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Interaction of dietary cholesterol and

triglycerides in the regulation of hepatic low

density lipoprotein transport in the hamster.

D K Spady, J M Dietschy

J Clin Invest. 1988;81(2):300-309. https://doi.org/10.1172/JCI113321.

These studies report the effects of dietary cholesterol and triglyceride on rates of receptor-dependent and receptor-inreceptor-dependent LDL transport in the liver of the hamster. In animals fed diets enriched with 0.1, 0.25, or 1% cholesterol for 1 mo, receptor-dependent LDL transport in the liver was suppressed by 43, 63, and 77%, respectively, and there were reciprocal changes in plasma LDL-cholesterol concentrations. In addition, dietary

triglycerides modified the effect of dietary cholesterol on hepatic LDL transport and plasma LDL concentrations so that at each level of cholesterol intake, polyunsaturated triglycerides diminished and saturated triglycerides accentuated the effect of dietary cholesterol. When animals were raised from weaning on diets containing small amounts of cholesterol, the decline in receptor-dependent LDL transport was nearly abolished by the addition of polyunsaturated or monounsaturated triglycerides, but was markedly augmented by the addition of saturated lipids. When animals raised on diets containing cholesterol and saturated triglycerides were returned to the low cholesterol, low triglyceride control diet, hepatic receptor-dependent LDL transport and plasma LDL-cholesterol concentrations returned essentially to normal within 2 wk. Neither receptor-independent LDL transport nor the receptor-dependent uptake of asialofetuin was significantly altered by dietary

cholesterol or triglyceride suggesting that the effect of these lipids on hepatic LDL receptor activity was specific and not due to a generalized alteration in the physiochemical

properties of hepatic membranes. […]

Research Article

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Interaction of Dietary Cholesterol

and Triglycerides

in

the Regulation

of

Hepatic

Low

Density Lipoprotein

Transport in the

Hamster

David K. Spady and JohnM. Dietschy

Department ofInternalMedicine, The University ofTexasHealthScience CenteratDallas,

Southwestern MedicalSchool, Dallas, Texas 75235-9030

Abstract

Thesestudiesreport theeffectsofdietarycholesterol and

tri-glycerideonratesof receptor-dependent and

receptor-indepen-dent LDL transportintheliver ofthe hamster. Inanimalsfed

diets enriched with 0.1, 0.25,or1% cholesterol for 1 mo,

re-ceptor-dependent LDL transport in theliver was suppressed by 43, 63, and 77%, respectively, and there werereciprocal

changes in plasma LDL-cholesterol concentrations. In addi-tion, dietarytriglyceridesmodified the effect ofdietary choles-terol on hepatic LDL transportand plasma LDL

concentra-tions so thatat each level of cholesterol intake, polyunsatu-rated triglycerides diminished and saturated triglycerides accentuated the effect of dietary cholesterol. When animals

wereraisedfrom weaningon dietscontainingsmall amounts of

cholesterol, the decline inreceptor-dependent LDL transport was nearly abolished by the addition of polyunsaturated or

monounsaturated triglycerides, but wasmarkedly augmented by the addition of saturated lipids. When animals raised on

diets containing cholesterol and saturated triglycerides were

returnedto thelowcholesterol, lowtriglyceride controldiet, hepaticreceptor-dependent LDL transport andplasma LDL-cholesterol concentrations returned essentially to normal within 2 wk. Neitherreceptor-independentLDLtransportnor thereceptor-dependent uptakeofasialofetuinwassignificantly

altered by dietarycholesterol ortriglyceride suggesting that

theeffect of theselipidsonhepaticLDL receptoractivitywas

specific andnotduetoageneralized alterationin the

physio-chemicalpropertiesofhepaticmembranes. Thesestudies dem-onstrate the importantrole of saturated triglycerides in

aug-menting the effect of cholesterolinsuppressing hepatic LDL receptoractivityandelevatingLDL-cholesterollevels.

Introduction

It haslong beenrecognizedthatthe substitution of

polyunsat-urated fat for satpolyunsat-urated triglycerides in the diet results in a

reduction in plasma cholesterol levels (1-4). Although the mechanism(s) responsible for this effect remains poorly

un-derstood, therelationshipbetween variousdietary lipids and

plasma cholesterol levels inmanhasbeen studiedin

consider-abledetail (5-9).Fromthese studies itappearsthatthe

magni-AddressreprintrequeststoDr.Spady, Department of Internal Medi-cine, Universityof Texas Health Science CenteratDallas,5323Harry HinesBoulevard, Dallas,TX75235-0930.

Receivedfor publication27February1987andinrevisedform30 July1987.

tude of the change in plasma cholesterol levels that can be

achieved is dependent upon the ratio ofpolyunsaturated to saturated fatty acidsin the diet. Furthermore,saturated fatty

acidsareapproximately twiceasactive in raising plasma cho-lesterol levels as are polyunsaturated fatty acids in lowering

them. Thedifferential effects of polyunsaturatedand saturated

fatty acids on plasma cholesterol levels also appear to be

magnified by dietary cholesterol(6).

Although it has been suggested that the decline in plasma

cholesterol concentration producedbypolyunsaturated fatty

acids is related to an increase in fecal sterol excretion, the specific reason why fatty acids regulate plasma lipoprotein-cholesterol levels isnotknown.Anumber of mechanismshave

been proposed including alterations in (a) theabsorption of

cholesterol from the small intestine, (b) ratesofde novo

cho-lesterol synthesis, (c) the distribution ofcholesterol between

plasma and various extrahepatic pools, (d) the cholesterol contentofVLDLand LDL, and (e)the ratesofsynthesis or catabolismof plasmalipoproteins.Littledirect evidence exists,

however, to support anyofthesemechanisms. For example, it

isnot evenclearwhattheeffectofdietary triglycerides ison the synthesis anddegradation of LDL-cholesterol. Whereassome investigatorshave reported that the increasein plasma LDL levelsproduced byfeedingsaturated fat is accompaniedby a

fall in the fractional catabolic rate (FCR)' (10), others have found little or nochange in thisparameter(11, 12).

Further-more, since most LDLis cleared from the plasma via

recep-tor-dependenttransport, it isdifficulttointerpreta changein

the FCR for this lipoprotein under conditions where the

plasma LDL-cholesterol concentration has alsochanged (13, 14). Thus, on the basis of currently available data it is not possibleto determine whether the changes in plasma choles-terol levels brought about by triglyceride feedingresult from alterations intherateofreceptor-dependentLDLdegradation ortherateofsynthesisofLDLparticles.

