THE EffECT OF POLyu nSATURAT EDfATT YACIDS or:CHOLEST EROLMETABOLISM
By SEBELY PAL
A The s is Submit ted to the School ofGr a d ua t e st ud ies inPartia l Fu lf ilmentof the Requ ire ments
for the Deg r e e of Master of Science
DepartDentof Bioc he mistry Memori al University of Newfou ndland
St. John's, Newf oundland
Ma rc h, 1991
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS• ABBREVIATIONS • LIST OF TABLES • LIST OF FIGURES ABSTRACT• • • •
1. INTRODUCTION
1.1 Lipoprote insandcoronary arterydi s e a s e 1.2 Theories of atherogenesis . 1.3 Low densitylipoproteinsand coronary
artery disease . , . •. . . . 1.4 Di e t a r y fat and atherosclerosis 1. 5 Objectives • • • • .
2. MAT ERI ALS AND METHODS 2.1 Mate rials.. • . • .
2.1.1 Lipids . . . . 2.1.2 Ra d i oisoto pe s . 2. 1.3 Ra d i oc hemi c a1 s
Page
vi vii viii
6 11 15
17 17 17 17 17 2.2 Methods
2.2.1 Fibroblasts . . . •. . . . . 2.2.1. 1 pr e para t i o n of fatty
acid enrichmentmedium forhuma n fibrob l as ts 2.2.1. 2 Fatty acid enrichment in human fibroblasts . 2.2.1.3 Assessmen t of fatty
ac idenr ichment. . •. 2.2.1.4 De t e r mi nati on of
cellular esterif i c a t i o n inwhol e cell lysates ofhuma nfibrobla sts Macrophag es . . . •• . 2.2.2 .1 Preparationof fa tty
acidenric hment mediumfo r macro- phages(J774) . • . •
i
"
"
i 19
20 22
22
I i Page 2.2.2.2 Fattyacid enrichment
ofJ774macr:ophages • 23 2.2.3 Human fibroblastmicrosomes . . . .
2.2.3.1 preparatio nof microsomes fr om human fibroblasts enrichedwithcfccaa- pentaenoicacid and linoleic acid •. . . . .
23
23 2.2 .J.2 De terminationof fatty
acid enrichment in microsomes , is o l a t ed from human fibroblastsgrown inthe presenceof eicosapentaenoic acid
andlinol eic acid . . . . 24 2.2 .3.3 Determination of microsomal
ACATactivity. . . 25
2.2.4
2.2.5
Choleste ro lEfflux • • • . • • • 2.2.4.1 Radiolabellingof
endoge n ous cel lular cholestero l . . . . 2.2.4.2 Cholesterolefflux from
human fibroblasts enriched with various fattyacids. Lipoproteins • • • • . . . • . . • 2.2.5.1 Lipoprotei npreparation . 2.2.5.2 preparationof acetylated LOL • • • • • • • • • • •
26
26
26 27 27 28 2.2.6 Ana lysis
2.2.6.1 Oete~min~t io~ ~f ' c~liuia~
cholesterolcontent . . . Analysisof fatty acyl CoA pool in humanfibroblasts enrichedwithva ri o us fatt y acids
28 28
29
3. RESULTS• • • • • •
3.1 Fatty acid enrichement of fibroblasts.
32 32 3.1.1 Effect onchole s t e r ol level s 34
iii Page 3.2 Measurementofchol e s t erol esterificat ion
inlys a t e s ofhuman fibroblasts . . . • 38 3.2.1
3.2.2
3.2.3
Stimulation of cholestero l esterif icatio n by native LDL . Linear ityof cholesterol esterificat i o nwit hinc reasi ng proteinconcentra tionsand ti me in unen r i ched human fibroblasts Rateof cho lesterol este rif icat ion in humi'.:1 fibroblasts enriched wi th polyunsatura tedfattyacids . . . •
38
41
47 3.3 The effect of pol yunsa tu ratedfat tyaci d
enr ichment of human fibroblastson fattyacy l CoA pool size . . • . . . • . • • . . . . 50 3.4 ACAT activityinhuman fibroblas t microsomes. 53
3.4.1 J.4.2
Assayconditio ns • • . •. . • . • ACATact i v i t y in human fibroblast microsomesenrichedwith diffe re nt polyunsaturated fattyacids . • . .
53
58 3.5 Cholesterol esterifica tionin macrophages . . 63
J.5.1
J.5.2
3.5.3
Polyunsa tura tedfatt yacidenrichment of J774 macrophages . . . . . StimUla t ionof choleste rol eate r Lr fc atLoninmacro phages . • • The effe ct of pol yunsat u rated fatty acid enr i c hme nt of ma crop ha ge s on cholesterolmetabo l ism . •. . . .
63
66
69 3.6 The effect of polyuns a tu rated fa t ty acids
enrich me nt in huma n fibroblastson cho lestero l efflux . . . •. • . . . 72
iv Page 4. DISCUSSION. . . . . . . . . . . . . . . . . 81 4.1 The effect of modificationof cellularfatty
acids oncholestero l regulation . .. 81 4.:\.1
4.1.2 4.1.3 4.1.4
Fibroblasts. . . .. . . Human fibroblast rnicrosomes . . Macrophages .. . . . . .. . . Molecu larmechanisms . . . . . 4.1.4.1 Membrane fluidity . . 4.1.4 .2 Substratespecificity. . 4.1.4 . 3 Other possiblemechanisms
87 87 90 92 92 95 96
4.2 Cholesterolefflux 100
4.2.1 The effectof fattyacidenrichment onrev e r se cholesteroltransport
Molecular mechanisms 10 0
10 2 4.3 Conc lusion
REFERENCES • . .
108 112
A~KNOWLEDGEMENTS
It is my pleasure to expre s s my grat i t ud e to my supervisor, Dr. P.J. Davis, for hispatience , encoura gement , willingness to di s c u s s pr ob l e ms and hi s advice duri ng the entireresearch project.
r am very grat,e f u l to Dr. G.R. Herzberg and Dr. W.S.
Davidson fo r theirsuggestions and advice. I would liketo extendmythanksto Donna Osborne fortyp i ng my thesis .
Mysincere appreciationtothe Schoolof Gradua testudies for their financial support and the bursary received from Dr. Davis' MRC grant is grateful lyack nowledged.
1.BBREVIATIONS
ACAT Acyl CoA:cholesterol acyl transferase
AGLDL Acetylated LDL
8511. Bovine serum albumin
CE Cholestaryl esters
CEH cytoplasmic cholesteryl ester hydrolase CHD Coronary heart disease
OHA Docosahexanenoic acid
OPA Docosapentaenoicacid
EPA Eicosapentaenoicacid
HOL High density lipoprotein
HMGR J-Hydroxy-3-methyIglutaryl co-nayme A
hr hour
LA Linoleic acid
LDL Low density lipoprotein LPDS Lipoprotein dl:!icient serum
mg milligram
PC Phosphotidylcholine
PE Phosphatidylethanolamine
PL Phospholipid
PUFA Polyunsaturated fatty acid
8M spingomyel in
tl/2 halftimes
VLDL Very high dens! ty lipoprotein
vi
Table 1
Tab le2
Ta bleJ
LISTOFTABLES
Fa t t yaci d cont e n t of phospholip id fractio n from human fi b r ob l a s t s inc u bate dwit,hlino l eic ac i dor eico sapenta e n o i c acidsupplemented medium . . . • . . • . . . • • Fa t tyacid contentof ph ospholipid frac tio n fr om human fibrobla sts mi c r o s omes isol a t ed from fibr ob l ast s incub ate d wi t h lino l e i c acidor eicosapent a enoi c ac id supp l e me nt ed me di um• • • . • . . • . • • . . . . Fatty acidcontent in pho sphotidyl- eth a nolami n e (PE) andphosphotidyl- chol ine (PC) fr ac tio nofmi c r o soma l pho s pholipids • . . . .
