean þ S.D. of the six individual animals in each group. Plasma HDL was separated from apoB-containing lipoproteins by dextran sulfate precipitation as described previously using 60 Wl of plasma for each mouse [37]. HDL-cholesterol (HDL-C) was subsequently determined using the Boehringer kit (Indianapolis, IN). Lipoprotein fractions were separated by either discontinuous [38] or continuous [39] gradient ultracentrifugation in a L8-70M ultracentrifuge (Beckman) according to a modi¢cation of the procedure of Lindgren et al. [38]. Plasma was collected (100 Wl per mouse) from six individual mice of each group and pooled to a total of 600 Wl of plasma per group. The highest expressing male founder hHLhi‡ and an age-matched C57BL/6 male control were bled 4 times and plasma was kept at 380³C until 600 Wl of plasma was available for analysis. Total cholesterol,

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phospholipid, triglycerides and glycerol were determined in triplicate from undiluted samples in each fraction with the same enzymatic kits that were used for plasma. 2.5. Determination of plasma apolipoprotein concentrations Plasma samples were pooled (as described above) from control, hHLmed‡ , hHLmed‡=‡ , and hHLhi‡ transgenic mice and concentrations of mouse apoAI, A-II and E were determined by radial immunodiffusion (RID) assays, essentially as described [40] but with the following modi¢cation for apoE determination (as communicated by B. Ishida, University of California, San Francisco, CA). Assay of mouse apoE by RID requires the presence of lipid to stabilize apoE conformation and Triton X-100 to unmask cryptic epitopes. Accordingly, diluent for apoE plasma samples and calibration standards contained 0.15 M NaCl, 1 mM EDTA, 0.2% NaN3 containing 10% (v/v) human plasma and 1% Triton X-100. Immunodi¡usion plates were incubated at 37³C for 24 h. Puri¢ed apoA-I, A-II or E (kindly provided by B. Ishida, UCSF) were included on each plate as calibration standards and RID immunoprecipitates were stained with Coomassie blue R250. The lower limit of sensitivity of this RID assay is 0.10 mg/dl. ApoAIV did not stain well upon RID analysis and therefore no quantitative data were obtainable. 2.6. Agarose gel electrophoresis and immunoblotting Fresh plasma samples (kept brie£y on ice at 4³C) were drawn and loaded onto agarose gels (Paragon Lipo Kit, Beckman) (within 30 min of sampling). Duplicate samples were electrophoresed simultaneously on two separate gels, and after electrophoresis one of the gels was directly transferred to polyvinylidene di£uoride (PVDF) microporous membranes (Immobilon, Millipore, Bedford, MA) for immunoblotting while the second gel was ¢xed and stained (5 min) with a neutral lipid stain. The gel and corresponding immunoblots shown (e.g., in Fig. 8) are representative of (at least three) repeated analyses. Apolipoprotein composition of lipoproteins separated by agarose gel electrophoresis were qualitatively analyzed from immunoblots and identi¢ed by

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blotting with rabbit K-mouse apoA-I, apoA-II, apoA-IV (kindly provided by G. Castro, Institut Pasteur de Lille, France), rabbit K-mouse apoE and rabbit K-mouse apoB (recognizing both B48 and B100) (BioDesign, Kennebunk, ME), and a monoclonal antibody against mouse apoB100 (kindly provided by Drs. L. Flynn and S. Young, Gladstone Institute, San Francisco, CA). Visualization of immunodetected apolipoproteins was carried out using ECL Western blotting detection reagents (RPN 2106; Amersham Life Sciences) in combination with the appropriate primary and horseradish peroxidase linked secondary antibodies. 2.7. Polyacrylamide gel electrophoresis The apoprotein content of lipoproteins was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting of total plasma lipoproteins (d 9 1.21 g/ml) isolated from plasma by ultracentrifugation at d = 1.21 g/ml in a TLA100 rotor (using a Beckman tabletop TL100 ultracentrifuge) at 40 000 rpm for 18 h (10³C). Proteins were stained with 0.25% Coomassie R250. For detection of hHL and estimation of hHL mass, a monoclonal antibody HLX3-6 [41] (generously supplied by Dr. A. Bensadoun) was 125 I-radiolabeled using chloramine-T radiolabeling, [42] immunoreacted with plasma samples and detected by autoradiography. 2.8. HDL clearance study Human HDL was isolated by preparative ultracentrifugation (d = 1.09^1.21 g/ml) and dialyzed against a bu¡er containing 0.15 M NaCl, 0.01 M phosphate and 0.02% sodium azide (pH 7.4). HDL apoprotein was iodinated by a modi¢cation of the iodine monochloride method of McFarlane [43] using 125 I as tracer. The purity of the labeled HDL was assessed by polyacrylamide and agarose gel electrophoresis. Approx. 1U106 dpm (approx. 2.5 Wg) of 125 I-HDL was injected into the tail vein of hHLmed‡ and hHLmed‡=‡ transgenic and C57BL/6 control mice (n = 4 for all three groups). Mice were fasted throughout the 27 h study period but had free access to water. Blood was withdrawn from the retroorbital plexus at 10 min, 1 h, 3 h, 6 h, 9 h, 24 h and 27 h after tracer injection. Aliquots of plasma were assayed for