The hamster hasprovedtobe agood model in whichto

study these regulatory mechanismssince itsconcentration of

plasma LDL-cholesterol respondstochanges indietary lipid intake in a manner that isessentially identicalto that seen in man (15). Furthermore, thekinetics of LDL-cholesterol

pro-duction anddegradationhavebeen workedoutindetailinthis

species (13) so that it is possible, in quantitative terms, to

identifythe reasonwhyaparticular dietary or

pharmacologi-calmanipulationresultsinachangein thesteadystateplasma

LDL-cholesterol concentration. Usingthismodelwerecently

reportedthatsaturated triglycerides,but not

polyunsaturated

triglycerides,

suppressed receptor-dependentLDL transport in

1.Abbreviations usedinthis paper:FCR,fractional catabolic rate;Jd, receptor-dependentrate;

J1,

receptor-independentrate; Jm,maximal

transport velocity; Jt, production rate; methyl-hLDL, reductively methylatedLDLofhumanorigin.

J.Clin.Invest.

K TheAmericanSocietyfor ClinicalInvestigation,Inc.

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the liver of hamsters that were alsoreceivingsmallamountsof dietary cholesterol (15). Such alterations in hepaticLDL re-ceptoractivitywillsignificantlyaffectcirculating LDL-choles-terol levels since - 75% of LDL turnover is mediatedbyLDL

receptors(16-18)and - 85% of thisreceptor-dependentLDL

transport is found in the liver of allspeciesfor which data are available (19-21). The present studies were therefore under-taken in thisspecies (a)toexamine in detail the time and dose

dependency ofsuppression ofhepatic LDL transport by di-etarycholesterol andtriglycerides; (b) todetermine ifthe di-etarysuppressionofreceptor-dependentLDL transport in the

liver isreversible; and (c)todefine the relationship between

changesinreceptor-dependentLDL transport and other meta-bolic events within the liver cell.

Methods

Animals and diet. Male GoldenSyrianhamsters(CharlesRiver Lake-view,Newfield, NJ) were housed in colony cages and subjected to light cyclingforatleast3wkbeforeuseinspecific experiments.The control diet used in these studieswasground Wayne Lab Blox(Allied Mills, Chicago,IL), which contained 0.23 mg/g of cholesterolasdetermined bygas-liquidchromatography. The total fatcontentofthis dietwas5% and thefattyacidprofile,asdetermined bygas-liquid chromatography of themethylesters,was 14%of thefattyacid16:0,5%18:0,27%18:1, 46% 18:2, and 6% 18:3. The experimental dietswere prepared by addingvaryingamountsof cholesteroland/ortriglyceridetothis

con-troldiet.Inthosedietscontaining only cholesterol,the sterolwasfirst dissolved inwarmethanol andmixed with thegroundfoodfollowing which the solvent was evaporated. Inthosediets also containing tri-glyceride,the cholesterolwasfirst dissolved in the oilbeingaddedto

thefood.Inallcasesthedietsweresubjectedtoprolongedmixing using a mechanical food blender. The triglycerides used in these studies includedsafflower oil (iodine value of 140-150), olive oil(iodinevalue of 79-90), hydrogenatedcoconutoil(iodinevalue of<4) and tripal-mitin(iodinevalueof<4). When thefattyacid methylestersof these triglycerideswereanalyzedbygas-liquid chromatography, saffloweroil contained 79% of the 18:2fatty acid,olive oil contained 65% 18:1 and

coconut oil contained >90% satrated fatty acids of varying chain length. All dietswerefed ad lib. and allexperimentswerecarried out duringthemid-dark phase of thelightcycle.

Lipoprotein preparations. Hamster and human LDL was isolated from plasma by preparativeultracentrifugationin the density range of 1.020to 1.055g/ml and labeledwith either

[1251]tyramine

cellobiose (22)or'3'Iusingtheiodinemonochloridemethod(23). Both hamster and human LDL in thisdensityrangecontained almost exclusively apoprotein Blooonpolyacrylamidegels. Thewhole-animalturnoverof each LDL preparationwasidentical regardless of whether it was la-beled with[125I]tyraminecellobioseor13'Ialone (unpublished obser-vations from this laboratory). The human LDL was alsosubjectedto reductive methylation (methyl-hLDL) to fully block its interaction with the LDL receptor (20, 24). Thus, the

[125I]tyramine

cellobiose-la-beled homologous hamsterLDLwas used to quantitate the rate oftotal

LDLtransportby theliver while the similarly labeled methyl-hLDL

wasutilized to measure the receptor-independent component of this transport process (13, 20). Alllipoproteins were used within 24 h of preparation and were filtered through a0.45-AmMillipore filter imme-diatelybeforeuse.

Determination ofhepatic LDL uptake rates in vivo. Rates ofhepatic

LDLclearance were determined using a primed-continuous infusion of[1251]tyramine cellobiose-labeled LDL (19, 20). The radioactivity present in the priming dose relative to the radioactivity subsequently infused each hour was adjusted so as to maintain a constant specific activity of theLDLin plasma throughout the experimental period. The infusions of[r251]tyraminecellobiose-labeled LDL were continued for

4hatwhich timeeachanimalwasadministeredabolusof13"'-labeled

LDL and killed 10 min later by exsanguination through the abdominal

aorta.Samples of various organswerequickly rinsed and weighedand, along with aliquots of plasma, were assayed for radioactivity. The tissue space achieved by LDL at 10 min

("'lI

dpm per g of tissue dividedbythe

IIII

dpmper yA ofplasma) andat4 h(the125Idpm per g of tissue divided by the '25Idpm perAlofplasma)wasthen calculated and hasthe units of microliters per gram (25). The increase in tissue space with time represents theclearance of LDL by the liver andwas

expressedasthe microliter of plasma cleared of its LDLcontentper hour per gram of liver. This clearance rate was also multiplied by the plasma LDL-cholesterol concentration togive theabsolute mass of LDL-cholesterol taken up per hour by each gram of liver (20).

Determination ofhepaticsterol synthesis rates in vivo. As pre-viously described (26, 27), animals were killed 1 h after the intravenous administration of [3H]water (- 50 mCi). Rates of sterol synthesiswere

expressedasthe nanomoles of[3H]waterincorporated into digitonin precipitable sterols per hour per gram of liver.

