high density lipoprotein metabolism in low density ... · hdl metabolism in ldl receptor-deﬁ...

1914 Journal of Lipid Research Volume 55, 2014

Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

This article is available online at http://www.jlr.org

Specifi c plasma membrane receptors play critical roles in lipoprotein metabolism ( 1 ). A well-established mole-cule is the LDL receptor (LDLR) ( 2 ). This protein medi-ates the cellular internalization of cholesterol-rich LDL particles, for instance by the liver ( 3 ). Another membrane protein, scavenger receptor class B type I (SR-BI), has a dominant role in the physiology of HDL ( 4 ). This receptor facilitates the selective uptake of HDL-associated choles-teryl esters (CEs) by liver and adrenals (i.e., cellular lipid uptake without holo-particle internalization) ( 5, 6 ).

The physiological signifi cance of these receptors is il-lustrated by mutations in the respective genes. In mice, LDLR defi ciency is associated with an increase in LDL cholesterol as well as accelerated atherosclerosis ( 7, 8 ). With respect to HDL, induced mutations in the murine gene encoding SR-BI induce an increase in plasma HDL cholesterol and accelerated atherosclerosis ( 9, 10 ). How-ever, besides an increase in LDL cholesterol, a defi ciency of the LDLR can modulate non-LDL lipoproteins in plasma as well. For instance, a rise in plasma HDL cho-lesterol has been reported in LDLR-defi cient mice in some studies but not in others ( 7, 11, 12 ). These obser-vations suggest that an LDLR defi ciency can affect the metabolism of more than one lipoprotein fraction, at least to some extent. However, with respect to the mech-anism, it is not known how an LDLR defi ciency modifi es HDL cholesterol.

Abstract The LDL receptor (LDLR) and scavenger recep-tor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-defi cient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein ( 125 I) and in the cholesteryl ester (CE) moiety ([ 3 H]). The metabolism of 125 I-/[ 3 H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([ 3 H]). In contrast, in LDLR � / � mice, selective HDL CE uptake is signifi cantly reduced in liver and adrenals. In he-patocytes isolated from LDLR � / � mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by im-munoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR � / � mice. In summary, an LDLR defi ciency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice. —Rinninger, F., M. Heine, R. Singaraja, M. Hayden, M. Brundert, R. Ramakrishnan, and J. Heeren. High density lipoprotein metabolism in low den-sity lipoprotein receptor-defi cient mice. J. Lipid Res . 2014. 55: 1914–1924.

Supplementary key words scavenger receptor class B type I • choles-teryl ester • selective uptake

This study was supported by Research Grant Ri 436/8-1 from Deutsche Forsc-hungsgemeinschaft (Bonn) and funds from German Diabetes Association, Wer-ner Otto Stiftung (Hamburg), and Gertraud und Heinz Rose Stiftung (Hamburg). J. Heeren is supported by EU FP7 project RESOLVE Grant FP7-HEALTH-2012-305707.

Manuscript received 7 March 2014 and in revised form 2 June 2014.

Published, JLR Papers in Press, June 22, 2014 DOI 10.1194/jlr.M048819

High density lipoprotein metabolism in low density lipoprotein receptor-defi cient mice 1

Franz Rinninger , 2 , * Markus Heine , † Roshni Singaraja , §, ** Michael Hayden , †† May Brundert , * Rajasekhar Ramakrishnan , §§ and Joerg Heeren †

Department of Medicine* and Department of Biochemistry and Molecular Cell Biology, † University Hospital Hamburg Eppendorf , 20246 Hamburg, Germany ; Translational Laboratories in Genetic Medicine, Agency for Science, Technology and Research § and Department of Medicine,** National University of Singapore , Singapore 117609; Centre for Molecular Medicine and Therapeutics and Child and Family Research Institute, †† University of British Columbia , Vancouver, British Columbia V5Z 4H4, Canada ; and Department of Pediatrics, §§ College of Physicians and Surgeons of Columbia University , New York, NY 10032

Abbreviations: CD36 , cluster of differentiation 36; CE, cholesteryl ester; CEt, cholesteryl oleyl ether; CETP, cholesteryl ester transfer pro-tein; FPLC, fast performance LC; LDLR, LDL receptor; LRP1, LDL re-ceptor-related protein 1; organ-FCR, organ fractional catabolic rate; plasma-FCR, plasma fractional catabolic rate; SR-BI, scavenger receptor class B type I; TC, tyramine cellobiose.

1 This study is dedicated to Professor Heiner Greten, MD. 2 To whom correspondence should be addressed. e-mail: [email protected]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of fi ve fi gures and three tables.

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HDL metabolism in LDL receptor-defi cient mice 1915

the CE moiety ( 6, 13 ). [ 3 H]CEt was introduced in 125 I-TC-HDL by exchange from donor liposomal particles, which contained [ 3 H]CEt using human plasma cholesteryl ester transfer protein (CETP) ( 15 ). 125 I-TC-LDL was prepared as outlined ( 13 ). The fi nal 125 I-TC-/[ 3 H]CEt-WT-HDL, 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL, and 125 I-TC-LDL preparations were extensively dialyzed against PBS (pH 7.4, 4°C) containing EDTA (1 mM).

HDL metabolism in mice For plasma decay analysis of radiolabeled WT-HDL or

LDLR � / � HDL, mice were fasted for 4 h before tracer injection ( 5 ). Then 125 I-TC-/[ 3 H]CEt-WT-HDL or 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL (30 µg HDL protein per mouse) was injected via tail vein, and thereafter blood samples (30 µl per time point) were collected periodically (10 and 30 min; 2, 5, 9, 22, and 24 h) for 24 h after injection. Animals were fasted throughout the 24 h study period but had unlimited access to water. Plasma aliquots were directly assayed for 125 I radioactivity, and [ 3 H] was analyzed after lipid extraction ( 16 ). Computer modeling was used to fi t (by method of least squares) multiexponential curves, arising from a com-mon two-pool model, simultaneously to both tracers’ plasma de-cay data, and to calculate plasma fractional catabolic rates (plasma-FCRs) for each tracer ( 17 ). The modeling was done separately for the data from each mouse, so that individual plasma-FCRs for both tracers were calculated for each animal. In some cases, HDL metabolism in mice was explored for a 2 h in-terval only.

