Dietary polyunsaturated fatty acids enhance the uptake of high-density lipoprotein cholesterol ester by rat adipocytes13

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1 Dietry polyunsturted ftty cids enhnce the uptke of high-density lipoprotein cholesterol ester by rt dipocytes13 Ev Zsigmond, Bessie Fong, ndaubie Angel ABSTRACT Compred with diets high in sturted nd monounsturted ftty cids (2% lrd by weight), diets high in polyunsturted ftty cids (2% sunflower oil) lter the ftty cid composition of rt dipocyte plsm membrnes nd enhnce HDL2 binding We exmined the effect ofthese two diets on HDL1 nd HDL2 polipoprotein nd cholesterol uptke by dipocytes isolted from perirenl nd epididyml dipose tissue of mle Wistr rts Consistent with selective cellulr uptke, HDL esterified cholesterol uptke ws 3-1-fold higher thn predicted from HDL polipoproteins ssocited with dipocytes Dietry polyunsturted ftty cids significntly enhnced polipoprotein nd esterified-cholesterol uptkes from HDL2 by perirenb dipocytes This effect ofdietry ft composition ws dipose-region (perirenl > epididyml) nd HDLsubfrction (HDL2 > HDL1) specific Thus, diet-induced chnges known to lter membrne phospholipid composition nd increse HDL2 binding re lso ssocited with enhnced HDL2-esterified-chobesterob uptke by dipocytes Am J Clin Nutr 199;52: KEY WORDS Dietry ftty cids, polyunsturted ftty cids, ft cell, high-density-bipoprotein-cholesterol-ester uptke, selective uptke of cholesterol, plsm cholesterol concentrtions Introduction It hs been proposed tht high-density lipoprotein prevents the formtion of theromtous lesions by competing for the uptke of low-density lipoproteins (LDL) by endothelil cells of the rteril wll (1) or by promoting reverse cholesterol trnsport (2) HDL hs dditionl functions s it delivers chobesterol to sites of steroidogenesis, including the gonds nd drenls (3), nd provides cholesterol to heptocytes (4), cultured drenl cells (5), lutel cells (6), nd grnulos cells (7) in rts without concomitnt internliztion nd degrdtion of the entire HDL prticle Adipose tissue is n importnt site for cholesterol storge nd this dynmic cholesterol pool is involved in cholesterol uptke, mobiliztion, nd exchnge by interctions with plsm lipoproteins (8) Humn dipocytes specificlly bind HDL2 nd HDL3 polipoproteins (9) nd disply preferentil uptke of HDL-cholesterol ester compred with HDL polipoproteins (1) Furthermore, the selective nd optiml delivery of cholesterol ester ppers to be dependent on polipoprotein (po) A- I (1 1) Although ft-cell weight, obesity, nd dipose region re fctors tht ffect HDL interctions with dipocytes (1 2, 13), it is not known whether the selective uptke of HDL-cholesterol ester is responsive to specific chnges in dietry ftty cid composition In mn, dietry polyunsturted ftty cids ffect both the plsm lipoprotein concentrtions nd the interction of lipoproteins with cells Studies ofldl metbolism in nimls support n ntitherogenic role of dietry polyunsturted ftty cids by demonstrting enhnced heptic LDL-receptor ctivity, incresed cholesterol-ester content of the liver, nd lower plsm cholesterol concentrtions ( 1 4) The response of plsm HDL cholesterol to dietry polyunsturted ftty cids is vrible nd studies hve shown either no chnge ( 1 5) or reduction in plsm HDL-cholesterob concentrtions ( I 6) fter these diets Despite the vrible chnges in plsm HDL cholesterob, dietry polyunsturted ftty cids seem to provide protection ginst the development oftherosclerosis, possibly by cusing redistribution ofcholesterol between plsm lipoproteins nd vrious tissues, including dipose tissue (17) Although HDL binding to rt dipocytes ppers to be medited by protein-recognition site (1 8), membrne lipids my lso be involved in the regultion of HDL binding (19) In previous studies with rt dipocytes, we (2) observed enhnced HDL2-binding ctivity to purified plsm membrnes fter feeding diets contining sunflower-seed oil high in pobyunsturted ftty cids compred with lrd, which is enriched in sturted nd monounsturted ftty cids The higher HDL2- binding ctivity ws ssocited with specific chnges in the ftty cid composition of dipocyte membrne phosphobipids In the present study of similr diets with 2% (wt:wt) sunflower oil or lrd, to lter the structurl lipid components of dipocyte membrnes, we exmined the effects of dietry ftty cids on cellulr uptkes of HDL polipoproteins nd HDL esterified cholesterol s well s the trnsfer of HDL free cholesterol to isolted rt dipocytes I From the Institute of Medicl Science, Deprtment of Medicine, Fculty ofmedicine, University of Toronto 2 Supported by grnts from the Ontrio Hert nd Stroke Foundtion nd the Medicl Reserch Council of Cnd nd by n Ontrio Ministry ofhelth student scholrship (EZ) 3 Address reprint requests to A Angel, Room 7368, Medicl Sciences Building, University oftoronto, Toronto, Ontrio M5S-lA8, Cnd ReceivedJnury 31, 1989 Accepted for publiction July Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216 Am J C/in Nuir 199;52: Printed in USA 199 Americn Society for Clinicl Nutrition 289