Analytic procedures. Plasma LDL-cholesterol concentrations were determined by simultaneously centrifuging plasma at densities of 1.020 and 1.063 g/ml. The cholesterol content of the top one-third of eachtube,aswellasthe totalplasma cholesterol concentration,was

assayed colorimetrically as previously described (28). The hepatic

content of free and esterified cholesterolwasmeasured using silicic acid/celite columns (28).

Calculations. Whereappropriate, data arepresented as mean

values± 1SEM. In order to relate changes in hepatic LDL clearance to changes in LDL receptor number or, more appropriately,tothe maxi-mal transport velocity (J") for the receptor-dependent system, the experimentally determined clearancerates weresuperimposed upon the kineticcurvesdefining theratesof LDL uptake in the liverasa function of the plasma LDL-cholesterol concentration in control hamsters. Thesecurves wereconstructed for the liver (e.g., Fig. 1) using the following, previously published transport parameters: P* 0.080+0.025Ag/hper g permg/dl;Km 108.7±5.7 mg/dl; and J'- 126.0±9.6gg/hper g( 13). Utilizing these curves, LDL clearance

ratesmeasuredatanyplasma LDL-cholesterol concentration could be converted directlyto J' values and these valuesarepresented asa

percentage of the appropriate control values in each experiment.

CHOLESTEROL DAYS ON 0.5% CONTENT CHOLESTEROL OF DIET FED CHOESTEO FOR 30DAY5SIE

16O 0 C 0 Control

z 120 *

erow%

i o 7

30Dy

c'r. ..Tot LDL.

.ii1

nq Tn

f

oL801010ao04080 2 6 0

0

60J 20

20B 408 2100 04D 02100

PLASMA LDL-CHOLESTEROL CONCENTRATION (mg/dlI)

Figure 1. Hepatic LDL clearance and LDL-cholesterol uptake in ani-mals fed varying amounts of cholesterol for 30 d (A and B) or 0.5% cholesterol for varying periods of time (C and D). The shaded areas represent the kinetic curves for total (stippled) and receptor-indepen-dent (hatched) LDL transport (mean values+2 SD) in the liver of normal animals with a constant receptor number (Jm). The

individ-ualpoints superimposedonthesekineticcurvesrepresent the

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Results

Wefirst examined the effect of adding cholesterolalonetothe dietand the results of these studiesaresummarized in TableI. Control animalswerefed the low cholesterol, lowtriglyceride

rodentdiet and experimental animalswere fed the samediet thatwaseither enriched with varyingamountsof cholesterol (0.1,0.25,or 1%) and fed for 30 d,orthatwasenriched with

0.5%cholesterol and fed for varying periods of time (4, 7,or30 d).Althoughnotshown,therateof weight gainwasessentially

identical in allgroupsof animals. Liver weight, however, pro-gressively increasedas increasingamounts ofdietary choles-terolwerefedorwhen thesameamountofdietary cholesterol wasadministeredforlonger periods of time (column 1). Simi-larly,the hepatic cholesterylestercontent, whichwas0.8mg/g in control animals, increasedas afunction of both theamount

ofcholesterol in the diet and the timeonthe diet (column 2).

Asshown in columns 3 and 4, dietary cholesterol produceda dose-dependent and time-dependent suppression in rates of hepatic LDL clearance and reciprocal elevations in plasma LDL-cholesterol concentrations. Although the data are not shown, therateofmethyl-hLDL clearance

(receptor-indepen-dent transport) equaled I 1±3,l/hper gincontrolanimals and

wasnotsignificantly altered by cholesterol feeding.

Because the circulating levels of LDL-cholesterol increased inall animals fed cholesterol, these changesinLDL clearance couldnotbe equateddirectlywithdown-regulation of

recep-tor-dependent LDLtransportsince saturationofthisreceptor systemcould,atleast partially, explainthesechanges. To dis-tinguish between thesetwopossibilities, thekinetics of

recep-tor-dependent and receptor-independentLDL transport in the liver of normal hamstersweredefinedovertherangeof

LDL-cholesterol levelsseeninthecholesterol fed animals. Thiswas

accomplished by measuringratesofhepaticLDLclearance in control hamsters whose plasma LDL-cholesterol levels were

acutely raised and maintainedatvaluesrangingfrom 25to200 mg/dl by adding varyingamounts of unlabeled homologous

LDLtotheprimed-continuousinfusionsof radiolabeledLDL (13). These studies were performed using both homologous LDL(total LDL transport)andmethyl-hLDL (receptor-inde-pendent transport).The datawereanalyzed using least-squares nonlinear regression analyses (13) and the best-fit curves±2 SD arepresented in Fig. 1. The upper panels show the

rela-tionship betweenratesoftotal(stippled areas) and

receptor-in-dependent (hatchedareas)hepatic LDL clearance and plasma

LDL-cholesterol concentrations under circumstances where the LDL receptornumber, i.e.,Jmforthe receptor-dependent system (13),was constant. These same data are presented in thelower panel as total and receptor-independent

LDL-cho-lesteroluptake (microgramsperhour per gram). In both cases, the area between the two curves represents the

receptor-de-pendentcomponentof LDL transport.

Theobserved rates of LDL transport in the cholesterol-fed animals are superimposed upon these kinetic curves for LDL transport in control animals. As seen in A, rates of hepatic LDLclearance in animals fed cholesterol for 30 d fell signifi-cantly below those rates that would be expected in normal animals at similar plasma LDL-cholesterolconcentrations. It can be calculated from this analysis that receptor-dependent LDL clearance per gram of liver was suppressed by - 45, 70,

and 85% in the animals fed 0.1, 0.25, and 1.0% cholesterol, respectively. Since the suppression of hepatic LDL clearance was accompanied by a rise in plasma LDL levels, the actual massofLDL-cholesterol taken up per hour per gram of liver in these same animals remained remarkably constant at -25

jig

(B). However, whereas > 90% of LDL transport was receptor dependent in the control animals, receptor-independent

mechanisms became increasingly important in the choles-terol-fed animals andactually accountedfor mostofthe LDL-cholesterol uptake that took place in the animals fed 1.0% cholesterol. The time dependency ofthese changes is illus-trated in C and D. When animals were fed a 0.5% cholesterol diet for only 4 d, theplasma LDL-cholesterol concentration doubled, the LDL clearance rate decreasedslightly, but there was no change in the absolute rate of receptor-dependent

transport (C) indicating that this initial rise in the plasma LDL-cholesterolconcentration wasdue entirely toincreased LDL-cholesterol production.As a consequence,theamountof

LDL-cholesterol taken upby the liverat 4 d wasnearly dou-bled (D). However, with longer periods of feeding there was

progressive suppressionofreceptor-dependentLDLuptaketo those values seenat30 d ofcholesterolfeeding.

