The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice.

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.

Human hepatic lipase (HL) is a central component of lipoprotein metabolism (1,2). HL is synthesized and secreted by the liver, where it is anchored to the surface of hepatocytes and sinusoidal endothelial cells via heparan sulfate proteoglycans (HSPGs) (3)(4)(5)(6). HL hydrolyzes triglycerides and phospholipids in remnants (chylomicron remnants and IDL) and LDL to yield particles that are depleted in triglycerides and phospholipids and are more optimal for receptor-mediated uptake (2,(7)(8)(9). HL may also play a role in reverse cholesterol transport by hydrolyzing phospholipids in HDL, which converts HDL2 to HDL3 (10,11).
The significant role of HL in lipoprotein metabolism is apparent from human studies and data generated in animals. For example, plasma of HL-deficient patients contains high levels of apolipoprotein B (apoB)-containing lipoproteins and HDL (12)(13)(14)(15)(16)(17). Infusion of anti-HL antibodies in rats and monkeys increases levels of apoBcontaining lipoproteins and HDL (18)(19)(20). Expression of moderate and high levels of wild-type HL in mice and rabbits reduces levels of apoB-containing lipoproteins and HDL (6,(21)(22)(23)(24). Taken together, these studies indicate a major role for HL in determining the plasma levels of apoB-containing lipoproteins and HDL.
HL regulates plasma levels of apoB-containing lipoproteins using both catalytic and bridging functions (6,25). In particular, wild-type HL (reflecting both catalytic and bridging functions) reduces plasma levels of both apoB-48-and apoB-100-containing lipoproteins. However, the contribution of the bridging function to their reduction is not clear. Previous in vitro studies demonstrated roles for the bridging function in the cellular uptake of chylomicrons, remnants, and LDL. Cells transfected with wild-type HL and studied at 4 Њ C to abolish catalytic activity showed enhanced uptake of human chylomicrons, and cells incubated in the presence of heat-inactivated HL displayed increased binding and uptake of remnants (26,27). Cells transfected with a mutant, catalytically inactive hepatic li-pase (ciHL) displayed more association with 125 I-labeled LDL than did control transfected cells, thus suggesting that the bridging function facilitated LDL receptor (LDLR)-mediated uptake of LDL (26,28).
The effect of the bridging function on plasma levels of apoB-containing lipoproteins has also been studied in vivo in apoE-deficient and LDLR-deficient mice. In apoE-deficient mice (which have high levels of apoB-48-containing lipoproteins), overexpression of ciHL reduced levels of VLDL, remnants, and LDL (6,29). In LDLR-deficient mice (which have high levels of both apoB-48-and apoB-100-containing lipoproteins), overexpression of ciHL also reduced remnants and LDL (30). This latter finding indicated that the bridging function uses an LDLR-independent pathway to reduce apoB levels, inasmuch as both apoB-48-and apoB-100containing lipoproteins are catabolized in part via the LDLR (which is absent in LDLR-deficient mice) (31).
On the basis of this information, we hypothesized that the bridging function of HL facilitates removal of both apoB-48and apoB-100-containing lipoproteins. Furthermore, we hypothesized that the bridging function facilitates particle removal by whole-particle uptake. To test our hypotheses, we expressed ciHL in LDLR-deficient mice that were genetically modified to express only apoB-48 or apoB-100 and determined the effect of the bridging function on levels of apoB-100-and apoB-48-containing lipoproteins (31). We also assessed the dependence of the bridging function on particle composition. To do this, we examined the response of plasma lipids and apolipoproteins to a cholesterolenriched, high-fat (Western) diet. To control for any contribution from endogenous mouse HL, we also expressed ciHL in LDLR-deficient mice that lacked mouse HL.

