Regulation of rat biliary cholesterol secretion
by agents that alter intrahepatic cholesterol
metabolism. Evidence for a distinct biliary
precursor pool.
B G Stone, … , W Y Craig, A D Cooper
J Clin Invest.
1985;
76(5)
:1773-1781.
https://doi.org/10.1172/JCI112168
.
Propensity for cholesterol gallstone formation is determined in part by biliary cholesterol
content relative to bile salts and phospholipid. We examined the hypothesis that the rate of
biliary cholesterol secretion can be controlled by availability of an hepatic metabolically
active free cholesterol pool whose size is determined in part by rates of sterol synthesis, as
reflected by activity of the primary rate-limiting enzyme 3-hydroxy-3-methylglutaryl
coenzyme A (HMG CoA) reductase and of sterol esterification, as reflected by the activity of
the enzyme acyl coenzyme A/cholesterol acyltransferase (ACAT). Rats were prepared with
biliary, venous, and duodenal catheters. The enterohepatic circulation of biliary lipids was
maintained constant by infusion of a bile salt, lecithin, cholesterol replacement solution.
Administration of 25-hydroxycholesterol decreased HMG CoA reductase activity, increased
ACAT activity, and decreased biliary cholesterol output 26% by 1 h. By 2 h, ACAT activity
and biliary cholesterol secretion were at control levels. Administration of mevinolin, a
competitive inhibitor of HMG CoA reductase, had no effect on ACAT activity and decreased
biliary cholesterol secretion 16%. Administration of progesterone, an inhibitor of ACAT, had
no effect on HMG CoA reductase and increased biliary cholesterol output 32% at 1 h. By 2
h, all parameters were near control levels. None of these agents had any significant effect
on biliary bile salt or phospholipid secretion. Thus, acutely altering rates […]
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Regulation
of
Rat Biliary Cholesterol Secretion by Agents
That Alter Intrahepatic Cholesterol Metabolism
Evidence fora DistinctBiliary Precursor Pool
BradfordG.Stone, SandraK.Erickson, WendyY.Craig,and AllenD. Cooper DepartmentofMedicine, Stanford University School ofMedicine, Stanford, California 94305
Abstract
Propensityfor cholesterol gallstoneformation is determined in partbybiliary cholesterolcontentrelativetobile salts and phos-pholipid. We examined the hypothesisthat the rateof biliary cholesterol secretioncanbecontrolledby
availability
ofan he-paticmetabolicallyactive free cholesterolpoolwhose size is de-termined in part by rates of sterol synthesis, as reflected by activityof theprimary rate-limitingenzyme 3-hydroxy-3-meth-ylglutarylcoenzymeA(HMGCoA)reductaseandofsteroles-terification,asreflected bytheactivityof the enzyme
acyl
coen-zymeA/cholesterol acyltransferase (ACAT).Ratswereprepared withbiliary,venous, andduodenal catheters. Theenterohepatic circulationofbiliary lipidswasmaintainedconstantbyinfusion ofabile salt, lecithin,cholesterolreplacementsolution. Admin-istration of25-hydroxycholesterol decreasedHMG CoA reduc-tase activity, increased ACAT activity, and decreased biliary cholesterol output 26%by1 h.By2h,ACATactivityand biliary cholesterol secretion were at control levels. Administration of mevinolin,acompetitive inhibitorof HMGCoA reductase,had no effect on ACAT activity and decreased
biliary
cholesterol secretion 16%. Administration ofprogesterone, aninhibitorof ACAT, had no effect on HMG CoA reductase and increased biliary cholesterol output 32% at 1 h. By2 h,all parameters were near control levels. Noneofthese agents had anysignificant effectonbiliary bilesaltorphospholipidsecretion.Thus,acutelyalteringrates ofesterification and/or synthesis
canhave profound effectsonbiliarycholesterolsecretion inde-pendentofthe otherbiliary lipids. Theseexperimentssuggest theexistence of ametabolically active poolof free cholesterol thatserves as aprecursorpoolfor biliarycholesterolsecretion. Furthermore, the size of this precursor pool is determined in partbothbyratesof cholesterolsynthesisandesterificationand isakey determinant of biliary cholesterol secretion.
Introduction
Cholesterol gallstone disease iscommonin Westernpopulations.
Normally
thehydrophobic
lipid, cholesterol,
is maintained inPortions ofthiswork werepresentedat the annualmeetingsofthe
Amer-ican Federation for ClinicalResearch, 1983, and at the American
As-sociation for the Study of Liver Disease, 1983, and werepublished in
abstractform in Clin. Res. 31:289 and in Hepatology. 3:217. Dr.Stone'spresent address is Division ofGastroenterology,University ofPittsburgh,PA 15261. Address correspondencetoDrs.Ericksonand
Cooper.
Receivedfor publication 5 February 1985 and in revised form 15
July1985.
solution in the bile
through
formation of mixed micellescom-posed
of bile salt and lecithin(1).
It is established thatgallstones
canform when the cholesterolcontentofthe micelle relativeto
phospholipid
andbile salt exceeds certain limits(2), and that suchlithogenic
bile is derived from the liverasopposedtoform-ing
in thegallbladder (3, 4).Therateofbiliary lipidsecretion isdependent
in partontherateofbile salt secretion(5,6).However,
thiscannotaccountfor all ofthe metabolic observations. Under different metabolic circumstances therecanbe either decreased bile sale secretion withaconstantcholesterol outputorincreased cholesterol secretion with amaintained bile sale output
(7-9).
Thephysiologicbasis for these abnormalities remains unknown. Cholesterol secreted into the bilecan be derived from atleast threesources:hepatic newly
synthesized sterol,cholesterol de-rived fromplasma lipoproteins,
and sterol frompreformedhe-patic
stores.Although
all of thesesourcesappeartobe available forbiliary
secretion,the relative contributions of each and howthey
areregulated
remain unclear.Onepostulatewasthat therateof cholesterol synethesis in the liver was an important determinant ofbiliary cholesterol secretion. Thiswasbasedonthe observations of several inves-tigators (10-12) that patients with cholesterol gallstones had higherlevels of the enzyme, 3-hydroxy-3-methylglutaryl
coen-zyme A
(HMG CoA)'
reductase,thanpatientswithoutgallstones.