Whenthe datain Fig. 1 are corrected forchanges inliver weight(TableI), theeffect of dietary cholesterolonwholeliver

LDL receptor activity can be determined and these values, along withthe plasmaLDL-cholesterol levels, are plotted in

Table I.Effect ofDietaryCholesterolonHepaticCholesterylEsterContent, CholesterolSynthesis,

andLDL

Tranisport,

andonPlasma LDL-Cholesterol Concentrations

Hepaticcholesteryl Timeondiet Liverweight estercontent d None 0.10 0.25 1.0 0.5 0.5 0.5 30 30 30 4 7 30 g 5.3±0.2 5.8±0.1 6.4±0.2 7.9±0.3 5.7±0.2 6.3±0.2 7.3±0.3 mg/g 0.8±0.1 11±1 26±1 88±6 9±1 27±2 48±4 Hepaticcholesterol synthesis

nmol/hper g

48±3 8±2 6±1 6±1 9±1 6±2 6±1

HepaticLDL Plasma LDL-cholesterol clearance concentration

pl/hper g

125±8 65±4 32±2 18±2 98±5 46±4 28±3 mg/dl 22±3 43±4 72±5 126±13 41±3 62±3 96±5

Groupsof animalswerefed diets enrichedwith0.10, 0.25,or1.0% cholesterol for 30 d or with dietscontaining0.5% cholesterol for 4, 7,or

30 d. Each valuerepresentsthemean±1SEM fordata obtained in sixanimals.

Cholesterol addedto

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-100

80

> 608

-Jr

S K

40

I-J> 40

J

< 20 0 125

o 0%

ns F

sA-CHOLESTEROL

CONTENT OFDIET FED

FOR 30 DAYS

A

-0

,B9.

DAYS ON 0.5% CHOLESTEROL

DIET C

D

LLJ 100- 0

~-z

nJf4.-

00

Cfl_j

I-9j

z25-0'-0o 0.250.50.75 1.0 0 10 20 30

DIETARY DAYS ON 0.5%

CHOLESTEROL DIETARY

CONTENT(%) CHOLESTEROL

Figure 2. Whole liver receptor-dependent LDL clearance andplasma LDL-cholesterol concentrations in animals fedvaryingamountsof cholesterol for 30 d(Aand B)or0.5%cholesterol forvarying periods of time(CandD).Whole liverreceptor-dependentLDL clearance wasobtained by multiplying the percent reduction in receptor-de-pendent LDL clearance per gram of tissue by the whole liverweight and thenexpressing the valuesaspercentages of the control value.

Fig. 2as afunction of thecontentof cholesterol in the diet(A

andB)orthe numberofdaysthat the 0.5% cholesterol dietwas fed (Cand D). Since cholesterol increasedliver weightsas a

functionofthedose and timeonthediet,thesuppression of

hepatic LDL receptor activity was somewhat less when

ex-pressedperwhole liver than when expressedper gramof tissue.

Inthe second groupofexperimentswenextexamined the effect ofdietary triglycerides alone on ratesofhepatic LDL transport. Intheseexperiments, hamsterswerefed either the controlground rodent dietorthesamediet enriched with 20%

safflower oil, 20%oliveoil,or 20%hydrogenatedcoconutoil

foronemonth.Weight gainamongtheanimalsfedthevarious

triglycerideswasidenticalbut - 15% greaterthanseenin the control animals. Asshown in TableII, the liverweightsand

hepaticcholesterylestercontents werenotaffectedby triglyc-eridefeeding (columns I and2). Ratesof hepaticcholesterol

synthesis, however, increased byapproximately five-, three-,

andtwofold, respectively,in animals fed saffloweroil,oliveoil,

andhydrogenatedcoconutoil. Rates ofhepaticLDL clearance

were notsignificantlyaltered by thetriglyceride diets.Plasma

LDL-cholesterol levelswereonly modestlyaffectedby

supple-menting thediet withtriglyceridealone.Saffloweroil and olive oil reduced plasma LDL levels by 28 and 20%, respectively,

while hydrogenated coconut oil increased plasma LDL-cho-lesterol levels 64%. The absolute magnitudeof thesechanges, however, was verysmall comparedtothatseenwhen choles-terolwaspresentin thediet.

Thethird series of experimentswasundertakentoexamine theinteraction ofdietary triglycerideand cholesterol in

regu-lating hepaticLDL transport. Accordingly, hamsterswerefed dietssupplemented with 0.06, 0.12,or0.24% cholesterol with and withoutsafflower oil (20%)or hydrogenatedcoconutoil (20%) and the resultsofthesestudiesaresummarizedin Table

III. Atall levels of dietary cholesterol, hepatic cholesterylester

levels (column 2)werehighestinanimalsfed cholesterol alone, intermediate when safflower oil was added andlowest when

hydrogenatedcoconut oilwasadded. The hepatic cholesteryl

ester contentequaled 0.6 mg/g in controlanimals(column2). Inanimals fed 0.06% cholesterol alone, the hepaticcholesteryl ester content equaled 7.8 mg/g but this content was signifi-cantly lower in those hamsters simultaneously given safflower

oil(2.6 mg/g) orhydrogenated coconutoil (1.4 mg/g). Simi-larly, in those animals fed the 0.12 and 0.24%cholesteroldiets, hepatic cholesterylester contents werehighest intheanimals

fedonly cholesterol,werelower in thoseanimals fedsafflower oilandwerethe lowestwhen the hamstersweregiven hydroge-nated coconut oil. Rates of hepatic cholesterol synthesis

equaled44 nmol/hper gin control animals(column 3). On

the 0.06% cholesteroldiet, ratesofsynthesisweresuppressed - 80% butthissuppressionwaslargely reversed in the animals fed either safflower oilorhydrogenatedcoconutoil.Similarly, whenfed 0.12% or0.24% cholesterol, the suppression in

he-paticsterolsynthesiswasgreaterin the absence oftriglyceride than in the presence of these oils. The rate of hepatic LDL

clearance equaled 117 ,il/hper gin controlanimalsand was

progressively suppressed when cholesterol was added to the

diet. However,ateachof the three levels of cholesterol feeding, hepatic LDLclearancewasactually increased by adding saf-floweroiltothedietandwasstrikingly suppressedbyfeeding hydrogenated coconut oil (column 4). These changes in he-patic LDL clearance were faithfully reflected by reciprocal changes in thecirculatingplasma LDL-cholesterol

concentra-tions(column5). Thus,atanylevelof cholesterolfeeding,the

TableII. EffectofDietaryTriglycerideonHepaticCholesterylEsterContent, CholesterolSynthesis andLDL Transport, andonPlasmaLDL-CholesterolConcentrations