Genetically modified mice
Mice that were genetically modified to express only mouse apoB-100 ( apob 100/100 ) (31) (a gift from Dr. Stephen G. Young, Gladstone Institute of Cardiovascular Disease, San Francisco, CA) were bred with Ldlr Ϫ / Ϫ apob ϩ / ϩ mice (that have the wild-type mouse apoB gene), and the resulting offspring were bred with each other to yield LDLR-deficient mice that were homozygous for apoB-100 and that no longer had the wild-type mouse apoB gene. The resulting Ldlr Ϫ / Ϫ apob 100/100 mice were bred with Ldlr Ϫ / Ϫ apob ϩ / ϩ mice that are transgenic for a human ciHL (HLS145G). Also, LDLR-deficient mice that were genetically modified to express only mouse apoB-48 ( Ldlr Ϫ / Ϫ apob 48/48 ) (31) (also a gift from Dr. Stephen G. Young) were bred with Ldlr Ϫ / Ϫ apob ϩ / ϩ mice that are transgenic for human ciHL ( Ldlr Ϫ / Ϫ apob ϩ / ϩ HL S145G ). The resulting littermates were bred to achieve homozygosity for both the genetically modified mouse apoB gene (to yield either Ldlr Ϫ / Ϫ apob 48/48 or apob 100/100 ) and the gene-targeted mouse LDLR gene as well as heterozygosity for the ciHL transgene (30). The wild-type mouse apoB gene was absent in these mice. The ciHL transgene was reported previously and consists of a mutant human HL cDNA in which a glycine replaces serine at position 145 in the catalytic triad (HLS145G), resulting in expression of ciHL (6).
Liver-specific expression of the HLS145G transgene was conferred by sequences in the human apoE gene as described (6): 3 kb of the 5 Ј -flanking sequence, the first exon, the first intron, and the first six untranslated nucleotides of the second exon, a polylinker for cDNA insertion, the nontranslated portion of the fourth exon, 0.1 kb of 3 Ј -flanking sequence, and the first hepatic control region of the apoE locus.
All mice were male. Groups of littermates were analyzed within each of the Ldlr Ϫ/Ϫ apob 48/48 , Ldlr Ϫ/Ϫ apob 100/100 , and Ldlr Ϫ/Ϫ hl Ϫ/Ϫ apob ϩ/ϩ genotypes. After weaning at 21 days, mice were fed a chow diet and were housed in a full-barrier facility with a 12 h light-dark cycle. All studies were approved by the Institutional Animal Care and Use Committee of the University of Washington.

Expression of human ciHL
Plasma samples from mice fed a chow diet were collected in tubes containing ethylene diaminetetraacetic acid prior to and by guest, on July 20, 2018 www.jlr.org Downloaded from 10 min after tail vein injection of heparin (150 U/kg body weight) and were kept frozen at Ϫ80ЊC until analysis for protein expression. Western blot analysis of pre-and postheparin plasma was performed with a monospecific polyclonal rabbit antihuman HL antiserum (6).

Lipase assays
Triglyceride lipase activities were quantitated in two separate assays in triplicate with glycerol [ 1 -14C]trioleate-labeled triolein emulsion as a substrate in the presence of 1 M NaCl (35).

Lipoprotein analysis
Plasma was obtained by orbital vein bleeding after a 4 h fast. Mouse plasma lipoproteins were fractionated by fast-protein liquid chromatography (FPLC) on a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech, Piscataway, NJ) as described previously (6). The cholesterol and triglyceride levels in whole plasma as well as in eluted fractions of plasma were determined with standard enzymatic assays (cholesterol: Abbot Spectrum, Abbott Park, IL; triglycerides: GPO-PAP kit, Boehringer Mannheim, Indianapolis, IN). The cholesterol and triglyceride concentrations in the eluted fractions (of plasma) were then corrected against the cholesterol and triglyceride concentrations in whole plasma to account for variability in the efficiency of the Superose 6 column (recovery). The recovery ranged from 70% to 100%.

Western analysis of plasma apoB-48 and apoB-100
Plasmas from 4-5 mice of each genotype were pooled and subjected to Western blot analysis. The pooled plasmas were applied in quadruplicate or quintuplicate to 4% polyacrylamide-sodium dodecyl sulfate gels. Three to four individual gels were run (duplicate gels run for the Ldlr Ϫ/Ϫ apob 48/48 mice on a chow diet) to facilitate comparisons between genotypes and to ensure reproducibility. After blotting, nitrocellulose membranes were incubated with a rabbit anti-mouse apoB antibody that reacts with both apoB-48 and apoB-100 (34), incubated with biotinylated second antibody, and developed with an ECL kit (Amersham Pharmacia Biotech). Immunoblots were analyzed by densitometry on a GelDoc 2000 (Bio-Rad, Hercules, CA) using the Quantity One software package (Bio-Rad). Reproducibility of densitometry results was assessed by repeat measurements of individual blots.