vii Page
32a
3Ja
3Jb
Figure 1
Fi g u r e 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
viii LIST OF FIGURES
Page Choiester y l esterand unesterified cho.i.esterol content of human skin fibroblastsenrichedwith linoleic acid or doccsapen taeno icacid 36 Choleste rolcont entof cells
incubated wit h (+LOL)andwithout
withoutLOL (-L OL) 39
Linearity of rHC]- ol e oy l CoA incorpor ationinto cho lesteryl este rswithtime, in whole cell
lysates 43
Li ne a rity of incorporationof {'4C) -ole oyl CoA int.ocholesteryl esterswith increasingprotein
concentrationinwhole cel ls 45 Cholesterylesterification1n
fibroblastsenriched with linoleic acid ordoc os a pe nt a e noi c acidas measured byinc o r po r ati o n of radiolabelledoleoyl CoA into cholestery l esterin wholQcell
lysates 48
Acyl CoA pool size in human fibroblas t incubatedwitheither linoleic acid supplemented medium or edccsepe nteencdcacid suppleme nted
medium 51
ACAT activity inhuman fibr o bl a s t microsomeswithincreasing time 54 ACAT activityin microsornes with increasing protei nconcentration 56 Acyl CoA:cholestero lacyl tr a ns f e r a s e (ACAT) activity in microsomes is ol a t e d from human fibrolba lstsenriched with linoleic:acid ordoc os a pen t a e noi c
acid 59
Fig ure 10
Figure 11
Figu re12
Figure13
Figu re14 a
Figure14 b
Fi gur e15
Figure16
Cholesterol conte nt (unes terified ) inhumanfibroblastsmicrosomcs enrichedwith doc o s ap e ntae noic
acid orlinoleicacid 6~
Relative pe r c e nt of ld-3 PUFAs and
"'-6PUFAsin macr o phagesenrich e d withdoc os ap e n t aen o icacid or
l inolei cacid 64
Cholesterol conte nt of macrophages inc ubate d wit heit he rLOLor
acetYlat·~dLOL 67
Thecholestero l con tent in macrophages enri c h ed with eit he rlinol e i cacid
ordocosapentaeno icacid 70
Efflux ofradi olabelle d cho leste rol from huma n fibr obl asts inc ubated with li nol ei c acid or docosapentaen oic acid inthE:! presence ofHOL• 73 EfflUX of radiolabelledcho les terol from hu man fibroblasts in c uba t e d wit h li nol e i c ac idor docosapentaenoic
acid inthepresenceof HDL• 75
Ha lf times (T 1/2 ) for cholesterol effl ux fromhu ma nfibr obl a s t s en r i c he dwi th linoleicacid or
doc os ape nt a en oic acid 77
Ho t-med i a t e dnetrelease of cho l esterol into the cult u r e medium by fi brobl asts enriche dli nol eic acidor docosa pen tae no icacidafter 2.4hour.s incuba tio n wit h250pg/ml
HOL . 79
ABSTRACT
At he r o s c l e r o si s is characte rized by dep osi t i on of cholestero l and esterified choleste ro l in cells of the vasCUl a r in tima. These lip i d laden cells are called foam cells due tothe irmicroscopic appearanceand ariseprimar ily from smooth muscle cel ls and monocyte macrophages . It is believed th a t the transformation of thesecells int o foa m cell o is a key step in the ini tiation of at h eros c le r os i s. Therefore, tobette r und ers t a nd thepr oc ess of atherogenesis it is important to investiga te the mechanisms that ma y regul a techolest erolesterificatio nand hen ce theformationof foam cells .
Polyu n s atu r ated fatt y acids (PUFAs ) in the die t can subst an tiall yreduce the incidence of coro na r y he art disease. Howeve r , die t ar y w-~ poly uns a t urated fa t t y acids fo und vri maril y in mari ne oilsha v e be en shown to be better in reduc ing the ris k of athe r os cle r osis than othe r polyunsatu ratedfa ttyacids . Un f ort unately ,it is unclear ho w w-3 PUFAs are inv o lved indel ay ingth e at herogen icprocess.
It is pos s i bl eth a t thesefat tyacidsma ybe antia the r ogenic by in h ib i t ing chol es terol este ri fica t ion or increasing choles terol remova l fr om ce lls the re byinh i b it ing theinit ia l ste poffoam cell forma tion .
Themajor object iveof thisstudywas to inves tiga t e the impact of di ffere n t polyuns a tura tedfat ty ac i d enrichmentof nonhepatic tissues on cho lesterol eater-LrIcet Ion and
xi cholesterol efflux. Human fibroblasts and macrophages were errrLched with either linoleic acid (18:3, Col-G) e LcoaaperrtaenoLc acid (20:5, (,1-3)bysupplementingthe cUlture medium. The incorporation of radiolabelledoIeayl CoA into choleste rol esters was reducedby44%when human fibroblasts were enr-Lched withlJ-) polyunsaturated fatty acidscompared with cells enriched with foI-6 polyunsaturated fa t t y acids.
Also, ma c r op h a g e s enriched with C<l-3PUFAs had significantly lower (52%)total cholesterolcontent than those enriched with w-6 PUFAs. Cholesterol efflux was measured in cells enriched
wi t h Ll-3 PUFAs andw-6 PUFAs. In the presenceof HOL, the
rate of efflux of radiolabelled cholesterol from cells enriched withW-3 PUFAs was substantially faster (2x) than efflux from cells enrichedwith W-6 PUFA enriched cells. The data suggests that w-3 PUFAs may be antiatherogenic by inhibiting cholesterol esterificat ion and by accelerating cholesterol efflux from nonhepaticcells.
1. INTRODUCTION
Cholesterol was first identified almost two centuries ago and was characterized as "the waxy, white component of gallstones" (Stallones, 1980). In 181 6, Chevreul, a French scientist, gave cholesterol its original name "c ho l e s t e ri n e" , from the Greek words chole (bile ) and seeres (solid) (Bloch, 1987 ). Later,this compoundwas found to have a reactive hydroxylgroup and the name was changedto
"cholesterol " (Bloch, 1987). In the early1900s, extensive efforts focused on the structure of the sterol molecule. At the time these structural studies werein progress,remarkable findings aboutthe role ofcholesterol in atherosclerosis were also being made. In 1906, Aschoff first drew attention to the highcontent of cholesterol in atheroscleroticaortas and in 1910, Windaus showed that the cholesterol deposited in atherosclerotic arte rieswas chieflypresent as cholesteryl esters (Goodman, 1989).
Cholesterolhas long beenrecognized as an essentia l component in many biological systems. It appears to play key structural and, possibly functional, roles in cellular membranes (Gibbons et al. ,1982). Cholesterolis alsoa major precursor for many steroid hormones and bileacids. Bec:aU3C of t.he obvious biologicalimportance of cholesterol ,intensive efforts were made to elucida te th e synthetic pathlo'al' of cholesterol in mammals, CUlminat ing with the Nobe l prize
awardedto Konrad Blochfordefiningth ebio synt he ti c pathway fromacetate to choleste ro l (Bloch, 19 87 ) .
Itistherol eof chole sterol in th e eeveacp ae ne of coronary arterydi seas e,however tha thasspur re d the greates t inte r e s t incholesterolmet a bo li sm. Coronary arte ry disea s e hasbeencal led the"bl ackplague"ofthe20t hcentury (Havel, 1988 ). Atheros cleros i s is the le ad i ng cause of premature naturaldeat hinNorthAmericatod a y and itis widelyaccepted thatanelevated plasmacholeste ro llevelis one of the major risk fac tors foratherosclerosis (Small,198 8).
1.1 Lipoproteins and CoronaryArteryDisease Cho l estero l, like most lipids , is transported in body fluids by eee eceere e u r a r compl e xes cdll e dli p opr ote ins (St ryer, 1988 ). Cons i d e r a bl e interest in li popr o tein meta bo lisllIhasbeenge nerated inthe pastfew eacadesbecause of the appe ren c caus alrelationship s betweenthe levels of certainserumlipoproteins andthe inc idenc eof cardiovascular disease (Vance, 1990). Fo r examp l e,low dens i t y l ipoprote in s (LOL) carry a major portionof chole sterol in the form of cholesteryl ester in human pl asma and there is a st rong positive rela tionshipbetweenthe level 0':LDLin pl a s ma and the ris k of coronary artery disease (xcxeaa xe , 19 87 ; Nor doy an d Goodn i ght, 199 0r Va nce , 1990). LDL is the major lipoprotein resp onsible for the deliv ery of cholesterol to peripheraltissues including tho se of the vasculature. The
factthatcholestero linthe arte rialwallis derive dfr omLDL has led to th is lipoprot ein being re f er r ed to llS -bad ch o l e sterol " or -bad lipoprotei n "(Brown andGolds tein, 1986:
Steinbe rg, 19 8 3 ) .
The high density lipopro teins (HDL) arc also cholesterol ri c hparti cle s . Evi denceBuqqests thatHOLplays an importa n t role in re ve rse cholestero l transpor t (the mo vementof cho leste rol fromncrr-hepatiLe tissuesto the liver fo r cataboli s m and excre t i on ) (Glo ms et, 19 6 8) . Theret or e , high le v e ls of HDL appea r to lower the relat i v e riskof co r ona ry di s e a s e and ar e thouqht to be ant i a the rogenlc (Ca s telli et aI., 1977: Fr ick et aI., 1.969 : Nor do y and Goodnight , 19 90 , Riechl and Miller, 19 89) .
It appearsthatthe depositionof choles te rol wit h i n theart!!ri a l wallplays a centra l roleinthepathoge nesisof ath e ro s cle r o t i c plaque (Goodman,1989;Steinberg, 1983; Vance, 1990). The ability ofLDLto deliver cholesterol to arteries andof HDLtoremove cholest ero l from arte ries co u ldaccount for the cl o s e rel at i ons hi pbetwe e nthe s ep Le sae lipoprotei n le v els and coronary disease (Hord o y and Goodnight, 1990 : Riechl and Miller , 19 81) .
1. 2 Theori e s of Atheroge ne sis
A stri kin gfeat ureof ather o s cl ero s isisthemas sive accumul ationof esterif ied chol e s t erol that for ms "the heart of " the atheromatouspl ague. The name, "a t her oma" itsel f
stems fro mthe Greekwor d me ani nggrue l, whi chwas first used by pat holog istsin the 18thcen t ury todes c r i b e a thi c ke ned area in the wall of a major arteryWhich exu ded a yellow gr umou s lipidupon sect ioning. Thislipidisno wknownto be nearly pure Cholcsteryl es t e r (Gol d s t e i nand Brown, 1977).