Triglycerideaddedto Hepaticcholesteryl Hepaticcholesterol Hepatic LDL PlasmaLDL-cholesterol control diet Liverweight ester content synthesis clearance concentration

g mg/g nmo//hper g 1l/hper g mg/dl

None 5.1±0.2 0.8±0.1 34±2 115±6 25±2

Safflower oil 5.5±0.3 0.6±0.1 160±10 138±8 18±2

Olive oil 5.6±0.3 0.9±0.2 106±7 114±7 20±3

Coconut oil 5.3±0.4 0.7±0.1 78±6 116±7 41±3

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TableIII.EffectofDietaryTriglycerideat Varying DietaryCholesterol Levels on Hepatic Cholesteryl Ester Content, Cholesterol Synthesis, andLDL Transport,andonPlasma LDL-Cholesterol Concentrations

Cholesteroladded to Triglycerideadded to Hepaticcholesteryl Hepatic cholesterol Hepatic LDL PlasmaLDL-cholesterol

controldiet controldiet Liver weight ester content synthesis clearance concentration

% g mg/g nmol/h per g ill/hper g mg/dl

None None 5.1±0.3 0.6±0.1 44±3 117±9 23±2

0.06 None 5.3±0.2 7.8±1 9±1 77±6 40±3

Saffloweroil 5.4±0.2 2.6±0.2 36±3 103±7 32±2

Coconutoil 5.3±0.4 1.4±0.1 31±2 32±3 86±5

0.12 None 5.8±0.3 13±1 7±1 50±4 63±4

Saffloweroil 5.6±0.3 10±1 20±1 54±4 57±4

Coconut oil 5.8±0.6 6±1 18±2 17±2 156±9

0.24 None 6.5±0.5 30±2 7±1 38±3 86±7

Safflower oil 6.6±0.4 23±2 12±1 41±3 71±5

Coconutoil 6.2±0.6 8±1 10±2 14±2 198±12

Groups of animalswerefed diets enriched with0.06, 0.12,or0.24% cholesterol either aloneor incombination with 20%safflower oilor20% hydrogenatedcoconutoil. Alldietswerefed for 30 d. Each value represents themean±1 SEM for data obtained insix animals.

additionofsaffloweroil tended to lower theplasma

LDL-cho-lesterol concentration while feeding the saturated hydroge-natedcoconutoil markedly increased these levels.

When the rates of hepatic LDL clearance shown in Table III were superimposed on theappropriate kinetic curves for

LDL transport in normal hamsters, the percentagereduction inreceptor-dependent LDLtransportcould be calculated and thesevalues, inturn, could be corrected for theslight

differ-ences in liver size found in the various groups. InFig. 3 are

shown themeanchanges in whole liverLDL receptoractivity

and plasmaLDL-cholesterol concentrationsas a function of

0.06 0.12 0.18 o.2

DIETARY CHOLESTEROL CONTENT (%)

Figure 3. Whole liverreceptor-dependentLDLclearance andplasma LDL-cholesterolconcentrationin animalsfed diets enriched with 0.06,0.12or0.24% cholesterol with and without 20% safflower oilor

hydrogenatedcoconutoil. Whole liverreceptor-dependentLDL

clearancewasobtainedbymultiplyingthe percentage reduction in receptor-dependent LDL clearance per g of tissuebythe wholeliver weights and is expressedas apercentage of the controlvalue.

thedietary cholesterol content in animals fed diets containing

either no added triglyceride, safflower oil, or hydrogenated

coconut oil. As is apparent, ateach level of dietary cholesterol thesuppressionof whole liverreceptor-dependentLDL trans-port was less in the presence ofsaffloweroil and markedly

greater inthe presence of hydrogenated coconut oil. The most

dramatic differences inwhole liverreceptor-dependent LDL

clearance, however,occurred inanimals fed the 0.06%

choles-terol dietwhere safflower oilnearlyabolishedthesuppressive

effect of dietary cholesterol while hydrogenated coconut oil markedly augmentedit.AsshowninB, plasma

LDL-choles-terol concentrations progressively rose with increasing

amountsofdietary cholesterol; however, this risewaslessin

the presence ofsafflower oil but significantly greater in the

presenceof hydrogenatedcoconutoil.

Since the mostdramatic modulation ofhepatic LDL re-ceptoractivity by dietary triglyceride occurred inanimals fed the 0.06% cholesterol diet and since this level ofdietary cho-lesterol is similartothat ofWesternman(- 150mgof

cho-lesterolper 1,000 kcal ofdiet), afourthgroupof studieswas

undertaken to examine thetime-course for the inhibition of hepatic LDL transport and its reversibility in animals fed 0.06% cholesterol forprolongedperiods of time in thepresence

and absence of added saffloweroil, oliveoil,orhydrogenated

coconutoil.Groupsof animalswerefed thesediets for either4 d orfor 1,2,or4mo. All animalswerekilledat - 5moof age. Total bodyweight gainwas identicalamong the animals fed

the threetriglyceride-containing diets,butweightgainin these animalswas somewhatgreater thanin control animalsorin animalsthatreceived cholesterol alone.Asshown inTableIV,

hepatic cholesterylesterlevels increased with timein all

ani-malsfed the 0.06% cholesteroldiet(column 2);however, the

increasewasgreater in animalsreceivingolive oiland much less inanimalsreceiving safflower oilorhydrogenatedcoconut

oilso thatafter4mothehepatic cholesterylester content was 12.7 mg/g inanimalsfed0.06% cholesterolalone,butequaled 18,2.8,and 1.9 mg/g whenoliveoil, saffloweroil,or

hydroge-nated coconut oil, respectively, was added. Rates ofhepatic LDL clearance fell in all animals fed the 0.06% cholesterol

(7)

Table IV. Time-coursefortheChangesinHepaticCholesterylEster Content and LDL Clearance, andforPlasma LDL-Cholesterol Concentrations in Animals Fed 0.06% Cholesterol PlusSafflowerOil, Olive OilorHydrogenated Coconut Oilfor Prolonged Time Periods

