Use of an anti-low density lipoprotein receptor
antibody to quantify the role of the LDL receptor
in the removal of chylomicron remnants in the
mouse in vivo.
S Y Choi, … , M J Kirven, A D Cooper
J Clin Invest.
1991;
88(4)
:1173-1181.
https://doi.org/10.1172/JCI115419
.
Lipoproteins are removed from the plasma by LDL receptor-dependent and -independent
pathways. The relative contribution of these has been established for LDL by using modified
lipoproteins, but this has not been possible for apoE-rich lipoproteins, such as chylomicron
remnants. To do this, we used a monospecific antibody to the rat LDL receptor. The
antibody was injected intravenously into mice followed by 125I-lipoproteins. Blood samples
were obtained sequentially and radioactivity measured to determine the plasma clearance
of the lipoproteins. The animals were then sacrificed and the tissues removed, dried, and
the radioactivity measured to determine tissue uptake. An albumin space was also
measured to correct for blood trapping. With 125I-human LDL, approximately 50% of the
injected dose was cleared in 180 min. This was reduced to 30% by the antibody and this
was identical to the disappearance of reductively methylated LDL. This is a lower estimate
of LDL-mediated uptake (40%) than in other species. LDL uptake per gram tissue was
similar for the liver and the adrenal gland and was approximately 50% LDL
receptor-dependent in both tissues. With 125I-chylomicron remnants, clearance was much more
rapid with approximately 50% cleared in 5 min. By agarose gel electrophoresis, radioactivity
was not transferred from chylomicron remnants to other lipoprotein classes. Chylomicron
remnants with label on only apoB or in 3H-cholesterol esters showed a similar […]
Research Article
Use of
anAnti-Low Density Lipoprotein Receptor
Antibody
to
Quantify
the Role of the LDL
Receptor
in the Removal
of
Chylomicron
Remnants
in the Mouse In
Vivo
SungshinY.Choi,*LorenG.Fong,* MelissaJ.Kirven,*and Allen D.Cooper"t
*Research Institute, Palo Alto Medical Foundation, Palo Alto, California 94301; andtDepartment ofMedicine,
Stanford University, Stanford, California 94305
Abstract
Lipoproteinsareremoved from the plasma by LDL receptor-dependent and-independentpathways. The relative
contribu-tionofthese has beenestablishedfor LDL by using modified lipoproteins, but this has not been possible for apoE-rich
lipo-proteins,such as chylomicron remnants. To do this, we used a
monospecific antibodyto the rat LDL receptor. The antibody was
injected
intravenously into mice followed by'25I-lipopro-teins. Bloodsamples were obtained sequentially and
radioactiv-itymeasured todeterminethe plasma clearance of the
lipopro-teins. The animals were then sacrificed and the tissues re-moved, dried, and the radioactivity measured to determine
tissue uptake. Analbuminspace was also measured to correct
forblood trapping. With '25I-humanLDL, - 50% of the
in-jected dosewascleared in180min.Thiswas reduced to 30% by the antibody andthis was identical to the disappearance of
reductivelymethylated LDL. This is a lower estimate of
LDL-mediateduptake(40%)than in other species. LDL uptake per gramtissue was similarforthe liver and the adrenal gland and was - 50% LDL receptor-dependent in both tissues. With
125I-chylomicron remnants, clearance was much more rapid
with - 50%cleared in 5 min. By agarose gel electrophoresis,
radioactivitywas nottransferred fromchylomicron remnants to
otherlipoprotein classes. Chylomicronremnantswith labelon
onlyapoBorin3H-cholesterolestersshowedasimilarpattern.
Combining the estimates of the three labeling procedures,
- 35%ofthe30sand25% ofthe5minchylomicronremnant
disappearance was LDL receptor dependent. The liver, per gramtissue,took up five times as muchradioactivity as the
adrenalgland.At 5min,atleast 50% of thiswasLDL
receptor-dependentin liver and65%inadrenalgland.Weconclude that the LDL receptor
plays
amajor,and somewhatsimilar quanti-tative rolein the clearanceof bothLDLandchylomicron rem-nantsinthemouse.However,atleast in the mouse,non-LDL receptor-mediated lipoprotein clearanceis quantitatively im-portantandisalso veryrapid for chylomicronremnants.Thus,for
chylomicron
remnants,it caneasily compensatefor LDLAddress correspondencetoAllenD.Cooper, M.D., ResearchInstitute,
PAMF, 860 Bryant St., Palo Alto, CA 94301.
Receivedforpublication13March1991andinrevisedform14 June 1991.
1.Abbreviations used in thispaper:DLT, dilactitol125I-tyramine;FH, familial hypercholesterolemia; HRP, horseradish peroxidase; LRP, LDLreceptor-like protein;RM, reductively methylated.
receptors if they are blocked or absent.Further,thetissue dis-tribution of lipoproteinuptake may be directedby factorsother than LDL receptor density. (J. Clin. Invest. 1991. 88:1173-1181.) Key words: lipoprotein- LDL receptor .chylomicron
remnant * cholesterol-triglyceride-LDL
Introduction
Becausethediscovery ofastrong
positive correlation
between plasmaLDLand theincidence ofcoronaryheart disease(1), thedeterminants of the level ofLDLin theserumhave been studied intensively. The classic studies ofBrownandGoldstein identifiedareceptor onthe surfaceof
mostcells(2) thatiniti-atestheuptake of intact plasmaLDL
particles
into the cell via endocytosis,andthusplaysamajor role inmaintaining
choles-terol homeostasis.Although thereceptorrecognizes
bothapoB
andapoE, ithasamuchgreater
affinity
forapoE
thanapoB (3).
Thenumberofreceptorsexpressedonthesurface ofacellis regulatedbythecellularcholesterolcontent; LDL receptorsare
downregulated
whensubstantial
amountsof
cholesterolare de-liveredtothe cells, thuspreventing
them fromaccumulating
excess cholesterol (4).
Quantitatively,
themajor
site ofLDLreceptor-mediated lipoprotein degradation
is the liver. How-ever, evenin the completeabsenceofLDL receptors, alarge
fraction ofLDLcontinuestobe takenupby hepatocytes (5), indicating thatthereisatleastonemechanism other thanthe LDL receptorthatcanplayarole in thecatabolism ofLDL.