Tissue sites of uptake of HDL-associated tracers were deter-mined 2 h or 24 h after injection of radiolabeled WT-HDL or LDLR � / � HDL ( 5 ). Finally, animals were anesthetized, the abdo-men and chest were opened, and a catheter was inserted into the heart. The inferior vena cava was cut, and the mice were perfused extensively with saline (50 ml per animal). After perfusion, liver, adrenals, kidneys, brain, heart, lungs, spleen, stomach, intestine, and carcass from each mouse were harvested and homogenized. Homogenates from each tissue and from carcass were directly assayed for 125 I radioactivity, and aliquots were analyzed for [ 3 H] after lipid extraction ( 16 ).

Total radioactivity recovered from all tissues and from the car-cass of each mouse was calculated ( 5 ). The fraction of total tracer uptake attributed to a specifi c organ was calculated as the radio-activity recovered in that organ divided by the total radioactivity recovered from all tissues and carcass. Thus the % of recovered extravascular radioactivity in tissues is determined 2 h or 24 h af-ter injection of labeled HDL.

To allow comparison of the specifi c activities of various tissues in HDL internalization and to directly compare the rates of up-take of the apo component and the CE moiety of HDL, the data are expressed as organ fractional catabolic rates (organ-FCRs) ( 5 ). These rates are calculated as follows: (Organ-FCR in Tissue X) = (Plasma-FCR) × (Fraction [%] of Total Body Tracer Recov-ery in Tissue X). This organ-FCR represents the fraction of the plasma pool of either HDL tracer cleared by an organ per hour. 125 I-TC represents the uptake of HDL holo-particles by tissues ( 18 ). Selective HDL CE uptake is calculated as the difference in organ-FCR between [ 3 H]CEt and 125 I-TC.

LDL metabolism in mice 125 I-TC-LDL metabolism in plasma and tissue sites of uptake of

this lipoprotein were investigated in mice analogously as out-lined for radiolabeled HDL.

Preparation of murine hepatocytes Primary hepatocytes were isolated from murine liver by

perfusion (37°C, 18 min) with Hanks’ balanced salt solution

In the current study, HDL metabolism was explored in LDLR-defi cient mice and compared with WT animals that express functional LDLR ( 7 ). To explore the fate of dis-tinct lipoprotein components, HDL particles were radiola-beled in both the protein as well as in the lipid moiety ( 13 ). In metabolic studies using double radiolabeled HDL, liver and adrenals of WT mice selectively take up HDL-associated CE. In LDLR � / � mice, however, selective HDL CE uptake is signifi cantly reduced in liver and adrenals. In parallel, uptake of radiolabeled HDL by isolated primary hepatocytes in vitro was explored. Compared with WT and in line with the in vivo studies, an LDLR defi ciency is as-sociated with a decrease in selective HDL CE uptake. No-tably, SR-BI expression in liver and adrenal membranes was identical in mice with and without LDLR even though HDL selective CE uptake was reduced. In summary, we demonstrate that LDLR expression has a substantial im-pact on HDL metabolism in vivo and in vitro. Remarkably, regulation of selective HDL CE uptake occurs indepen-dent from SR-BI protein expression in tissues.

MATERIALS AND METHODS

Materials Primers were purchased from Metabion. Taq-DNA polymerase,

culture media, sera, and supplements for cell culture were sup-plied by Invitrogen. Six-well tissue culture plates were obtained from Becton Dickinson. Collagenase was from Worthington. 125 Iodine, [ 3 H]cholesteryl oleyl ether ([ 3 H]CEt), and ECL re-agent were purchased from GE Healthcare. Protease inhibitor cocktail “complete” and enzymatic assays for cholesterol, HDL cholesterol, and triglyceride were supplied by Roche. Assays for phospholipid were obtained from Wako. BSA, tyloxapol, and standard laboratory chemicals were purchased from Sigma Al-drich. Scintillation cocktail was from PerkinElmer. Nitrocellulose membrane was obtained from Schleicher and Schuell. Films were supplied by Kodak. Rodent chow was purchased from Ssniff.

Mice Mice lacking a functional LDLR gene (LDLR � / � ) were pur-

chased from The Jackson Laboratory (Bar Harbor, ME) ( 7 ). Male LDLR � / � mice on a C57BL/6J genetic background and the respective male littermate controls (WT) were used. The geno-type of each mouse was analyzed by PCR from genomic DNA iso-lated from tail biopsies ( 7 ). Mice were maintained on a standard laboratory chow diet with unlimited access to food and water. The age of the rodents used in this study was between 20 and 40 weeks. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University Hospital Hamburg.

Lipoprotein preparation Mice were fasted 4 h before blood harvest. Murine HDL (d =

1.063–1.21 g/ml) was isolated from WT (WT-HDL) plasma or from LDLR-defi cient (LDLR � / � HDL) plasma by sequential ul-tracentrifugation ( 14 ). LDL (d = 1.020–1.050 g/ml) was pre-pared from plasma of LDLR � / � mice.

Murine WT-HDL or LDLR � / � HDL was double labeled with 125 I-tyramine cellobiose ( 125 I-TC) in the apo and with [ 3 H]CEt in


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RESULTS

As expected, total plasma cholesterol was signifi cantly higher in male LDLR � / � mice (284%) compared with their WT littermates (100%) ( Table 1 ). HDL cholesterol was elevated (111%) in LDLR-defi cient male mice com-pared with WT animals (100%); however, this difference was not statistically signifi cant. Triglycerides were signifi -cantly higher (235%) in LDLR � / � mice compared with WT (100%). Plasma lipids were also analyzed in female mice ( Table 1 ). In female LDLR � / � animals, total choles-terol and triglycerides increased to a similar extent com-pared with male LDLR � / � mice, whereas no increase in HDL cholesterol was detected.