This enzymecatalyzesthe rate-limiting step of cholesterol syn-thesis
(13)
andin mostinstancesaccuratelyreflects therateof cholesterolsynthesis (14). Moreover, feedingthe bile salt cheno-deoxycholatereduced theenzyme'sactivity and decreasedbiliary
cholesterol excretion (11, 12). However, a thorough series of studies
by Turley
andDietschy (6)intheratsuggestedthat therateofhepatic cholesterolsynthesis persedoesnotdetermine the rateofbiliarycholesterol secretion. Moreover, ithas been shown that chenodeoxycholate's mechanism of action is not
throughdirect inhibition of HMG CoA reductase (15).
Lastly,
under normal circumstances, only 10-20% of biliary cholesterol is derived fromnewsynthesis(16, 17).
Only free cholesterol is secreted in bile, but in the cell the sterol isin both the free and esterified form. The balance between thetwois controlledbytherateof esterification of free choles-terol, catalyzed by the enzyme acyl coenzyme A/cholesterol acyltransferase(ACAT),and by therateofhydrolysis of choles-terolestersboth in the lysosomal and extralysosomal
compart-ments.Nervi and colleagues ( 18) have investigated the relation-shipbetween therateof cholesterol esterification via ACAT and biliarycholesterol secretion.Theyobserved that decreased ACAT activity induced by chronic administration of progesterone to ratscorrelated with increased biliary cholesterolcontent.
Taken
together,
these observationssuggestedthat there isa1.Abbreviationsused in this paper: ACAT, acyl coenzyme A/cholesterol
acyltransferase: DMSO, dimethylsulfoxide;GLC, gas-liquid chromatog-raphy;HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A.
J.Clin. Invest.
©The American Society forClinicalInvestigation,Inc.
0021-9738/85/11/1773/09 $1.00
pool of free cholesterol that can serve as a precursor for biliary cholesterol. The existence of such a pool of cholesterol has been suggested by Schwartz et al. ( 19). However, the nature and reg-ulation ofthis pool remain obscure. Synthesis and esterification mightbe important determinants of the biliary precursor pool. Previous work has relied on chronic administration of choles-tyramine(6), cholesterol (6, 20), bile salts (6, 20), or progesterone (18) to uncoverrelationshipsbetween metabolic processes and biliary cholesterol secretion, making it difficult to distinguish betweenthe direct effect oftheagent and the role of compen-satory changes. In the present study, a rat model was developed that allowed us to assess the effect ofacute changes in cholesterol metabolismonbiliary cholesterol secretion. Although there are numerous differences between human and rat sterol metabolism, therathas been extensively studied andprovides a model for elucidating principlesthat may be applicable to man. Specifically, the effect ofacute alterations of hepatic cholesterol synthesis andesterification alone and together on biliary lipid secretion were studied.
Methods
Materials
Chemicals. 4-'4C-Cholesteryl oleate (52.5 mCi/mmol),
1,2,6,7-3H-pro-gesterone(99 Ci/mmol), 1,2-3H-cholesterol(40-60 Ci/mmol),
3-hydroxy-3-methyl-[3-'4C]glutaric
acid(57.6mCi/mmol), 3H-water(100mCi/g),25-hydroxy26,37-3H-cholesterol(87Ci/mmol),andD,L-5-3H-mevalonic
acid(dibenzylethylenediamine salt,5Ci/mmol)wereobtained fromNew
EnglandNuclear(Boston,MA). I-'4C-OleoylcoenzymeA(56-60 mCi/
mmol)wasfrom Amersham Corp.(Arlington Heights,IL).Cholesteryl oleate, oleicanhydride,oleoyl coenzyme A,/1-NAD,
fl-NADP,
glucose-6-phosphate,taurocholate (Na salt, 98%pure), a-phosphatidylcholine,cholesterol,3-hydroxy-3-methylglutarylcoenzyme A,
D,L-mevalonolac-tone, 3a-hydroxysteroid dehydrogenase, and glucose-6-phosphate de-hydrogenasewerefromSigmaChemical Co.(St. Louis, MO). 5a-Cho-lestane wasfromAppliedScienceDiv.,Milton Ray Co. LaboratoryGroup (State College,PA),25-hydroxycholesteroland progesteronewerefrom
Steraloids,Inc.(Wilton, NH); Mylar-backedsilicagel chromatography
sheetswerefromEastman Kodak Co.(Rochester, NY)andsilicagelH was from E. Merck(Darmstadt,FederalRepublicofGermany).
Liqui-fluor andAquasolwerefrom NewEnglandNuclear.Instagelwasfrom Hewlett-Packard Co. (DownersGrove, IL). Polyethylene tubingwasfrom Clay-Adams, Div. ofBecton-Dickinson& Co.(Parsippany,NJ). Mevi-nolinwas agiftof Mr.AlfredAlberts of the Merck Institute for
Thera-peuticResearch(Rahway, NJ).All otherchemicalswereanalyticalgrade. Animals. MaleSpragueDawley rats(Simonsen, Gilroy, CA)were
housed under normallightingconditions(lightson6 a.m.;lightsoff 6 p.m.) andfedstandardratchow ad lib.They weightedbetween 300 and 380 gatthetime ofuse.