Furthermore, other
lipoproteins, including chylomicron
rem-nants,seem tohave bothLDL
receptor-dependent
and-inde-pendent
mechanisms oftransport(6).
Chemically-modified
forms of LDL, such asreductively
methylatedLDL(7)and
1,2-cyclohexanedione-modified
LDL(8), havebeen usedtodetermine the relative
contribution
ofLDLreceptor-dependent and -independent pathwaystoLDL
metabolism. However,it hasbeendifficulttodefinitively
sepa-ratethecontributions ofLDL receptorandnon-LDL
recep-tor-mediatedtransportfor
lipoproteins
other than LDL. The development ofananti-LDLreceptorantibody
whichspecifi-cally blocks theLDL receptor
(9)
allowedustoapproach
this problem.Wehave observed(10)
thatthedegradation
ofchylo-micronremnants inisolated
primary
rathepatocytes
in vitro could be almostcompletely
abolishedby
theaddition oftheanti-LDLreceptor
antibody,
suggesting
thatprocessing
ofchy-lomicronremnantsby hepatocytescanbedependentuponthe
LDL receptor. These results are not
entirely
consistent with observations in Watanabe rabbits(11)
and inpatients
with fa-milialhypercholesterolemia
(FH)
(12) in whichclearance ofchylomicron
remnants wasrapid, although
LDL receptorswereabsent, thussuggesting that eitherthe LDL receptor hasa
limited roleinremnantremoval orthatanalternate mecha-nism cancompensate foranabsence ofLDL receptors. The
J.Clin.Invest.
©TheAmericanSocietyfor ClinicalInvestigation,Inc.
0021-9738/91/10/1173/09 $2.00
recent description (13) of an LDL receptor-related protein (LRP) that can bind lipoproteins that are enriched in apoE (14) and is not subject to down regulation by cholesterol provides a potential mediator of such an alternate pathway.
Todirectlyexamine the role of LDL receptor-dependent and-independent pathways to the plasma clearance and tissue uptake of chylomicronremnants invivo, micewereinjected
with anti-LDLreceptorantibody, and the plasma removal of subsequently injected lipoproteins was measured. The results suggest that the LDL receptor plays a major quantitative role in the clearance and tissue uptake of both LDL and chylomicron remnantsin thenormal mouse in vivo.
Methods
Animals. Female Swiss Webster mice, weighing 27-30 g, were pur-chasedfromSimonsen Laboratories and housed with a 12-h light cycle (7 am-7 pm) and free access to a standard mouse chow and tap water.
Sprague-Dawley-derivedratsfrom the same source were fed a standard diet until they were used as lymph donors.
Reagents. Radioiodine ('25I-carrier-free) was purchased from
AmershamCorp., Arlington, IL, and tissue culture media were from Gibco, Grand Island, NY. Fatty acid-poor bovine albumin (BSA) were obtained fromBehringDiagnostics, San Diego, CA.Allother chemi-cals were obtainedfromSigma Chemical Co., St. Louis, MO, or J. T. BakerChemical Co., Phillipsburg, NJ.
Preparationoflipoproteins.Human LDL (d 1.0 19-1.063 g/ml) was
isolatedfrom EDTA-containing plasma by density gradient
ultracentri-fugation (15)and radiolabeledwith Na '25I usingIodogen (22). The
acetylationandreductive methylation (RM)ofLDLwereperformedas
describedby Basuetal.(17) andWeisgraberetal. (18),respectively. Dilactitol '25I-tyramine (DLT)-LDLwas preparedusing the method of Strobeletal.(19).Chylomicronswere prepared by the method previ-ously described (20), andchylomicronremnantswere prepared by the modificationofthe methodofRedgrave and Martin (21) previously
described (22). Chylomicronremnants wereradiolabeled usingthree
differentprocedures. Formostoftheexperiments,isolated
chylomi-cron remnants were iodinated byamodification (22)of the iodine monochloride method(23).Inonegroup ofexperiments, chylomicron
remnants wereisolatedfromratsthat had beeninjectedwith
chylomi-cronsthat had beenpreviously radiolabeled bytheiodine monochlo-ride method. In another group ofexperiments, chylomicronremnants
werelabeled with3H-cholesterolesterby the method ofFielding (24).
Alllipoproteinswerescreened forbiologic activityasdescribed below.
Inaddition, the degree oflipidiodinationwasmonitoredonallbatches
aspreviously describedandfell within the rangepreviouslyreported
(22). Selected batcheswereanalyzed byPAGE inasystemcontaining
0.5% SDS withorwithoutautoradiography,andapoproteincontents
andiodinationpatternsweresimilartopreviously reportedvalues(25).
Total protein concentration of lipoproteinswasdetermined bythe method ofLowryetal.(26).
Anti-LDL receptorantibodies.Theanti-LDL receptor antibodies used have beenpreviouslydescribed andcharacterized (9,27).
Mono-specificityand lackofcross-reactivitywith theLRPhave been
previ-ouslydocumented(27).
Degradation of lipoproteins by mouse macrophages. Resident
mouseperitonealmacrophageswereharvested from micebyperitoneal
lavage andresuspendedin DMEMsupplementedwith 10% FCSat a
densityof 1.25 X 106 peritoneal cells/ml. 1 mlof the cellsuspension wasadded to24-welltissue cultureplates (Costar,Cambridge,MA)and incubatedat37°C for 2 h. Nonadherent cellswereremovedby rinsing
threetimeswithDMEMcontaining0.5% BSA and 10 mM N-2-hy-droxyethylpiperazine-N'-2-ethane-sulfonicacid (Hepes) (pH 7.4).The cellswerethenincubated with '25I-labeledlipoproteins (10 ug/ml)in the presenceorabsenceofunlabeledlipoproteinsoranti-LDL receptor
antibodyat
370C
for4h. Theextentoflipoprotein degradationwasassessedby measuringtheamountofTCA andsilver nitrate soluble
radioactivitypresent in the incubation medium. The small amount of
degradation productsgenerated in the absence of cellswasalso
mea-sured and subtracted from thecorrespondingsamples incubated with cells.