To determine the distribution of cholesterol with re-spect to lipoprotein fractions, murine plasma was frac-tionated by FPLC ( Fig. 1 ) ( 25 ). The major change due to the LDLR defi ciency was an increase in cholesterol corresponding to particles of the LDL/IDL fractions. However, in LDLR � / � mice, a small increase in plasma cholesterol in the size range of HDL could be detected. These results on plasma lipids and on HDL cholesterol in LDLR � / � mice are in line with previous studies ( 7, 12 ). The compositional analysis of HDL isolated by sequen-tial ultracentrifugation from murine plasma showed that LDLR � / � HDL is signifi cantly enriched in triglycerides and depleted in phospholipids compared with WT-HDL ( Table 2 ).

The metabolism of 125 I-TC-/[ 3 H]CEt-WT-HDL was in-vestigated in WT and in LDLR � / � mice ( Fig. 2 ). This murine HDL preparation was injected intravenously in mice, and thereafter, blood samples were harvested dur-ing a 24 h interval ( 5 ). In these studies, [ 3 H]CEt repre-sents the metabolism of HDL-associated CE, and 125 I-TC shows HDL holo-particle clearance ( 18 ). The difference between both tracers ([ 3 H]CEt � 125 I-TC ) represents se-lective CE removal. In WT mice, the plasma decay of HDL-associated [ 3 H]CEt is faster compared with 125 I-TC; the difference in decay between both tracers yields se-lective CE removal from the HDL plasma pool by tissues in WT animals. In parallel, 125 I-TC-/[ 3 H]CEt-WT-HDL was injected in LDLR � / � mice. In this case, no difference in plasma decay between both HDL tracers can be de-tected. These data suggest that selective CE removal from the HDL plasma pool does not occur in LDLR � / � mice.

From decay curves shown in Fig. 2 , plasma-FCRs for both HDL-associated tracers were calculated ( Fig. 3 ) ( 17 ). In WT animals, the plasma-FCR for WT-HDL-associated [ 3 H]CEt is substantially higher compared with 125 I-TC; the difference between both rates ([ 3 H]CEt � 125 I-TC) yields selective CE removal from the plasma HDL pool by tissues. In LDLR � / � mice, no signifi cant difference in plasma-FCR for 125 I-TC is detected compared with WT. In contrast, in LDLR � / � mice, a substantial reduction in plasma-FCR for [ 3 H]CEt is observed. Calculation of the difference be-tween [ 3 H]CEt and 125 I-TC yields no difference, again showing that there is no selective CE removal from plasma WT-HDL by tissues in LDLR � / � mice.

supplemented with collagenase (0.3 mg/ml, type I), HEPES (10 mM), and protease inhibitor mixture “complete” ( 19 ). There-after, these cells were seeded (37°C, 2.0 h) in DMEM contain-ing FBS (5%, v/v), penicillin (100 µg/ml), and streptomycin (100 µg/ml). Finally, the culture medium was aspirated, and the cells were washed (PBS, 3×). Hepatocytes were used for 125 I-TC-/[ 3 H]CEt-WT-HDL uptake or 125 I-TC-WT-HDL binding assays.

Uptake and binding assay for radiolabeled HDL To determine uptake of radiolabeled HDL, hepatocytes were

incubated (37°C, 2.0 h) in DMEM containing BSA (5 mg/ml), penicillin (100 µg/ml), streptomycin (100 µg/ml), and 125 I-TC-/[ 3 H]CEt-WT-HDL ( 20 ). Finally, cells were harvested by trypsin/EDTA (1×, trypsin 0.05%, EDTA 0.53 mM) treatment, and cellu-lar uptake of HDL tracers was measured. 125 I was directly radioas-sayed, and [ 3 H] was analyzed after lipid extraction ( 16 ). Uptake of 125 I-TC-/[ 3 H]CEt-WT-HDL by cells is shown in terms of appar-ent HDL particle uptake, expressed as HDL protein ( 5, 20 ). This is done to compare the uptake of both tracers on a common ba-sis. The uptake of HDL holo-particles is represented by 125 I-TC, and the difference between [ 3 H]CEt and 125 I-TC yields apparent selective HDL CE uptake by cells ( 18 ).

To investigate binding of radiolabeled HDL, hepatocytes were incubated (4°C, 2.0 h) in medium containing 125 I-TC-WT-HDL ( 21 ). Thereafter, the medium was removed, and the cells were washed (PBS, 4°C). 125 I-TC-WT-HDL binding to the cells was fi -nally analyzed as outlined ( 21 ).

Immunoblots Membrane fractions from murine liver or adrenals were pre-

pared ( 22 ). Protease inhibitor cocktail “complete” was present during the entire preparation. Membrane fractions were boiled (93°C, 10 min) and separated by SDS polyacrylamide gel (10%) electrophoresis under reducing conditions (mercaptoethanol) and thereafter transferred to a nitrocellulose membrane ( 6 ). Fi-nally, the blots were probed with anti-SR-BI (rabbit polyclonal antibody to murine SR-BI, Novus Biologicals), anti-LDL receptor-related protein 1 (LRP1) (sheep anti-LRP1 antibody, raised against a synthetic peptide from human LRP1) ( 23 ), anti-cluster of differentiation 36 (CD36) (rabbit polyclonal antiserum against murine CD36) ( 24 ), anti-ABCA1 (rabbit polyclonal antibody, No-vus Biologicals), or anti- � -actin (monoclonal anti- � -actin, mouse, Sigma Aldrich) as primary antibodies. � -Actin was used as load-ing control. Finally, the blots were incubated with respective horseradish peroxidase-conjugated secondary antibodies (Sigma Aldrich) and developed with ECL. Blots were exposed to Bio Max MR fi lms and quantifi ed using Image Quant software, version 5.2 (GE Healthcare).