Methods
Preparationof animals andexperimental protocol.Groupsof age- and weight-matched animalswereused in allexperiments.Under ether
anes-thesia,polyethylene catheters(PE-1O)wereinserted into the bile duct andbilewasallowedtodrainbygravity. Polyethylenetubes
(PE-100),
through whichabilereplacementsolutionwasinfusedat a rateof1.4
ml/htomaintaintheenterohepaticcirculation,wereplaced intraduo-denally.Inaddition, theywerefitted with either femoral vein catheters
(PE-1O)for25-hydroxycholesterolorprogesterone administrationor in-tragastric tubes (PE-50) formevalonalactoneormevinolin administration. After surgery the animalswereplaced in individualrestrainingcages and
allowedfreeaccess tofood andwater.Aftera21-h recoveryperiodthe
protocoloutlined in Fig. 1 wasfollowed. First a 1-h bilesamplewas
t
~~T
bile solution solution
replacement injection injection
Figure 1. Experimental protocol for studies ofbiliarylipidsecretion. Under etheranesthesia,each300-380-gratwasfittedwith a biliary
drainagecatheter.Anintraduodenal tube was placed, through which a bilereplacementsolution of 24 mM taurocholate, 3 mM lecithin, and 0.45 mM cholesterolwasinfusedat1.4 ml h-' tomaintain the
entero-hepatic circulation.Inaddition,each rat was fitted with a femoral vein catheter foradministrationof25-hydroxycholesterolorprogesterone
orwithanintragastrictubeforadministration of mevinolin or meva-lonate.Aftera21-h recovery period, during which the animals were allowed freeaccess tofood and water,a1-h bile collectionwas ob-tained. Then, either the test agent or theappropriatevehicle alone was
administeredandtwoconsecutive 1-h bile samples collected. This was followedbya21-h recoveryperiodwithbile replacement, and the se-quencewasrepeated,except that the solution(agentorvehicle alone) thatwasnotgiventheday beforewasadministered.The orderof ad-ministrationofthe test agentorvehicle alonewasdetermined
ran-domly; thus,eachanimal servedasitsowncontrol.
obtained, and theneither thetestagent dissolved in its vehicleorthe vehicle alone was administered. Two
1-h
bile samples were then collected. The animalswereallowedtorecoverfor another 21-h period, a1-hbilesamplewascollected, and the protocolwasrepeated. At this time the solutionnotgiventheday beforewasadministered andtwosubsequent
1-hsampleswerethenobtained. The order of administration of the agent
orvehicle alonewasdetermined randomly,allowingeach animalto serve ashisowncontrol.
Bilereplacementsolution. Sodium taurocholate (98% pure), 2.4 mmol, 0.3 mmolL-a-phosphatidyl-choline (derivedfrom egg yolk, 60% pure), and 45.0Mimolcholesterol(recrystalized sequentiallyfromglacialacetic
acid, ethanol,andacetone)weredissolved inchloroform/methanol (2:
1). Thechloroform/methanolwasremoved undervacuumusingarotary
evaporator,leavingauniformlayer ofbilesalt, cholesterol, and phos-pholipid.Thiswasredissolved in 100 ml of normalsaline,andformed
anopticallyclear solution with thefollowing concentrations: 24 mM
taurocholate,3 mMlecithin,and 0.45 mMcholesterol. Thissolution
wasstoredat 15'C and used within 1 wk ofpreparation.
Test agentpreparation and administration. 25-Hydroxycholesterol
wasdissolved in 100%ethanolat afinalconcentration of 20mg/ml,and 6.25mg/kg body weightwasinjected intravenously throughthe femoral catheter.Progesteronewasdissolved in 100% ethanolat100mg/ml,and 31.25 mg/kg body weightwasinjected intravenously toeach animal. Mevalonolactone(I g/ml in normalsaline)wasadministeredat adose
of 200mg/animal throughanintragastrictube. Mevinolin(17.5 mg/ml
indimethylsulfoxide) wasdiluted with normal salinetoafinal
concen-tration of 3.5mg/ml,andadose of 1mg/kg bodyweightwasadministered
intragastrically.Thecontrol solutionsweretheabove mentioned vehicles
aloneadministered inthesame mannerasthetestsolution.
Preparationofmicrosomes.Animalswerekilledbyexsanguination,
the liversremoved,rinsed in iced normalsaline,and microsomes pre-pared,asdescribedpreviously (21). Samplesof liverwerehomogenized
in0.25Msucrose, 1 mMEDTA,pH 7.2,and thehomogenate
centrifuged
at 10,000 g for 10 min. The resultantsupernatantwas
centrifuged
at105,000 g for 60 min and washedbyresuspensionandrecentrifugation.
The final microsomalpelletwasthenresuspendedin thesamebufferat
Assay
of
ACATactivity. ACATactivity
was assayedasdescribed previously (21). Tothe assay mixturecontaining
150usg
microsomal protein wasadded 5 nmol of'4C-oleoyl coenzyme A(specificactivity -24,000 dpm/nmol)toinitiate the reaction. The assaywasterminated after4minbythe addition ofchloroform/methanol(2:1)followedby 3H-cholesteryloleate(8,000dpm)asinternal standard toestimatere-covery. Theproductwasisolated and the results calculatedasdescribed
previously (2 1).
AssayofHMG CoAreductaseactivity.HMG CoA reductaseactivity
wasassayedasdescribedpreviously (22).Microsomespreparedasabove
weresuspendedat afinal concentration of 2.5 mgprotein/mlin buffer
containing0.1 Msucrose, 50 mMKCI,40 mMKH2PO4,and 30 mM
EDTA,pH 7.4. The assay mixture contained 10 mmoldithiothreitol,
15 mmol EDTA, 35 mmol NaCl, 15 mmol
NADP',
2 U glucose-6-phosphatedehydrogenase,and 500 ,ug microsomalprotein.The reactionwasinitiatedbytheaddition of 85 nmolof
D,L-3-14C-HMG
CoA and terminated after 20 min with 10 N NaOH. D,L-3H-mevalonolactone (90,000 dpm)wasaddedtoestimate recovery. Theproductwasisolated,and results calculatedasdescribedpreviously (22).
Measurement
of
[3H]OH
incorporationintocholesterolinvivo.An-imalswerepreparedasdescribed above andconstantly infusedwith the bilereplacementsolution. Aftera21-h recoveryperiod,thetestagent
orvehicle alone and 50 mCi of[3H]OH adjustedtoisotonicity were
deliveredthroughtheappropriatecatheters. After 2h,theanimalswere
exsanguinated,andthespecific activityofplasmawaterwasdetermined,
accordinglytoJeske andDietschy (23).Theliverwasrinsed with iced saline in situ and400-800-mg samplesweresaponifiedin alcoholic KOH
at700C. '4C-Cholesteryloleatewasaddedto assessrecovery. The
non-saponifiable
lipids
wereextractedwith 3X6mlofpetroleumether,the etherextractswashedtwice with water, andseparatedbythin-layerchro-matographyonsilicagelHbydevelopingwithbenzene/ethylacetate5:
1(24).Thecholesterol bandswereidentified,scrapedinto scintillation vials and counted. The rate of
3H-incorporation
into cholesterolwasthencalculatedbythe methodof Jeske andDietschy(23).