The literature pertaining to the developmen t of atherosclerot ic le sions , exemplifies the emerg ence of two disti nc t schoo ls of thought abou t the biochemica l and physi ol ogi cal basis unde r ly i ng the earl y stages in athe r ogenes is . These two hypoth ese s ha v e gene ra l l y be e n termed the "e ndothe lial inju ry hypothesis " and the "lipid in filtratio n hypot h esis " (stei nberg , 1983) .
The endothelia l in j ur y hypothe s is sugges ts that damage to the mono la yerof endothelial cel ls lining theartery mayini t i ate plague formation (Ros s andGlomset, 197 6). Loss '.:If the struc t ural in tegrityof theendothel i um may cau se the re l e as e of pl atelet derived gr o wthfactors (PDGF) which cou l d stimula tesmo o t h muscle cell pT.,lifera ti o n and secretionof ot her gr owt h factors. Thus,repea tedepiso desof endothe lial damage and smoot h muscle cei.j, prolifera t io n can le ad to dev el o p ment oflesion s (Ros sand Gl omset , 1976; Ross, 1981). According to the classi ca l l ip id inf ilt ra t ion theor y, li poprot eins, espec ia llyLOL,inf! l tratethear te ria l inti ma pr i or to endoth e l ial da mage (Small, 1977 ; Ste i nb e rg, 19 8 3). Endotheli a l cel ls, smoot hmuscl e cells andmo nocyte ma c r oph a ges in th e intim a appea r to accumUlate l ipid s,
part i c u l a r l y cholesterol, from th e s e LOL particles and possib ly from chylomicron remnants, very low density li popro t e ins (VLDL) and ,B-VLDL (steinberg, 1983). Thus, th e lipid infiltration hypothes is implies that circu lati ng chol e s t e r ol (principallyin th e for m ofLOL)is centra l to the atherogenicprocess ,andat lowerpl2l.sma levels, atherogenesis do es no t proceed (Steinberg , 19 8 3 ).
It is now unders tood that ath erosc lerosis is a
"multifa c t or i al diseas e " involvi ng a complex ar ray or circul ati ng bloodprotei ns,li p op r o t e i ns and cel ls and their interactions with the cells and matrix proteins of the arteria l wall (Witztum, 1990 ). The resul t is the"u n if i e d"
hy pothe s i spr opo s e d by Ste inberg,whichstates tha t ava r i ety of factors, including endothelal in ju ry and lipid infil tr a t i o n , combi neto produce arterial lesions(Steinbe rg, 1983). Infact, i tis now recognizedthat endot heli a l injury maynot bethe in itial even t, but rathe r a subsequent even t th a t makes an important contributio n tothe developme ntan d progr ession of fat ty st reaks within the arterial wall (stein b erg, 1983; Witztum ,1990). Ev i d en c e suggests tha t in re s po n s e to el evatedplasma LOL, theearliesteven tapp ears to be the focal eccuuarae Lc n of LDL wit hin the int i ma of an appa re n tl y no r mal artery (Sch wenke and Carew, 1989). This accumu latio nofLOL appears to bethefirs t ide nt i f i abl e even t inthe lesion pr one sitesofthe aorta. Mon ocyte macro ph ages the n adhere to th e lumi nal sur face of the over l yi n g
endotheliumatthe sites of LDL eccumufetIc n, Su bsequent to endothel ium binding, monocyte ma croph a ge s migrate intto-the subintima l space under an int a ct endot helium. Here they take onthe classic appearance of foamcells. Th i s ter m arises from the foam- like appearance of these cel ls in elect ron microg raphsresulting fromenrichment ofthe macrophageswith cholesterylesters (steinbe rg,1983). As more mono cytesente r the int i ma,more foam cel lsare generated. Incre a s e s in the sizeand numberof these macrophageseventual lyresultsinthe rupture of the endothe l ium. At thi s stage, macrophages are exposed tothe circula tingbloo d elements,allowing platelet adhe r enc eandagg r e g a t i onto occur. Release of factorssuch a,sPDGF serve bot h to recruit smooth musclecel ls int o the int i mallay e r and to stimula te thei r proli fera tion (s t einbe r g, 1983; Witztum,1990). The accumu latedcho leste rol , cells and de br i s cons ti tutesanatheroscleroticplaquewh ich intime can na rr owthe lumen of thear teryandle a dto occl usion (Brown and Gol dstein, 198 6 ). ~~h at ev e rthe relative contributio nsof lipid in f il tra t i onand endothelia l damage tothede ve l o p ment of arteria llesions, the accumu l ationof choleste r y l es t e rin the arterial intima is a crucial eventin athe roge nes is.
1. 3 Low DensityLipoprotei n sandCoronary Artery Disease In a severelydi s e a s e d arte ry, it isnot unusu alto much as a lO-fo ld increa s e in the content of unesterifiedcholesterol and mor e than a 50-foldinc r ea s e in
thecholesterylester content (St. Clair , 1976). The close relationship between elevated cholesteryl ester in the arteries and athe r os cl eros is , has generated considerablo int erestin themec ha ni s m ofdeliveryofLOL cholesterol and theaccumul ati onof cholesteryl este rsinnon-h epatictissues.
The mechanismof LOLuptake fr om the circulation by the LOL receptor pathway was esta b li s hed by Brownand Goldstein. They were awarded the Nobelprizein 1985 for theirinvesti ga t ions of receptor-mediate dendocytosi sof LOL(Br own and Goldstein , 1986). The discoveryof theLOLreceptorhas had a profound impact on cell bi ol og y , on clinic al inve stigations and ther apeutic s ,andon the stu d y of atheros clerosi s ingeneral (arcvnandGol dstein,1986;steinberg, 1988) .
LOL is a large spheri cal pa r t i cle whose fluid core contains neutral lipid, mostly cho l e s te r yl ester (Brown and Gol dstein, 1986). This co r e of cholesteryl ester is surrounded bya monolaye r of Phospholipid and unester ified cholesterol molec ule s. Embedded in the phospholipid is a large proteinmolecule, apop r o tei n B- 100. The st udi es of Brownand Gold steinsuggestedthatLOLwas taken up by cells after apoprotein8-100wasrecog niz e d and boundby aspec i fic receptor on the cell au rrece (Bro wn and Goldstein, 1986). These recepto rsareclust e re d inspec ia l i ze dre g i o ns of the cell membra ne known as clathrincoatedpits. Afterbinding of LDL, the pits form invagination~ and pinch off to form membrane bo un d structures in the cytoplasm, called coated
ves ic les or endo s omes. Ac id i f i cati onof thesevesicles is believed to pcomote separat ionof ligan d and the rece pt or.
The re ceptor re c ycle s bac k to the cell su r fa ce and th e endo s ome con t ain i ng the LOLfus es with ly s os ome s (Br own and Gol ds tei n , 1986) . Lysosoma l acid lipaseshydrol yze the LDL compone nts, liberating unest e ri ! i ed cholestero l whic h enters thece llular choleste r o lpool(5). Ac c umul at i onofcholest ero l inhibits a ke yenzyme, 3-hYd r ox y-3 -me th y lglu t a ryl coenzyme A red u cta s e (HHGR) , in the pat hway of endogenou s cho lestero l biosy nthesis to prevent the synthesis of add i tional cholestero l. The in flux of chol es t er ol also reduces the numb er of LDLre c e pt ors by suppressi ng t ra ns cription ofthe rece ptor gene (Brown and Goldstein, 1986). Final ly, LDL derivedch o leste ro lpromotes the sto rageof chol e s t e rolin the cel l by activating ac y l CoA :cho lesterol acyl tra nsferase (ACAT ). This enzyme in the me mbrane of the endo p lasmic retic u lum , este ri fies afatt y acidto the ,8-0Hofcholeste ro l and the re SUlting chol es t e ry l este r s are deposi t ed as cytop lasm icdroplets(Brownan d Gol ds tei n , 1986) . Th is hi ghly raqu Lac ed LDL-receptor pathway described by Br ow n an d Goldstein does not, however, fu lly account fo r the accu mulat ionand deposi t io n oflarge quant i t ies of cholester o l incell s that is associated with at heromaand ther e fo r ethe releva nceofthispa t hwa y toa t.he r-c q enesLehas been questio ned (Brown and Gol d ste in , 1986 ). Severa l line s of evidence suggest that the uptakeof nat i ve LOL by mac rophag es inthe
arte r ial ....all doe s not to any signi fica nt extent (steinber g , 1983;Ste inberg, 1988:Witz.tum, 1990). Exposure of macrophaqes to LOLin culture le a dsto a decrease inthe numberof LOL receptors and thUS, as in othe rce lls, up t ake of LOL is downrag ulated.
Itha s been shown in cel l cultures thatmacrophag es canno t be transformedinto foam cellssimply by exposure to hig h levels of nati v e LOL (Witztum, 1990 ). Fur t hermo r e , humans wi t hhomozygous familialhy p e rchoLe s t.e roLe a f a (HFH) and the rece p t orde f i cien t WHHLrabbit (an anima l model fo r HFH) develop severe and rapid atherosclerosiswith marked foam cell formationdespi teato t al absenceof functiona l LOL receptors (Witztum , 1990) . Ifthe LDL receptorisnot involvedin the mec hanismby which macrophagesta ke up excessiveamou ntsof LDL then the obviousquestion is wh at are the mechan isms for foamce l l formation and theatherogenicity associated with high pl a s ma LOL?