Miscellaneous Routinely, all mice were fasted for 4 h before blood harvest for

analytical or preparative purposes. Total cholesterol, HDL cho-lesterol, and triglycerides from plasma were measured using en-zymatic assays. Plasma lipoproteins were fractionated by fast performance LC (FPLC) ( 25 ). In order to measure VLDL pro-duction, the nonionic detergent Tyloxapol (Triton WR 1339) was used to inhibit VLDL catabolism ( 26 ). Protein was analyzed as outlined ( 27 ).

Statistics Values are means ± SEM. All statistical analyses were per-

formed using Student’s t -test. Probability values <0.05 were con-sidered statistically signifi cant.


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Fig. 1. FPLC analysis of plasma cholesterol from WT or from LDLR � / � mice. After fasting for 4 h, blood was harvested from 4 WT or from 4 LDLR � / � male mice. Finally, the pooled plasma was subjected to FPLC, and cholesterol was analyzed in each fraction as outlined in Materials and Methods. Shown is a representative ex-periment from a total of n = 2.

TABLE 1. Plasma cholesterol, HDL cholesterol, and triglycerides of WT or LDLR � / � mice

Total Cholesterol HDL Cholesterol Triglycerides

Males mg/dl mg/dl mg/dl WT 93.3 ± 3.8 (30) 83.8 ± 3.6 (27) 105.8 ± 7.0 (20) LDLR � / � 264.8 ± 16.9 (31) 92.7 ± 4.7 (28) 249.1 ± 26.1 (23) P <0.0001 0.0705 <0.0001Females WT 89.9 ± 4.4 (30) 78.2 ± 3.0 (30) 122.5 ± 8.2 (24) LDLR � / � 229.7 ± 8.8 (22) 75.1 ± 3.3(22) 161.2 ± 8.5 (17) P <0.0001 0.251 0.0014

Male or female WT or LDLR � / � mice were fasting for 4 h. Thereafter, blood was harvested and plasma was analyzed as outlined in Materials and Methods. Values are means ± SEM; the number of mice is given in parentheses.

Organ-specifi c HDL catabolism was determined. Twenty-four hours after 125 I-TC-/[ 3 H]CEt-WT-HDL injec-tion, tracer content of each tissue was analyzed, and HDL uptake was calculated and expressed in terms of organ-FCRs ( 5 ). HDL tracer uptake by the liver is shown in Fig. 3 . In WT mice, the hepatic organ-FCR for [ 3 H]CEt is higher compared with 125 I-TC; the difference between both rates ([ 3 H]CEt � 125 I-TC) yields selective CE uptake by the liver from WT-HDL. This result is in line with earlier studies in WT mice in which similar levels of selective CE uptake from HDL by the liver were observed ( 6, 31 ). In LDLR � / � mice, the hepatic uptake rate for 125 I-TC was similar to the respective rate in WT animals ( Fig. 3 ). However, in LDLR � / � mice, the hepatic organ-FCR for [ 3 H]CEt de-creased signifi cantly compared with WT, indicating that although holo-particle uptake was similar in both LDLR � / � and WT murine livers, selective CE uptake from WT-HDL ([ 3 H]CEt � 125 I-TC) was decreased signifi cantly in livers of LDLR � / � mice.

Uptake of 125 I-TC-/[ 3 H]CEt-WT-HDL by adrenal glands was explored ( Fig. 3 ). In WT adrenals, the organ-FCR for [ 3 H]CEt is substantially higher compared with 125 I-TC, and the difference between both rates yields substantial selec-tive CE uptake from HDL ([ 3 H]CEt � 125 I-TC) in glands with LDLR protein expression ( 5, 6 ). In adrenals from LDLR � / � mice, the organ-FCR for 125 I-TC was not signifi -cantly different compared with WT, indicating normal

holo-particle uptake. Similar to the results for liver, adre-nal organ-FCR for [ 3 H]CEt decreased signifi cantly in LDLR � / � glands compared with WT. This decrease in HDL lipid internalization by LDLR � / � glands yielded a signifi -cant reduction in selective CE uptake ([ 3 H]CEt � 125 I-TC). Uptake of 125 I-TC-/[ 3 H]CEt-WT-HDL by murine kidneys was investigated ( Fig. 3 ). The kidney organ-FCR for HDL-associated 125 I-TC is higher compared with the respective rate for [ 3 H]CEt, and this result is consistent with a physi-ological role of the kidneys in HDL apo catabolism ( 28 ). Kidney organ-FCRs for both HDL-associated tracers did not differ between WT and LDLR � / � mice.

The composition of WT-HDL and LDLR � / � HDL is dif-ferent ( Table 2 ). Therefore, the metabolism of 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL was explored by the identical ap-proach as with labeled WT-HDL ( Fig. 4 ; supplementary Fig. I). In WT mice, the plasma-FCR for LDLR � / � HDL-associated [ 3 H]CEt is substantially higher compared with 125 I-TC; the difference between both tracers ([ 3 H]CEt � 125 I-TC) yields selective CE removal from the plasma HDL pool by tissues ( 17 ). Comparing WT and LDLR � / � mice, no signifi cant difference in plasma-FCR for 125 I-TC is de-tected. In LDLR � / � mice, however, a signifi cant reduction in plasma-FCR for [ 3 H]CEt is observed. The difference be-tween [ 3 H]CEt and 125 I-TC yields a signifi cant reduction in selective CE removal from the plasma LDLR � / � HDL pool by tissues in LDLR � / � mice.