Biliary lipiddetermination. Bile saltsweremeasuredaccordingto
Turley and Dietschy
(25).
Biliary cholesterol was determinedcolori-metrically accordingtoMann(26)aftersaponificationof thebilesamples (27).Phospholipidphosphoruswasassayedaccordingtothe methodof Bartlett(28)after totallipidextraction(29).
Other determinations. Microsomalprotein wasdeterminedbythe biuret method(30) usingbovineserumalbuminas areference standard. Microsomal cholesterolwasdeterminedusing gas-liquid chromatography (GLC),asdescribedpreviously (22).Fordetermination of freecholesterol,
0.25mlof microsomescontaining2.5-5 mgproteinand37.5 mg 5-a-cholestaneas aninternal standardwereextracted,accordingtoFolchet
al. (29). The chloroform layerwastakentodrynessanddissolved in
tetrachloroethylene.AI-Ml samplewasanalyzed byGLC. Total choles-terolwassubsequentlymeasuredin the remainder of the sample by the methodofIshikawaetal.(31).Theamountsof free and total cholesterol
werequantitated by comparingthecholesterol peak areas to that of the added 5-a-cholestane. The cholesterolester content wascalculatedas
thedifference between the total and free cholesterol in thesamesample. Statisticalanalyses.Thesignificanceofchangesinbiliarylipidswas
assessedusingapairedttest.For eachindividualanimal,changes between the1-hsampleandthefirst and second hoursamplesafteradministration
of thetestsolutionwere comparedwith those changes evoked by the vehicle administration alone.Allotherstatistical analyses used the group
Itest.
Results
The
working hypothesis being
tested in thesestudiesis that there isanhepatic pool
offree cholesterolfromwhichbiliary choles-terolis derived.Although
it isestablishedthat therateof cho-lesterolsynthesis
perse doesnotdetermine therate of biliary cholesterol output underallcircumstances(6),
thesize ofthisTableI.EffectofAdministrationof the
Various Agents on ACA TActivity
Vehicle control
pmolcholesteryl Post I h Post 2 h oleate mg
Agent protein-'min- %of controlactivity
25-Hydroxycholesterol 50.3±5.3 (9) 142.9*(5) 107.0 (4) Mevalonate 90.3±6.9 (7) 145.4* (4) 159.5* (3) Mevinolin 84.9±8.9 (7) 108.3 (5) 100.3 (5)
Livermicrosomes werepreparedandACAT activity determinedasdescribedin
Methods. The controlvalues are the averages of the 1- and 2-h ACATvaluesin
microsomes preparedfrom animalsgiventhe vehiclealone.The vehicles wereas
follows:intravenous ethanol(25-hydroxycholesterol); intragastricnormal saline
(mevalonate); intragastric DMSO/normalsaline1:10(mevinolin). Mean±SEare
given. The number of determinations are inparentheses.
*Different from control by groupedt test,P<0.05.
precursor pool could be regulated in part by the rate ofcholesterol synthesis under certain conditions. Moreover, because only free cholesterol is secreted into the bile, therateofcholesterolester
formation could beadeterminant of the pool size. Changes in cholesterol input into this potential precursor pool by variations in synthesis rate could be balanced by changes in the rate of cholesterol ester formation, thus maintaining the free cholesterol pool andbiliary cholesterol outputconstant.This wouldexplain the observed lack ofconcordance betweenachange in anysingle metabolic parameter and the rate of biliary cholesterol secretion. To test thishypothesis, we used a number of compounds that are known toalter hepatic cholesterol metabolism.
Effect
of25-hydroxycholesterolonbiliary cholesteroloutput.Thecompound25-hydroxycholesterol has been shown to both increase cholesterol ester formation (32) and decreasehepatic HMG CoA reductase activity (33, 34), the rate-limiting step of cholesterol synthesis. These changes should deplete thisbiliary precursor pool, and if the size of this pool is a determinant, shouldcauseadecrease in biliarycholesterolsecretion.
First,toassesswhetherthis compound rapidly affected these enzyme activities in vito, a 6.25-mg/kg bolus of 25-hydroxy-cholesterol was administered intravenously to paired groups of animals. The activities of ACAT and HMG CoA reductase were determined in hepatic microsomes 1 and 2 h after the bolus had beenadministered. There was a 42.9% increase in the activity
Table II.Effect ofAdministration ofthe Various AgentsonHMGCoAReductase Activity
Vehicle control
Post I h Post2 h
pmolmevalonate
Agent mg-'min' %of controlactivity
25-Hydroxycholesterol 145±19 (9) 41.9* (5) 66.0*(4) Mevalonate 95±14(7) 23.1*(4) 19.9*(3) Progesterone 157±5 (5) 97.6 (4) 103.2 (3)
Microsomes werepreparedand HMG CoA reductaseactivitymeasured as
de-scribedinMethods. Thecontrol valuesarethe averages of the 1 and 2 h reduc-taseactivityinmicrosomes preparedfrom animalsgiventhe vehiclealone.
Mean±SE aregiven.The numberof determinationsareinparentheses.
of ACAT (P < 0.05) 1 h after the bolus was administered (Table
I).