2 29 ZSIGMOND ET AL Methods Dietry tretments nd preprtion ofdipocvtes Mle Wistr rts weighing 26 ± 1 g (1 ± SD) were fed semipurified test diets (Tekbd diets, Ahrln Sprgue-Dwbey, Inc, Mdison, WI) contining 48% sucrose, 16% csein, 1 1% fiber, 3% minerl mix, 1% vitmin mix, nd 2% sunflower oil or lrd (wt:wt) for 23 ± 3 d Rts were individully housed nd hd free ccess to wter nd the test diets throughout the experimentl period Food intke nd niml weights were mesured throughout the dietry tretment Clorie intkes were clculted s the men dily intke over the 23-d feeding period The cre of nimls ws in ccordnce with the University of Toronto niml-cre guidelines Freshly isolted dipocytes from rt perirenl nd epididyml dipose tissue were prepred by modified method of Rodbell (2 1), previously described by Fong et b (9) Adipocyte dimeters were mesured with light microscope (22) oltion nd 25llbeling of HDL HDL1 (density kg/l) nd HDL2 (density kg/l) were purified from the plsm of mle Wistr rts weighing 3 ± 1 g fed Purin Rt Chow (B&W Feed & Seed Ltd New Hmburg, Ontrio) by KBr density ultrcentrifugtion (23) Lipoproteins were wshed once by ultrcentrifugtion nd dilyzed ginst solution of 1 5 mol NC1/L nd 4% EDTA (ph 86) The chrcteriztion of HDL1 nd HDL2 prticles ws previously described (18) Briefly, HDL1 prticles re lrger nd contin more esterified cholesterol thn do HDL2 prticles The mjor polipoprotein of both HDL frctions is po A-I Apo E nd po A-IV re lso present in both frctions but po E ws reltively higher in HDL1 thn in po AIV-enriched HDL2 prticles HDL1 nd HDL2 frctions were iodinted by modifiction of the iodine monochboride method (24) nd were used within 14 d oftheir preprtion Biologicllbeling ofrt HDL with cholesterol ester Esterified cholesterol in HDL ws lbeled in vivo with [3H]- cholesterol ccording to the method ofgbss et l(4) Two mle Wistr rts (weighing 32 nd 325 g) on lbortory rt-food diet were fsted overnight nd given 1 85 X b Bq of[3h]cholesterol (Amershm, Okvibbe, Ontrio) vi gstric tube Plsm ws obtined from the rts under ether nesthesi by crdic puncture nd HDL1 nd HDL2 frctions were isolted s previously described (18) The biologiclly lbeled [3H]- HDL-chobesterob-ester frctions were incubted with 185 x l Bq of [ 4C]cholesterol (NEN, Boston) in the presence of 4 mmob dithiobis 2-nitrobenzoic cid/l for 4 h t 4 #{176}C to obtin prticles with specific 4C trcer in free cholesterol Lipoproteins were wshed by ultrcentrifugtion t density of 1 21 kg/l nd dilyzed Lipids ofhdl prticles were extrcted with chloroform nd methnol (2: 1, vol:vol) nd free chobesterol nd esterified cholesterol were seprted by Sephdex LH/ 2 [Phrmci Fine Chemicls (Cnd), Bie d Urfe, Quebec) column chromtogrphy (25) 3H- 4C-lbeled HDL frctions were used in cellulr uptke experiments within 14 d of their preprtion The totl cholesterol, free cholesterol, nd phospholipid content of 3H- 4C-lbeled HDL1 nd HDL2 frctions were determined by use ofssy kits from Syntex Dignostics (Kingston, Ontrio) Esterified-cholesterob content ws clculted s the difference between totl cholesterol nd free cholesterol Protein ws mesured ccording to the method oflowry et l (26) Reltive to protein content, the mss rtios (expressed s g/ tg) of esterified nd free cholesterol in HDL1 were 4 ± 1 nd 1 ± 1 (1 ± rnge of duplicte determintions) HDL2 contined 14 ± 1 g esterified chobesterob/g protein nd 1 ± 1 zg free cholesterol4tg protein HDL1 nd HDL2 contined 48 ± 7 nd 25 ± 3 g phosphobipids/tg protein, respectively [ 251]HDL nd [3H- 4C]HDL uptke olted rt epididyml nd perirenl dipocytes (- 1 mg lipid/vil) were incubted t 37 C for 2 h in 15 ml Krebs- Ringer bicrbonte buffer (KRB) contining 5% bovine serum lbumin (BSA) (ph 74), 5 mmob glucose/l, nd 1251 or 4C-lbeled HDL (5-3 mg/l) After the incubtion, dipocytes nd the medium were seprted nd the cells were wshed four times with Krebs ringer buffer (KRBA) contining 1% BSA Adipocytes incubted with 251-bbeled HDL were dentured by dding 1 ml 1% trichborocetic cid (TCA) nd processed s described by Fong et b (9) TCA-insoluble 125! rdioctivity ws mesured to determine cell-ssocited uptke of HDL polipoproteins After n extrction (27) the lipid weights were mesured nd together with the ft-cell-dimeter determintions, the cell-ssocited uptke of [ 251]HDL ws clculted per million cells Cell-ssocited uptke ws lso mesured in the presence of25-fold excess protein ofunlbebed HDL Prllel ssys were performed with the incubted dipocytes of the sme rt to determine [3H- 4C]HDL uptke by dipocytes In these experiments cells were wshed four times with KRBA, extrcted with 5 ml of ether nd ethnol (3: 1, vol:vol), nd extrcted with S ml diethyl ether The two orgnic phses were combined nd evported nd the oily residues were diluted to 2 ml with hexne nd chloroform (55:45, vob:vol) A subsmple of the redissolved smple (2 tl) ws trnsferred to weighed scintilltion vils, dried down, nd weighed to determine lipid weights Aqusob (NEN) ws then dded nd [3H]- nd [ 4C]cholesterob rdioctivities were mesured in liquid scintilltion counter The remining dipocyte extrcts were dded to Sephdex LH/2 column nd ebuted with hexne nd chloroform (55:45, vol:vol) to seprte free cholesterol nd esterified cholesterol The collected frctions were dried nd dissolved in 5 ml of qusol to determine 3H nd 4C counts ssocited with the esterified cholesterol nd free cholesterol frctions To nlyze the esterified nd free cholesterol in the medium fter incubtion with cells, the medium ws extrcted by the procedure of Fobch et b (27) nd its neutrl lipid clsses were seprted by thin-lyer chromtogrphy (silic gel H) by use of solvent system ofheptne:isopropyl ether:cetic cid (12:8: 8, vol:vol:vol) for 3 mm Esterified cholesterol nd free cholesterol were scrped from the plte nd their 3H nd 4C counts mesured in liquid scintilltion counter [ 251]HDL degrdtion After incubtion with cells, [ 251]HDL degrdtion products (iodopeptides) were determined in the medium by mesuring the nonchboroform extrctble TCA-solubbe rdioctivity Nocell control flsks contining [ 251]HDL nd buffer were incu- Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