Brown and Golds t ein hav e reported tha t acetyla ted LOListake n up by monocyte-macrophaqesbya pathwa y that is not down regulated (Goldsteinet al., 1979). Acetyl LOL is takenupra pi dl y by macrophagcsinculture,tra ns f or ming these cel lsintolip i d- l ade n cells that resemble foa m cells.inYi.YQ.
The acet ylLOL is takenup by a saturable,specific pr oc e s s , implying the pr esenceof an acetyl-LOL re c eptorthathas now been designated "t he scavenger recept or" (Steinbe rg, 1986 ; witz tum, 1990). The scavenger receptor pa t hwa y does no t
10
appear to be regulated by the cholestero l content of the cells. Other chemically modified forms of LOL have also bee n shown to compete .... ith acetylLOL for the scavenge r recep tor . These Lncl ude methylated LOL (Gajdusek at al., 19 80 ) , acetoacetylat edLDL(Fog elman at a1., 19 8 0 ) andmalonalde hyde - conjugated LOt (Fogelman at al . , 19 8 0 ). These chemically modifie d rcrmsof LOLall ha v ethe lysineepsilonamino groups of apo- B -IOO blocked.
Endothelial cells ,arterial smoothmusc lecellsand macrophagescanmodify LOL by anoxidativemechan ismtoa form tha t becomes recognhed by the high affinity acetyl LOt receptor (Leake and Esfahani, 19 89) . Oxidized LOt is also taken up by the s e cells and re s ults in chol este r yl este r accumul a tion (ste i nberg ,1988 ) . Evidenceis mountingthatLOt can be oxidized within atherosclerotic lesions in Y.i.Y2 (Steinb recher et al., 1984; witz tum , 1990). It isthought thatoxidationof phospholipidswithi n the plasmamembrane of cel lspropagatestheoxid a t i ve chain re a c t ion in LOL.
Thus,hig h pl a s ma LOt levels ma ybesuf fi cient as a sing le initiating factor fo r the deve lopment of at herosc lerosis (steinberg, 1988, Witz tum, 19 90) . Inc r e a s ed LDL levelscou ld cause increasedLDL infiltratio n into the intima and enhancedoxidativemOdificati onby smo othmuscle eLl s ,endot h el i a l cells and monocyte ma c r oph ag es (Steinberg, 1988; Witztu m, 1990). Uptake of thi s oxidized LDL via the scavenger receptors would lea d to the accumu lation of
11
cholestery l esters wi thin the smoot h muscle cells and macrophages. The deposition of cho lesteryl esters wi thi n thes ece ll s inthe arterialwal lmaytrans formthe cells into foa m ce lls . that eventua l l y lead to the formation of atheroscleroticlesions (Steinbe rg, 1988 ). There f o r e ,i tis theini.tia l stage ofatherosclerosis,involvi ngthe deposition of cho lesteryles t e r s and the format io nof foam cella, that must beinhib i tedto preventcoronary artery disease (Witz tum, 1990). Moreover, since it is now apparent tha t arterial lesionsare detect edeve ninth e firstyear or two of li f e, th e effortsto reduceatherogenesismustbeinitiated in young childr e n and be maintainedthroughout one's life (Witztum, 19 9 0 )•
1.4 Di e t a r y Fat and Athe rosclerosis
Although many factors can infl ue nce the re lati ve risk of coro naryarterydisease, the effectsot dieta ryfat on atherog e nesis have re c e i ve d much of the atte ntio n.
r-el ations hip be t we en dietary fa t and coronary disease was suggestedas earlyas 191 6 (Goodman, 1989 ). Inrecentyea rs, ahost oflargelip i d cli nicst ud i e s have clea rl y de mon s t r ate d th a t ind ividuals co nsumi ng diets richin sat ura tedfattyacids exhibit a muchhig he r incidence of coronaryart ery disease th a n individual s eating diets ri c h in polyunsa t ura ted fatty acids (PUFAs) (Goodnight et al., 19 82: Grundy and Ahrens , 1970; Mc Namara , 1987; Neste1,1987). Thesefindingshav ebe e n
12
confirmed by controlledfeeding trials in severa lexperimenta l animals (Balasubrarnaniam et a L,, 198 5 ; Bi eberdorfand Wilson, 1965, Garg etal. , 1988; Krause andHartman, 19 8 4 ). It has been establishedthatthe biochemicalbasisforthe reduction in atherogeneoisby dietary polyunsaturatedfatty acid lies in the marked decrease inpla s ma cholesterol, particularly LDL cholesterol, observed whensa t ura t e dfatt y acids in the diet are re pl ace dbypolyunsaturatedfatty acids (Baudet et a1.. 1984; Shepherd et a1., 1980).
re appears that differe nt types of polyunsatura ted fatty acids may differ in th e i r effectiveness in preve n ting arterial lesions. As early as 1952, Nelson observedthat feedi ngcoronarydisease patients largeamounts of fatt y fish signifi.,;antly impr o ve d the 20-year survival ratesfrom at to 35% (Nelson,197 2 ). AlthoughNelson'swork went virtua llyunnoticed, in 1970 Bang, Oyerberg and coworkers re pc r -te d that theincidence of coronary artery dis e a s e among the Eskimopopulationof NorthernGreenlandwas le s s than10%
of tha t in Eski mos living in Denmark (Bang and Oyerberg, 1972 ). They suggesttha t the absenceof atheroscl erosisin the Green land Eskimos mi g h t be attributable to th e high co n t e nt of (,1-3 polyunsatu rated fatty acids in theEs kimo' s marinebas e ddiet. Thesefattyacidshave the firstdouble bond located threecarbons from the terminal methyl group. In contrast,the1.l-6polyunsaturatp.dfattyac i d inve get a b l e s and ve ge t a b le oils, the majorsources at pOlyunsaturated fatty
13
aci ds inmost We sterncountries, have the firstdoub lebond inserted at carbon 6 fromthe terminal met hy l qroup(Harri s , 1985:Zibo handHllle r, 1990). Th eability of dietsrichin W-J polyuns aturated fatty acids to pro tec t against atherosclerosishassi n ce been confi rme d inseveralcontrolled animal studies (Dav is et al.. 198 .; Wiener at aI., 1ge 6 ), al though some repor ts have SU99 s sted th a t diet a ry Iol-J polyunsatura t e d fatty acids do not re duce arte r i a l lesi ons (Thi e ry and Sledel,19 8 7 ) .
Die t aryw-J fattyac id s have avarie tyof effec tson pl asm a lipi d and lipopro teins (Nestel, 1990). Exten s ive re s earch has focused on tri g l ycer i de metabolism sdnce 10/-)
PUFAs ha ve been shown to consi stent ly lowe r plas ma t rl ",lyc e ride level s in vi rt ua ll y all human and anil llal experime n ts (Ha rris , 1989; Land, 1986; Ne stel , 1990) . Fish oi lfe edinghas be e n sho wn to di mini shLfpoq e nes.L s , incre a s e ketog ene s i s andtatty ac idox i d at ionandre sul t inanoverall re d uct i on intrigl yce rid e secretionby theliver andinlower pla sma VLDL (Bro nsgeest-schon te et al. , 19811Wong et a1., 1985; Lang and Da vi s ,1990l.
Numer ous st ud i e s have reported bothinc reases and dec re a s es in plasma LDL cho leste ro l upon fish oil su p p l e me n tati o nin human sand expe rimental ani ma ls (Goodnight et a1., 19821Har r is, 1989 ; Nestel, 1987 ;Nestel, 1990). In ge ne r a l thoug h, it wouldappea r thatpl a s matotalcholesterol and LOt. cn e re et.ere t le vels in an imals fed 101-3 PUFAs are
14
similar tothe le v e l s in animalsfedw-6 PUFAs (Har ris , 19 89 ; Nestel, 1990) . The effectof w-J PUFAs on fIDLle v el s hav e also be e nincons isten t ,butitwouldappear th a t HOLlevels in plasma are not altered by fish oil supplemented diets when compared with vegetableoi l di ets (Kromhoutet al. , 19R51 Nestel,198 7;No r d o y and Goodnight, 199 0 ).
The usefUlness of fish oils in treating hypercholesterolemia is doubtfUl , excep t Wh e n cholesterol is in VLDL as in type V hyper l ipop rotel netnia (Neste!, 19 8 7 ) . Yet, most existing kncwj edqe confirms that dietary fish oils offe r considerably greater protection against the developmentof atherosclerosis. Therefore, th e beneficial effects of w-3 PUFAs are probably no t mediated thr o ug h alterationsin li popr ot e i nle v el s.
Evidence suggests that w-3PUFAs af f e c t cholesterol metabolism,specificallycholesterolesterificationinhe patic and non-hepatictissues . CaCo-2cel ls, (derived fr om huma n colon adenocarcinoma) in wh ich the membr ane fatty acid composition has been enr-Iched with eicosapentane no icacid had significantlylower rates of cholesteroleste rificationthan didcells modifiedby palmitic acid (Murthy ct; al., 1988 ). similar findings have alsobeen reported inhe patoc yte s and liver microsomes that have been enriched with eicosapentanenoicacid (Rustan at aLI 1988). prel imina ry experiments have also suggested that dietaryw-3 PUFAs lower thees t e r i fi c a t i on of cholestero linli ve r microsomesis olate d
15
from rabbits fed fish oil supplemented diets. Liver microsomes isolated from fish oil fed rabbits contained significantly le s s cholesteryl ester by mass than li v er microsornes from rabbits fed vegetableoil supplementeddiet!':
(Goodyear and Davis,personal communication).