To explore the metabolism of LDLR � / � HDL by tis-sues, tracer content was analyzed 24 h after injection of 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL ( 5 ). In WT mice, the he-patic organ-FCR for [ 3 H]CEt is higher than the one due to 125 I-TC; the difference between both rates ([ 3 H]CEt � 125 I-TC) yields selective CE uptake by the liver from LDLR � / � HDL ( Fig. 4 ). In LDLR � / � mice, the hepatic up-take rate for 125 I-TC was similar to the respective rate in WT animals. However, in these LDLR-defi cient mice, the hepatic organ-FCR for [ 3 H]CEt decreased signifi cantly compared with WT. Although holo-particle uptake was similar in livers of both genotypes, selective CE uptake from LDLR � / � HDL ([ 3 H]CEt � 125 I-TC) decreased sig-nifi cantly in the livers of LDLR � / � mice.

HDL-associated apos can exchange in plasma between lipoprotein fractions ( 1 ). In some species, CE can be trans-ferred from HDL to more buoyant lipoproteins, and this reaction is mediated by CETP ( 15 ). However, mice have no CETP activity in the circulation, and therefore, no


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us to determine the reutilization of radiolabeled tracers for VLDL production ( 26 ). In WT and in LDLR � / � mice, Tyloxapol induced a substantial increase in plasma triglyc-eride, indicating an inhibition of VLDL clearance. With respect to tracer recovery in lipoprotein fractions, Tyloxa-pol induced a substantial increase in [ 3 H]CEt tracer con-tent in the VLDL gradient fraction in LDLR � / � mice and only a small increase in WT littermates ( 14 ). In contrast, Tyloxapol had virtually no effect on the distribution of the 125 I-TC tracer between the VLDL and the non-VLDL frac-tions. This experiment indicates that, following uptake of 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL by the liver, there is sub-stantial resecretion of the [ 3 H]CEt tracer in VLDL parti-cles in LDLR � / � mice. This observation explains at least to some extent the recovery of the initially HDL-associated [ 3 H]CEt tracer in non-HDL lipoproteins during the time course in LDLR � / � mice.

To ensure that the observed differences in HDL catabo-lism in LDLR � / � mice are independent from the resecre-tion of lipid tracers into non-HDL lipoprotein fractions, we determined the plasma decay and tissue uptake of ra-diolabeled HDL in a short-term 2 h study. In this period, all HDL-associated tracers remained within the plasma HDL fraction. 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL was in-jected in WT or in LDLR � / � mice (supplementary Fig. II). In WT animals, the initial plasma-FCR for LDLR � / � HDL-associated [ 3 H]CEt is substantially higher compared with 125 I-TC; the difference between both rates ([ 3 H]CEt � 125 I-TC) yields selective CE removal from the plasma HDL pool by tissues. In LDLR � / � mice, a quantitative minor difference in initial plasma-FCR for 125 I-TC is detected

mechanism exists that mediates a transfer of HDL-associ-ated lipids to more buoyant lipoproteins ( 36 ). To address the question of whether initially HDL-associated tracers can be transferred into non-HDL lipoproteins in plasma during a 24 h period, 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL was injected in WT and in LDLR � / � mice (supplementary Table I). After 40 min, 8 h, and 24 h, blood was harvested and lipoproteins were separated using FPLC. Tracer in fractions corresponding to triglyceride-rich lipoproteins, LDL, and HDL were analyzed. In WT mice, during the 24 h time course both LDLR � / � HDL-associated tracers re-mained predominantly in the HDL fraction. In LDLR � / � mice, however, [ 3 H]CEt tracer initially associated with LDLR � / � HDL was detected after 8 h and after 24 h to a substantial extent in fractions with a lower density than HDL. Qualitatively identical results were obtained with 125 I-TC-/[ 3 H]CEt-WT-HDL in both groups of mice (data not shown). Because there is no CETP in murine plasma, we investigated the mechanism of this [ 3 H]CEt recovery in non-HDL lipoproteins (supplementary Table II). We spec-ulated that HDL-derived radiolabeled tracers are partially reused for VLDL assembly and secretion. In contrast to the WT situation, the LDLR-dependent uptake of VLDL and LDL is reduced in LDLR � / � mice, and consequently, small amounts of recycled tracers can be found in non-HDL lipoproteins. To test this hypothesis, 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL was injected in WT or in LDLR � / � mice (supplementary Table II). First, blood was harvested from these animals 24 h after tracer injection. Thereafter, Tyloxapol (Triton WR 1339), an inhibitor of lipoprotein catabolism, was injected. This experimental setup allows

TABLE 2. Chemical composition of murine HDL

WT LDLR � / � P

% of Total Mass % of Total Mass

Cholesterol 21.1 ± 0.3 20.4 ± 0.4 0.25Phospholipids 28.0 ± 0.6 24.2 ± 0.5 0.0039Triglycerides 3.4 ± 0.07 7.9 ± 0.1 <0.0001Protein 47.5 ± 0.9 47.6 ± 1.0 0.98

Blood was harvested in parallel from fasted (4 h) WT or LDLR � / � male mice. Subsequently, from plasma, HDL (d = 1.063–1.21 g/ml) was isolated by sequential ultracentrifugation. Thereafter, cholesterol, phospholipids, triglycerides, and protein were analyzed. Values are means ± SEM of n = 5 independent determinations; four independent preparations yielded qualitatively identical results.

Fig. 2. Plasma decay kinetics of 125 I-TC-/[ 3 H]CEt-WT-HDL in WT mice or in LDLR � / � mice. 125 I-TC-/[ 3 H]CEt-WT-HDL was injected intravenously in a WT or LDLR � / � male mouse. Thereafter, during a 24 h interval, periodic blood samples were harvested, and plasma was analyzed for 125 I-TC ( 125 I) and [ 3 H]CEt ([ 3 H]). The y -axis represents the fraction of the tracer in plasma (%). Shown are typical experiments of n = 13 WT mice and n = 9 LDLR � / � mice.