3 HIGH-P:S DIET AND HDL-CHOLESTEROL UPTAKE 291 TABLE 1 Rt weights nd dipose morphometry5 E I- E I H- HDL1 (A) EC 1 Sunflower-oil diet Lrd diet Weight (g) 3836 ± ± 69 Ft-pd weight (g) Epididyml 79 ± 3 85 ± 9 Perirenl 8 ± I 2 82 ± 9 Ft-cell weight (Lg) Epididyml 25 ± 2 24 ± 3 Perirenl 36 ± 3 35 ± 4 5i±SEMn=4 bted simultneously to clculte cell-independent degrdtion [ 251]HDL degrdtion in the bsence ofcells (< 1% of totl) ws subtrcted from [ 25IJHDL degrdtion in the presence of cells Dt nlysis EC Frction FC Number H- HDL2 ( EC FC Frction 3H4CHDL2 (B) 3H4HDL2 (D) FC Specific ctivity of [ 25IJHDL ws clculted s cpm/zg of HDL protein The uptke ofesterified cholesterol nd trnsfer of free cholesterol from HDL to the dipocytes were determined by dividing the decys per minute (dpm) of cell-ssocited 3H-lbeled esterified cholesterol nd 4C-lbeled free cholesterol by the respective specific ctivities of3h-esterified cholesterol nd 4C-free cholesterol ofthe lignd (dpm/g esterified cholesterol or free cholesterol) HDL cholesterol uptke or trnsfer to dipocytes ws expressed s microgrms esterified cholesterol or free cholesterol per million cells to give n estimte of mss trnsfer Cellulr uptkes of esterified cholesterol nd free cholesterol were lso expressed proportionlly to the totl mount of esterified or free cholesterol dded to the cells in the incubtion medium To clculte selective uptke of HDL esterified cholesterol by dipocytes, the clculted mss of cell-ssocited esterified cholesterol ws divided by the clculted mss ofcell-ssocited [ 251]HDL protein Similr clcultions were mde to determine HDL-free cholesterol trnsfer to dipocytes reltive to cell-ssocited [ 251]HDL uptke Sttisticl comprisons between dietry tretment groups were nlyzed by unpired I tests E V 1- Results Feeding rts semipurified diets contining 2% (wt:wt) lrd or sunflower oil for 23 ± 3 d resulted in stedy weight gin with no significnt weight differences between the two dietry groups (Tble 1) The weight of the epididyml nd perirenl ft pds, s well s the ft-cell weights ofthe isolted dipocytes, E 4! I EC Number FC 3H 3H 4c Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, Frction Number 1 2 Frction Number 3 FIG 1 Distribution of 3H nd 4C rdioctivity in esterified cholesterol nd free cholesterol oflbeled rt HDL1 nd H DL2 frctions The 3H counts ssocited with esterified cholesterol nd free cholesterol of biologiclly lbeled HDL nd HDL2 re shown in pnels A nd C, respectively Pnels B nd D represent the distribution of3h nd 4C counts in esterified cholesterol nd free cholesterol ofdoubly lbeled HDL1 nd HDL2, respectively

4 292 ZSIGMOND ET AL 6 Sunflower oil EPIDIDYMAL 5) ) v : - to V Toll +co$d 1 5) : 8 U ) Sunflower HDL1 1 oil 125l-HDL1 (tg/mi) (jig/mi) PERIRENAL 5) -7-- = - ) l-HDL1 (rig/mi) i-HDL1 FIG 2 Cell-ssocited uptke of [ 251]HDL1 by rt epididyml nd perirenl dipocytes from sunflower-oil nd lrd-fed rts HDL1 uptke represents the TCA-precipitble 251 counts ssocited with the cells fter the incubtion in the bsence (U) or presence of 125 zg unlbeled HDL () Figures show one representtive experiment from three different experiments Ech vlue represents the men ofduplicte determintions were similr in rts on the sunflower-oil nd lrd diets Perirenb ft cells were lrger thn epididyml cells for both dietry groups Thus, ech diet ws similrly efficient in supporting totb body nd dipose orgn growth The distribution of 3H rdioctivity in esterified cholesterol nd free cholesterol ofbiologiclly lbeled rt HDL is presented in Figure 1 Ninety percent of 3H counts were recovered in esterified cholesterol of HDL1 (Fig 1, A) nd 8 1% of totl 3H rdioctivity ws ssocited with esterified cholesterol of HDL2 (Fig 1, In vitro incubtion of the biologiclly 3H-lbeled HDL with [ 4C]cholesterol in the presence of becithin-cholesterol cyl trnsferse (LCAT) inhibitor resulted in doubly Ibeled HDL1 (Fig 1, B) nd HDL2 (Fig 1, D) prticles Becuse ll the 4C rdioctivity ws recovered in HDL-free cholesterol, 4C counts represent specific trcer of free cholesterol Tritium ws used s the mrker for HDL estenfied cholesterol The dose-response reltionship ofcell-ssocited [ 25I]HDL1 nd HDL2 uptkes by epididyml nd perirenl dipocytes from sunflower- nd lrd-fed rts re shown in Figures 2 nd 3, respectively Curves in ech figure show one representtive experiment from three different experiments Cell-ssocited uptke of [ 251]HDL represents the sum of HDL bound to the surfce nd internlized by the cell Totl cell-ssocited uptke ws mesured in the presence of 5-3 zg/ml 2I-lbeled (pg/mi) 3 blt +cold HDL Prllel ssys were performed in the presence of 25-fold protein excess of unlbeled totl HDL [ 251]HDL1 uptke by dipocytes did not rech sturtion within the concentrtions tested nd it ws significntly inhibited by the ddition of unlbebed HDL At lignd concentrtion of 5 mg/l, the cell-ssocited uptke of [ 25I]HDL1 by epididyml nd perirenb cells ofsunflower-oib-fed rts ws 73 ± 21 nd 144 ± 33 ;L,I 16 cells (1 ± SEM, n = 3), respectively Cell-ssocited uptke of[ 251]HDL1 (5 mg/l) by epididyml nd perirenb cells ws 28 ± 39 nd 144 ± 33 g/b6 cells (n = 3), respectively, for lrd-fed nimls At higher lignd concentrtions the cell-ssocited uptke of [ 251]HDL1 by epididymb nd perirenl dipocytes ws higher fter feedinglrd thn fter sunflower-oil feeding (Fig 2) In n identicl experimentl design, the cell-ssocited uptke of [ 251]HDL2 ws lso nonsturble within 5-3 mg/l HDL2 concentrtion, nd > 5% oftotl uptke ws inhibited by the presence of25-fold excess unlbeled HDL (Fig 3) After sunflower-oil feeding, the cell-ssocited uptke of [ 251]HDL2 (5 mg/l) by epididyml nd perirenl cells ws 1 34 ± 26 nd 244 ± 57 jg/b6 cells (n = 3), respectively The cellssocited uptke of [ 251]HDL2 (5 mg/l) by lrd-fed rts ws 15 ± 9 g/l6 cells (n = 3) by epididyml dipocytes nd 18 ± 9 g/ 16 cells (n = 3) by perirenl cells At Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