Disappearanceoflipoproteinsfrom plasma.Micewereinjected
in-travenouslywith 20Ag protein of '25I-labeledLDLorchylomicron
remnantsin0.1 mlPBSvia the tail vein. Blood samples were with-drawnatvarioustimepointsthereafter intoheparinized capillarytubes
bypuncturingtheretroorbitalplexus (28). Radioactivityin 10Mlof bloodwasthenmeasured inagammacounter.Theamountof
lipopro-teinremainingin the plasmawasexpressedas apercentage ofthe calcu-lated initial bloodconcentration(To).Theinitial plasma concentration
wasestimatedforLDLbyextrapolatingthe amount of lipoprotein at timezerofromthefirsttwomeasured values or, forchylomicron
rem-nants,bycalculatingthe correctedtheoretical initial plasma concentra-tion basedontheamountinjected,theapproximateblood volume(9%
ofbodyweight)and the recovery ofinjectedmaterialasdetermined in the LDLremovalexperiments(the difference between the extrapolated initial concentration and theinjecteddosedividedby the blood vol-ume;seeResults andDiscussion).
Determinationofspecifictissue spaces. After the last blood sample
wascollected, theanimalsweresacrificed and various tissues removed, rinsedinPBS, anddried inan oven.Theweightand theamountof
radioactivitywasthen measured. Tissue spaceswerecalculatedby the methodofSpadyandDietschy (29) usingthefollowingformula: tissue space (microliters plasma per gram dry tissue weight) = [cpm in
tissue/(grams dry weight X cpm/microlitersplasma)]. The cpm per microlitersplasmawascalculatedfromthe average of theamountof
radioactivityin the 0- and 5-minor0- and180-minsamplesfor
chylo-micronremnantsandLDL, respectively.Atissue space for
'25I-BSA
wasdeterminedtocorrect fornonspecifictrappingoflipoproteins.The albumin space forspecifictissue spaces oflipoproteinswasthen calcu-latedbysubtractingthe'25I-albumin
space from thelipoproteintissue spaces(29).The resultswereexpressedasthemicroliters ofplasmaper gram of dry tissue weight. Liver weights in grams were LDL, 0.397±0.06;LDLplus antibody, 0.389±0.032;remnants,0.329±0.04;remnantsplus antibody,0.345±0.026.
Immunoblotting.Membranes frommouseliver and adrenalglands
and liver fromethinylestradiol-treatedrats werepreparedand solubi-lizedasdescribed earlier(27). The solubilized membranes(200
Mg)
wereseparatedon6%polyacrylamide gels bythe method of Laemmli
(30)undernonreducingconditions.Immunoblottingfor the LDL re-ceptorwasperformedaspreviouslydescribed(9).
Briefly,
proteins
weretransferredtonitrocellulose paperbyblotting asdescribed, and the
portionof the papercontainingthemolecularweightstandardswascut
off and stained with 0.1% amido black. The remainder ofthe paperwas
incubated with 1
Mg/ml
polyclonalrat LDL receptor rabbitanti-serumfor 1 hat roomtemperature,washed,and thenincubated with horseradish peroxidase (HRP)conjugatedgoat anti-rabbitIgG.The HRPconjugated IgGwasdevelopedwith the
Immuno-Blotfm
(GAR-HRO)assay kit(Bio-Rad Laboratories, Inc., Richmond,
CA).
The ni-trocellulose paperwasdried and then scanned with adensitometer(HoeferScientificInstruments,SanFrancisco, CA).
Statistics. Statistical analysiswasdone by
nonpaired
Student'st test.Results
Characterization
of
lipoproteins.
Thelipoproteins
used wereroutinely
characterizedby
assessing
theirapoprotein
composi-tions and
measuring
theirrateofdegradation by
mousemacro-phages.
Theapoprotein compositions
ofLDL,
reductively
methylated LDL,
andchylomicron
remnantswereanalyzed by
SDS-PAGE
(data
notshown).
Theapoprotein
patternswereTofunctionally test the lipoproteins,they were incubated with
mouse peritoneal macrophages. Specific degradation of both 125I-LDL and '251-chylomicron remnants could be demon-strated. As previously reported (31), chylomicron remnants weredegraded at a slower rate than LDL; however, degradation ofboth LDL and chylomicron remnants were suppressed by >70% byan excess of unlabeled lipoproteins (not shown) or of anti-LDL receptor antibody (Fig. 1 a). In contrast, the
degrada-400
300-200
100
-01
200
r-._.
0 c
= °
m
-1 00
0 = ._
0
300
200
-100
-0
-T
~~~~~~~~~~~~~~~~..
--....
~~~~~~~~~~~~~~~....
-. ...-... ... ,LD L LDL* PCAb C R CRF+PCAb
-n
LDL* LDL*+LDL RM-LDL
IE'
--.C R* CR*+CR CR*+aLDL
Figure 1.Degradation of lipoproteins bymousemacrophages. Mouse
peritoneal macrophageswereincubated with 10 Mg/ml'251-labeled
LDL, reductively methylated (RM)-LDLorchylomicronremnants
(CR)inthepresenceorabsence of unlabeledlipoproteins (500 Mg/ml)
oranti-LDLreceptorantibody(500 ug/ml)at37'C for4h.
Degra-dationof '251-labeledlipoproteinswasdeterminedasdescribedunder
Methods.(A)Effect ofpolyclonalanti-LDLreceptorantibodies (PCAb)onthedegradationoflabeledlipoprotein. LDL**,CR**are thespecific (total-nonspecific) degradationoflabeled LDLor chy-lomicronremnants. LDL*+PCAb,CR* +PCAbarethetotal
deg-radationof LDLorchylomicronremnantsinthepresenceofexcess
anti-LDLreceptorantibody. (B) Comparisonoftotaldegradationof '251-LDL(LDL*)withthat of 1251-RM-LDL (RM-LDL)and nonspe-cificdegradationof1251-LDL (LDL*+LDL);(C) Comparisonoftotal
degradationof1251I-CR(CR*)inthepresenceofunlabeledCR(CR*
+CR)oracetyl-LDL (500 Mg/ml,CR*+aLDL).*P<0.05.