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a less complex system compared with an animal, hepato-cytes were isolated from WT and LDLR � / � mice. Follow-ing a 4 h seeding period, liver cells were incubated with medium containing 125 I-TC-/[ 3 H]CEt-WT-HDL. Uptake of 125 I-TC-/[ 3 H]CEt-WT-HDL by WT hepatocytes is shown in Fig. 5 . The uptake of both HDL-associated tracers in-creased in a dose-dependent manner, but internalization of [ 3 H]CEt was higher compared with 125 I-TC; the differ-ence in uptake between both tracers yields selective CE uptake from HDL by WT hepatocytes. The uptake of both HDL tracers into hepatocytes isolated from LDLR � / � mice was signifi cantly lower compared with WT cells. Ac-cordingly, WT-HDL holo-particle uptake ( 125 I-TC) and se-lective CE uptake ([ 3 H]CEt � 125 I-TC) were decreased in the LDLR-defi cient hepatocytes. In contrast to uptake, no

compared with WT. In LDLR � / � mice, a signifi cant reduc-tion in initial plasma-FCR for [ 3 H]CEt and a signifi cant decrease in selective CE removal ([ 3 H]CEt � 125 I-TC) from plasma LDLR � / � HDL by tissues are detected during the 2 h period. In WT mice, 2 h after 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL injection we observed selective CE uptake ([ 3 H]CEt � 125 I-TC) by the liver. In LDLR-defi cient mice, however, selective CE uptake from LDLR � / � HDL ([ 3 H]CEt � 125 I-TC) is decreased signifi cantly. In summary, plasma decay and liver uptake of HDL-associated tracers during a 2 h period are qualitatively consistent with the results for the 24 h experiments.

The liver is composed of distinct cell types; however, he-patocytes quantitatively dominate in this organ ( 29 ). To investigate the role of the LDLR in cellular HDL uptake in

Fig. 3. Plasma-FCRs and tissue tracer uptake rates for 125 I-TC-/[ 3 H]CEt-WT-HDL in WT mice or LDLR � / � mice. 125 I-TC-/[ 3 H]CEt-WT-HDL was injected intravenously in WT or LDLR � / � male mice. A: During the subsequent 24 h interval, blood was harvested periodically to determine the plasma decay of both tracers. 125 I-TC ( 125 I) and [ 3 H]CEt ([ 3 H]) were analyzed, and plasma-FCRs for 125 I-TC ( 125 I) and [ 3 H]CEt ([ 3 H]) were calculated. The difference in plasma-FCRs between [ 3 H]CEt and 125 I-TC was calculated. Twenty-four hours after tracer injec-tion, the animals were euthanized, and tissues were analyzed for both tracers. Liver (B), adrenal (C), and kidney (D) organ-FCRs for 125 I-TC ( 125 I) and [ 3 H]CEt ([ 3 H]) and the difference in organ-FCRs between [ 3 H]CEt and 125 I-TC were calculated. All calculations were done as described in Materials and Methods. A: Values are means ± SEM of n = 13 (WT) or n = 9 (LDLR � / � ) mice. B–D: Values are means ± SEM of n = 7 (WT) or n = 5 (LDLR � / � ) mice. An independent experiment yielded qualitatively identical results.


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Fig. 4. Plasma-FCRs and liver tracer uptake rates for 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL in WT mice or in LDLR � / � mice. 125 I-TC-/[ 3 H]CEt-LDLR � / � HDL was injected intravenously in WT or in LDLR � / � male mice. During the subsequent 24 h interval, blood was drawn periodi-cally, and 24 h after tracer injection, the animals were euthanized and tissues were harvested. Plasma-FCRs and liver organ-FCRs for 125 I-TC ( 125 I), for [ 3 H]CEt ([ 3 H]), and for selective uptake ([ 3 H]CEt � 125 I-TC) were analyzed and calculated as outlined in Fig. 3 . All values are means ± SEM of n = 5 (WT) or n = 5 (LDLR � / � ) mice. Where no error bars are shown, the SEM is on the respective line.

Fig. 5. Uptake of 125 I-TC-/[ 3 H]CEt-WT-HDL by hepatocytes isolated from WT or LDLR � / � mice. Hepato-cytes were isolated from a WT or an LDLR � / � male mouse. These cells were incubated (37°C, 2.0 h) in me-dium containing 125 I-TC-/[ 3 H]CEt-WT-HDL, and the respective concentrations are given in the abscissae. Finally, cells were harvested, and apparent HDL particle uptake was analyzed as outlined in Materials and Methods. Values are means of n = 3 (WT) or n = 2 (LDLR � / � ) independent experiments; within each experi-ment, incubations were done in triplicates. Comparing all data from WT and LDLR � / � hepatocytes, P < 0.05; two (WT) and two (LDLR � / � ) independent similar experiments yielded qualitatively identical results.

signifi cant differences in 125 I-TC-WT-HDL binding to he-patocytes isolated from WT and LDLR � / � mice were de-tected (supplementary Fig. III), indicating that double labeled HDL does not interact directly with LDLR.

To validate our experimental model, we investigated the metabolism of 125 I-TC-LDL in WT and in LDLR � / � mice in a manner analogous as outlined for 125 I-TC-/[ 3 H]CEt-HDL (supplementary Fig. IV). Following intravenous injection of murine 125 I-TC-LDL, periodic blood samples were harvested during a 24 h period. The plasma decay of 125 I-TC, representing LDL clearance, was substantially attenuated in LDLR-defi cient mice compared with WT animals. As expected, the plasma-FCR for 125 I-TC-LDL decreased signifi cantly in LDLR-defi cient mice compared with WT (supplementary Table III). The organ-FCRs for

125 I-TC-LDL uptake by liver and adrenals were signifi cantly reduced in LDLR � / � mice compared with the respective tissues with LDLR expression (WT). In summary, these re-sults demonstrate the appropriateness of the experimen-tal model used in the current study ( 7 ).