5 HIGH-P:S DIET AND HDL-CHOLESTEROL UPTAKE 293 EPIDIDYMAL U, - : to V ), ) C,, _J U, = -to, Sunflower Sunflower oil l-HDL2(tg/ml) 1 oil 125i-HDL2(pg/ml) 3 PERIRENAL ) 6 = 2 ( 4 2 ) Lrd 3 Lrd l-HDL(pg/ml) l-HDL2(pg/mi) FIG 3 Cell-ssocited uptke of [ 25I]HDL2 by rt epididyml nd perirenl dipocytes from sunflower-oil nd lrd-fed rts HDL2 uptke represents the TCA-precipitble 1251 counts ssocited with the cells fter the incubtion in the bsence (U) or presence of 125 tg unlbeled HDL () Figures show one representtive experiment from three different experiments Ech vlue represents the men ofduplicte determintions higher lignd concentrtions, dipocytes from the perirenl dipose region of the sunflower-oil-fed rts showed greter totl cell-ssocited [ 25I]HDL2 uptke thn did cells from the lrd group No similr diet effect ws observed in the epididyml ft cells Tble 2 shows the totl cell-ssocited uptke of [ 251]HDL1 nd [3H]HDL1 esterified cholesterol nd trnsfer of[14c]hdl1 free cholesterol to epididyml nd perirenl dipocytes from sunflower- nd lrd-fed rts Rts on the two different diets were pired ccording to weight nd for ech experiment the ft from one rt ws used for the ssys of [ 251]HDL nd [3H- 4C]HDL cell-ssocited uptkes Mss trnsfer of HDL polipoproteins nd HDL cholesterol ws clculted by dividing the rdioctivities ssocited with the cells by the specific ctivities ofthe trcer isotopes in the lignd nd expressing it per million cells The specific ctivity of 25I-lbeled HDL1 ws dpm/jzg protein nd the specific ctivities for HDL1 esterified cholesterol nd free cholesterol were H dpm/zg nd C dpm/zg, respectively The ddition of25-fold excess protein of unlbeled HDL resulted in 4-65% reduction in totl uptke ofhdl esterified nd HDL free cholesterol uptke (dt not shown) All the vlues nd clcultions reported in this study represent totl cell-ssocited uptkes ofhdl polipoproteins nd HDL esterified nd HDL free cholesterol by ft cells 3 S Totl +cold U Totl +cold Mss trnsfer of HDL2 polipoproteins nd cholesterol shown in Tble 2 were clculted by use ofspecific ctivities of [ 251]HDL2 (2 58 cpm/zg), [3H]HDL2 esterified cholesterol ( I 879 dpm/zg esterified cholesterol), nd [ 4C]HDL2 free cholesterol ( dpm/zg free cholesterol) The presence of 25- fold protein excess of unlbeled HDL reduced esterified-cholesterol uptke nd free-cholesterol trnsfer by 25-6% (dt not shown) No significnt dietry differences were observed in HDL2-esterified-cholesterol uptke by epididyml cells from sunflower- nd lrd-fed nimls In perirenl ft cells, HDL2- esterified-chobesterol uptke ws enhnced fter feeding the polyunsturted ftty cid diet Degrdtion of HDL1 nd HDL2, mesured s the ccumultion of polipoprotein degrdtion products in the postincubtion medium, remined unffected by dietry ft tretment (Tble 2) Becuse cell-ssocited uptke of HDL2 pobipoproteins ws significntly higher thn tht of HDLI polipoproteins, the rtio of HDL polipoprotein degrdtion vs cellssocited uptke ws significntly higher for HDL1 (2-4) thn for HDL2 (5-9) (Tble 2) Clculted mss trnsfer of esterified cholesterol ws I 7-27-fold higher from HDL1 thn from HDL2 but free cholesterol trnsfer from HDL1 nd HDL2 ws not significntly different Becuse HDL1 nd HDL2 contin greter mounts of esterifled thn of free cholesterol, the reltive mount of esterified Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

6 294 ZSIGMOND ET AL TABLE 2 Effect ofdietry lipids on the cell-ssocited uptke ofhdl polipoproteins nd HDL cholesterol nd on the degrdtion ofhdl polipoproteins by isolteddipocytes5 HDL1 Sunflower oil Lrd HDL2 Sunflower oil Lrd g/1o6 cells Epididyml dipocytes Apolipoprotein uptke 45 ± 6 53 ± ± ± 1 Esterified-choiesterol uptke 237 ± ± ± ± 27 Free-cholesterol uptke 18 ± 3 13 ± ± 2 9 ± 2 Apolipoprotein degrdtion 1 17 ± ± ± ± 48 Rtio ofpolipoprotein degrdtion to polipoprotein uptke Perirenl dipocytes Apolipoprotein uptke 77 ± ± ± ± 36 Esterified-cholesterol uptke 245 ± ± ± ±19 Free-cholesterol uptke 1 9 ± ± ± 2 13 ±2 Apolipoprotein degrdtion 32 ± ± ± 45 18±5 Rtio ofpolipoprotein degrdtion to polipoprotein uptke S i± SEM offour experiments with duplicte mesurements nd free cholesterol ssocited with dipocytes cnnot be directly compred by expressing the dt s microgrm of HDL uptke per million cells Therefore, the effect of substrte concentrtion on cellulr uptke or trnsfer ws exmined by clculting the mss of cell-ssocited HDL polipoprotein nd esterified-cholesterol uptke or free cholesterol trnsferred to cells, s percentge ofmount dded to the rection mixture The results demonstrte tht s percentge ofmount dded, five- to sevenfold more HDL1 free cholesterol (Fig 4, thn HDLI esterifled cholesterol (Fig 4, B) ws ssocited with dipocytes fter the incubtion Twelve to 19% of the HDL1- free-cholesterol trcer dded to the incubtion medium ws ssocited with dipocytes wheres only 18-4% of the HDL1 esterified cholesterol ws cell-ssocited The increse in [ 4C]- HDL1-free-cholesterol rdioctivity in the cell corresponded to 2% decrese of 4C counts in the postincubtion medium (dt not shown) Becuse the movement of free cholesterol between HDL nd cells is known to be bidirectionl, the trnsfer of [ 4C]HDL free cholesterol to dipocytes represents only one component of free-cholesterol exchnge The percentge of HDL1 polipoproteins, esterified cholesterob, nd free cholesterol ssocited with dipocytes s percentge of totl mount of HDL1 dded ws not significntly ffected by dietry tretment or dipose region (Fig 4) These results re consistent with those shown in Tble 2, which showed similr cell-ssocited uptkes ofhdl polipoproteins nd cholesterol (expressed s tg,/milbion cells) by dipocytes from sunflower-oil nd lrd-fed rts Becuse ft-cell weights from epididymb nd perirenl regions were similr for the two dietry tretment groups, HDL uptke my be expressed s n bsolute mount tken up per million cells (Tble 2), or expressed s proportion of the mount of HDL esterified nd HDL free cholesterol dded to cells (Fig 4) In Figure 5 HDL2 preferentilly trnsferred two- to fourfold more [ 4CIHDL2 free cholesterol thn [3H]HDL2-esterified cholesterol counts into ft cells when expressed s percentge ofech lipoprotein constituent present in the medium In contrst to HDL1, the cellulr uptke ofesterified cholesterol from HDL2 reltive to the totl mount dded ws significntly higher in perirenb dipocytes from sunflower-fed rts thn from lrd-fed nimls (Fig 5, B) (p < 1) This diet-induced difference ws observed in perirenl but not in epididyml dipocytes Becuse the ft-cell weights of perirenl dipocytes were similr in sunflower-oil (36 ± 3 g) nd lrd-fed rts (35 ± 