Inhibition of cholesterol eseeetrtcetacn by w-J PUFAsan the cells of the intima, such as the smooth muscle cells and monocyte-macrophages, 'Would result in reduced cholesteryl ester deposition, delayed foam cell development and inhibition of the initial stage of atherosclerotic lesions. However, investigations of the effects ofl&l-J PUFAs on cholesterol esterificationin mammalsha v e been limited to int e sti ne , li v er and cellsderived from these t\,lO tissues.
Intestinalcells and hepatocytesare involvedin the packaging andsecretion ofli pop r o t e i ns andthe r e f o r e may not adequately representcholesterol metabolism innon-hepatic tissue. It is,therefore,important tha t the possibility of sucheffects of w-3 PUFAs on no n- he pa t i c cells be examined.
1.5 Objectives
My objectivevas to studythe effectof w-3 and w-6 PUFA enrichment of non-hepaticcells (human fibroblasts (0551 ) and mouse macrophages (J"774), on cholesterol metabolism.
Experiments involved specific fatty acid enric hment of the cel lularlipids in humanfibroblasts andmousemacrop hagesand SUbsequent mass andradiochemicalmeasurement of cholest eryl
rs
esterifica tio n after tat ty acid enri ch ment. To study the eff ectof fatty acid enrichme nt oncholest e r ol meta bol ism, effluxof chol e ster olfro m "'-) and "'-6 purAenri ch e dce llswas also me asured. Thi sinvolvedenri ch i nqfibrobla stswiththe s e fat ty acids and radiolab e llinq th e s e cells with ac e t at e to la b eltb echole sterol poolenablingusto measu r e chole ster ol effl uxin thepre s e nc e ofanacc e ptor (HDL).
17 2. MATERIALS AND ME:THODS
2. 1 Materials 2.1.1 Lipids
All lipids were obtained from Sigma. Chemical Company,st.Louis,MO,USA. These were used without further purification . Lipid purity was checked using thin layer chromatography (t.Le.) and gas liquid chromatography
(9.1.C.) •
2.1. 2 Rad i ois otope s
[oleoyl-l -UCj oi ecya-ccenayne A, (58.3mel/ mmel) , and (3H] - a c e tic acid sodiumsalt, (1 mel/mmol) were obta ined from DuPont-NEN, Lachine. Quebe c.
2.1.3 Chemicals
Unless specified, all chemicals used were reagent grade. Ammonium acetate, ammonium sulphate, cupric sulphate, chloroform (HPLC grade). hexane, methan ol(HPLC grade), sodium phosphate (d i b a s i c ), potassium bromide, potassium sodium tartrate (crystal) and sodium ch l o r i d e were purchased from Fisher Scientific company. Tris, EDTA (disodium salt), ophthaldialdehyde, bovine se r um albumin, cholesteroland DL- dithlothreitol were productsof SigmaChemical Company, st.
Louis, 110,USA.
18 2. 2 Met hods
2.2.1 Fib r o bl a s t s
Normalskin fibro blasts(Detroit551,American Type Cu l tureCol lection , Ro c kv ill e, MD, USA)we r egrowninEag l e' s Minim umEssential Medium(HEM)contai ning 10 % Feta lCalfSe rum (FCS). (3%)bicarbona te, andpentroo Iu/ml)-strep(lOO1J9/ml)- funigizone (25~g/ml) (Flow Laboratories, Inc••Mc Lea n, VA, USA). Cellswereseededat5 x10 5 cells in 25 cm 2 flasksor in17 5cmz flasks andweregrown at 37cCir'1n CO2 and ai r.
2.2.1.1 Prepar ation of Fa t t y Acid Enrichm e nt Med ium fo r Human Fib r ob lasts
Fattyacid enrichmentmedium for humanfibrobl as t s was preparedusing a modificationof the procedu re re p ortedby Spector (Spector at aI., 1981 ) . The sodium saltsof va rious fa tty acids in phosphate buf f e r ed sa line (PBS, Flow Laboratories, Inc.) wer eaddedto Fetal CalfSerumtha t had be e n dial yzed aga i nst PBS, pH 7.4for 24hours. Thi ssolution wa s the n added toMEMto give final concentrat ions of lO\: FeS and 60 pg/ml of fattyacidsalt.
2.2.1 .2 FattyAcidEnrichmen t in Human Fibroblasts Cel lswere grownto 2 X108cel ls in25 cml fla s ks in MEM conta ining bicarbonate, 10% Fe s and pe n-strep- fun i g lz o ne. This mediumwas thenre mo ve dand rep lacedbythe fat ty acid enrichment me d ium, prepared as described above.
19
The cellswere then incubated at 370C for 120 hours in this medium. To activatethe enzyme ACAT, cells were loaded with cholesterol in the form of LOL (Brown and Goldstein, 1983). Dialyzed human LOL(100/Jgof LOL protein/ml)was added to the enrichment medium18 hours before cellswere harvested (at 102 hours). Human fibroblastswere washed twoti me s with PBS and collectedin1ml of PBS with a rubber policeman. Cellswere pellatedat 12,000x 9 for 1 minute and the supernatant wa s discarded. The cells were resuspended in a known volume of either PBS or ACAT buffer (50 mMTris. 10 mMEDTA, pH 7.4) (Gaviganand Knight, 1983). Cell protein was estimated by the procedure of Lowry et a1. (Lowry et al., 1951).
2.2.1.3 Assessment of Fatty Acid Enrichment
To determinethe degreeof fa t t y acid enrichment in human fibroblasts , cellswere harvested and pelleted. They werethen resuspended in 250til of PBS. Cellular lipids were extracted by using the procedurereported by 81 igh and Dyer, first by the addition of 750 til of chloroform:methanol (1:2, v/v). The protein was then pelleted at 12,000 x 9 for 5 minutes and discarded. Chloroform:water(1:1) (500 til) was addedtothe supernatant. The top layer was discarded after centrifugation at 12,000 x 9 for 5 minutes and the lower fr a c t i on was evaporated under Nz gas. The li p i ds were dissolved in 50 pI of chloroform:methanol (1 : 1 ) and were applied to silicagel plates (Rediplate, Fisher Scientific,
20
Dartmouth ,N.S.) asa singlespce. The plateswer e de velope d in aneutr allip i d solve ntsys tem(hexane:diet hyl ethe r:a c et i c acid, 85:15:2,v/v/v). Thelipidspotswereidentifiedon the ch r omatograms under uv light af ter staini ng with 2'.7'_ di chlaro fl u o r e sce in . The polar li p id (ori gi n ) was scr a ped fromthe plate and was extractedfromthe sil ica wi t h 200111 of ch loro form:met han ol (1:1, vJv) .
The methyl esterderivativesof th efa tty acidsfrom the pol ar lip i d fr a c tio n were anal yz e d by ga s li quid chroma tog raphy(g. 1.c.) (Keoughand Davis, 1979 ). separation of methyl esters was ca r r i ed out on an HI P 5890 ga s chromatograph fitted wi th a Supelcowax 10 fu s e d capil lary column (JOm,0.53mmtOand1.0 pm filmth ickness) us i nghe lium as the car riergas (f low ra t e was 15 ml/min) and opera ted iso t herma l lyat atempe r a t u r e of19 00C. Thefattyacidswere quantified, usi ng he pt a de c an o ic acid (50/.Ig) asthe inte r nal standa r d.
2.2.1.4 Determi nationof Cellular Es t eri f i c atio n in Whole CellLysatesof HumanFibroblasts
Followi nga five -dayenrichment with eit he r linol eic acidor eicosapentaenoicacid,huma n fibroblas tswascol lected indiv idua llybyscrapingthe fla s ks wi t h aru bber polic eman.
Cellswere pelleted at 12, 000 x gandresuspe nded in 60p1of ACAT buffer (50mMTris, 10roMEDTA,pH 7.4). A 10 /.11 aliquot of cellsuspe nsionwas the n ta ke n for pro t eindete rmi n ati o n
21
(Lo....ryet al•• 19 51) .
The measu remen t of cholesterol esterificatio n act i v i ty in wh o le cel l lysat'Js requ ired the us e of a radiolabelledfatty acid cca, A stud y ha sshown that the best substrate for ACAT activity me as u r eme n ts is oleoyl eoA followedbypalm i t oy l , stearyl andllnoleoylCoAs in orderof decreasingspec ificity(Suckling andSt ra ng e, 1985). Oleoyl eoA was used In the se studies. The human fibroblasts were lysedby suspensionin hypotonic ACAT buffer. The exte nt of cell lysi s was determined by measuring cytop l asm ic lac t a te dehydrogena se act i v i t y andtr y pa n blueexclusion.