HDL selective CE uptake by the liver and adrenals in vivo and by hepatocytes in vitro is reduced in LDLR � / � mice compared with WT animals. Because SR-BI mediates the selective CE uptake from HDL, we next addressed whether a downregulation of this receptor is responsible for the decrease in HDL CE uptake detected in tissues of LDLR � / � rodents ( 4 ). Murine liver membranes were im-munoblotted using antibodies specifi c for SR-BI or LDLR ( Fig. 6 ). The signal for SR-BI protein was identical in mem-branes prepared from WT or LDLR � / � liver, suggesting


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( Fig. 7 ). The signal for ABCA1 was identical in membranes isolated from WT and LDLR � / � mice. This result argues against an altered HDL biogenesis in LDLR-defi cient mice compared with WT, and this is true at least for the liver.

DISCUSSION

A major consequence of a defi ciency of functional LDLR in mice is an increase in plasma LDL cholesterol ( 7 ). Distinct from this change, a small or a more substan-tial increase in HDL cholesterol in LDLR � / � mice has been reported ( 7, 11, 12 ). We found a minor increase in plasma HDL cholesterol in male LDLR � / � mice compared with WT littermates, although this difference was not sta-tistically signifi cant. The explanation for these differences in HDL cholesterol may be the different genetic back-grounds of the mice or the feeding conditions. In contrast to HDL levels, the composition of LDLR � / � HDL is signifi -cantly different compared with WT-HDL (i.e., HDL from LDLR-defi cient mice is enriched in triglyceride and de-pleted in phospholipid).

Mechanisms underlying the increased plasma HDL and LDL cholesterol in LDLR � / � mice were explored in this study in vivo. As expected, the decay of 125 I-TC-LDL in the circulation was decreased in LDLR � / � mice compared with WT animals, and this result is in line with an earlier investigation ( 7 ). The liver is a major organ for LDL ca-tabolism in vivo, and adrenals internalize LDL for ste-roidogenesis ( 3, 35 ). As predicted, the LDLR defi ciency yielded a decreased rate of 125 I-TC-LDL uptake by liver and adrenal glands in this study.

that altered SR-BI protein expression did not contribute to the reduction in selective uptake of HDL CE in the LDLR � / � mice. As expected, no signal for the LDLR was detected in membranes isolated from LDLR � / � liver con-fi rming the correct genotype. Besides SR-BI, scavenger re-ceptor CD36 and LRP1 have been implicated in selective HDL CE uptake in rodent liver and in hepatocytes, re-spectively ( 30–32 ). To determine whether these receptors played a role in the diminished selective CE uptake of LDLR � / � mice, expression levels of CD36 and LRP1 were determined by immunoblots ( Fig. 6 ). In liver membranes from WT or LDLR � / � mice, the signals for CD36 and LRP1 proteins were nearly identical. Thus, these data sug-gest that altered protein expression of neither SR-BI, CD36, nor LRP1 accounts for the reduced HDL CE uptake observed in the livers of LDLR � / � mice. Similarly, no obvi-ous differences in SR-BI, CD36, or LRP1 signals were de-tected in adrenal membrane preparations isolated from WT or from LDLR � / � mice (supplementary Fig. V).

ABCA1 is a membrane protein that controls the rate-limiting step in HDL particle assembly by mediating the effl ux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles ( 33 ). ABCA1 expressed by the liver has a substantial quantitative effect on HDL biogenesis and on HDL levels in plasma ( 34 ). In the studies presented previously, HDL selective CE uptake by tissues is reduced without concomitant in-crease in plasma HDL cholesterol, suggesting diminished HDL biogenesis. To test the hypothesis that a reduced he-patic expression of ABCA1 is responsible for a diminished HDL formation, the expression of ABCA1 protein was ex-plored in liver membranes from WT or LDLR � / � mice

Fig. 6. SR-BI, LDLR, LRP1, and CD36 expression in liver membranes prepared from WT or LDLR � / � mice. Membrane fractions were isolated from livers originating from WT or LDLR � / � male mice. The indicated mass of protein was subjected to electrophoresis and trans-fer to a membrane. Finally, the proteins were immunoblotted using SR-BI- or LDLR-specifi c (A ), LRP1-specifi c (B), and CD36- or � -actin-specifi c (C) antibodies. � -actin was used as loading control. Representative blots are shown. D: Three independent blots were quantifi ed by densitometric scanning; P < 0.05 for all blots.


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isolated LDLR-defi cient hepatocytes, a decrease in uptake of HDL-associated 125 I-TC was shown, suggesting reduced HDL holo-particle internalization. In parallel, 125 I-TC-HDL binding (4°C) to WT liver cells or to LDLR � / � hepa-tocytes was identical. This observation suggests that the reduced HDL holo-particle uptake of LDLR � / � hepato-cytes is not due to reduced HDL binding to the cell mem-brane. Thus, the interaction between HDL and the plasma membrane is not necessarily followed by hepatocellular HDL uptake. Compared with these results obtained in vi-tro, the HDL experiments performed in vivo are presum-ably physiologically more relevant. In summary, an LDLR defi ciency has no substantial effect on HDL holo-particle metabolism in plasma and by tissues.

These observations on HDL holo-particle metabolism are in strong contrast to the selective CE pathway. A lack of LDLR is associated with a signifi cant decrease of selec-tive CE uptake from HDL by liver and adrenals. This was true for WT-HDL and for LDLR � / � HDL, as well as for the 2 h and the 24 h metabolic studies. Quantitatively, the decrease in selective CE uptake by liver and adrenals in LDLR � / � mice was smaller in the case of radiolabeled LDLR � / � HDL compared with WT-HDL. The explanation for this difference may be the different composition of both HDL preparations; a similar result has been obtained previously ( 6 ). Consistent with these observations on HDL metabolism in vivo, in cultured murine hepatocytes with an LDLR defi ciency, a signifi cant decrease in selective HDL CE uptake is observed compared with WT liver cells. This regulation of the HDL CE selective uptake pathway in the absence of LDLR has not been established previously, and our data represent a novel fi nding.