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125

I-HDL radioactivity. HDL fractional catabolic rates (FCR) were calculated from the plasma decay curves of 125 I-HDL assuming a two-pool model as described by Matthews [44]. 2.9. Statistical analysis HL activity and mouse lipoprotein and lipid data are expressed as means þ S.D. Data medians were compared with the nonparametric Mann-Whitney statistic test between controls and hHL transgenic mice. The exception was the highest expressing male hHLhi‡ for which (taking into account n = 1 for this line) no statistical test was performed. Instead, the data for hHLhi‡ are reported as the mean þ S.D. of three determinations. Statistical signi¢cance was de¢ned as P 6 0.05. 3. Results 3.1. Tissue distribution of HL mRNA and post-heparin HL activities Analysis of the expression of the endogenous mHL and hHL transgene by Northern blot analysis was carried out using total and highly enriched poly(A)‡ RNA from various tissues (heart, lung, adrenal, testes, intestine, spleen, adipose, brain, liver) and species-speci¢c probes. Expression of the endogenous mHL gene was primarily liver-speci¢c; however, low levels of mHL mRNA were also found in the adrenal glands of nontransgenic C57BL/6 control mice (Fig. 1, top panel). In transgenic mice, in addition to the high liver expression, abundant levels of the approx. 1.6 kb full-length transgene mRNA were detected in the adrenals and testes of all transgenic mouse lines examined (Fig. 1, lower panel, rep-

Fig. 1. Northern blot analysis of poly(A)‡ mRNA isolated from representative C57BL/6 control (top) and hHLmed‡ transgenic (bottom) mouse tissues. Approx. 5 Wg of mRNA from the spleen, adrenal, testes and liver were hybridized with the mHL, hHL and GAPDH 32 P-labeled cDNA probes as indicated. The amount of RNA loaded was approximately equivalent as determined by GAPDH expression (lower image of top and bottom panels).

resentative hHLmed‡ mouse RNA samples are shown). Human HL expression in the transgenic mice was accompanied by signi¢cant increases in total postheparin plasma HL activity (Fig. 2). The HL activities were 68 þ 11 (in male control mice), 273 þ 37 (hHLmed‡ ), 342 þ 77 (hHLmed‡=‡ ) and 1568 þ 151 nmol FFA/min/ml (hHLhi‡ ) (the HL activity reported in transgenic mice represents the sum of endogenous and human HL activity). Pairwise comparisons were signi¢cant between both hHLmed‡ and hHLmed‡=‡ transgenic groups and controls

Table 1 Concentration (in mg/dl) of apolipoproteins remaining in plasma of HL3/3, control and hHL transgenic mice after a 6 h fast Micea

HL3/3

C57BL/6

hHLmed‡

hHLmed‡=‡

hHLhi‡

A-I A-II E

121 þ 12 37 þ 3 1 þ 0.5*

115 þ 9 33 þ 3 8þ1

98 þ 6 33 þ 2 10 þ 2

71 þ 4* 28 þ 2 7þ1

27 þ 2** 17 þ 2* 2 þ 0.7*

Values are means þ S.D. (n = 3 determinations). Means are signi¢cantly di¡erent from controls (*P 6 0.05; **P 6 0.005). a HL3/3, hepatic lipase-de¢cient mice; C57BL/6, control mice, hHLmed‡ , hHLmed‡=‡ and hHLhi‡ , transgenic mice.