I.
,0
oE
o .
03
0
-a.
ML.
, es
-1
0
100I
80
60
40-20
0 30 60 90 120
Time (minutes)
150 180
Figure 2. Effect of anti-LDL receptor antibody on the clearance of
125I-LDLfrom the blood of mice in vivo. Micewereinjected intrave-nously with0.1mlrabbitanti-LDL receptor IgG (0.5 mg) or saline. After 1 h, they were injected intravenously with 20
Og
of1251-human LDLor125I-RM-LDLvia the lateral tail vein, and blood samples (10,ul)taken for determination ofradioactivity. Resultsareexpressedas the amountof radiolabel remaining in thecirculationas apercent of the initial plasma concentration which was calculated by extrapo-lating the amount of LDL or RM-LDL in the first two samples (i.e.,
1 and 2.5 min) back to time zero. Each point represents the mean±SE. (n) LDL (n=8); (o) LDL+anti-LDL receptor antibody (n=4);(o) RM-LDL (n=8). *P<0.05.
tion ofreductively methylated
1251I-LDL (RM-LDL)
wassignifi-cantlylower than
1251I-LDL
(Fig. 1 b), indicatingthat thereduc-tivemethylationwaseffective in
making
theparticlesapoorligand forthe LDL receptor pathway. Inaddition, lipoproteins
were screenedforuptake by the acetyl LDL receptor. The up-take by these cells was low compared to acetyl LDL (not shown),was notblockedbyanexcessof unlabeledacetylLDL
(Fig. 1 c), butwas blockedby the anti-LDLreceptorantibody (Fig. 1 a), demonstrating that they hadnotbeen convertedto
ligands for the acetyl LDL receptor by isolation, storage or
iodination.
Effect
of
anti-LDL receptorantibody
onthe
disappearance
of '25I-LDL from plasma.
Theappropriate
concentration toblockLDL receptor
activity
in vivowasdeterminedby
com-paring
the plasmaclearance of1251I-LDL, 12'I-RM-LDL,
and125I-LDL
plus anti-LDLreceptorantibody.
Aftertheinjection
of
1251-LDL
or125I-RM-LDL,
samples of bloodwereobtained andthe amountofradioactivity
determined.Forthesestudies,
theinitial
lipoprotein
concentrationwasdeterminedby
extrap-olating the firsttwotime
points
backtotimezero. From thesedata, itwascalculated that87%ofthe
injected
dose ofLDLand RM-LDLreached thecirculation. Both LDL and RM-LDL wereremovedslowlyfromtheplasma. 3 hafterinjection,
the plasma concentrations of125I-LDL
and125I-RM-LDL
were50 and70%,
respectively,
of their initial concentrations(Fig.
2). Injection of 0.5mganti-LDLreceptor
antibody
1 hbeforethe
injection
of'25I-LDL
reduced thedisappearance
of125I1
LDL to30%,aresult
virtually
identicaltothatwithRM-LDL.Nonimmune
IgG
didnotaffect theclearance of1251-LDL
orin laterexperiments
of'25I-chylomicron
remnants(data
notshown); thereforeinmost
experiments,
asalineinjection
wasused as a control. Theseresultsdemonstrate thatthe
antirecep-torantibodyblocks the interaction ofhuman LDL with the
high-affinity
LDL receptorsaseffectively
asdid reductive meth-ylation oftheLDL.Theeffect ofvarying
amountsofanti-LDLC,01=
. £
a.M
CSt
0
a. D.
r c
_ .
C,U)--6
-o2
C C,
0.
r-C
0
-J
.-.'
cio
a as
-°" 0a
.viX
U
_-4 Q
V-0
E0
0
0at
o 100
Time (minutes)
receptorantibodywasalsoexamined.The degreeofinhibition
was dependent upon the amountofantibody injected(Fig. 3).
Themaximum effect wasobtained with0.5 mg per mouse.
Injection of> 1.0 mg actually resultedinreducedblockade,
therefore, 0.5 mgantibodywas usedinall subsequent experi-ments.Theresult that- 40%ofLDL uptakeinthefirst2 hin
the mouse is dependent on the LDL receptor is somewhat lower thanpreviousestimates with RM-LDL andautologous LDLin therat(32)orrabbit (33).
Effect
of
anti-LDL receptorantibody
on LDL uptakeby
tissues. The
specific
tissue spaces ofLDL were examined in severalorgans to assessthemajor tissue sites ofLDLuptake in the mouse. After the final blood sample, the liver, adrenalgland, spleen,
bothkidneys
andapiece
of smallintestine
were removedand countedforradioactivity. The dry tissue weightwas measured to reduce the variability due to the different
amountsoforganmoisture. Thiswasalsocorrectedfor
nonspe-cific
trapping
oflipoproteins
bysubtracting
the tissue spacedetermined for albumin. The differences betweenthe 1251-la-beledLDLandthe
"25I-albumin
spacesfor each tissuearere-05
v
0 d
2000-(n =
0 .U
U( >.
m' 1 000
--a
0
un
E
Cl
Ej-,
O
-I
I-:
Ii
LIVER SPLEEN KIDNEY AD GL SI
Figure 4.LDL-specifictissue spaces. '25I-DLT-LDL(20pg/mouse) wasinjectedIhafter saline or 0.5 mg anti-LDL receptor antibody and theanimals sacrificed3 h later.Tissueswereremoved, rinsed in
saline,blotted dry, placed inan oven(80°C) overnight, and then
weighed.Theradioactivity of the tissues was then measured.Asimilar
experimentwasconducted3 hafterinjectionof
'25I-albumin.
Tissue spaceswerecalculatedasmicroliters plasmaper gramdrytissueweightasdescribed in Methods. Thelipoproteinminusthe albumin
tissue space isthe LDL-specifictissue space. Resultsareexpressedas
mean±SEof valuesfromthree animals in each group. (m)CR; (m)CR
+anti-LDL receptorantibody.