By 2 h, the activity had returned to the control value. HMG CoA reductase activity decreased to 41.9% of control at 1 h (P < 0.01) with a return to 66% of control by 2 h (Table II). Thus, 25-hydroxycholesteroladministrationacutely elicited the metabolic responses in vivo that have previously been demon-strated in vitro.The acute effects of the administration of this sterol on biliary lipid composition were examined using the experimental model and the protocol described in Methods and Fig. 1. There was no significant change inbile flow during the course of the ex-periment (Table III). A small and nonsignificant change in bile salt output (Fig. 2 A) occurred in the first hourafter adminis-tration of 25-hydroxycholesterol compared with the preadmin-istration value. A similar change was observed with administra-tion of
the
vehicle alone, suggesting that this change was not due toadministration of 25-hydroxycholesterol. A decrease in phospholipid secretion (Fig. 2 B) greater than that seen with vehicle administration alone was also observedwithinthefirst hour after the administration of 25-hydroxycholesterol. How-ever, this effect was not statistically significant.In contrast, there was a significant decrease in biliary cho-lesterol output (P < 0.05) in the first hour after 25-hydroxycho-lesterol administration (Fig. 2C).This decrease in biliary cho-lesterol output was not seen with administration of the control vehicle alone and is, therefore, an effect of the 25-hydroxycho-lesterol. By the second hour, biliary cholesterol output had re-turned to thepreadministration value. The values from the in-dividual experiments are shown in the insert (Fig. 2 C) and demonstrate the consistency of this phenomenon. The fall in cholesterol output was seen in every animal and the return to-ward control in the second hour in all but one animal. The striking
temporal
relationship between the reducedbiliary cho-lesterol output and the changes in intrahepatic enzymeactivities responsible for modulating the free cholesterol concentration suggest that the observed decrease is due todiminished precursor pool size.To demonstrate that the change in biliarycholesteroloutput was not due tosubstitutionof25-hydroxycholesterol or oneof
Table III. Effect ofAdministration of the Various Agents on Bile Flow
Preadmin-Agent istration Post I h Post2h
ml x 100g bodywt-' x h-'
Control (all vehicles) (22) 0.33±0.01 0.33±0.02 0.33±0.01
25-Hydroxycholesterol (5) 0:34±0.02 0.34±0.02 0.34±0.02
Mevalonate (6) 0.32+-.03 0.36±0.04 0.34±0.04
Mevinolin (6) 0.35±0.03 0.33±0.03 0.34±0.03
Progesterone (5) 0.38±0.02 0.43±0.02* 0.41±0.03
The experimental protocol was that described in thelegendtoFig.1.The bile
flow rate for the hour before administration of the agent(preadministration)is
compared with the first andsecond hour after the agent was given.Thecontrol
values are the flow rates associated with administration of thevehicle alone and
represent the average of all the individualcontrols1 h before and1 and2 hafter
the appropriate vehicle was administered. Eachanimal received the agentorthe
vehicle alone and the order ofadministration was random. Therefore, changesin flow rates due to the agentadministration arecompared with flow rate changes
induced in the same animal by administrationof thevehicle. Mean±SE is
shown. The number of determinations are inparentheses.
*Different from control by pairedl test, P<0.05.
a:
N
tz 40
0
I-~i
0
a
40
a:
A
CONTROL 25 ON CHOLESTEROL
/5-
/2-
9-
6-
3-pre post post pre post post
control I h 2 h 25OH I h 2 h
B
CONTROL 25 OH CHOLESTEROL
2.0
'.5-1.0
158~~~~~~~~r pot post
0.XSU
-Dre D~OS t wlot
coro pus
pos2
control I h 2 h0.18
a:
10
40
a:
I.-,
40
0
a
0.15
Ooogr
006r
0031-pre post post
25 OH I h 2h
pre post post pre post post
control Ih 2h 25OH I h 2 h
Figure2. Effect of25-hydroxycholesterol administrationofbiliary lipidsecretion. The animalswerepreparedasdescribedinthelegend
toFig. 1. The bilesampleswereanalyzedfor the contentof bilesalt,
cholesterol,andphospholipid.The effect of administrationof ethanol
aloneis shownonthe leftof eachpair. On theright,theeffectof
ad-ministrationofa6.25-mg/kgbolus of25-hydroxycholesterol in
ethanolis shown(A) Effectonbile saltsecretion.(B)Effecton
phos-pholipid secretion. (C)Effectoncholesterolsecretion (insetshows
in-dividual experiments). Eachpointis the mean±SEof five
determina-tions.*Differentfromcontrol valueby pairedttest,P<0.005.
itsmetabolites forthe cholesterolmoleculeinbile,radiolabeled
25-hydroxycholesterol wasadministeredand theappearanceof
radiolabelinthe bilemonitored.Thesampleswere alsoanalyzed
byGLC fortheappearanceof25-hydroxycholesterol or
metab-olites. Neither a significant amount of25-hydroxycholesterol
nor nonpolar metabolites were detected (data not shown).
Therefore,it isunlikelythat25-hydroxycholesterol or oneof its
25 OH CHOLESTEROL
C CONTROL
upcaa75
Table IV. Effect of the VariousAgents on LiverMicrosomal Cholesterol Content
Agent Total Free Ester
g cholesterol mg protein'
Control (19) 22.20±0.59 20.07±0.77 2.67±0.39 25-Hydroxycholesterol
Ih(5) 20.95±0.55 17.93±0.75* 3.02±0.39 2 h (5) 19.49±1.08* 16.80±1.07* 2.69±0.40 Mevalonate
Ih (4) 22.62±2.02 19.30±0.82 2.95±1.26
2 h (4) 26.92±1.42* 21.17±0.26 5.74±1.46* Progesterone
Ih(3) 22.05±4.33 22.17±4.43 0.10±0.10t
2 h(3) 25.30±1.62 24.23±1.48 1.18±0.76 Microsomes were prepared andtotal,free,andestercholesterolcontents were
determinedasdescribed in Methods. The control valuesarethe averages of all
themicrosomal cholesterolcontentsmeasuredafteradministrationof the
var-ious controlvehiclesat Iand2 h. The total and freecholesterolcontentswere
measured by GLC asdescribed inMethods;ester wascalculatedasthe
differ-ence. Allvaluesarethemean±SE. The number of animalsaregivenin paren-theses.