To mea s ure cho l est ero l ester ifica tion in enriched human fibro bl a sts, ACAT buffer containing 0.5 mg/mi Bovine Se rum Albumi n (85A) and 0.2 IICi/ ml ot rHCJ-oleoy l CoA (58.3 mCi/mmo l ) was add ed tocell lysates (4 0119)togi v e a final vo lum e of 250pI (Billhe i mer, 19 85;Li ch en steinand B...-e e h ner-, 19 8 0 ). This mixt u r e was incubatedat 37DCfor 5minute s .snd the re a c t i on was st o pp e d by the ad d itio n of 750 p l o t ch lo r o fo rm: lIIet hano l (1:2, v/v).
The proteinfractionwas pell eted at 12 , 0 0 0 x g and discarded . The supe r n a t a nt was retainedand theli p i d s were extractedby theadditionof 500 /.11of chloroform:water (1:1 ). The upp o r lay e r wali di s c a r de d and th e lowor po r ti on wa D evaporate d using
" 1
gas. The lipid residue was dissolved in 100 III of chloro form and was chroma t ograp hed on si lic a gel platesinhexane:diethylether:aceticacid (85: 15:2, v/v/V).22
The lip i d s were visualizedin iod inevapor. The cholesteryl ester sp ot was scraped and co u n ted fo r the presence of cholesteryl-(I~C] -oleate in a liqu i d scinti l lation cou nter (withau tomatedque nc hcorr ection). Therateof ch o lest e ro l este rif icationwasexpre s sed as picomolesof cholesteryle~C) olea teformed per milligram of totalextr ac t pro tein perhour .
2 .2.2 Mac ro phages
Mouse macrophages (J7 7 4 . American Type Cultur e collect ion, Rockv il le, MD, U.S.A.) were gr ownin RPMI 164 0 (FlowLaborator ies , Inc . , Mclean,VA. , U.S .A . ) contain ing10 ' Fe S , bi c a rb on ate , and pen-strep-fugi z one in 25 cmz fl asks and weregrownat31°Cin4%COzandair .
2.2.2.1 Preparation of Fatty Ac i d Enri c hment Me d i um for Ma croph a g es (J77 4)
In the latter pa rtof thi s st ud y, webecameawareof a simple r method for enri Ch i ng macroph a g es. ratt y acid enric he d medi umfor macrophageswas pr e pared byadding the sodiumsalt of various fattyacids in anetha no l solut ion to RPMI (containing 10%FeB and pen-strep-fungltone) togive a fina l concentra tio nof 60t£g/ mlof fattyacid salt. Eth anol concen trations in the medium did not exceed 0.5 % and 0.5%
etha no l wa s adde dtomediumin contr olflasks toensure tha t ce llgrowt hvasnot affected.
23
2.2.2.2 Fatty Acid Enrichmentof J774 Macro ph ages Ma c r oph ag e s were grown until they wer e confluence in RPMI containing bicarbonate, 10\ FeB andpen- strep -fungizone. The medium was re p l a c e d by fatty ac i d enri chme nt medium, prepared as descri be dabove. Th e cells we r e in c ubated at 37°C for 36 hours in thi s medium. To stimulate ch ole s t e r ol esterification in macrophages, cells were inCUbated for 18 hours with 50 Itg/mI acetylat e d LDL (Gol dste in et aj,I 197 9 ). Macrop hageswerewashe dtwo t.i me s wi th PBSand the ncollectedin1ml of PBS with the aidof a rubb erpol i c ema n. Cellswere pelletedat 12,000 x 9 for 1 minu t e and the supe rnat ant was disc ard e d. The cel ls we r e resuspe nded in a known vol ume of PBS. Cell pro t e in was es timate d by the procedure of Lowry (Lowr yet aL,, 1951 ). Fat t y ac i d enrichment inmacrophageswas assessed by thesa me metho d used to analyze the fattyacid composition in hu man fibr o blasts.
2.2 .3 Hu ma nFibrob l a s t Mi c r o somes
2.2.3. 1 Pr e pa r at io n of Microsomes from Human Fibrob lasts
Enri c he d with Eicosapentaenoic Acid and Li no leic Acid
Microsomes fr om human fib r ob l ast s wereisolated as described before (Davis and poz nanaky, 1986). Ce lls were grown in 175 cm1fla s ksand en rich ed wit h various fatty acids (asdescribedpreviously). Cells were washed two timeswith
2<
PBSand collected by scraping wi t h a rubber policeman. Cells we r e pelleted at 3500x 9 for5minutes, the supernata ntwa s discarded and the pelletwas resuspendedin 10 ttll of 0.88 M sucr ose /50mMTri.s/ c.S %BSA(Fr a c t i o n V, SI g ma) , pH 7.4.
Th ecell suepensIcn washomog e n iz e d by hand (8 - 10 str ok e s) in aDou nce Homogenize r . Nucl e i andunbrokencells we reremov ed bycentrifugati onat 500x 9 for 10 minutes and the supernatantwas centrifugedat 20,00 0x 9 for20 minute s toremovemitoc h o nd r i a. The postmitochondrialsupernatant was dilute dwit h 2.5vo l ume of bufferA (50 mM phosphate/ I OrnM dlthiotreit al , pH 7.4) andcent r ifuged at 105,000 x 9 for 2 hours. The micr osom alpelletswere resuspended in bufferAat 105, 00 0x9for90 minutesand suspen de d in ACAT bu f f er.
NADPH-cytochromec reductase activity was determined in microsome sisolatedfrom human fibroblasts incubated with various fattyacids (Lake, 1987).
2.2.3.2 Determination of Fat ty Acid En ri chment in Microsomes, Is olate d from Human Fibroblasts Grown in the Presence of Eico s a penta e n o ic Acid and Linolei cAcid
Microsomes from fatty acid enri ched cells were isolatedby the proceduredescribed in the previoussection . To determinethe ext e n t of fattyacid enrichment, microsomes (100,:19) wereext r ac t e d in 750 ttlof methanol:chloroform(2:1 ) and pelleted at 12,000 x 9 (p ell e t was discarded) .
25
Chloro!o rrnlwate r (1: 1 ) was adde dto thesupernata n t andagain centri fugedat 12,0 00 x g. The lowe r fractionwasreta ined andev a p o ra t e d unde r a stre amof nitrog en ga s. The lipi d s weredissolv e din100pI of ch loroform:methanol (1:1). Sa 111 w-asappli e dto a si l ica gelplate and developed in aneutral lipid solv e nt syst e m(see section2,2.1. 3) to est imate the fat t y acid compositionof the tota l polarlipidfra ct i on. To de t e rm inethe degre eoffat tyacidenrichmen t inthe difteren t phos ph olip i d classes , 50 pIof totallipidextrac twas applied to a silica gel pl a te as a singlespo t and develo ped in a polar lipid solvent system (chlorofo rm:methanol:water , 65=25:4, v/v/vj. The phosphotidylcho l i ne spo ts and phospha t i dyl e t ha nol a mi ne spots were ident if i e d on the chroma tog rams unde r uv light aft er sta i n ing with 2',7' dlchlor of l uo r e s c e in. The lipidspots were scraped fromthe pla t e and extracted from the si lica gel wi th 200 ~I of chloroform:methanol (1: 1, vyvj , Thefattyacidme thylesters wer e ana ly z e d by ga s liq uid chromatog r a ph y as de s cribe d previ o us ly.
2.2.3.3 Determination of Microsomal ACAT Activi ty ACAT activ itywasdetermined inmicro so mesisolated fromhuma n fi b r ob l a sts incubatedwith various fatt y acids.
ACATa~ t iv itywas me a s ur e d by add i ng 15 0 ~l otACATbuff e r co ntain i ng 0.3~CiofCUCl-ole oyl CoA (58.3mCl/mmel) and1.3 mg eSA to100~lof micr osames poIJg of protein) (Li c hen stein
2.
and Breehner, 1980). This mixture wasinc ubat ed at 37 ~Cfor 20 minutes andthe reactionwas terminatedbyth e addit ion of methano l: c hloroforrn (2:1). Lipi d s were extracte d and chromatoqraphed as descri bed previously. Cholestero l ester spots were scraped and counted in a li qu i d scintil lation counter.
2.2.4 Choles terol Efflux
2.2.4. 1 Radiolabellingof Endogenous CellularCholesterol Theendogenous chol estero lpool in humanfibroblasts was radiola bell ed bytheadditionof 1Jlcijrnlof (JH]-a c et ic acid, sodium sal t (1 ,uei/ pmol), tothe medium us e d to enrich cells with fattyacid. The enrichmentmediumco nt ai n i ng the radiolabelledco mp ound was incubatedwi th the cells for 5 days, at which point. the medium was replaced by MEM
(containing bicarbona te, pen-strep- fu nigizo n e and 5%
lipoproteindeficiant serum(LPDS) (Phillips etal. , 1980 ).
2.2.4.2 Cholesterol Efflu)( from Human FibroblastsEnriched withVarious Fatty Acids
Choleste rol e rrfux was measured in cel ls tha t were enriche d with l inoleic acid and eicosapentae noic ac id. Endogenous cellu la rchol esterolwasra di ol abelled as ou t line d above . Effluxexperimentswe r einiti a t e dby the addi t ion of an acceptor particle, high density lipoprotein (250 /JgHOL protein/ml) in fresh culturemedium (Lund- Ka t z et al., 1982).