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Fig. 2. Total HL activity (mouse plus human) was assayed in post-heparin plasma from control males and females (white boxes) and independently derived hHL transgenic lines (grey boxes) expressing moderate (hHLmed‡ ), moderately high (hHLmed‡=‡ ), and high HL (hHLhi‡ ) activity. Determinations were performed in triplicate for the control, hHLmed‡ , and hHLmed‡=‡ groups and the means are expressed as nanomoles of fatty acids (FA) released per minute per milliliter of plasma (nmol FFA/min/ml) þ S.D. of the six individual animals in each of these groups. The data for the hHLhi‡ animal (n = 1) are reported as the mean þ S.D. of three independent determinations.

(P 6 0.01). A signi¢cant di¡erence was also noted in HL activity between male and female C57BL/6 nontransgenic control mice (68 þ 11 vs. 88 þ 13 nmol FFA/min/ml) (P 6 0.01). 3.2. E¡ect of increased hHL activity on plasma and lipoprotein lipid composition To assess the e¡ect of hHL overproduction on plasma lipid and lipoprotein lipid pro¢les, mice fed a regular chow diet were bled after a 6 h fast and plasma lipid and lipoprotein pro¢les were determined. We found that moderately high increases in heparin releasable HL activity, up to the 5-fold in-

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crease seen in hHLmed‡=‡ , were associated with modest yet signi¢cant decreases in plasma total and HDL cholesterol and phospholipids (19% and 18%, and 20%, respectively; Fig. 3) but no signi¢cant e¡ect on TG. The highest hHL expression, 23-fold increased in hHLhi‡ , had a more substantial e¡ect on plasma total and HDL cholesterol, PL and TG (77%, 64%, 60%, and 24% decrease, respectively; Fig. 3), and a signi¢cant decrease in lipoprotein lipid concentrations (Fig. 4). The high expression signi¢cantly diminished the cholesterol in apoE-rich and apoAI-rich mouse HDL (d = 1.032^1.097 g/ml) but not in the more dense HDL fractions (d = 1.134 g/ml; upper pro¢le of Fig. 4). The PL in apoE-rich HDL (d = 1.032^1.061) but not in the more apoA-I-rich lipoproteins (d = 1.097^1.134 g/ml) was also diminished in hHLhi‡ (Fig. 4, lower pro¢le). The TG content of a number of fractions of hHLhi‡ was diminished, most notable being that of the IDL (d = 1.013 g/ml) and LDL (d = 1.032 g/ml). 3.3. Analysis of plasma apolipoprotein concentrations and distribution 3.3.1. E¡ect of HL on the HDL apoprotein content The e¡ect of increasing HL activity on plasma apolipoprotein concentration was analyzed by quantitative RID analysis. In this analysis, HL-de¢cient mice (HL3/3) were included in the analysis as a point of reference [29]. The data for the concentrations of apoA-I, A-II and E are shown in Table 1. Overall, increases in HL activity were associated with a signi¢cant net decrease in apoA-I (77%), apoA-II

Fig. 3. Plasma lipid concentrations [total cholesterol (T-CH), HDL cholesterol (HDL-CH), phospholipid (PL) and triglyceride (TG)] represent the mean þ S.D. of six animals in each group (with the exception of the hHLhi‡ animal (n = 1), which represents the mean þ S.D. of three independent determinations). *Pairwise comparisons are signi¢cant between both hHLmed‡ and hHLmed‡=‡ transgenic groups and controls (P 6 0.01).

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Fig. 4. Lipid composition of lipoprotein density fractions isolated by continuous density gradient ultracentrifugation of C57BL/6 control vs. hHLhi‡ transgenic mice. Pooled plasma samples were used from the highest expressing male founder, hHLhi‡ (bled 4 times and pooled plasma was kept at 380³C until 600 Wl of plasma was available for analysis) and an agematched C57BL/6 male control (also frozen before ultracentrifugation). The data represent the mean of triplicate lipid determinations.