Figure 3.Effect of varying amountsofanti-LDL re-ceptorantibodyontheclearance of'25I-LDLfrom the bloodof mice in vivo. Theprotocolwasthesame
as inFig. 2, except that theamountof rabbit anti-LDL receptorIgG varied from 0.2to2.1 mg per mouse.(n) 1251-LDL;(-) 1251I-LDL+0.2 mgIg;(i) 1251-LDL+0.5 mgIgG;(o) 1251-LDL+ 1.0 mgIgG;
(i) 125I-LDL+ 1.3mgIgG;(o)'251-LDL+2.1 mgIgG.
ferredto as"specific tissuespaces"(29).DLT-LDL was usedin these studies to minimize the amount of degradation that
might haveoccurred, subsequent toendocytosis(34, 35). The
plasma clearance ofDLT-LDL wasidenticaltothatof
conven-tionally-labeledLDL(not shown).
Liverandadrenal glandwerethemostavid in
accumulat-ingboth nativeLDL and DLT-LDL ascompared to spleen,
kidney, and small intestine (Fig. 4). By mass, the liver
ac-countedfor the uptake of 85% of the
lipoprotein.
It was some-whatsurprisingthat theliverhad aspecific tissue space onlyslightlyless thanthatofthe adrenal.This
might
be duetothe fact that, in the mouse,the adrenal isso small thatwe wereunableto completely free it of
surrounding
adherenttissue,particularly
fat. Consistent withthis, immunoblots
of liver and adrenalwith equalamountsofapplied protein
confirmed that,on a
protein
basis,
the adrenalgland is about twofold richer than theliver inLDL receptors(Fig.
5). Afterinjection
ofanti-LDL receptorantibodies,thespecific tissuespacesof
"23I-DLT-LDL werereduced in both theliver and adrenal glandand to alesserextent, inthespleen,kidney, and small intestine. Based
1 2 3 4 Figure5. Immunoblots of
(kDa) mouseliver and adrenal
glandmembranes. Mem-braneswerepreparedfrom tissue
homogenates by
cen-trifugation
andsolubilizedwith CHAPSasdescribed in Methods. Proteinswere 116 I- separatedby electrophoresis
on6%polyacrylamidegels
and then transferredto
ni-trocellulose.Stripswere in-cubated with the anti-LDL receptorpolyclonalrabbit
serumfollowedby horse-radishperoxidase
conju-gatedgoat anti-rabbitIgG.
(Lane1)Molecularweight
standards(myosin,
205,000;
fl-galactosidase,
116,000); (lane2)liver membranes fromethinylestradiol-treated rats;(lane 3)liver
mem-branes from normalmice;(lane4)adrenalglandmembranes from normal mice.
on the data in Fig. 4, it was estimated that 45% of hepatic uptake and 55%ofadrenal uptake of LDL was LDL receptor-dependent. These values are also lower than those obtained with RM-LDL in rat (32) and rabbit (33). Taken together, these results suggest that the anti-LDL receptor antibody blocks LDLuptake, but may result in an underestimation of the con-tribution of the LDL receptor-dependent pathway.
Effectofanti-LDL receptor antibody on the disappearance of'25I-chylomicron remnants from plasma. In agreement with other studies,
'251-chylomicron
remnants were removed from the plasma at a veryrapidrate (20). Due to the rapid disappear-ance,the initialplasmaconcentrationwas calculated based ontheamountof chylomicronremnants injected,theestimated
total blood volumeand thepercentlossof injected material,as
determinedin the LDLexperiments.Using this to calculate the
To
radioactivity, - 33%oftheinjected particleswere removedin 0.5min and 48% in 2min (Fig. 6).Toexaminethe roleofthe LDLreceptor inthe removal of chylomicron remnants from thecirculation, micewerefirst injected with theanti-LDL re-ceptorantibodyat adosethat waspreviouslyshown to
maxi-mallyblock LDL receptor-dependent clearance ofLDL. At
this concentration,theantibody reducedthe veryrapid(30s) clearanceto25% and the 5minclearanceto31%(Fig. 6).Based onthis,at least 35% ofthe remnant disappearance could be
attributedtotheLDLreceptor.
A significant portion ofthe radioactivity ofthe
chylomi-cronremnants radiolabeled after isolation islocated on
pro-teins other thanapoB. Althoughthetime frame isveryshort,
someof these might exchange in plasma with other lipopro-teins; thus, itwaspossible that the radioactivity inplasma may represent labeled remnantapoproteins that hadredistributed
toother lipoproteinsand notchylomicronremnantsremaining
in thecirculation. Totestthis,plasmasampleswere analyzed
aftertheinjection of
'25I-chylomicron
remnantsbyagarosegelelectrophoresis.
If therewastransfer of labeledapoproteins
to otherlipoproteins, their radioactivity wouldbeassociated with fastermigrating lipoproteins that caneasilybe distinguishedfrom
chylomicron
remnants which remain at the origin.Figure 7. Agarose gel elec-trophoresis of plasma. To determine whether the ra-dioactivity of '25I-chylomi-cronremnantsredistributed toother lipoproteins, plasma samples (30
Ml)
from mice after injection of
'25I-chylomicron
remnant*;
weresubject
to agarose(0.7%) gel electrophoresis. Thegelwasthenexposed to x-ray film. (Lane 1)
hu-manLDL;
(lane 2)
acetyl-LDL;(lane 3) chylomicron
^ remnants;
(lane 4) plasma
sample at 1 min; (lane 5)
LDL a-LDL CR 1 1.5 4 plasmasample at 1.5 min; (min) (lane6) plasma sampleat
1 2 3 4 5 6 4min.
Plasmasampleswereseparated byagarosegel
electrophoresis
and autoradiograms prepared. The autoradiogram was
pur-poselyoverexposedtorevealanyradioactivityassociated with
nonchylomicron remnant lipoproteins. As expected, nonin-jected
chylomicron
remnants remainedattheorigin (Fig. 7,
lane 3). Inplasma samplescollectedup to 4 minafter
chylomi-cron remnant injection, there was no
significant
transferoflabeled apoprotein to other
lipoprotein
classes(Fig.
7, lanes 4-6). Alsoshownisthemigration
of labeledLDL(Fig. 7,
lane1) and acetyl-LDL (Fig. 7,lane2)forcomparison.