27
Attime intervalsup to 24hou r s , the med i umwascollected and the cells were washed twicewi t h PBS. The cells were~~ r a pe d frommultiwel l plateswith a rub b e r policeman sUbsequentto theaddition of PBS and were pellated bysp i nni ng at 12,000x 9 for 5minutes at24° C. Tho pellet was suspendedin 260 III of PBS. and 10 Itl was taken for protein determination (Lowry at a1., 1951) . The lipids were extracted by the addi t i o not' 750~lmetha no l:ch l o r of o rm (2:1) .protein pelleted at 12,00 0 x9 fo r 5 minutes at 24° C, and the supernatant retained. Chlorofo rm:wate r (1:1) (500 ttl ) wasaddedto the supe r n a ta nt and thelowerportion was saved andevapo r a t e d undera strea mofNzga s. The lipids were chromatographed on silicagelplates inhex a ne:diethye ther:aceticacid(85:15:2, v/ v/v ). The unesterified and esteri f ied cholest erol bands were identified in iodine vapo r and were scraped and the radioacti Vity determined by liqu id scintillation counting.
Thelipids in the medium were alsoextracted, chromatagraphed andcholesterol andcho l e s te ry l esters weredet e r mi ne d in the
2.2.5 Lipo proteins 2.2.5.1 Lipoprotein Preparation
Human plasma was obtained from the Canadian Red Crass, st. John's, NF. Human LOL Cd 1.019 - 1.063g/m1) and HOL Cd 1.125- 1. 215g/m1 )wer-e isolated fromthe plasma by Ul t r a c e nt r i f ug a t i o n ac c o r dingtothe met hodof Havel (Ha ve l ,
"
1~a8). Lip oproteinsweredial y zedagainst (150mMNaCl,50toM Tria,SmMEOTA,pH 7. 4 ) for 24hoursbefore use. Themethod of Lowry wa s used to determi ne the amount of li popr ot e i n- protein per mL (Lowry et a1., 1951).
2.2.5.2 PreparationofAcet yla t e d LOL
Human LOL waspr ep are d asdescribedabove . LOL was acetylated by adding1 ml of0.15 MNaOHco n ta i n i n g 16 mg of LOL proteinto 1 ml ofsatur a t e d solu t i on of sodiumacetate W"ithcontinuousst irring in an ice water bath (Basu et a1., 19 7 6 ). Aceticanhydride (2 ml) wa s added in mUltiplesma ll aliquots(.2 ml) over a periodof one hour at 4" C. After the lastaddition,the solutionwasle f t at4° C for an additional thirt yminutes. AcetylatedLOL solution was then dialyzed at 4"Cfor 24 hours against (0. 1 5 Na Cl, .3 mMEDTA, pH 7.4) . Lipoproteinproteinwas es t i mated by theLowrymethod (Lowry et a L, , 1951 ).
2.2 .6 Analyses
2.2.6.1 Determinationof Cellular Cho l esterolContent To quantifythe content of cellula r and microsomal cholesterol, human fibroblast cells and microsomes were is olatedand extracte d as aescribedpreviously . To determine th e cel lula r chol es":e rol contentof J774 rnacrophages,cel ls were iso l a t ed (asdesc ribedpre viously) and extracted (u sing the sa me procedure used to extract human fibroblasts).
2.
Extractedlip ids we r ech r olllatog raph edonsilica gel platesin hexane:di methyleth yle t her:acetic acid (85:15:2. v/v/v)• Cholesterol and chole&teryl ester- spo ts were identified in iodinevap or and collectedbysc r ap ing . Therecoveryoffr ee and un e s t e r i ! ied cholesterol was detet'lllinedby cholesterol an a l ysis using the O~ phth a l d e h ydeme t h od (RUdel and Horris, 1973). 900pIofo-phtha ldehydereagent,with a concentration of0.5mg/ml In glacia laceticacid, wasaddedto each sample.
The so lutionWasmixedthorough l y and incuba tedfo r 10 minute s at room te mpe ratu r e. Concentrate dSUlf u r i c acid (450pI) was added andthe solutionsimmedia telymixed. Theab s orban c e was read at 550 nm, 10 minutes aft er the addition of the concen trated sulfuri cacid(RUde l an dMor ris, 1973).
2.2 .6.2 Anal ys i s of Fa t ty Acyl CoA Pool in Human Fibrobl a s t s Enric hed wit hVario usFa t t yAc i d s The fa t ty acyl CoA pool size was measured to investigatewhe the rfatty acid enrich me ntof hUlRanfibroblast influencedchole ste ro l esterif icat ion by the mo difi c a t ionof metabolicpool s. Thefatty acyl CoApool size wasestimated using a modified versionof the proc edure established by Bartl e t t (watnough et a1., 19 89 ). Human fibroblasts ....ere en r iched with variou s fatt yacid s andwereth e nha r vested by scra pin g, pel letedat12 , 0 00 x 9 for 5minutes at24° C and resuspendedin 1 ml of PBS. Thesu s pensionwas quenchedwith 200/11of acetic acidand20nmolof [I· C)-o l e oylCoA wasthen
'0
addedas an inte r nal standa rdto estimatepercent re c ove ry.
A por t ion (50 1t1) was removed for determi na t ion of acid soluble rad i oac t i vity . Each sample was tra n sfe r r e d to an extraction tube containing 100 III of saturated (NH4)25°4and placed in a boiling water bath for two minu tes and the n al lowed to cool to room temperature. Each sample was extractedtwice with10 volumes of diethylether to removefree fatty acids and then extracted for 1 hour with 8 ml of methanol:chloroform (2:1, vjv) with continuous agitation. After centrifugation (40,000x 9 for 10 minutesat 4°C) the super natantwas reta ined . Thepelletwasresus pe nde d in3 rol of rnethanol:ch loroform (2:1, v/v) and recentrifuged. Th e solvent was removed from therecombined supe rnatantswit h a stream of Nz gas at 241> C until the volume was 2 ml. The pH was adjusted to 6-7 by adding 2.0rol of 1 M- a mrnon ium acetate, and the sample was freezedried overnigh t. The residuewas extractedwith 2.4 ml of methanol ,centrifugedat 90,OCOx 9 and the supernatant reta i n e d. The pellet was re su spe nde din 0.8 rol of methanol, recentrifuged and the combined supernatants diluted to a final volume of 4.0 rol with 0.8 ml of dedor.Lzed water. This was applied to a DEAE-sephacel (acetate form) column pour e d in a 10 ml disposablepipette. The columnwas washedwith5 rol of methano l:water(4:1,v/v). The fraction contai ni ng acyl CoA was eluted with 6 ml of met ha no l:water (4:1, v/v) containing 0.5M-ammoni urn ac e t ate and 10roMacetic acid. This fractionwas then dil u t e d with 2-
31
3ml of de i on i ze d wate r and freeze driedove rnigh t. The residuewas dissolved in 1 ml of chl o ro f orm: me thanol (1:2 ), centrifuged at 12,000 x 9 for 10 minutes at 24°C and the pel let discarded. The supernatant was retainedand a 200#11 aliq uo t was assayed for radioactivity in a liqu id scin ti llationcounte r to determine acy l eoA recovery. Th e remainder of the supernatant was evaporated underastre am of nitrogen at 24°C, 5 /.19of heptadecanoic acid was added and the lipi d S were thentransmethylatedovernight andana lyzedby 9.1.C . (under same conditionsdescribed previously) . The acyl eoA pool size was quantifiedusing heptadecanoicacid as an internalst a nd a r d.
32 J. RESULTS
3.1 FattyAcidEnrichme ntof HumanFi br obl a st s Huma n fibr obl a s t s wer e incub a t ed with eith er li nol eic acid orei c osap ent ane n o i c acid en r ichm e n tmed ium for fi ve days. Ce ll s incubatedwithmediumsupp lem e nted with60 pg / ml of l in ol e i c acid (18:2 , "-6) had a hi g her relativ e conten t of linoleic acid andlittlec.I-Junsatura tedfat tyacid (Tabl e 1). When cells wereincubatedwit h eicosapentanenoic acidenrichment med i um, thecellu la r phospholipidsco n tained much le s s linoleic acid (T abl e 1). Howe v er , cellular phosphOlipidSwere not enriched witheicosapentaneno i caci d but rat her were enriched withtheimmediate elon ga tion pr oduct of EPA, doco sapentaenoic acid (22:5, W-J) (Ta b le1). Th e relative co n t ent of docosah exaenoic acid (22:6, w-3) was simila r at appr o x i ma t e l y 4.1% inthe cel ls incuba tedwi th eithe r lino leic acid or eicosapenaenoic. The r e were no signi ficant differences in the re l a tive conten ts o£ other fatty acids in cell u larphospholipids (datanot shown ).
Cel lular proteinwas simi lar in flasksof cells incubat ed with either linOl e i c or eicosapen taenoic acid supplemented medium but varied from batch to batc h from approx imately30 119-100 #9 of protein perfl as k. The average cell number per flask for both groups wasestimated tobe approximate ly2 x106cel ls/flask.