(48%) and apoE (75%) when comparing the C57BL/6 control plasma apoprotein concentrations to those of hHLhi‡ (Table 1). Extremely low levels of total plasma apoE were found in HL3/3 mice compared to controls and low expressing hHL transgenic mice (approx. 80% less in HL3/3). Mouse plasma apolipoprotein content of plasma lipoproteins (d 9 1.21 g/ml) was also analyzed by

SDS-PAGE, agarose gel electrophoresis and immunoblotting. Increased hHL was associated with decreased plasma levels of apoA-I, apoA-II, apoE and the apoC proteins in hHLmed‡ , hHLmed‡=‡ and hHLhi‡ transgenic mice (not shown). Analysis of apoprotein density distributions associated mainly with HDL (apoA-I, apoE and apoA-IV) was also carried out by sucrose density gradient ultracentrifugation fraction, SDS-PAGE, and immunoblotting. Fig. 5 shows that increasing HL activity results in a skewing of the apoA-I distribution towards the higher density lipoprotein fractions and an overall reduction in apoA-I levels with increased titer of HL. In contrast, the apoE distribution remained relatively constant while the total level of lipoproteinassociated apoE was reduced in all density fractions. The distribution and levels of apoA-IV remained relatively unchanged with increasing levels of hHL activity. Plasma lipoproteins analyzed by 15% (Fig. 6, lower panel) and 5% (Fig. 6, upper panel) SDS-PAGE were transferred to PVDF membrane for immunoblot analysis of apoproteins A-IV, E, and B100/48. The levels of apoA-IV are substantially increased in HL3/3 mice compared to C57BL/6 controls (approx. 3^4-fold), but remain virtually constant across control and hHL transgenic mice (Fig. 6, lower panel). In contrast to apoA-IV, increased hHL activity appeared inversely associated with plasma apoB48

Fig. 5. Sucrose density gradient pro¢les of apoproteins A-I, E and A-IV isolated from HL3/3, control, and hHLmed‡ and hHLmed‡=‡ transgenic mice. Pooled plasma samples were fractionated as described, concentrated on fumed silica [39] and resolved by 15% SDSPAGE and apoproteins visualized by immunoblotting. The average densities of each fraction (mean value of three gradients) are as follows : fractions: 1, d = 1.01; 3, d = 1.02; 5, d = 1.04; 7, d = 1.07; 9, d = 1.12; 10, d = 1.16; 12, d = 1.22.

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Fig. 6. Plasma lipoproteins (d 9 1.21) analyzed by 5% (upper panel) and 15% (lower panel) SDS-PAGE and transferred to PVDF membrane for immunoblot analysis of apoproteins B100 (550 kDa), B48 (220 kDa), A-IV (43 kDa), and E (34 kDa).

levels. HL3/3 mice had relatively high apoB48 that is progressively diminished with the increased HL activity of controls and transgenic mice. ApoE was very low in HL3/3 mice, relatively higher in control and low expressing hHL transgenic mice, and substantially reduced in the highest expressing sample, again supporting the previously described plasma RID results (Table 1). We hypothesized that the diminished HDL-associated apoprotein concentrations (A-I, A-II and E) observed with increased titers of HL activity were associated with enhanced clearance rates from the plasma compartment. To evaluate the e¡ect of hHL on the removal of HDL-associated protein in vivo, human HDL was labeled with 125 I and injected into the tail vein of hHL transgenic and C57BL/6 control mice. The disappearance of radioactivity from plasma was followed for 27 h. The plasma clearance rate for exogenously labeled human HDL (Fig. 7) was signi¢cantly increased in the hHLmed‡ and hHLmed‡=‡ transgenic mice compared to C57BL/ 6 controls (FCR values of 0.708, 0.875 and 0.472, pool/day, respectively). 3.3.2. E¡ect of HL on apoB composition Immunoblotting with a polyclonal K-apoB48/B100 antiserum (Fig. 6, upper panel) revealed signi¢cantly diminished apoB100 in HL3/3 mice compared to