Cholesteroladdedto Triglycerideaddedto Hepaticcholesteryl Hepatic LDL Plasma LDL-cholesterol

controldiet control diet Timeondiets Liverweight ester content clearance concentration

% g mg/g Ai/h per g mg/dl

None None 5.0±0.3 0.7±0.1 116±8 25±2

0.06 None 4 d 4.9±0.3 2.1±0.1 100±7 29±2

1 mo 5.3±0.3 8.2±1 79±6 35±2

2mo 5.8±0.2 9.6±1 61±6 40±3

4mo 6.6±0.5 12.7±1 34±4 43±3

0.06 Safflower oil 4 d 4.8±0.3 1.7±0.2 112±8 28±2 1 mo 5.3±0.2 2.8±0.2 103±8 30±3 2mo 5.9±0.4 2.6±0.1 94±7 34±2 4mo 6.8±0.4 2.8±0.3 81±7 35±2

0.06 Olive oil 4 d 5.2±0.4 2.8±0.2 110±8 32±2

1 mo 5.3±0.4 9.9±1 102±6 38±3

2mo 6.3±0.3 13.2±1 87±6 38±3

4mo 7.4±0.5 18.0±2 74±7 39±3

0.06 Coconut oil 4 d 5.2±0.3 1.0±0.1 86±6 45±3

1 mo 5.2±0.3 1.6±0.2 35±2 76±5 2mo 6.0±0.2 1.6±0.2 30±2 87±6

4mo 7.2±0.4 1.9±0.2 22±2 86±6

Groupsof animalswerefeddiets enriched with 0.06% cholesterol aloneorincombination with 20% saffloweroil,20% oliveoil,or20% hy-drogenatedcoconutoil for4dorfor 1,2,or 4 mo.Each value represents themean±1 SEM for data obtained in six animals.

whensafflower oil orolive oil was added to thedietbut was

muchmorewhenhydrogenatedcoconutoilwasadministered (column 3).ThesechangesinhepaticLDLclearance,in turn, were reflected by appropriate, reciprocal changes in plasma

LDL-cholesterol levels (column 4).

Whenthehepatic LDL clearanceratesshowninTable IV were compared to theappropriate kineticcurves and corrected

fordifferences in liverweight, thepercentage changes in

he-paticLDL receptoractivitywereobtained.Asshownin Fig.4, in thoseanimals raised for4 mo on adietcontaining0.06%

cholesterol alone there was aprogressivedecline in receptor-dependentLDLtransportto 50%of the control value anda

corresponding reciprocal rise in the plasma LDL-cholesterol

concentration (B). However, the addition of either safflower oilorolive oiltothischolesterol-containingdiet almosttotally

abolished the suppressive effect of the dietary cholesterol on

receptor-dependent LDL transport in the liver and largely

blunted theriseincirculatingLDL-cholesterol.In contrast, the

addition of hydrogenated coconut oil to the diet markedly

enhancedsuppressionof receptor-dependent hepaticLDL

up-takeandincreasedtheplasma LDL-cholesterol concentrations

to levels normally seen in adult man. Finally, after 4 mo,

animals receiving each ofthe four dietary regimens were

re-turnedtocontrolrodent dietcontaining neither added choles-terol nortriglyceride.Asisalsoapparent in Fig. 4, at the end of 2 wk thedramaticchangesin receptor-dependentLDL trans-port and theplasma LDL-cholesterollevels had all reverted to the normal valuesfound in the control animals. Thus, these

diet-induced changeswererapidlyreversible.

Since thehydrogenated coconut oil contained significant amounts offatty acids ofshortand medium chain length, it was possible that the dramatic effect of this triglyceride on LDLmetabolismwas related more to the chain length of the

fatty acids than to the degree of saturation. Therefore, the effect ofpuretripalmitinonLDL metabolism was next stud-ied,asshown in Fig. 5. Hamsters were fed the control ground rodent chow containing 0.06% cholesterol plus either oliveoil

(20%),orolive oil (12%) plus tripalmitin (8%). As isapparent, when olive oil was partially replaced by tripalmitin, whole liver receptor-dependent LDL transportdeclined significantlyand

plasma LDL-cholesterol concentrations increased. Diets con-taining greater proportions of tripalmitin resulted in

steator-' -T iI-T

A Saffloweroil

,,z100 -t

->_60 \ j_''

Ioo I i

I >040 Noaddedtriglyceride ,' 20 Hydrogented coconut oil

Or |0'

B

-J

100-OZlO

80z

ir60

40-_j 20

1 2 3 4 5

TIME ON DIET(Months)

Figure4.Whole liver receptor-dependent LDL clearance and plasma LDL-cholesterol concentrations in animals fed diets enriched with 0.06% cholesterol with or without 20%saffloweroil, olive oil, or hy-drogenatedcoconutoil as afunction of time on the diet. The dashed lines represent the changes observed when animals werereturnedto

(8)

>>_3 80 A

w 60

_j Olive oil

(12%)plus 0

38;i 40- Tripalmitin(8%)

20-0'

°z

B

0O

60-gZ2W 20

-J

0 1 2 3 4

TIME ON DIET ( Months)

FigureS5.Whole liver receptor-dependent LDL clearance and plasma LDL-cholesterol concentrations in animals fed diets enriched with 0.06% cholesterol and either 20% olive oil or 12% olive oil plus 8% tripalmitin.

rheaanddiminishedweightgain andsoit was notpossibleto studyhigherconcentrations of dietarytripalmitin.

In a final study the mechanism by which saturated and

polyunsaturated triglyceridesmodulated LDL metabolism was

investigated by examining the effect ofthese triglycerides on

the hepatic uptake of asialofetuin, a compound that is also removed from the plasma by a receptor-dependent pathway

similar to that oftheLDL receptor pathway (29,30).As shown in TableV,the hepaticclearanceofasialofetuinwas

approxi-mately40-foldhigher thanthatofLDL. However, wholeliver asialofetuin clearance was totally unaffected by feeding diets

containingcholesterol andtriglycerides(column 3). Thus, the

receptor-dependent uptake of thiscompound continued at a constant rateunder circumstances wherethe feedingof satu-ratedandunsaturated dietarytriglycerides resulted in marked alterationsinreceptor-dependentLDL transport into the liver.

Discussion

In man (14), aswell as in the hamster (13), the steady-state concentration ofplasma LDL-cholesterol is dictated by the

balance between the rate of LDLentryinto the plasma

com-partment (the LDL production rate, Jt) and the rate ofLDL

removal by the varioustissues of the body through both

recep-tor-independent(Ji)and receptor-dependent (Jd) mechanisms.