Microsomal phos phol ipids from human fibroblas t s
)~J
~
:;l§
N~~ .,
~ ~
~
~
0
~
~
§
00
3
~i~
~
..;~
~
0
~
l
~
~33
incubated withme d i umsupplemented with either li noleic acid or etcceapcn eeencLe acid were analyzedfor thei r fat tyacid conten t (Ta ble 2). Mi crosoma l membra ne phosp holipids from humanfibr obl a st s incubate dwi th linoleicsupplement e dmedium were enri c he d with l in o lea t e (15.5 %of total mic rosomal Phosph o lipidfattyacid) . However,whe n ei cosapentae n oic acid was added tothe medium, the cells elon ga tedth isfat tyacid to docosapen taenoic acid that appeared in the micros omal phospholipids (9.4% of tot al mi cr o s omal phospholip i d fat ty ac i d) . Therelativecontent of docoaehexaenol.cacid was 2.3\
and1.9\ in microsornesfro m cel lsin c u bat edwith l inol e i cacid and ei cosapenta e noic acid, re s pe c ti vely , In sUbseque n t experi ments , whenreferring to cellsthathavebe e ninc uba t e d ineicosapentaenoicaci d suppleme ntedmedium, they will be referred to ascel ls enriched withdo cos a p en t a e n o i c acid.
To fur t h e r investigatethe incor porat ionof lIJ-3and w-6PUFAsin t ocel lula r li pid s , the phOSPholipid frac t io n of microsomes(lsolatedfrom cellsincubated wi th ei therl inol eic acidor eicos ape ntaenoicacid supplementedmedi u m )was further separated by thin laye r chromatography into differen t Phos pho lipi d clas s e s. Table3 shows the re l at iveperce ntsof
"'-3an dL1-6 PUFAs ineit herphosphotidylethanolamine (PE)or phos ph otidyl c h oline(PC) fraction ofmicrosomalphosph olipi d s. No diff e rence was observedin the conten t of ",-6 PUFAs in ei ther thePCor thePEfractionof microsomal phosp h olipi ds when cells incubate d wi th li noleic acid
Fatty acid Cont.nt inphosphotidyl. t " 'no l •• ln. (PE)and phosphotidylcholine (PC)fr . c tionof 1Il1c rosOIl& lphospho lip l .b. lIicro s ola4lS"e reis ol at ed fromcellsincubatedwi th eithe rlInole ic ac id CIA)oreico sape nu a noicacidCEPA)su pp lelllenud me d i um (n..2).
FATTYACI D "ICIl.OSOKlS ISOLATEDFROKCELLS KICROSOKES ISOLATEDFROK
INCUBATED VITHLA CELLSINCUBATED VITa EPA
(:It01'TOTALPATTYACI D) (X-OF TOTALFATTYACI D)
PCFRACTION PEFRACTION PCFRACTION PEFRACTION
...-6 P'lJFAs 13.S 12.7 10.7 13.0 '-' 3.c 3. '
,.,
"".]purAs 6.2 6.0 0 11.] 10.6 8.8 11. 0 21.5 31.4
wc-
34
eicosapentaenoi c supplemented Iled i um. Howev er, a grea t er percen tageof11I-3PtlFAs werein c or po r a t e d into the PEfraction of mi c r os oma l pho s pholip id s when cells we r e incubated with either linoleicor eicos a pe ntae no icacid supplementedmediulI, Microsomalmarker enzyaeactivity ,NADPH-cytoc h rolle
~ re duc t ase was measured in mteresca e s is olated from huma n fibrob lasts incubated with different polyunsatu ra ted fat ty acids. The activity of NADPH-cytoc hr ome sr. reduct a se was simila rin microsome s enrichedwith either /01-3 or/01-6 purAs (t he activ i t y of NADPH-cytochrome SO reductase wa s 74.4 nmoles/mgpro t ein and 72.9 nrnoles/rng protein, re s pe ctively [Exper iment 1J and"'0.7nmoles/ mgprotein and 52.7 nmoles/mg protein,respe ctivelY (Experimen t 2]).
3.1. 1 Effecton Chole sterol Levels
Cellular cholest e r ol lev e l s,bothunesterifiedand es terifiedwere me asured in human fi br oblaststo assess the effect sof fattyacidmodif ication once llul ar cho l e ste ro l. The cholesterylQsterleve lsincel lsinCUb ate d with li n o leic acid supp lemented me dium and eic osapen ta e no i c ac id supple ment e d mediumweresig n i f i c ant ly different. Fi g ure 1 clear l y showsthat Incubet.Icnof cel lswitheicosape ntae noic acidde cre a s e d thecellularcholesteryleste r conte nt in the s e cells by 56%compared....ith cel lsenriched with linol eic acid . However,th e r e appearedtobenosi g n ific a n t diffcrl"nce in the level of treecholesterol in cellsinc uba t e dwith either ratt y
J5
aci d (Fig ure 1). sta tis t icalanalysiswas doneby two-sided stude nt t·tes t in this experimen t and all subsequent experime n ts the r eaf t e r .
Figure 1:
36
Cholestery l ester (op e n bar) anduneste r ified chole s te r ol (cl os ed bar) conten t ofhumanskin fibroblasts enr i c hed with linol e i c acid or docosapentaenoic acid. Errorbars are S.D.
*p <O.005 (n .. 4) , s>o.oi (not signif icant) student t- test(two sided).
- t
I
I
!
~
'0uc'§~ '"
o.c:u.,-1
C' -+
r
",0:::0-....~--'
i
O'[JL. .
-
~u..-,:·u0 0
~;>;:~
'-1 x 4
0Qjn::::u.~s:r --' o'[J
----,
:§~
;
i
Ii I --I i
-I.
a a a a a a
a a a a a a
C?
co to.,.
Na
ci ci ci ci ci
(U!~f Ojd DW/ low n ) l OtJ31 S3 l 0H 8
38
3.2 Measurements of Cholesterol Esterific a tion In Lys ate s of HumanFibroblast
3.2.1 Stimulat ionof Cholesteryl Esteri!ica tionbyNat i ve LOL
We could not detect cholesteryl este rifi cation in fibroblaststha.twereincubated inHEM+ 5\ LPDS (d a t a no t shown) . To stimulate cholesterol esterifica tio n in human fi b rob l a s ts, the cells we re inc uba t ed wit h 100 ltg/tol of LOL pr otein for18ho u rs(Brown, 197 5 ) . Cells incu batedwithHtH +5\LPDSandLDLcontained 40%mo r e cho lesterolestercompared wit h cells that were inc ubated with only MEMand 5\ LPDS (Figure 2).
Figure2
39
Cholesterol contentof cells incubated with (+LDL) and wit hout LDL (-LDL) . Open bars represent une sterified cholest ero l (UC) and crossed ba rsrepresentcholestery lester (CE) content. Error bars are S.D. *p<O.OOl (n = 4).
o
0 0 0 0,.... (0 LO V n
a
w o
U::J
o w
U ::J
-l
o
-l
+
- l
o
- l I
(N1310tJd ~V1 \~n) l OtJ31S3l 0 H8
41
3.;L2 Llneari ty of Cholesterol Esterification with In c r e a s i ng Protein Concentrations and Time in Unenriched Human Fibroblasts
TOinvestigatethe effect of w-3 and lil-6PUFAs on cholesteryl esterification, incorporationof[HC] _ ole o yl eoA into cholesteryl esterswas measured. Figure 3 shows that esterificationactivityin wholecell Iysates appearedto be linear withincreasingtime up to10minutes. ACATact ivity in Whole cells als o increased in a linear fashion wit h increasing amounts of protein, up to 60 jJgof cell protein (figure 4). In all sub s eq ue nt measurements of cholesterol esteri!ication,approximately40 119 of cellproteinwas used per assay and assay mixtures wer e incubatedfor 5 minutes.
Bovine Se r um Albumin was added to the re a c t io n mixture to mainta in a molar ra t i o of oleayl CoA:albuminof 4:1. serum albumin is requiredin the assay medium topr e vent the high concentration of oleoyl CoA from inhibiti ng ACAT presumablybecauseof thede t erg e n t-l ike effects of oleoyl CoA that might disruptthece llme mb r a n e (Bilheimer, 19 8 5 ).
Estimation of cell lys i s by the hy poton i c ACAT buffer wasdo ne by measuringcytoplasmicla cta t e dehydrogenase (LDH) activity. Asmall butsig n i f i c a nt differencewas found in LDH activity between1J-3and141-6PUFA enrichedcell lys a t e suspe nsions. (LORacti v i t y was 58.9±10.1nmoles/min/mland 42.9 ±6.05 nmoles/min/ml, respectively. (n >=4), p<0.05.) Th e number of viable cells in 111-3 and 141-6 PUFA enriched
42
fibro bla s t s we r e silllilarwhenstained wi t h tr ypanblue(56,86
±1.50% and 511.6::!: 4.59%were stained wi th trypan bl ue, respective ly . )
Figure 3:
43
Linear ity of [1·C]-oleoyl CoA incorporation into cholesteryl esters with time, in who le cell lysates (human fibroblasts) .
o
o
0 0
000
0 0 0 0
0
';!. ~
lI)N
0CO
0r-,
0<D
0 lI)
0
...
~en c
0
i
n :::;w f=
0 N
~
0 0
Figure4:
45
Lin e a ri tyof incorporationof [HC) -o leoyl CoA into cholesteryl esters with increasing prote inconc entrat i on inwho lece l l s.