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controls. The increased HL activity in controls and hHLmed‡ mice was associated with appreciably increased amounts of apoB100 over that seen in the HL3/3 mice. We observed an average 3-fold increase in the levels of apoB100 (HL3/3 compared to C57BL/6) upon densitometric scanning of four gels using plasma and lipoprotein samples prepared from di¡erent animals on separate occasions. In contrast, increased HL activity from moderate (4-fold) in hHLmed‡ to moderately higher levels (5-fold) in hHLmed‡=‡ was consistently associated with modest decreases in plasma apoB100 (1.4-fold). The relative amount of plasma apoB100 in the highest expressing line (Fig. 6, upper panel) is obscured by the greatly retarded electrophoretic migration (arrow). The cause of this anomalous migration, which occurred consistently, increased with increasing hHL, and was nearly absent in HL3/3 mice, remains unclear. Densitometric scanning and summing of the distinct apoB100 band together with the aggregated (di¡use band) indicates that the amount of apoB100 (in hHLhi‡ ) actually increases (approx. 1.5-fold) over the C57BL/6 controls. The extent of the reduction of apoB100 in HL3/3 mice observed by SDS-PAGE was somewhat unexpected and therefore we examined plasma samples to corroborate these ¢ndings. The apoprotein composi-

Fig. 7. Radiolabel HDL decay curve in control and transgenic mouse. Mice were injected with 125 I-HDL (1U106 dpm) via the tail vein and blood (approx. 50 Wl) was withdrawn from the retroorbital plexus for determination of radioactivity. Values are the means þ S.D. for C57BL/6 control (b), hHLmed‡ (F), and hHLmed‡=‡ (R) transgenic mice (n = 4 for each group). Pairwise comparisons are signi¢cant between both hHLmed‡ and hHLmed‡=‡ transgenic groups and controls (P 6 0.01 and 0.008, respectively).

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Fig. 8. The neutral lipid and apoprotein distribution on plasma lipoproteins analyzed qualitatively by agarose gel electrophoresis and immunoblotting of plasma from HL3/3, C57BL/6 control, hHLmed‡ , hHLmed‡=‡ , and hHLhi‡ transgenic mice after fasting. The e¡ect of progressive increases in HL activity (from the HL3/3 to hHLhi‡ ) is displayed from left to right across the panels shown. In the upper panel the neutral lipid stained plasma lipoprotein pro¢les are shown. The lower panel was immunoblot speci¢cally for apoB100.

tion and distribution amongst plasma lipoproteins was resolved by agarose gel electrophoresis and immunoblotting (Fig. 8). The e¡ect of increasing HL activity on neutral lipid-stained plasma lipoprotein pro¢les is shown for reference (Fig. 8, top panel). As expected, HL3/3 mice had relatively high levels of K-migrating lipoproteins (apoAI- and apoE-containing HDL, not shown). The relative amount and migration of K-migrating neutral lipid material de-

creased with the increasing HL activity of hHLmed‡ and hHLhi‡ transgenic mice. The apoB100 distribution was inferred from immunoblotting with a monoclonal antibody that reacts speci¢cally with murine apoB100 (prepared and kindly supplied by L. Flynn and S. Young, Gladstone Institute, San Francisco, CA). ApoB100-containing (L-migrating) particles increased in the plasma of HL3/3 to control and hHLmed‡ transgenic mice. These L-migrating particles, based on immunoblot analysis, represent apoB100-containing LDL. This analysis corroborates the di¡erences observed in plasma apoB100 levels between HL3/3, control and hHLmed‡ transgenic mice noted by SDS-PAGE (Fig. 6). In addition, agarose gel electrophoresis also reveals consistent changes in the electrophoretic mobility of (native) LDL (Fig. 8, lower panel). Increases in HL activity result in a faster, more negative LDL particle migration [average surface potential increase from 34.4 mV (HL3/3) to 34.9 mV (hHLhi‡ )]. 3.3.3. E¡ect of strain variation It should be noted that the HL3/3 mice were originally generated using embryos from the 129 strain and subsequently bred for a number of generations (at least six) into the C57BL/6 strain. Strain variation has previously been associated with di¡erences in the levels of apoA-IV mRNA in the liver (6^ 8-fold) and intestine (2-fold) of 129 and C57BL/6 strains [45]. This variation in expression results in strain 129 exhibiting a 3-fold higher concentration of apoA-IV protein in the circulation. We therefore considered the possibility that the di¡erences ob-

Fig. 9. Plasma lipoproteins (d 9 1.21) isolated from three representative B6129F2/J, C57BL/6J and HL-de¢cient mice analyzed by 5 and 15% SDS-PAGE and transferred to PVDF membranes for immunoblot analysis of apoproteins B100 (550 kDa), B48 (220 kDa), A-IV (43 kDa), and E (34 kDa).