Because thereis
nonapoprotein protein
onchylomicron
remnants, particles containinga corelabelwerealso studied.
Chylomicron
remnants werelabeled with 3H-cholesterolesterand their disappearance measured as above.
Although
the amountofradioactivity
that couldbeinjected
limited theaccu-racyofthestudy,theresultsweresimilartothosewith
iodin-ated chylomicron remnants. The clearance of 3H-cholesterol ester-labeled
chylomicron
remnants wasrapidwith - 80%re-moved after5 min andthe
plasma
clearancewassuppressed
bythe anti-LDLreceptor
antibody
(Fig. 8).
Tostudy
remnant100
80
60
40
20
c ^
c
E-6
_ 6.
OV c
c cl
0 -.
I-r-=
c
_
0_
IL)
-0 2 3 4
Time (minutes)
Figure 6. Effect of anti-LDL receptorantibodyonthe clearance of
123I-chylomicron
remnantsfrom the blood of mice in vivo. The pro-tocolwasthesame asinFig.2 except that the animalsweresacrificed 5minafter the injection ofradiolabeled lipoproteins,and thecon-centrationattime0wasestimated from the actualamountinjected
correctedforloss,asdeterminedin theLDLclearanceexperiments,
dividedbytheestimated blood volume. Eachpointrepresents the mean±SE. (o) CR (n=5);
(i)
CR+anti-LDL receptorantibody(n=6). *P<0.05.
c
=
-_ L
c
- 0
O a
O 0
Io-,
C a 0
-u _
a.-(0
0.
Ot
%.
100
80'
60'
40'
20
* *
S *Z *
60 120 180 240
time (s)
300
Figure 8. Effect of anti-LDL receptorantibodyonthe clearance of
3H-chylomicronremnantsfrom the blood of mice in vivo. The pro-tocolwasthesame asinFig.4except thatchylomicronremnants
werelabeled with3H-cholesterolester. Eachpointrepresents the
removal over a longertime period without the possibility of exchange of radiolabel, the removal of chylomicron remnants were preparedwith the label primarily on apoB. This was done by labeling chylomicrons with 1251, injecting these particles into
eviscerated rats and isolating remnants. In these particles, >80% of theradioactivitywas onthe nonexchangable apoB as assessed by radioautography of polyacrylamide gels (not
shown). The clearance of these particleswassimilar, although
somewhat morecompletethan that ofinvitrolabeled particles.
After1 h, at least 25%ofremnantdisappearance was still due to the LDL receptor(Fig. 9).
Thekinetics ofclearance ofremnantsissomewhat complex and does notobeyasingleprocesskineticmodel(not shown). Withthis preparation,thetime forremovalof50%ofthe parti-cles was - 7min in control and- 21min in antibody-treated mice, consistentwith theLDLreceptorplayingamajorrole in
their clearance. Itshould benoted, however, thateveninthe
antibody-treated mice, remnant clearance was much more
rapidthan LDL clearance and by 1 h, b
completion
in both groups. Thissugges receptor clearance pathway (6) hasa hinantsthanforLDL, and the LDL recept
z
azE
L
wo
oI0Z
zo
0-J
F t
I.
ZILL
oS
I--0-~
0
(0
-I
a.
100
80
60
40
20 0
(I)
I-z
4c
z
ao
11.0 z o
oZ 0
z0
O
C
I
C.)
Figure 5
chylomi
term.Ti
the poss
were
prn
the
radii
scribed i
pointrel
remnani
1 0 20 30 TIME (MIN)
cn
_
u _~ co 3
en C
0
-Q C"
C
C:
E ArtiLDL RAb
11VER ADRENALSPLEEN KIDNEYSM. INT.
Figure10.Effect of anti-LDL receptor antibody on chylomicron remnant specific tissue spaces. The same protocolwas followed as
describedin the legend to Fig. 4, except that the animals were sacri-ficed 5 min after the injection of lipoprotein. Each point is the
mean±SE. (o) CR (n=3);(n)CR + anti-LDLreceptor antibody, (n
=4). *P<0.05.
iad proceededto near way can remove most of a bolus ofremnants in a relatively its that the non-LDL shorttime.
igheraffinity for rem- Effect ofanti-LDL receptor antibody onchylomicron
rem-tor-independent path- nantuptake bytissues. The liver, adrenalglands, spleen, kid-ney, and a piece of small intestine wereremoved and counted
forradioactivity5minaftertheinjection ofchylomicron rem-nants. In contrast to LDL, the specific
125I-chylomicron
rem-a Remnants nant tissue space in the adrenal gland was significantly lower - Anti-LDL RAb than that in the liver (Fig. 10). The injection of anti-LDLre-ceptorantibody blockedthe uptake of
'25I-chylomicron
rem-nants intheliver,adrenal gland, and spleenby at least 50% atthe 5-min timepoint. This was similar to its effect on LDL
uptakeand suggests that the LDL receptor normally plays a
major quantitativerole in therapid removalofchylomicron remnants in thesetissues that is similar to thatforLDL. How-ever, in the 1-h clearanceexperiment of Fig. 9,the remnant space in the liver ofantibody-treated animals was nowonly 40 50 60 - 25% less than in the control animals. This is consistent with thepostulate that the non-LDL receptor pathwaycan com-pensateforthe LDL receptor over alonger timeperiod.
Discussion
\
Remn-
Ants-LDLR b These experiments establish for the first time that the LDLB0_ \ >receptor
plays
asignificant
role in theremovalofchylomicron
remnantsin the normal animal in vivo. This isnot
necessarily
so _ an unexpected conclusion because these particles contain a
large
amount ofapoE
and have beenshown to bind and be40- taken up by the LDL receptor ina variety of cultured cells,
including fibroblasts (36), macrophages (37),hepatomas
(38),
20
-and isolated primary liver cells (10). However, a number of lines of evidence arguedthat the LDL receptor
might
beless0 1 2 3 4 * importantforremnantremovalinvivothanthis receptor is for
TIME (MIN) LDL uptake. First, chylomicron remnants are removed from
blood muchmorerapidlythanLDL; second,theparticlesare
).Effect of anti-LDL receptor antibody on clearance of1231. removed by the liver almost exclusively (39), even though the icron remnants from the bloodof mice in vivo over thelong adrenal gland has a higher density of LDLreceptors (29); third,
ostudyremnantremovalover alonger timeperiodwithout earlier studiesfromthis
laboratory
(22)
foundnormaluptake
;ibility
ofexchangeofradioactivity, chylomicron
remnantsa
.n.i
1pared
from'251-chylomicrons.