*Different fromcontrolbygroupedItest,P<0.05. * Different from controlbygroupedttest, P<0.001.
metabolites replaced the cholesterol in bile. These data
dem-onstratethat
25-hydroxycholesterol,
whichbothstimulates cho-lesterolesterificationandconcomitantly
blockscholesterol syn-thesis, inducedadecreaseinbiliary
cholesterol output without alteringbiliary
bilesaltorphospholipid
secretion.The microsomal free cholesterol content was slightly but
significantly
decreased in thetreated
groupascompared with controlatboth Iand2 haftertreatment(TableIV). Thisdecrease suggests thattheremay bedepletion
ofasmallbiliary
precursor pool recovered in the microsomal fraction thatis,
however, overshadowed byalargestructuralcholesterol pool.Taken together, these results suggest that 25-hydroxycho-lesterol, by increasing cholesterol ester formation while
con-comitantly
decreasing
cholesterolsynthesis,
causeddepletion of thefree cholesterol pool available forbiliary
secretion.Effect of
mevalonateonbiliary
lipidoutput. Ithas been sug-gestedthat theactivities ofHMGCoAreductase(35)orACAT (36) maydetermine the rate of biliarycholesterol output. To demonstratethatit is thebalancebetween cholesterol synthesis and esterification that controls the size ofthe precursor pool and not the activities ofthe enzymes per se, mevalonate was administered.Prior
workfrom this laboratory (21, 34)has dem-onstrated thata 200-mg intragastric bolus of mevalonatede-creases
hepatic
HMG CoA reductaseactivity
and stimulates ACATactivity. Thus, administration ofmevalonate evokes thesameenzymeactivitychanges as25-hydroxycholesterol. Unlike 25-hydroxycholesterol, however, mevalonate administration shouldnotdepletethemetabolicallyactivefreecholesterol pool because mevalonate itselfis converted to cholesterol.
Afterintragastric administration of a 200-mg bolus of mev-alonate,thechangesin ACAT activity (TableI)andHMG CoA reductase of activity (TableII)were comparable to those induced by25-hydroxycholesterol administration. There were no signif-icant changes in bileflow,bile salt, or phospholipid output, nor
were there any changes inbiliarycholesterol output 1 or2 h after the administration of mevalonate (Table III and Fig.
3).
Therewas astatistically significant increase in microsomal cho-lesterolester content2 h after the administration of mevalonate (Table IV)withnochangein microsomal free cholesterol.
Concurrent administration of 6.25 mg/kg 25-hydroxycho-lesterol and 200 mg of mevalonate resulted inaconstant amount ofbiliary cholesterol secretion overthe 2-h collection period (0.22±0.03) gmolcholesterol secretedh-' 100
g-'
vs.0.22±0.22,qmol
cholesterol secretedh-'100g-',n =4). Thus, the decreasedfree cholesterol poolinducedby25-hydroxycholesterol
admin-;t It N
In9
6.
C) 3.
'a
2.5
4o
2.0-CZ
'a
To20
a
1.5-X
1.0-I.
0
A CONTROL MEVALONATE
pre post post pre post post
control lh 2 h M VA lh 2 h
B CONTROL MEVALONATE
pre post post pre post post control Ih 2 h MVA I h 2 h
C CONTROL MEVALONATE
t 0.25
to
N
Q 0.20
0.
1-E 0.150
(a
zt 0
pre post post
control / h 2 h pre
post post MVA I h 2 h
Figure3.Effect of mevalonateonbiliary lipid secretion.The same protocolas inthe legend to Fig. 2 was used, except that a 200-mg
bolusof mevalonolactone innormalsalinewasadministered through
anindwellinggastric catheter. The vehiclewasnormalsaline. (A) Ef-fectonbilesaltsecretion. (B) Effectonphospholipid secretion. (C) Ef-fectonbiliarylipid secretion.Eachpoint is the mean±SE of six
exper-iments.
istration can be repleted by mevalonate,whose conversion to cholesterol is not affected by the sterol, withconcomitant res-toration of biliary cholesterol secretion.
These results demonstrate that theactivitiesof theenzymes per se do not regulate biliary cholesterolcontent. Rather, they suggest that it is the net effect ofchangesin thevarious processes that regulate intrahepatic cholesterolmetabolism, which deter-mines the size of thebiliarycholesterolprecursor pool itself which appears to be a determinant ofbiliarycholesterol output.
Effect
of mevinolin on binary lipid output. To determinewhether biliary cholesterol output could be acutely altered by change in the rate of cholesterol synthesis alone, thecompound mevinolin wasadministered. Mevinolinis acompetitive inhib-itor of HMG CoAreductase,andisreported to inhibit cholesterol synthesis in rat liver within 1 hofadministration at a dose of 1 mg/kg body weight (37). Theeffect ofmevinolin administration on cholesterol esterification rate has not been reported. If the rate of ester formation isnotaffected,then this mode of egress of free cholesterolfromthebiliaryprecursor pool will be main-tained. The decreased input intotheprecursorpool, as a result of decreasedsynthesis,wouldcause a net decrease in cholesterol pool size andthus inbiliary cholesteroloutput. Mevinolin,when administered at the above dose, did not alter ACAT activity (Table I). HMGCoAreductaseactivity was notdetermined be-cause,althoughmevinolininhibits sterolsynthesis invivo,HMG CoA reductaseactivity whenassayed in vitro ishigherthan in controls(38).Mevinolinhad a markedinhibitory effecton cho-lesterol synthesis as measured by
[3H]OH
incorporation. The rate ofcholesterol synthesis was depressed in the mevinolin-treated animals to 24% that of matched controls (303 nmol[3H]OH
incorporated g-' h-' in the treated compared with 1,277±348inthe controls, P < 0.01). In addition, there was a decrease in the amount ofradiolabeledcholesterol excreted in the bileover thesame2-h period(1 1.3±0.8 nmol3H-cholesterol/
gliverh-'vs.28.9±6.9in the controls, n = 3, P < 0.05). Inter-estingly, the ratioofradiolabeled cholesterol excreted in thebile to the amount of radiolabeled in theliverwasthesame(3.4%) inboththecontrol and treated animals. That the percentage of newlysynthesizedcholesterol that is excreted as biliary choles-terol isconstant despite varying synthetic rates has also been demonstratedby others(16, 17).
No significant change in bile flow was observed after ad-ministrationofmevinolin(Table III), nor wasthereasignificant change in bile salt orphospholipid output
(Fig.
4,A andB).