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Fig. 10. Distribution of plasma lipoproteins containing apoB isolated from HL3/3, control, hHLmed‡ and hHLmed‡=‡ transgenic mice analyzed by continuous sucrose gradient density centrifugation. Pooled plasma samples were fractionated as described, concentrated on fumed silica, and resolved by 5% SDS-PAGE and apoB100/48 visualized by immunoblotting. d 6 1.006, chylomicron remnants, VLDL; 1.006 6 d 6 1.02, IDL; 1.02 6 d 6 1.04, LDL, large HDL; 1.04 6 d 6 1.08, LDL, HDL1 , HDL2 ; 1.08 6 d 6 1.10, apoA-I-rich HDL; 1.10 6 d 6 1.21, HDL2 , HDL3 ; d = 1.21, free apoproteins [63].

served in apoB100/B48, apoE and apoA-IV, between the HL3/3 mice and the C57BL/6 controls were in£uenced by genetic di¡erences associated with strain variation and not necessarily the HL de¢ciency. To examine this possibility, apoprotein levels were compared in the hybrid B6129F2/J, pure C57BL/6 and the HL3/3 mice by SDS-PAGE (Fig. 9). The variation in apoA-IV levels previously noted between the 129 and C57 strains also appears to exist between the hybrid B6129F2/J and C57BL/6 strains in our current analysis. Based on densitometric scanning of three separate gels (nine individual animals from each strain were examined in total; three animals of each strain are represented in Fig. 9) we observed an approx. 4-fold increased apoA-IV level in the B6129F2/J compared to the C57BL/6 mice. HL3/3 mice have similar apoA-IV levels as the B6129F2/J hybrid. In contrast, the levels of apoB100/48 and apoE are indistinguishable between the B6129F2/J and C57BL/6J mice. HL de¢ciency

appears to be associated with signi¢cantly reduced apoB100 and apoE (3^4-fold) and increased apoB48 (2-fold). This analysis suggests that, unlike apoA-IV, the noted di¡erences in apoB100/B48 and apoE are associated with HL and most likely not due to strain variation. 3.3.4. E¡ect of HL on the density distribution of LpB lipoproteins Our analysis demonstrated that apoB100 was diminished in HL3/3 mice, increased in controls and moderately expressing hHLmed‡ mice. One possible explanation for this observation could be that the lack of HL results in reduced VLDL to LDL conversion in the mouse. To determine whether HL activity in£uenced the lipoprotein density distribution, we again utilized continuous sucrose density gradient centrifugation, SDS-PAGE and immunoblotting of plasma lipoproteins from HL3/3, nontransgenic control, and hHLmed‡ and hHLmed‡=‡ mice

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(Fig. 10). This analysis demonstrates that, in the absence of HL, the distribution of apoB100 and apoB48 lipoproteins is skewed towards the more buoyant (VLDL) fractions (Fig. 10, top panel, fractions 2^4). In HL3/3 mice there is an accumulation of IDL in fraction 3 with some buoyant LDL visible in fraction 4 (d = 1.02^1.03 g/ml). The apoB48 lipoproteins are found mainly in fractions 1^5. In contrast, the distribution of apoB100/LDL from nontransgenic control mice shifts down (right in Fig. 10) towards the more dense fractions. This is most evident in the apoB48-containing particles that become distributed throughout fractions 1^10. The expression of hHL drives the LpB100 distribution in hHLmed‡ transgenic mice further towards the more dense LDL populations (fractions 3^6; d = 1.03^1.06 g/ml). Likewise, the distribution of apoB48 is also skewed towards the higher density fractions, mainly 3^10. In the homozygote, hHLmed‡=‡ , the proportion of LpB100 increases in the more dense LDL fractions (4 and 5). The distribution of apoB48 is completely depleted in the buoyant fractions (1 and 2) and becomes strikingly skewed towards the more dense fractions (6^10). It is also of interest to note that fractionation of plasma lipoproteins from the transgenics consistently resulted in the appearance of two fragments that are compatible with the welldescribed kallikrein proteolytic cleavage products (apoB74 and apoB26) in fractions 3^6 [46,47]. This suggests that hHL-mediated lipolysis of plasma LDL may increase the susceptibility of apoB100 to proteolysis. Furthermore, the higher hHL activity of hHLmed‡=‡ mice resulted in the presence of apoB100/B48 in the bottom fractions (11 and 12). We speculate that this may be characteristic of apoB aggregates that have pelleted along with the lipid-poor fraction. Overall, the distributions of LpB (and LpAI particles; Fig. 5) in mice with and without HL, and with increased hHL indicates that increased HL activity promotes a change in the distribution of buoyant lipoproteins to more dense, lipid-poor particles. 4. Discussion The utility of expressing human lipoprotein-associated enzymes and apoproteins to address issues re-