Theseparticles
have mostof by the perfusedlver of rats fed
an atherogecdlet,
acondlton
oactivityinapoproteinB. Theprotocolwas the same as de- inwhich
the LDLreceptor
ismoderately downregulated;
inFig. 2. (a) Entire time course; (b)first 6minonly.Each fourth, the Watanaberabbit, which has a defective LDL recep-presents the mean±SE. n=4for remnants(o)and n=6 for tor,removes
chylomicron
remnantsrapidly (11)
andfifth, hu-ts+anti-LDLreceptor antibody (v). *P<0.05. manswith FH have notbeenreported
tobecomeunusually
1 0
a
6 4
hypertriglyceridemic orhypercholesterolemic after a
fat-con-taining meal.
Toassess the role ofthe LDL receptor in chylomicron rem-nantremoval, it was necessary to create an acute blockade of LDLreceptorfunctionbecause in anychronic model, such as a
receptor-deficient rabbit, compensatorychanges could occur,
and the expansion ofendogenous lipoprotein pools makes even simple kinetic studies difficult tointerpret. The mouse was chosen because its small blood volume allowed us to
achievehigh antibodyconcentrationswithout the need for
pro-hibitiveamountsof antibody. Lipoprotein metabolism in the mousehas notbeen studied in the same detail as in several
otherspecies, suchas rats,rabbits,andhumans. However, the mouse has analogous lipoproteins and apoproteins to these
species (40, 41), includingapoAI, AII,
B,
C, and E, it has a receptor similar tothe LDL receptor whichrecognizes both apoB andapoE (37), and itseems to be a reasonable modelbecause nounusual metabolic featureshave been identified.
Thelimitations of thisstudy, causedby the small size of the
animals,
werethatwith3H-cholesterol
ester-containing parti-cles, it wasdifficult to quantifytheradioactivity inthe laterblood samples,anditwasdifficulttofreethe adrenal glandof all adherenttissue. The lattermayaccountforthefindingthat LDLuptakeper gramtissuedryweightwassimilar in liverand
adrenal, whereas inmostotherspecies the adrenal is richer in
LDL receptors. In
fact,
onimmunoblots,
when similaramountsofproteinareapplied, the adrenal glandappears to have a greater amount ofLDL receptor thanliver, although this difference may not be as great asinsome other
species.
Nonetheless, because thesameprotocolwasusedwith thetwo
lipoprotein types in these
studies,
the relative differencebe-tween
hepatic
and adrenaltissuespaceswithLDLand chylo-micronremnantsstill remains.Inaddition, theuseof humanLDLandrat
chylomicron
remnants represents a
compromise;
theuseofhomologous
li-poproteins
was notfeasible because of the limited blood vol-ume of themouse. Inprevious
cell culture studies from this laboratory (38), humanLDLhad onlyaslightly
loweraffinity
for the rat LDL receptorthan rat
LDL;
however,
in in vivo studiesby
Dietschy
andcolleagues
(32),
there was a greaterdegree ofLDL
receptor-mediated uptake
ofratlipoproteins
inratthanof human
lipoprotein
in thatspecies.
Thismaybe dueto an
ability
of the ratparticles
toacquire apoE. Thus,
ourchoice of
lipoproteins
mayaccountforsomeof thequantita-tivedifferences between this
study
and those in therat(32).
The
antibody
used haspreviously
beenthoroughly
charac-terized(9, 10, 27).
Itdoesnot cross-reactwith theLRPorother proteins and inhibitsuptake of bothapoB-
andapoE-contain-inglipoproteins inratandmurinecells. In
general,
onWesternblotsof noninduced
liver,
the liverreceptor, itsdimer,
andaprecursor are seen. Smallerbands are
probably
degradation
products.Allbandsareinduced by
ethinyl
estradioltreatmentand reactwithamonoclonal anti-LDLreceptor
antibody (27).
Interestingly,
whereas itcompletely inhibitschylomicron
rem-nant and
fl-VLDL
uptake in fibroblast(37)
andtumorlines (10), it isonly 80-90% effectiveinmacrophages (37)
andhe-patic
cells(10).
Adose-response
experiment
ofLDLclearance with antibody showedanincreaseinblockade withupto0.5mg
antibody injected
andthen,
aparadoxical
fall.Thisamountshould
provide
anantibody
concentration of 180Ag/ml.
Basedonavailable information, this isroughlycomparableona
pro-tein basisto the apoB content ofmouse blood. Because the
antibody isamore potentcompetitorforLDLreceptor
bind-ing than LDL, the antibody isprobably present in excess of LDL,butperhaps not an adequate excess to completely com-peteforLDLuptake. BecauseapoE isahigher affinity ligand
thanapoB, the degree of blockade ofapoE-mediated uptake maybe less thanthatofapoB-mediateduptake.Thismay be
somewhat
mitigated
by the fact that theendogenous apoE level isrelativelylow. Thus, because the blockade may not becom-plete, thecontribution ofthe LDL receptor to the removalof thetwo typesof particles estimated by this method shouldbe
takenas aminimum value.ForLDL, the estimatesthat 35%of plasmadisappearance,45%ofliver uptake,and 55%ofadrenal
uptakeis duetothe LDL receptor arelower than thosethat
havebeenobtained inotherspecies using labeled residualized homologousLDLand RM-LDL. Thediscrepancy between
tis-sue
uptake
andplasma clearancesuggeststhateither theesti-mateof initial concentrationwasinaccurateortherewas some
sequestration
ofparticles.