4 gig; I ± SEM), the effect ofdiet on cellulr uptke of HDL esterifled cholesterol cnnot be ttributed to differences in ft-cell size HDL2 free cholesterol trnsfer to perirenl nd epididyml cells showed only smll increse s result ofpolyunsturted ftty cid feeding(fig 5, The disproportionte uptke of HDL esterified cholesterol nd trnsfer nd/or exchnge of HDL free cholesterol compred with the cell-ssocited uptke of HDL polipoproteins were lso exmined Figure 6 (pnel A) shows 6- to 1-fold greter cellulr uptke of HDL,-esterified cholesterol vs protein when compred with the rtio of esterified cholesterol to protein of the initil HDL1 prticle (4 pg/mg) The preferentil trnsfer of HDL1 free cholesterol vs protein (Fig 6, B) showed 42- to 6-fold increse when compred with the expected vlue ifthe entire HDL1 prticle ws tken up Cellulr uptke of HDL2 esterified cholesterol nd trnsfer ofhdl2 free cholesterol vs cell-ssocited uptke ofhdl2 protein ws 3- to 5-fold nd 9- to 1 1-fold higher, respectively, thn tht predicted from the composition ofthe originl HDL2 prtide (Fig 7) These results suggest selective cellulr uptke of both HDL nd HDL2 cholesterol ester over tht of HDL protein, with HDL1 preferentilly delivering more esterified chobesterob thn HDL2 Plsm HDL cholesterol nd phosphobipid concentrtions of rts fed sunflower oil were significntly different from those of lrd-fed rts Both HDL1 esterified nd free cholesterol incresed in the plsm fter feeding sunflower oil compred with brd (Tble 3) The HDL1 phospholipid concentrtions in the Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

7 HIGH-P:S DIET AND HDL-CHOLESTEROL UPTAKE 295 -I cecs u_ (A) (B) ( u) Epididyml Perireni Epididyml Perireni Phm FIG 4 Cell-ssocited uptke of[ 251]HDL1 (A) nd [3H]HDL1 esterified cholesterol (B) nd trnsfer of [ 4C]HDL1 free cholesterol to ft cells ( s percentge of HDL polipoprotein, esterifled cholesterol, or free cholesterol dded to the incubtion medium Br grphs show i± SEM for four different experiments with duplicte mesurements SF sunflower oil; LA, lrd plsm were lso higher fter feeding the polyunsturted ftty cid diet Becuse both HDL1 free cholesterol nd phospholipids incresed in the sunflower-oil-treted rts, the rtio of free cholesterol to phospholipids remined similr to the rtio in the plsm of lrd-fed nimls In contrst to HDL1, plsm HDL2 cholesterol ws only slightly ffected by diet HDL2 totl cholesterol nd HDL2 esterifled nd free cholesterol showed consistent but insignificnt decrese s result of feeding sunflower oil compred with lrd (Tble 3) Plsm HDL2 phospholipid decresed fter the diet contining more polyunsturted ftty cid The decrese in HDL2 free cholesterol nd HDL2 phospholipids in the plsm ofsunflower-oil-fed rts resulted in rtio of free cholesterol to phospholipid of 13, which ws similr to the rtio in the plsm of rts consuming lrd Sixty to 65% ofthe totl plsm cholesterol ws ssocited with the HDL frctions nd > 8% of HDL1 nd HDL2 totl cholesterol ws in the esterifled form There were no significnt diet-induced differences in plsm HDL1 nd HDL2 triglyceride concentrtions (dt not shown) The mss rtios ofhdl cholesterol to HDL pobipoproteins C 4 -J U (A) (B) #{128},, w - c3c,) 1;;,) ( Epididyml Perlrenl Epididyml -I Perirenl FIG 5 Cell-ssocited uptke of[ 25I]HDL2(A)nd [3H]HDL2 esterified cholesterol (B) nd trnsfer of [ 4C]HDL free cholesterol to ft cells ( s percentge ofhdl polipoprotein, esterified cholesterol, or free cholesterol dded to the incubtion medium Br grphs show 1± SEM for four different experiments with duplicte mesurements SF, sunflower oil; LA, lrd Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

8 296 ZSIGMOND ET AL (A) 5 (29) nd tht the trnsfer process occurs by queous diffusion In more recent studies with rt lutel cells, evidence ws pre- -3- o 3 ) 15 ) 5 2 1= ) U) w -3- : (B) 5 C HDL1 t; I E Selective Cellulr Uptke of HDL1 Esterified Cholesterol Epididyml III- Perirenl Trnsfer of HDL1 Free Cholesterol to cells Epididyml Perirenl FIG 6 Selected cellulr uptke of HDL1 esterified cholesterol (A) nd trnsfer of HDL free cholesterol to cells (B) reltive to cell-ssocited uptke of[125i]hdl1 Br grphs show 1± SEM for four different experiments with duplicte mesurements SF, sunflower oil; LA, lrd nd HDL phospholipids to HDL polipoproteins were lso mesured (Tble 4) HDL1 isolted from the plsm of rts on sunflower-oil diet contined significntly greter mounts of totl nd esterified cholesterol The rtio of phospholipid to protein ws higher fter feeding sunflower oil but this difference ws not sttisticlly significnt The cholesterol nd phospholipid contents of HDL2 reltive to HDL2 polipoprotein were not significntly ffected by dietry ft tretment (Tble 4) The finding tht dietry sunflower oil resulted in n increse in the mss rtio of HDL1 cholesterol to HDL1 protein (Tble 4), s well s n increse in HDL, cholesterol per liter ofplsm (Tble 3), suggests tht polyunsturted ftty cid feeding increses HDL1 cholesterol per prticle nd does not increse the number ofhdl1 prticles in the plsm Discussion In this study we report selective uptke of esterified chobesterol from HDL1 nd HDL2 by rt epididymb nd perirenl dipocytes Furthermore, we present evidence tht dietry lipid composition ffects uptke of HDL esterified cholesterol s ssessed by the unidirectionl ccumultion of 3H rdioctivity in ft cells The interprettion of [ 4C]HDL-free-cholesterol trnsfer to dipocytes is more complex Becuse the methodology empboyed in the present study does not mesure bidirectionl cholesterol flux between HDL nd ft cells, the ccumultion of the free-cholesterol rdioctive trcer in dipocytes my involve free-cholesterol exchnge nd/or net trnsfer Rpid nd energy-independent, free-cholesterol exchnge ws previously reported in humn dipocytes (28) In cultured cells it is well documented tht the trnsfer offree cholesterol is bidirectionl (A) Selective Cellulr Uptke of HDL2 Esterified Cholesterol C 1 - -S4 8 ) In ) 6 4 ;= ) U) w S4 (B) 1 - c,j -J c3 ;; 6 :, :: HDL2 HDL2 Epididyml Perirenl Trnsfer of HDL2 Free Cholesterol to cells Epididyml I, II LI Perirenl FIG 7 Selective cellulr uptke of HDL2 esterifled cholesterol (A) nd trnsfer of HDL2 free cholesterol to cells (B) reltive to cell-ssocited uptke of 251-HDL2 Br grphs show men ± SEM for four different experiments with duplicte mesurements SF, sunflower oil; LA, lrd Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