lated to lipoprotein metabolism has been validated in numerous transgenic mouse models. These studies have supported e¡orts to elucidate the functional importance of speci¢c interactions between plasma apoproteins and enzymes [27,37,48^50], corroborated the e¡ects on lipoprotein levels, physical properties [4,27,30,37,40,48^50] as well as determined their direct in£uence on atherogenesis [51^53]. In the course of examining a series of mice with varying levels of HL activity we have found that, in addition to corroborating the important role that HL plays in plasma HDL metabolism, it also appears to act as a key determinant of plasma LDL levels. Numerous studies have described the activities and speci¢cities of HL towards plasma HDL, while far fewer have focused on its in£uence on intermediate and low density lipoproteins [12,13,17,30,54,55]. We ¢nd that increasing hHL production substantially reduces the concentrations of lipoproteins containing apoA-I, apoA-II, the C apoproteins, apoE, and apoB48. The clearance study presented suggests that hHL enhances the rate of removal of HDL from the plasma compartment. Transgenic rabbits overproducing hHL also have notable reductions in the amount of lipoprotein-associated A-I, E and C-III [30]. Previous in vitro evidence, using a lipid monolayer system, supports a stimulatory role for apoE on HL phospholipase activity [22]. Thuren et al. [22] have shown that apoE-rich human HDL is a more potent HL stimulator compared to apoE-poor HDL, capable of enhancing HL phospholipase activity 2^3-fold. Likewise, Dugi et al. [48] have also reported a pronounced reduction of apoE-containing HDL in LCAT transgenic mice expressing hHL via adenovirus infection. HL appears to have higher a¤nity for HDL in which apoA-I is the most abundant protein [56]. These results are in accord with our plasma apolipoprotein concentration, distribution and clearance results suggesting that both apoA-I- and apoErich HDL serve as good substrates for hHL. A most intriguing observation in the current study was the increased apoB100 associated with normal HL and moderate hHL gene expression. In the initial description of mice lacking HL, the authors noted that apoB-containing particles, normally seen in controls, were not detectable in the plasma of HL3/3 homozygotes [29]. Lipoproteins containing apoB in the density ranges 1.04^1.06, 1.06^1.08, 1.08^1.1 and

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1.1^1.21 g/ml were absent in HL3/3 mice. Our results indicate that the diminished apoB100 associated with HL de¢ciency appears to result in mainly diminished LDL. Sucrose density gradient centrifugation distributions indicate that the low levels of apoB100/LDL may be linked to diminished conversion from the more buoyant VLDL fraction. Increased clearance of VLDL perhaps via an apoE-, LpL- or HSPG-mediated pathway could ultimately explain the low apoB100 and apoE levels in HL3/3 mouse plasma. Alternatively, limited HDL and remnant removal could reduce lipid and/or free fatty acid £ux to the liver and subsequently down-regulate VLDL secretion. The relative contribution of these alternative mechanisms is currently under investigation. In humans, LpL activity is directed towards large VLDL catabolism and is also estimated to contribute approx. 50% to the conversion of smaller VLDL to IDL [13]. HL is reported responsible for the other 50% and essentially all of the conversion of IDL to LDL [13]. A kindred with HL de¢ciency resulting from compound heterozygosity for two HL gene mutations was shown to display an increase in the buoyant LDL fraction [57]. In one Swedish HL-de¢cient subject, plasma LDL was reduced by approx. 90% [13] in the presence of accumulated VLDL. Kinetic analysis revealed that the lack of HL resulted in reduced VLDL to IDL to LDL conversion in this subject. These human studies are compatible with our own observations in the mouse models described and corroborate a causal connection between diminished apoB100/LDL and HL de¢ciency. Conversely, the apparent association of increased HL activity with enhanced conversion of VLDL/IDL to higher density LDL subfractions in hHL transgenic mice further suggests that the liver lipase plays an important role in the generation of these potentially atherogenic particles [30]. In a comparison of LDL from normolipidemic and coronary artery disease (CAD) subjects, LDL buoyancy and size were found inversely associated with HL activity levels [55]. When the individuals were separated according to their LDL subclass patterns, pattern B subjects (characterized by a predominance of small, dense LDL particles) had signi¢cantly higher HL activity than pattern A subjects (characterized by a predominance of large LDL) in both CAD and normolipidemic groups.