SR-BI can bind and mediate selective CE uptake from both LDL and HDL ( 4 ). LDLR � / � mice have a substantial increase in cholesterol-rich lipoprotein particles in plasma. Therefore, the question has to be raised as to whether a competition between LDL and HDL for SR-BI-mediated selective lipid uptake in vivo explains the decreased selec-tive CE uptake in LDLR-defi cient mice. However, the ex-periments with murine hepatocytes in vitro are a strong argument against this possibility.

With respect to intravascular lipoprotein metabolism, it is established that some apos, for instance HDL-associated apoA-I, are mobile and exchange between lipoprotein fractions ( 1 ). Concerning CE, in murine plasma no CETP activity is detectable; therefore, a lipid exchange reaction is unlikely in the circulation ( 36 ). To address the issue of an exchange of HDL-associated tracers with non-HDL li-poprotein, FPLC analysis of plasma lipoproteins after HDL tracer injection was done. In WT and LDLR � / � mice, no transfer of 125 I-TC tracer (i.e., no apo transfer) out of the HDL fraction could be detected. In contrast, during the time course of 24 h after injection of radiolabeled LDLR � / � HDL, initially HDL-associated [ 3 H]CEt tracer could be detected in FPLC fractions corresponding to non-HDL lipoproteins in LDLR � / � mice. Considering the CETP defi ciency of mice ( 36 ), the hypothesis emerged of a resecretion of initially HDL-associated [ 3 H]CEt by the liver, for instance in VLDL particles. In fact, an increased

The protein and the lipid moieties of HDL particles can be metabolized at different rates in vivo and in vitro ( 5 ). A previous study used protein-iodinated HDL in LDLR � / � mice, and therefore, no information was obtained with re-spect to the turnover of the lipid component of HDL ( 7 ). To explore HDL metabolism in more detail, we radiola-beled the lipid and the protein moieties of these particles. With this approach, the fate of the distinct HDL compo-nents can be explored simultaneously ( 5 ). The composi-tion of WT-HDL and LDLR � / � HDL is different; therefore, HDL preparations from both WT and LDLR � / � mice were used for these studies. Using double radiolabeled murine WT-HDL or LDLR � / � HDL in WT or in LDLR � / � mice, the major fi ndings of this study are as follows: a ) selective CE removal from the plasma HDL pool by tissues is re-duced in rodents with an LDLR defi ciency; b ) selective CE uptake from HDL is diminished in liver and adrenals of LDLR � / � mice; and c ) the reduced uptake of HDL-associ-ated CE by tissues is not mediated by changes in mem-brane protein expression of SR-BI, CD36, or LRP1.

HDL-associated 125 I-TC tracks the metabolism of HDL holo-particles ( 18 ). In vivo, the plasma decay of HDL-asso-ciated 125 I-TC and liver and adrenal uptake of this tracer were not different between LDLR � / � and WT mice during the 24 h experiments. During the 2 h turnover, a quantita-tively very small decrease in 125 I-TC decay in plasma was detected in LDLR � / � mice; however, the biological rele-vance of this reduction presumably is minor. In vitro, in

Fig. 7. ABCA1 expression in liver membranes prepared from WT or LDLR � / � mice. Membrane fractions were isolated from livers originating from WT or LDLR � / � male mice. The indicated mass of protein was subjected to electrophoresis and transfer to a mem-brane. Finally, the proteins were immunoblotted using ABCA1- or � -actin-specifi c antibodies. � -actin was used as loading control. A: A typical blot is shown; three independent blots yielded qualitatively identical results. B: Densitometric scanning of three blots, P < 0.05.


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In this study, no signifi cant change in HDL cholesterol in LDLR � / � mice was detected when compared with WT rodents. Generally, decreased HDL catabolism as observed here results in an increase in plasma HDL cholesterol ( 6 ). How can this discrepancy in our fi ndings be explained? The steady-state concentration of plasma HDL cholesterol is the result of HDL biogenesis and HDL catabolism ( 1 ). A quantitatively dominant organ in HDL catabolism in ro-dents is the liver, and HDL lipid uptake by this organ is decreased in LDLR � / � mice ( 5 ). A possible explanation for the discrepancy between the essentially unchanged HDL cholesterol in plasma and the decreased selective HDL CE uptake by tissues is that HDL synthesis is reduced in LDLR � / � mice. Hepatic ABCA1 is a key regulator of plasma HDL cholesterol ( 34 ). Therefore, the hypothesis was tested as to whether a reduced hepatic expression of ABCA1 protein mediates a decrease in cellular lipid effl ux in LDLR � / � mice. However, ABCA1 protein expression was unchanged in LDLR-defi cient liver membranes com-pared with those from WT mice. This fi nding suggests that a difference in cholesterol effl ux and a modifi ed HDL bio-genesis between both groups of animals is unlikely.

LDLR � / � mice are a frequently used model for studies on atherosclerosis ( 8, 41 ). Usually, this increased athero-sclerotic burden is attributed to the increase in plasma cholesterol contained in apoB-containing lipoproteins. However, our studies point to an additional mechanism that may be relevant for the observed susceptibility for ath-erosclerosis. Substantial changes in HDL metabolism are detected in the presence of an LDLR defi ciency, and HDL plays a key role in reverse cholesterol transport, i.e., the fl ux of lipid from peripheral tissues to the liver for excre-tion via bile ( 42 ) . Therefore, it is suggested that the LDLR modulates both LDL-mediated cholesterol delivery to cells as well as HDL-mediated reverse cholesterol transport to the liver, and both pathways may be relevant for the patho-genesis of atherosclerosis at least in mice.

The authors thank S. Ehret, B. Schulz, and M. Thiel for technical assistance. Dr. Richard E. Morton and Diane Greene donated CETP for radiolabeling, and Dr. Martin Merkel injected mice intravenously.

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