9 HIGH-P:S DIET AND HDL-CHOLESTEROL UPTAKE 297 TABLE 3 Effect ofdietry ftty cid composition on plsm HDL cholesterol nd phospholipid concentrtions HDL1 Sunflower oil Lrd HDL2 Sunflower oil Lrd mg/lp/sm Totl cholesterol 4299 ± ± 262t 52 1 ± ± 73 Esterified cholesterol ± ± 234t 433 ± ± 54 Free cholesterol 548 ± ± 34t 88 ± ± 23 Phospholipids 4278 ± ± l32t 637 ± ± llt 1± SEM ofthree duplicte determintions t Significntly different from vlues ofsunflower-oil-fed rts, p < 5 sented tht free cholesterol from HDL my be tken up by both receptor-dependent nd receptor-independent processes (6) The present study lso showed tht the mount of HDL esterifled cholesterol nd HDL free cholesterol ssocited with dipocytes fter the incubtion ws not in the sme proportion s tht present in the HDL prticles HDL1 nd HDL2 prticles re enriched in esterified cholesterol compred with free cholesterol, which might explin the finding tht mss trnsfer of esterified cholesterol exceeded tht of free cholesterol (Tble 2) However, when compred with the mss of free cholesterol dded to the incubtion medium, proportiontely three- to sixfold more free cholesterol thn esterified cholesterol ws trnsferred to dipocytes (Figs 4 nd 5) Preferentil utiliztion of free cholesterol compred with esterified cholesterol ws previously reported in rt grnubos cells (7), drenocorticl cells (3), nd lutel cells (6) The uptke of HDL esterified cholesterol by humn dipocytes is selective process (1) with po A-I specificity (I 1) In rt dipocytes HDL binding ppers to be medited by protein-binding site (18) but it is not known whether HDL-polipoprotein binding is involved in cholesterol trnsfer Becuse of recent studies tht demonstrted n effect of dietry ftty cids on HDL2 binding to plsm membrnes ofrt dipocytes (2), s well s on the ftty cid composition of membrne phospholipids, it ws of interest to exmine the effect of such diets on HDL-cholesterol trnsfer to dipocytes The results of the membrne-binding studies, which showed higher [l25i] HDL2 binding to perirenl nd epididyml plsm membrnes fter feeding with polyunsturted vs sturted nd monounsturted ftty cids (2), re lso consistent with higher HDL2 uptke following sunflower-oil feeding by perirenl but not epididyml dipocytes (Fig 3) Thus dietry lipid-dependent chnges in cellulr uptke of[ 25IJHDL2 cnnot be entirely ttributed to ltertions in membrne composition becuse other dipose-region-specific fctors my lso ply role (Cell-ssocited uptke of [ 25I]HDL includes both HDL binding to the plsm membrne nd internliztion, in contrst to binding of HDL prticles to purified plsm membrne preprtions, which represents only the first step of HDL interction with cells) Concurrent with the increse in the cell-ssocited uptke of [ 25I]HDL2 polipoproteins, esterifled-cholesterol uptke ws lso higher fter polyunsturted ftty cid feeding (Tble 2, Fig 5; p < 1) The diet-rebted chnges in esterifled-cholesterob uptke cnnot be ttributed to differences in ft-cell weight becuse dipocyte size in sunflower-oil nd lrd-fed rts ws similr (Tble 1) The observtion tht HDL2 cholesterol uptke by perirenl but not epididyml dipocytes responds to dietry ft tretment is consistent with results of other studies, which showed preferentil increse in the size nd number of perirenl dipocytes fter high-ft feeding (31) The mechnism ofenhnced HDL2 esterified cholesterol uptke by perirenl dipocytes fter polyunsturted ftty cid feeding is not known If the trnsfer of HDL esterified cholesterol is receptor dependent, then diet-induced chnges in membrne-lipid composition my influence the ctivity ofthis puttive receptor Enrichment of membrne phospholipids with polyunsturted ftty cids my result in n increse in membrne fluidity, which in turn my llow for protein conformtionl chnges required for optiml ctivity Alterntively, more subtle compositionl chnges in the nnulr lipid microenvironment or lipid-lipid interctions my directly ffect Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216 TABLE 4 Effect ofdietry ftty cid composition on plsm HDL composition* HDL1 Sunflower oil Lrd HDL2 Sunflower oil Lrd Totl cholesterol Esterified cholesterol Free cholesterol Phospholipids 5 1 ± 7 47 ± 7 5 ± 1 42 ± 3 41 ± 2* 38 ± 2t 3 ± 1 35 ± 3 27 ± 2 23 ± 2 4 ± 1 27 ± 2 23 ± 1 22 ± 1 2 ± 1 21 ± 1 * i± SEM ofthree experiments with duplicte mesurements Mesured in microgrms per microgrm protein t Significntly different from plsm HDL1 composition ofsunflower-oil-fed rts, p < 5

10 298 ZSIGMOND ET AL HDL binding to the dipocyte membrnes s well s chobesterol trnsfer between HDL nd ft cells In other cell systems, ltering membrne phospholipid composition by phospholipse tretment ws ssocited with incresed HDL esterified cholesterol uptke (32) Incresed membrne unsturtion hs lso been implicted in enhnced bipoprotein bipse ctivity in liver (33) s well s in higher heptic cycloenzyme A cholesterol cyltrnsferse (ACAT) ctivity (34) Thus, pobyunsturted ftty cids pper to ffect the ctivities of number of membrne-ssocited proteins The chnges in HDL polipoprotein nd esterifled cholesterol uptke by dipocytes fter high-ft diets ofdifferent composition were significntly greter for HDL2 thn for HDL1 These findings re reminiscent of previous studies tht used dog dipocytes, in which dietry-lipid modifictions resulted in significnt chnges in HDL2 but not in cellulr uptke of HDLC (35) HDL1 prticles in rts re similr to HDLC prticles from cholesterol-fed dogs becuse they re