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In contrast to normal levels of HL and moderately increased hHL activity, which are associated with signi¢cant increases in apoB100/LDL above the HL-null state in the mice examined, increased hHL activity from moderate to higher levels (i.e., the approx. 4^5-fold increase in HL activity seen in hHLmed‡ vs. hHLmed‡=‡ mice) diminished plasma apoB100. Hepatocytes are known to take up LDL in the absence of hepatic LDL receptors [58] and accelerated uptake of LDL has been noted in (Chinese hamster ovary) cells engineered to overproduce both apoE and HL [59]. These observations are consistent with the results obtained herein suggesting that HL is capable of in£uencing plasma LDL levels. However, at this time it is not clear whether hHL is directly promoting the removal of LDL or its (VLDL) precursors. Curiously, there does not appear to be a linear decrease in plasma LDL at the highest level of hHL activity expressed in the hHLhi‡ mouse. In fact, this animal has higher LDL than the controls. Inference from the analysis of one extremely highly expressing mouse must be weighed with caution. Nonetheless, it is possible that HLmediated lipolysis of apoB100/LDL-associated lipid results in altered apoB100 conformation, an increased tendency of apoB100 to aggregate when analyzed in vitro (consistent with what we have observed in Fig. 6, top panel, arrow). We note that evidence for incremental increases in apoB100 aggregation was also observed at the more moderate levels of expression found in hHLmed‡ and hHLmed‡=‡ mice (Fig. 6, top panel). Aggregation of LDL has been previously shown to be enhanced by in vitro phospholipase A2 [60] and phospholipase C treatment [61] as well as by enrichment with exogenous diacylglycerol [62]. Phospholipase A2 treated LDL bound nonspeci¢cally to cultured ¢broblast cells in the absence of uptake or degradation. Whether or not moderate to high hHL activity is capable of mediating similar structural and biological changes to LDL in vivo remains unknown. The mouse has notably low levels of plasma apoB100 and LDL and thus its utility as a model for studying e¡ects of HL on VLDL and LDL structure and metabolism is limited. Accordingly, we are currently extending our hHL mouse model by breeding the animals characterized herein to a human apoB transgenic model that should provide further insights

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into the in£uence HL has on LpB particle metabolism.

[7]

Acknowledgements [8]

The study was supported by operating grants from the Medical Research Council of Canada (MRC grant No. MT-12929) and the Heart and Stroke Foundation of Canada (HSFC grant No. NA-3184) to J.R.S, as well as generous support from the University of Ottawa Heart Institute Research Corporation. The Heart and Stroke Foundation of Canada also supports J.R.S as a Research Scholar. S.B. is supported by fellowship grants from Nestle¨-France, the Assistance Publique-Hoªpitaux de Paris and Alfediam. We would especially like to thank Yves L. Marcel and Zemin Yao for critical reading of the manuscript; Ross Milne for acute intellectual contributions, Vinita Chauhan, Heather Doelle, Yuwei Wong, Roger McLeod, Tracey Neville and Jim Burgess for excellent assistance; and the Animal Care Facility at the University of Ottawa Heart Institute including Dr. Marilyn Keaney, Richard Seymour, Joelle Mayer and Garnette Rodger.

[9]

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