Western analysis of plasma apoE and apoA-I
Plasma from 4-7 mice of each genotype was pooled, and the pooled plasmas were applied in quadruplicate or quintuplicate and fractionated on 12% polyacrylamide-sodium dodecyl sulfate gels (Bio-Rad) and transferred to nitrocellulose membranes. Three to four individual gels were run to facilitate comparisons between genotypes and to ensure reproducibility. The membranes were incubated with a goat anti-mouse apoE antibody (that also reacts with mouse apoA-I) (a gift from Dr. Karl H. Weisgraber, Gladstone Institute of Cardiovascular Disease), reacted with biotinylated second antibody, and analyzed as above.

Selective cholesteryl ester uptake assessment
The presence of a greater decrease in cholesterol than in apolipoprotein was used as an approximate measure of selective cholesteryl ester uptake. Thus, selective cholesteryl ester uptake was assumed to occur in VLDL, IDL, and LDL when lipoprotein cholesterol decreased to a greater extent than apoB-48 and/or apoB-100 levels. Likewise, HDL-selective cholesteryl ester uptake was assumed to occur when HDL-cholesterol (HDL-C) decreased but apoA-I levels remained virtually unchanged.

Diet study
To assess the dependence of the bridging function on lipoprotein lipid composition, six to nine animals of each of the Ldlr Ϫ/Ϫ apob 100/100 and Ldlr Ϫ/Ϫ hl Ϫ/Ϫ apob ϩ/ϩ genotypes were fed a cholesterol-enriched, high-fat (Western) diet [21% (w/w) fat and 0.15% (w/w) cholesterol] (TD 88137, Harlan, Teklad, Madison, WI) for 2 weeks. Fasted plasma lipoproteins were separated by FPLC, and plasma apolipoprotein levels were analyzed by Western blotting as described.

Statistical analysis
Data are presented as the mean Ϯ SD. Student's t-test for unequal variances was used to determine the statistical significance of differences.

Expression of the human ciHL transgene
PCR analysis for the ciHL transgene demonstrated a fragment of the expected 987 bp size in transgenic mice and its absence in nontransgenic mice. Western analysis of postheparin plasma confirmed the presence of the HL protein and demonstrated similar amounts of immunoreactive human HL in all transgenic mice (data not shown). The amounts of ciHL in postheparin plasma of all three genotypes were similar to the amounts of ciHL in the previously reported Ldlr Ϫ/Ϫ apob ϩ/ϩ HL S145G mice. There was no human HL in the nontransgenic mice. Absence of human HL catalytic activity in postheparin plasma was verified by the lack of increased HL activity (above that of the endogenous mouse HL) in all genotypes ( Table 1). Only background activity was present in the Ldlr Ϫ/Ϫ hl Ϫ/Ϫ apob ϩ/ϩ HL S145G mice.  48/48 HL S145G mice (n ϭ 6), P Ͻ 0.02]. The triglyceride levels were not changed significantly ( Table 2). Plasma lipoprotein profiles were determined by FPLC (Fig. 1). The FPLC cholesterol profiles in Ldlr Ϫ/Ϫ apob 48/48 mice showed minimal elevations of VLDL and IDL and major peaks of LDL and HDL.

Plasma apoB-48 and apoB-100 levels
We next sought to estimate the respective contributions of whole-particle uptake (reflected by similar decreases in both cholesterol and apoB-48 or apoB-100) and selective cholesteryl ester uptake (reflected by a decrease in cholesterol but minimal or no decrease in apoB-48 or apoB-100) to the ciHL-mediated lipoprotein reduction. To do so, we assessed plasma apoB-48 and apoB-100 levels by densitometric scanning of Western blots ( Table 4). The changes in apoB-48 and apoB-100 were then compared with the changes in lipoprotein cholesterol.
Plasma apoB-100 levels were not reduced significantly on either chow or Western diets in these mice (Table 4 and data not shown).

Plasma apoE levels
Next, using apoE as a marker of whole-particle uptake, we examined whether ciHL reduces cholesterol by mediating lipoprotein uptake.

Plasma apoA-I levels
To estimate whether the ciHL-mediated reduction in HDL-C occurred by whole-lipoprotein uptake (reflected by simultaneous reductions in cholesterol and apoA-I) or by selective cholesteryl ester uptake (reflected by reduction in cholesterol and minimal or no reduction in apoA-I), we assessed plasma apoA-I levels by densitometric scanning of Western blots.

DISCUSSION
Our studies in genetically modified mice demonstrate that the bridging function of HL enhances uptake of both apoB-48-and apoB-100-containing lipoproteins and suggest that this occurs by several mechanisms, including whole-particle uptake and selective cholesteryl ester uptake. In addition, our studies indicate that the bridging function is modulated by diet and endogenous (murine) HL in these mouse models.