Inallspeciesthat have been examined thus far, - 80% of the receptor-dependent LDL transport that is manifest in the

wholeanimalis found in the liver(19-21).Furthermore, this

hepatic receptor-dependenttransport, but not the receptor-in-dependentcomponent,issubject to regulation by dietary

ad-ditions suchascholesterol, triglyceride and bile acids ( 15, 31).

Thus, it becomes ofconsiderable importance to elucidatehow

the two major dietary lipids, i.e.,cholesterol andtriglyceride, interact to effect hepatic receptor-dependent LDL transport

and, ultimately, theconcentration of LDL-cholesterol inthe

plasma. Suchinformation is particularlyrelevant to the

situa-tion in Western manwherethe intake ofadiet highin lipids is

associated witha progressive increase inplasma

LDL-choles-terol levelsand ahigh incidence of atherosclerotic complica-tions(32, 33).

Using thehamster, four major conclusionswere reached in

these studies concerning theinterrelationships that exist be-tweenthemajor dietary lipids, thefunctionallevels of

recep-tor-dependent LDL transportin the liverandthecirculating concentrations of plasma LDL-cholesterol. First, feeding

cho-lesterol alone caused a marked rise in the concentration of cholesterylestersin theliver and suppression of boththe rate

of cholesterol synthesis and receptor-dependent LDLuptake. Second, saturatedtriglycerides augmented the suppressive ef-fect of the cholesterol on hepatic LDL receptoractivityand markedly elevated the circulating plasma LDL-cholesterol

levels. In contrast,unsaturatedtriglycerides largelyprevented

the deleterious effect ofdietarycholesterolontheseparameters

ofhepatic metabolism. Third, feeding triglycerides disrupted

the classical metabolic responses to cholesterol feedingthat exist in the liver with respect to receptoractivity, cholesteryl

ester levels, and ratesof cholesterol synthesis. Saturated

tri-glycerides, for example, markedly lowered cholesteryl ester

levels and increased rates ofsterol synthesis under

circum-stances wherereceptor-dependent LDL transport was mark-edly suppressed. Finally, the detrimental effects of feeding cholesterol and saturated lipids on receptor-dependent LDL transportin theliver could bereadilyreversedbyreturningthe

experimental animalto adiet low in these lipids.

The firstsetof data provided by these studieswas adetailed time-course for the effects of cholesterol feedingaloneon

he-paticLDL transportandcirculatinglevelsof LDL-cholesterol.

Immediatelyafterbeginningcholesterolfeeding,therewas an apparent increase in therate of LDL-cholesterol production

Table V.EffectofDietaryCholesterolandTriglycerideonHepaticAsialofetuin Transport

Cholesterol addedto Triglycerideaddedto Hepaticasialofetuin Hepaticasialofetuin control diet control diet Timeondiets clearance Liverweight clearance % mo ml/hperg g ml/hper whole liver

0.06 None 1 4.1±0.2 4.9±0.2 20.1±2

3 3.0±0.2 6.5±0.4 19.5±1

0.06 Saffloweroil 1 4.3±0.2 5.4±0.2 23.0±2

3 3.0±0.3 7.4±0.3 22.2±2

0.06 Coconutoil 1 4.0±0.3 5.3±0.3 21.3±2

3 3.3±0.3 6.7±0.4 22.1±2

Groups of animalswerefed diets enrichedwith 0.06% cholesterol aloneorin combination with 20%safflower oil and 20%hydrogenated

(9)

such thatat4 d theplasma LDL-cholesterol concentration had nearlydoubledyetthere had beennochangeinhepaticLDL

transport (D, Fig. 1). Asaconsequenceof this alteration,the amount of LDL-cholesterol taken up by the liver 4 d after

commencingcholesterolfeedingalsonearlydoubled. The liver

cellapparentlyrespondedtothis increased influx of sterol by reducing receptor-dependent LDLuptake until, at30d, the absoluteuptake of LDL-cholesterol had beenreturnedtothat

level seenin the animals fed the control diet.Thus,30 d after the initiation of cholesterol feeding, the rate of

receptor-de-pendentLDLuptakebythe liver had beenadjustedsothat the

amount of LDL-cholesterol taken up remained constant at

- 25 ,ug/hper gregardlessof the load ofdietarycholesterol (B, Fig. 1).Atthesametime,therewasalsoamarkedincrease in the level ofcholesterylestersin the liver cell andsuppressionof therateofcholesterolsynthesis (Table I). Thus,in this situa-tion the liver of the hamsterrespondedin the classicalmanner toincreased cholesterol influx withsuppressionof cholesterol synthesis and receptor-dependent LDL transport and an in-creasedlevel ofcholesterylesterformation.Itshould be noted thattheseresponses weredesignedtomaintain sterol

homeo-stasis onlyin the liver cell. Unfortunately, theseadjustments

by the liverweredetrimentaltotheanimalas awhole inthat the plasma LDL-cholesterol concentration necessarily in-creased.

The precise mechanism by which dietary sterol regulates theLDL receptorpathway isnotknown,although presumably long-termregulation is mediated by cholesterol,or someother

product ofmevalonate metabolism, atthe transcriptional

and/or translational level. Itis alsopossiblethatexcessdietary

cholesterol couldaffect the LDL-receptor pathway by disturb-ingthecholesterol/phospholipid ratio and,perhaps,the fluid-ity ofhepatic membranes. However, in these studies dietary cholesterol had littleeffectonthehepatictransportof asialo-fetuin, a glycoprotein thatis taken up and degraded by the liver throughareceptor-dependent pathwaysimilartothatof LDL uptake(29, 30). Thus, these results are consistent with

theview that enhanced uptakeof dietarycholesterol leadsto expansion ofsome putative intracellular, regulatory pool of sterolwhich,inturn,suppressesthesynthesis (or increasesthe degradation)of therate-limitingenzymes for cholesterol

bio-synthesisandtheLDL receptorand expands the poolof cho-lesteryl esters (34, 35). Unless some otheradaptive response occursin otherorgansinthebody,thesechanges intheliver

will necessarily be associated withanincrease inthecirculating levelsofLDL-cholesterol.