The latterpossibility
which could include somemacrophage uptakeseemsquite plausible.Esti-matesforLDLreceptor-mediatedLDLremoval
from
plasma in humans have been 67-80% (42); in therabbit, 62-76% (33); and in therat,57-75%(32).Interestingly,
inourstudy, RM-LDL clearance gavethesame estimate of receptor-mediated uptakeasdidantibody
blockade.Ingeneral,
adrenalhas had thehighest
per gramreceptor-mediated uptake
and theliver,
second
(29, 32).
Takentogether,
it is reasonabletoconclude that themouseis similartootherspecies
withregard
toLDLreceptor-dependent uptake,although itmayrelymore
heavily
ontheLDL
receptor-independent
pathway
forlipoprotein
re-moval, and that the
antibody
blockade methodgives
atworst,amodestunderestimateofLDL
receptor-dependent
lipoprotein
transport.
When studying chylomicron remnants, another
problem
presented itself in thatremnantremovalwas so
rapid
itwas notpossible
toestimate the initial concentrationby
extrapolating
theinitial time
points
to zero.Tocircumventthis,
weexam-ined uptake
using
thetheoretical concentration(injected
dose dividedby
estimated bloodvolume)
asthe initialconcentra-tion. Whena
comparison
of thetwoapproaches
wasmade for LDL, itappearedthat - 13%of theinjected
materialwaslostbetween thetesttube andthecirculation.Becauseof thesmall
volume
injected,
eventhe smallamountremaining
in thesy-ringe
andneedle isasignificant
percentage. Wethenapplied
this correctiontothe initial theoreticalremnantconcentration.
Because
chylomicron
remnants arelarger
and tendtoaggregateandsticktosurfacesmore than
LDL,
this maybeamodest underestimate and would result inanunderestimate
ofLDLreceptor-dependent
clearanceattheinitial timepoint
because such lossesappearascomponentsofLDLreceptor-indepen-dentremoval.
Takentogether,allof theseconsiderationssuggestthatour estimates ofLDL
receptor-mediated
uptake
ofchylomicron
remnants represent minimum values.Thus,
thesimilarity
ofthe percentageofremnantand LDL
disappearance
fromthecirculation that is mediated
by
the LDL receptor(35%
for LDL, 25-35% forchylomicron
remnants)
andhepatic uptake
(40% for LDL and 53% for
chylomicron
remnants)
suggests that in the normalanimal,
theLDLreceptor isanimportant
mediatorofremnantremoval andmayevenplay
awas seen atthe 30-stimepoint.Further, even though theliver
hadahigher "specificspace" than theadrenal gland, this was due to an increase in the LDL receptor's contribution in
pro-portiontothe LDLreceptor-independent contribution, sug-gesting that some factor other than LDL receptor density is
responsible forthetissue distributionof uptake. This could be duesimply to the size of the fenestrations in the space of Disse, allowing chylomicron remnants access to the hepatic LDL re-ceptorandnot toadrenalLDL receptors.
Although the LDL receptor appears to play a major role in remnantremoval, the non-LDL receptor pathway(s) also plays asubstantial role andcanfullycompensateforthe LDL recep-tor when it is blockedby the anti-receptor antibody. Thus, even inthe blockadedanimal,thedisappearanceofremnants is
muchmorerapid than that ofotherlipoproteinsandisalmost
complete in<1h. Thiscanexplain the data of Rubinszteinet
al. (12) who did not find amarked impairment ofremnant removalinpatients with receptor-negative familial
hypercho-lesterolemia. Inthat study, the totalserum andchylomicron
remnant response tooralretinyl palmitatewas only modestly (- 20%), and notsignificantlygreater in FH than in normal
patients. In contrast,patients whowerehomozygous forapo E-2 had markedly greater responses as would be expected if both removal mechanisms relied
primarily
onapo E as the recognition signal.Arolefor theLDLreceptoris furthersup-ported by the observation thatremnantremovalis accelerated byestrogen treatmentwhich increasesLDL receptor(43), but
not LRP(unpublished observation)levels.
This, then, leaves open thequestion of the nature of the
remnant removal mechanism when theLDL receptor is ab-sent,defective,ordownregulated. Asimple hypothesisis that
the
recently-described
LRP(13)
provides
thesecondary
path-way.Althoughit has been shown thatthis protein does bind apoE and apoB(14) andcan
initiate
internalization(44),com-paredto theLDL receptor, atleast incell culture, it doesso
withaloweraffinity and requires further enrichment ofalready
apoE-rich particles
withadditional
apoE (45,46).
Thus, under conditions where thiswastheonly
receptoroperative,
LDLclearances
might
bemarkedly delayed whereas,
eventhough
remnant clearance wasslower than
normal,
it could still bequite rapid by
comparison
to normal LDL removal. Thiswould alsoexplain why the remnantremovalcurves are not more divergentat later time
points.
Ifremnantremoval byLDL receptorhasa
T112
of 30sand non-LDLreceptorof 90s(threefold difference),
inaperfect experiment,
after 4.5 min, 99% would be clearedby the LDL receptorand 88%by the non-LDLreceptorpathway.
Becausethereissomeexchange
andsome
slowly
removed,
or evenresecretedradioactivity,
thiswouldaccountfor theplateau and
narrowing differences
seenatthelater
timepoints.
Thedisparity
between thetwoparticles
would befurther exaggerated ifthe
ability
toacquire
additional apoE isrequired
forbinding
becauseremnants aregood
sub-stratesfor apoE
acquisition,
whereasLDLdoesthispoorly
and in thecaseof humans, perhapsnotatall. Furthertesting
ofthishypothesis
willrequire complementary
studiestothese withananti-LRP
antibody
orstudies
inLRP-deficientanimals,
ifsuchcanbefound.
Acknowledaments
Wewishtothank Dr. S. Azhar forprovidingDLT-LDL,Ms. Jean Chenforassistancewithpreparingthe anti-LDL receptorantibody,
and Ms. JeanneGillfor assistance with themanuscript.
This work was supported by grants DK38318 and DK36659. Dr. Kirven and Dr. Choi are recipients of fellowships from the California Chapter ofthe American HeartAssociation.Preliminary reports ofthis research have been published in Clin. Res.(1990.38:1 53a) andArterio-sclerosis(1990. 10:766).
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