enriched in cholesterol ester nd po E, compred with the smller HDL2 prticles, which re preferentilly ffected by diet in both niml species These studies of cell-ssocited uptke of HDL by dipocytes re consistent with the observtions ofangelin et b (36), which showed tht the po E receptor of dog liver membrnes is not significntly ffected by dietry mnipultions HDL2 binds with lower ffinity to dipocyte plsm membrnes thn does HDL1 (2), suggesting tht HDL1 nd HDL2 prticles bind to seprte receptor sites or to different conformtions of binding domin of the dipocyte plsm membrne It is possible tht the low-ffinity HDL2 binding site is selectively enhnced by the increse in membrne polyunsturtion in similr fshion s suggested for the insulin receptor of rt dipocytes (37) The diet-induced chnges in membrne-lipid composition my be responsible for HDL2- esterified-cholesterol uptke by ltering HDL2 binding or by modifying the ctivity of cholesterol-ester trnsfer protein ssocited with ft cells Although both HDL1 nd HDL2 prticles re cpble of debivering esterified cholesterol to dipocytes, they do so t different rtes Reltive differences in polipoprotein composition of HDL1 nd HDL2 my determine the binding nd cholesterol-trnsport cpcities ofthe two subclsses Although po A-I is the mjor pobipoprotein ofboth frctions, HDL2 is enriched in po AIV compred with HDL1, which is enriched in po E Previous studies showed tht po A-I binding is involved in cholesterol-ester uptke (6 nd 1 1) but this does not exclude role for po AIV in the medition of cholesterol trnsport (38) Recent studies by Pittmn et b (39) demonstrted tht the density ofthe HDL prticles my lso be importnt in cholesterol delivery to cells HDL, is lrge, cholesterolester-enriched prticle (Tble 1) tht is formed in the plsm comprtment from HDL2 by LCAT (4), nd in terms of mss trnsfer it my be predominntly involved in cholesterol-ester delivery (Tble 2) Cell-ssocited uptke nd degrdtion of HDL1 polipoproteins differed from tht of HDL2 prticles Cell-ssocited uptke of HDL2 polipoproteins ws significntly higher thn HDL1 polipoprotein uptke by epididymb nd perirenl dipocytes (Tble 2) Despite the higher cell-ssocited uptke of HDL2 polipoproteins by dipocytes, the selective uptke of esterified cholesterol ws greter from HDL1 thn from HDL2 (Figs 6 nd 7), suggesting tht esterified-cholesterob uptke does not require internliztion of the HDL holo prticle In contrst, polipoprotein degrdtion reltive to cell-ssocited uptke ws significntly higher for HDL1 thn for HDL2 prticles in both epididyml nd perirenb ft cells (Tble 2) It is not cler yet whether the higher degrdtion of HDL1 polipoproteins contributes to the enhnced selective trnsfer of esterified cholesterol to dipocytes but HDL-polipoprotein degrdtion t the prticle surfce could fcilitte trnsfer of core lipids from HDL to cells In this study HDL1-estenfied- nd -free-cholesterol concentrtions incresed significntly in plsm in rts fter feeding polyunsturted ftty cids thn fter feeding diets contining sturted or monounsturted ftty cids The elevted concentrtions of plsm HDL1 cholesterol fter sunflower-oil feeding (Tble 3) coincided with the increse in the rtio of HDL1 esterified nd free cholesterol to HDL1 protein (Tble 4) These results suggest tht the effect ofpolyunsturted ftty cid-rich diets is n increse in the cholesterol content ofhdl1 prticles rther thn n increse in the number of HDL1 prtides in the plsm The physiologicl effects of diets rich in polyunsturted ftty cids on cholesterol metbolism in rts re still open to specultion The enhnced removl of esterified cholesterol from HDL2 by interctions with dipocytes from polyunsturted ftty cid-fed rts my result in HDL prticles tht re depleted ofesterified cholesterol nd hence my function more effectively in reverse cholesterol trnsport from tissues Incresed free-cholesterol efflux from peripherl cells, including dipocytes, my promote cholesterol esterifiction in plsm nd the formtion ofesterified-chobesterol-rich HDL1 prticles The concomitnt enrichment ofhdl1 with po E would trget the prticles for uptke by the liver, thus resulting in cholesterol excretion This hypothesis is consistent with the findings of ebevted HDL1 cholesterol concentrtions in the plsm of sunflower-oil-fed rts Thus, dipose tissue my ct s n intermedite storge orgn for cholesterol nd serve unique role in converting esterified cholesterol to free cholesterol, hence fcilitting the trnsport ofcholesterol to the liver for excretion In conclusion, reltive to the cell-ssocited uptke of HDL polipoproteins, rt dipocytes preferentilly tke up esterified cholesterol from both HDL1 nd HDL2 Diets rich in polyunsturted ftty cids significntly enhnce the uptke of HDL2 esterified cholesterol by perirenb dipocytes when compred with diets high in sturted or monounsturted ftty cids The effect of dietry ft on the uptke of cholesterol ester displyed regionl vritions nd ws more pronounced for HDL2 thn for HDL3 subfrctions Finlly, dietry ft tretment known to modify membrne structure nd HDL binding to dipocyte membrnes lso ppers to modulte HDL esterified cholesterol uptke by ft cells nd HDL cholesterol concentrtions in plsm El We re grteful to Shyl Berll nd Lur Sheu for their excellent technicl ssistnce Specil thnks to Christopher Prrish for helpful nd interesting discussions References 1 Crew TE, Koschinsky T, Hyes SB, Steinberg D A mechnism by which high density lipoprotein my slow the therogenic process Lncet l976;l:13l5-7 Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216