However, totalbiliary cholesterol mass outputdecreased 16% duringthe 2 haftertheadministration ofmevinolin
compared
with thepreadministration value (P <0.05, Fig. 4C). Thus,a
76%decrease incholesterolsynthesis resultedina 16% decrease inbiliarycholesteroloutput. Based on this, it can be calculated that 21% of the biliary cholesterol output in the control was
newly synthesized sterol, which was rapidly excreted. This
es-timate is in goodagreementwithpreviouslyreportedvaluesfor this parameter for rats in the basal state(16, 17).
Microsomal cholesterol contents were not
significantly
changed (data not shown). Thus,an acute decrease in therate
of cholesterol synthesis withoutacompensatory
change
ines-terification rate appears to decrease the size of the
metabolically
active biliary precursor pool, resultinginadecrease in
biliary
cholesteroloutput.
Effect ofprogesterone on binarylipidoutput. To
investigate
whether a change in the rateof cholesterolesterformation alone
o Is'
I.-X
6-303
:t25
22.0
o 1.5
C. 1
Q5
:t
%, 030
Tb a
90.25 W-J
0.2
(j
-j 0.15
A CONTROL MEVINOLIN
pre post post pre post post control I h 2h P4EV I h 2 h
BCONTROLMEVINOL IN
pre post post pre post post
control I h 2 h MEV I h 2 h
CCONTROL MEVINOLIN
0,p
0.30 <.0
s.LI
T~~~~~~~~~~~~~~
IOO
pre post post control Ih 2h
pre post post
MEV Ih 2h
Figure4.Effect ofmevinolinonbiliary lipidsecretion. Thesame
pro-tocolasinthelegendtoFig.2wasusedexceptthat 1 mg/kgof
mevi-nolininadimethylsulfoxide (DMSO)/saline(1:10)solutionorthe
DMSO/salinevehiclealonewasadministeredintragastrically. (A)
Ef-fectonbile sale secretion. (B)Effect onphospholipidsecretion.(C)
Ef-fectoncholesterolsecretion. Eachpointis the mean±SEof six
deter-minations. *Different fromcontrol valueby pairedttest, P<0.05.
couldaffectbiliarycholesterolsecretion,thecompound proges-terone was used. Chronic progesterone treatment in vivo
in-creases biliary cholesterol output anddecreases the activity of
hepaticACAT(18).ACATactivityis alsodecreasedinvitroby theaddition ofprogesteronetohepaticmicrosomes(21, 39),or
by inclusion in theculture medium ofhuman skinfibroblasts
(40). Thus, progesterone administration in our in vivo model
mightbeexpectedtoacutelydepressthe rate of cholesterolester
formationwithoutaffectingtherateof cholesterolsynthesis.The
netresultshouldbeanexpansion ofthebiliaryfreecholesterol
I
8 CONTROL MlEVINOLIN
pool with an increase inbiliary cholesterol output. Progesterone
administration did not change HMG CoA reductase activity
(Table II). ACATactivity wasdifficult to measure. Theeffectof progesterone in vitro is reversed by washing the microsomes (41). Progesterone administered in vivo was lost from micro-somes duringpreparation (data not shown). Moreover, the com-pound israpidly metabolized. 1 h after injection of 10 mgof progesterone, only 22 ng/mg protein was recovered in the mi-crosomal fraction. However, cholesterol esters weresubstantially reduced in microsomes 1 h after progesterone administration (Table IV), which suggests cholesterolesterification wasinhibited as expected.
Bile flow was significantly increased 1 hafter intravenous administration of progesterone as compared with the ethanol control (Table
III).
Thiseffect appears to be bile saltindependent, since nosignificant changein bilesaltsecretionwasfound(Fig.
5 A). Such a choleretic effect has been reported previously for the progesterone analogue pregnenolone- 16-a-carbonitrile(42). No significant change in phospholipid output was seen with either progesterone or the control vehicle alone (Fig. 5 B). A marked increase (P < 0.05) in biliary cholesterol output was observed in the first hour after progesterone administration (Fig. 5 C). By 2 h, biliary cholesterol secretion had returned to normal. The changes in biliary cholesterol secretion paralleled the changesin microsomal cholesterol ester content. These results suggest that a change in intrahepatic cholesterol esterification without a compensatory change in cholesterol synthesis can affect biliary cholesterol secretion independently of bile salt secretion and support the hypothesis thatesterification rates are animportant determinant of the biliary cholesterol precursorpool.
Discussion
There is considerable uncertainty regarding the mechanism and regulation of cholesterol secretion into the bile. It is generally accepted that the rate of bile salt secretion determined in part the rate of biliary cholesterol secretion. Under most circum-stances, as bile salt secretion increases, cholesterol secretion also increases. However, more cholesterol is secreted per molecule of bile salt at lowersecretoryrates than at higher rates (5). More-over, when different bile salt species are secreted at the same rate they induce secretion of different amounts of cholesterol (43). Thus, in addition to the rate of bile salt secretion, intra-hepatic metabolic factors seem to play an important role in de-termining the rate of cholesterol secretion into bile.
It has been suggested that the rates of cholesterol synthesis and esterification in the liver are important determinants of the rate of biliary cholesterol secretion. The activity of the enzyme HMG CoA reductase, which catalyzes the rate-limiting step of cholesterol synthesis, is elevated in patients with increased biliary cholesterol secretion and cholesterol gallstone disease (10-12). This led to the hypothesis that the rate of cholesterol synthesis determined the rate of biliary cholesterol secretion. It has been shown that under basal conditions, 10-20% of secreted choles-terol is derived within 30 min from newly synthesized sterol (16), and that synthesis and secretion varied together under some circumstances. Turley and Dietschy (6) demonstrated in the rat that the two are not necessarily related. However, this may not apply to man. Since 80% of biliary cholesterol is derived from preformed sources, factors other than the rate of cholesterol syn-thesis must beimportant in determiningbiliary secretion rate.
Cam
'I'.
U) U)
0z
2.
"I
2.
01,
o. U)
40
0.
Q .
(A
0
Qt.
To
:t0.