Asdemonstrated in thesecond groupofstudies, the

addi-tion oftriglyceridestothediet markedlyaltered the metabolic responseof the livertocholesteroltaken insimultaneously. At

eachlevel ofdietarycholesterol, polyunsaturated triglycerides

diminished,and saturatedtriglycerides markedly augmented,

theeffect of dietarycholesterolonhepatic receptor-dependent

LDL transport. For example, whole-liverreceptor-dependent

LDLtransportwassuppressed 20% inanimals fedthe diet

enriched with 0.06% cholesterol: however, this amount of

cholesterol suppressed receptor-dependent LDL uptake by

only5%whengivenin the presence of dietary unsaturated oils

but bynearly 70% when saturated triglycerides were adminis-tered to the animals. Similardifferential effects were seen at other levelsof cholesterol

feeding

although

thepercentage dif-ferenceswereless

(Fig.

3).

The dataobtainedin theanimalsfed 0.06% cholesterolare

particularly

relevanttothe human situa-tionsince thisloadof cholesterol

corresponds

to theintake of

- 150mgofcholesterolper1,000 kcal ofdietary intake. Thus, given this load of dietary cholesterol, the long-term effect of unsaturated lipids likesafflower and olive oil is to block, in somemanner, the deleterious effect ofdietary cholesterol in

suppressing receptor-dependent LDLtransportin the liverso thattheplasma LDL-cholesterol concentration remainsat the low,essentiallycontrol value(Fig. 4). Incontrast,the effectof

saturatedtriglycerides is to greatly augmentthe suppressive

effect ofdietary sterol on receptor-dependent LDL transport leading, in turn,to asignificant elevation in plasma LDL-cho-lesterol concentration. It is noteworthy that feeding this

com-bination of small quantities of cholesterol (0.06%) and larger

amounts of saturated triglycerides caused the plasma

LDL-cholesterollevelin the hamstertoincrease into therangeof80 to 100 mg/dl, which is commonly encountered in Western

manfed asimilar diet (Fig. 4).

Itis alsoimportanttorecognize that these profoundeffects ofsaturated and unsaturated triglycerideswereexerted

inde-pendently when combinations of oilswerefedaswouldoccur,

forexample, inamixed human diet. Thus, partial substitution of saturated triglycerides into a diet ofunsaturated lipids clearly caused a predictable decrease in hepatic

receptor-de-pendent LDL transport anda correspondingincrease in the plasmaLDL-cholesterol concentration (Fig. 5). In all of these studies commercial oilswereused thatcontainedamixture of different medium and long chain-length fatty acids. It will be

veryimportant in future researchtodeterminehow the inde-pendentvariablesoffattyacidchain-length,numbersof dou-ble bonds andposition of the double bonds specificallyalter thecharacteristics ofagiventriglyceridetoeitheralleviateor augment the effect ofdietary cholesterol on hepatic LDL transport.

It is difficultto explain how dietary triglycerides so

pro-foundly alter the hepatic response to dietary cholesterol al-though the third set ofexperimental dataprovides some

in-sights intotheunderlying mechanisms. Previously published

data have shown that after dietary cholesterol is deliveredto

the liver in thechylomicronremnant, asignificantamountof the sterol isimmediatelyesterified and stored in the liver cell

asrelatively inert, cholesterylesters(36).Atthesametime,the regulatorypool(s)ofsterol in thecellmustbeexpandedsince there is alsoinvariably suppressionof denovocholesterol

syn-thesis and, if theseevents areinadequatetomeetthechange in cholesterol homeostasis, suppression ofLDL receptor activity(18).

Clearly,these classical responses to the uptakeof dietary cholesterolare markedly altered by the simultaneous feeding ofdietary triglycerides. For example, at each levelofdietary cholesterol, hepatic cholesterylesterlevelswere much lower in

animalsfed hydrogenatedcoconutoil than in animals fed

saf-flower oil (Table III). Since thesaturatedfatty acidsare poor

substratesfor theesterificationreaction(37),it isconceivable that inthissituationlessintercellularcholesterol was seques-tered in theinertesterpool,morefreesterolenteredthe puta-tive regulatorypool and there was adisproportionate

suppres-sion ofLDL receptorsynthesis. Alternatively, whensafflower

oilwasfedagreaterproportion ofthe dietary cholesterol load wassequesteredintotheester pool, less free cholesterol entered the putative regulatory pool and there was derepression of

receptor-dependentLDLtransport.

This

relatively

simple explanation,

however, doesnot

ac-countfortheobservationthatsafflower oiland

hydrogenated

(10)

rela-tivetodietary cholesterolalone but had the opposite effect on

hepatic LDL receptor activity (Fig. 3). Thus, while there is

littledoubt that the major effect of dietary triglycerides is to

modifythe metabolic response of the liver cell to dietary cho-lesterol, it is not entirely clear how this effect is mediated. Whether such modification is the result of the changes in the rateof cholesterolentry into the cholesteryl ester pool,

alter-ationsin the partitioning of freecholesterol between various

membranecompartments or cytoplasmic carrier proteins or, even,changes in the fluidity of critical membranes within the

cell remainstobeelucidated.

Thereare twoimportant implications ofthese experiments

withrespect to humannutrition.First, these data suggest that

thedetrimental effect ofsaturated triglycerideson

LDL-cho-lesterolmetabolism could be minimized iftheintakeof

cho-lesterol could be reducedtoessentiallyzero. While it is nearly impossibletoaccomplish this through dietarymeans, there are anumberofnewpharmacologicalagentsthat holdthe

prom-ise of totally blocking sterol absorption and, so, effectively reducing the input of dietary cholesterolto near zero. Second,

given the likelihoodthatWestern diets willalwayscontain at

least several hundred milligrams of cholesterol each day, the

alternative way inwhich to enhancehepatic receptor-depen-dent LDL transport is to be certain that the diet contains

optimal amounts of those triglycerides that effectively block the deleterious effects of the dietary cholesterol. The

experi-mentalmeans are nowavailabletoworkoutthemechanism of action of thesedietaryfattyacids inprotectingLDL transport

and to determine theparticular chemical configuration that maximizes this effect. Hopefully, when identified, such oils could beproduced commercially and usedin theproduction

ofavarietyof foodproducts.

Acknowledgments

The authors wishtothank PeggyLoessbergand Mark Venturafor their excellenttechnical assistanceand Imogene Robison forpreparingthe manuscript.

This work was supported by U. S. Public Health Service Research grants HL-09610, AM-19329,and AM-01221 andbygrantsfromthe Moss Heart Fund and the Texas Affiliate of the AmericanHeart Asso-ciation(G155).

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(11)

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References

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