11 HIGH-P:S DIET AND HDL-CHOLESTEROL UPTAKE Orm JF, Albers JJ, Cheung MC, Biermn EL The effect of subfrctions of HDL on cholesterol efflux from cultured fibroblsts J Biol Chem 198 l;256: Andersen JM, Dietschy JM Reltive importnce of high nd low density lipoproteins in the regultion of cholesterol synthesis in drenl glnd, ovry, nd testis ofthe rt J Biol Chem l978;253: Glss C Pittmn RC, Civen M, Steinberg D Uptke of high density lipoprotein-ssocited po A-I nd cholesterol esters by 16 tissues ofthe rt in vivo nd by drenl cells nd heptocytes in vitro J Biol Chem l985;257: Leitersdorf E, Stein, Eisenberg 5, Stein Y Uptke of rt plsm high density lipoprotein subfrctions lbeled with [3H]cholesteryl linoleyl ether or with 251 by cultured rt heptocytes nd drenl cells Biochim Biophys Act 1984;796: Rjn VP, Menon KMJ Differentil uptke nd metbolism of free nd estenfied cholesterol from HDL in the ovry Biochim Biophys Act l988;959: Nestler JE, Bmberger M, Rothblt GH, Stuss JF Metbolism of HDL reconstituted with 3H cholesteryl ester nd 4C cholesterol in the rt with specil reference to the ovry Endocrinology 1985; 117: Angel A, Fong B Lipoprotein interctions nd cholesterol metbolism in humn ft cells Angel A, Hollenberg CH, Roncri DAK, eds In: The dipocyte nd obesity: cellulr nd moleculr mechnisms New York: Rven Press, Fong BS, Rodrigues P, Slter AM, Yip BP, Gregg RE, Angel A Chrcteriztion ofhigh density lipoprotein binding to humn dipocyte plsm membrnes J Clin Invest 1985;75: Despres JP, Fong BS, Jimenez J, Julien P, Angel A Selective uptke of HDL cholesterol ester by humn ft cells Am J Physiol l988;254:e Fong BS, Slter AM, Jimenez J, Angel A The role of polipoprotein Al nd polipoprotein All in high density binding to humn dipocyte plsm membrnes Biochim Biophys Act 1987;92: Angel A, Jimenez J, Despres JP, Julien P Zsigmond E, Fong B Effects ofobesity nd ft cell size on lipoprotein binding to humn dipocytes Berry EM, Blondheim SM, Elihou HE, Shfir E, eds In: Recent dvnces in obesity reserch London: John Libbey nd Co 1986: DespresJP, Fong BS, Julien P Jimenez J, Angel A Regionl vrition in high density lipoprotein metbolism in humn ft cells: effect ofcell size Am J Physiol l987;252:e Spdy DK, Dietschy JM Dietry sturted triglycerides suppress heptic LDL receptor ctivity in the hmster Proc Ntl Acd Sci USA 1985;82: Weisweiler P, Jnetschek P, Schwndt P Influence of polyunsturted ftty cids nd ft restriction on serum lipoproteins in humns Metbolism l985;34: Mttson FM, Grundy SM Comprison of the effects of dietry sturted, monounsturted nd polyunsturted ftty cids on plsm lipids nd lipoproteins in mn J Lipid Res l985;26:l Kruse BR, Hrtmn AD Adipose tissue nd cholesterol metbolism J Lipid Res l984;25:97-l 1 18 Zsigmond E, Fong B, Angel A Chrcteriztion of high density lipoprotein binding to rt dipocytes nd dipocyte plsm membrnes Biochem Cell Biol l988;66: Tbs I, Tll AR Mechnism of the ssocition of high density lipoprotein3 with endothelil cells, smooth muscle cells nd fibroblstsj BiolChem l984;259:l Zsigmond E, Fong B, Angel A HDL2 binding to dipocyte plsm membrnes is enhnced following high polyunsturted ft feeding Clin Res l987;35:3 (bstr) 2 1 Rodbell M Metbolism ofisolted ft cells J Biol Chem l967;242: DespresiP, Bouchrd C, Svrd R, Trembly A, Mrcotte M, Theriult G Effect of 2 week endurnce trining progrm on dipose tissue morphology nd lipolysis in mn nd women Metb Clin Exp 1984; 33: Jnsen H, Schoonderwoerd OC, Dlling-Thie GM Seprtion of rt plsm high density lipoprotein subfrctions by density grdient centrifugtion nd the effect of incubtion on these frctions Biochim BiophysAct l983;754: Shephrd J, Bedford DK, Morgn HG, Scott E Rdio-iodintion ofhumn low density lipoprotein: comprison offour methods Clin Chim Act 1976;66:97-l9 25 Kruse BR, Hrtmn AD Quntifiction ofdipoyte free nd esterified cholesterol using liquid gel chromtogrphy J Lipid Res 1978; 19: Lowry OH, Rosenbrough NJ, Frr AL, Rndll JJ Protein mesurement with folin phenol regent J Biol Chem 195 1; 193: Folch J, Lees M, Slone Stnley GH A simple method forthe isoltion nd purifiction of totl lipids from niml tissues J Biol Chem 1957;266: Angel A, Yuen R, Nettleton JA Exchnge of free cholesterol between LDL nd humn dipocytes Cn J Biochem 1981; 59: Rothblt OH, Phillips MC Cholesterol efflux: mechnism nd regultion Angel A, Frohlich J, eds In: Advnces in experimentl medicine nd biology; lipoprotein deficiency syndromes New York: Plenum Press, 1986: Owynne JT, Mhfee DD Esterifiction of cholesterol in HDL decreses its bility to support ACTH-stimulted steroidogenesis by rt drenocorticl cells J Biol Chem 1987;262: Fust IM, Johnson PR, Stern JP Diet-induced dipocyte number increse in dult rts: new model of obesity Am J Physiol 1978;235:E Collet X, Perret B, Chollet F, Hullin F, Chp H, Douste-Blzy L Uptke of HDL unesterified nd esterified cholesterol by humn endothelil cells: modultion by HDL phospholipolysis nd cell cholesterol content Biochim Biophys Act l988;958: Hulsmnn WC, Kort Wi Sturted ft feeding, hyperlipidemi nd hyperinsulinemi Biochim BiophysAct l983;754: Spector AA, Kduce TL, Dne RW Effect ofdietry ft sturtion on cylcoenzyme A: cholesterol cyltrnsferse ctivity ofrt liver microsomes J Lipid Res 198;2 1: Fong SB, Despres JP, Julien P, Angel A Interctions ofhigh density lipoprotein subclsses (HDL2 nd HDL) with dog dipocytes: selective effects ofcholesterol nd sturted ft feeding J Lipid Res 1988;29: Angelin B, Rviol CA, Innerrity TL, Mhley RW Regultion of heptic lipoprotein receptors in the dog Rpid regultion of polipoprotein B-E receptors, but not ofpolipoprotein E receptors by intestinl lipoproteins nd bile cids J Clin Invest l983;7 1: Vn Amelsvoort JM, Vn der Beck A, Stm JJ Effects ofthe type ofdietry ftty cid on the insulin receptor function in rt epididyml ft cells Ann Nutr Metb 1986; 3: Stein, Stein Y, Lefevre M, Roheim PS The role of polipoprotein AIV in reverse cholesterol trnsport studied with cultured cells nd liposomes derived from n ether nlog of phophtidylcholine Biochim Biophys Act 1986;878:7-l3 39 Pittmn RC, Glss CK, Atkinson D, Smll DM Synthetic high density lipoprotein prticles J Biol Chem 1987;262: Eisenberg S High density lipoprotein metbolism J Lipid Res 1984;25: Downloded from jcnnutritionorg t PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 216