40
~02
0
A CONTROL PROGESTERONE
5-
3-pre post post pre post post control h 2 h prog. b 2 h
B-CONTROL PROGESTERONE
.0
.0
pre post post pre post post control b 2 h prog. l h 2 h
CONTROL PROGESTERONE
'5
P <0.05
pre post post pre post post control lh 2 h prog. lh 2 h
Figure5. Effect ofprogesteroneonbiliarylipid secretion.The same protocol as in the legend to Fig. 2 was used, except that 31.25 mg/kg
progesterone inethanol or ethanol alone wasadministered
intrave-nously.(A)Effecton bile salesecretion.(B)Effectonphospholipid
se-cretion. (C) Effectoncholesterol secretion. Each point is the
mean±SEof fivedeterminations. *Different fromcontrolvalue by
pairedttest, P < 0.05.
However,it should beborninmindthatultimatelyallcholesterol
isderived from either de novosynthesis orthe diet.
It was suggested that the rate of cholesterol esterification
couldaffect cholesterol secretion because chronicprogesterone
administration inhibited hepatic cholesterol esterification and increased biliary cholesterol secretion (18). Other factors that
may beimportant include plasma lipoprotein cholesterol uptake
(44) andintracellular transportof cholesterol, about whichlittle
influencing the rates of entry and exit of cholesterol into the presecretory pool.
Inthe present work, we used an acute rat model to test the hypothesis that there is an active metabolic pool of cholesterol in the liver which, when altered, can affect biliary cholesterol secretion directly. The findings reported here and summarized in Table V are entirely consistent with this concept. The en-terohepatic circulation of bile salt, cholesterol, and lecithin was rigidly maintained by using a constant infusion of these com-pounds into the duodenum of bile-diverted animals. This min-imized any possible effects of changes in bile salt synthesis on these processes. In the face of this fixed bile salt return to the liver, none of the agents administered significantly altered bile saltsecretion. Thus, changesinbile salt-coupledcholesterol se-cretion could notaccount for the changes in cholesterol secretion evoked by the agents 25-hydroxycholesterol, mevinolin, or pro-gesterone. The lack of statistically significant changes in phos-pholipidsecretion is also consistent with these effects, being me-diated by changes in intrahepatic cholesterol metabolism alone. Thetime courseofchanges in secretion closely paralleled those of thechanges in the metabolic parameters. Thus, with 25-hy-droxycholesterol,HMG CoA reductaseactivitydecreased, ACAT activity increased,andcholesterolsecretion decreased within I hofdrug administration.
That enzyme activities per se are not the determinant of secretion is demonstrated by the experiment with mevalonate, wherethe enzyme activities are altered but the free cholesterol pool apparently is maintained withnochange in biliary choles-terolsecretion. The observation that changing either of two en-zymesinvolved in intrahepatic cholesterol metabolism (ACAT or HMGCoAreductase) independentlyaffectsbiliarycholesterol secretionsuggests that the metabolic regulationofsecretion is likelytobesubjectto avariety of interrelated processes influ-encing the rates of free cholesterol entry to orexit from the proposed precursor pool. No data is available concerning the sizeorlocation of this pool.That thebiliary cholesterolchanges occurredwithin 1 hofpharmacologic manipulationsuggestsit is small. Thatmicrosomal free and estercholesterolcontents in somecases mirrored the changes inbiliary cholesterol output suggests that the proposed precursorpoolis a small subset of microsomal cholesterol.However, no datapresentedhere allow furtherspeculation on it location.
In addition tosynthesis and esterification, other processes mayplaya role indetermining the sizeof thispool.Ithas been suggestedthatlipoproteins, especially high density lipoproteins, are animportantsourceofbiliarycholesterol(44).Others have foundthat
chylomicron
cholesterolisnot animportantsourceofbiliary cholesterolintherat(6).Intracellular cholesterolester
hydrolysis does not seem to be a regulated process (45, 46)
Table V.SummaryofEffect ofAlterationofCholesterol
Homeostasis onBiliaryCholesterolSecretion
Esteri- Biliary Agent Synthesis fication cholesterol
25-Hydroxycholesterol I t 1
Mevalonate I I
Mevinolin 1 - 1
Progesterone 1 T
(Stone, B. S., unpublished observations). Thus, its role is likely tobe passive.
Theexistence ofatightlyregulatedmetabolicallyactive free cholesterol poolwhich serves as theprecursor for biliary cho-lesterol may clarify some of the confusing species differences in the responseof biliarylipid output to a cholesterol-rich diet. For example, the rat that is relatively resistant to changes in biliary cholesterolsaturationhas arelativelyhigh levelofhepaticACAT activity, andthus may rapidly esterify even large amountsof influxing dietarycholesterol, thusmaintaininga constant pool size and biliary cholesterol secretion rate. In contrast, humans who aremore prone to cholesterol gallstone formation and who normally have ahigher mole percentageofcholesterol in the bile (47) have lower levelsof this enzyme (39, 48). This may allow thebiliaryprecursorpooltoexpand inresponse to arapid influx ofcholesterol,whichwould result in increased cholesterol secretion andsupersaturation ofthe bile. However, beforean extrapolation of this data, which was obtained from the rats, canbe made to man, furtherexperimentation in humans will be necessary.
The resultsof thisstudy,togetherwith evidencefrom other laboratories,supportthe conclusion that the rate of cholesterol secretionintobile is determinednot only by the rate of bile salt secretion,but alsoby intrahepatic factors includingratesof cho-lesterolsynthesis andesterification. Whatotherfactors are in-volved, where the precursor biliarycholesterol pool is located, and how cholesterol secretion is coupledto bilesalt secretion remain importantareasfor futureresearch.
Acknowledgments
Wewish to thank Ms. Elizabeth Bonney for performing some of the cholesteroldeterminations,Mr.Alfred Alberts for thegiftofmevinolin,
and Ms. JenniferFaravelli,Ms.Lynne Justen, Ms. Janis Wick, Ms. Jeanne
Gill,and Ms. ConnieKuruppu forpreparingthemanuscript.
This researchwassupported by research grants HL 05360 and AM
18774, traininggrant AM07056,andanindividual National Research
ServiceAward (HL06582)toDr.Stonefrom the National Institutes of Health.
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