Effects of simvastatin on the metabolism of polyunsaturated fatty acids and on glycerolipid, cholesterol, and de novo lipid synthesis in THP-1 cells

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1 Effects of simvsttin on the metbolism of polyunsturted ftty cids nd on glycerolipid, cholesterol, nd de novo lipid synthesis in THP-1 cells Ptrizi Rise,' Cludio Colombo, nd Cludio Glli Institute of Phrmcologicl Sciences, University of Miln, vi Blzretti 9, Milno, Itly Abstrct In the monocytic THP-1 cells, the 3hydroxy-3- methylglutvl-coenzyme A (HMG-CoA) reductse inhibitor simvsttin (5 p ~ enhnces ) the conversion of exogenous linoleic (18:2 n-6) nd eicospentenoic (20:5 n-3) cids to their long-chin polyunsturted ftty cid (LGPUFA) derivtives, nd this effect is ssocited with chnges in the desturtion steps. In ddition, formtion of monounsturted ftty cids from endogenously synthesized precursors is incresed. These metbolic chnges led to elevted LGPUFA nd ftty cid (FA) unsturtion in cells. The effects of simvsttin on FA metbolism re ssocited with incresed synthesis of triglycerides from glycerol. The dose-effect reltionships for the ctivity of simvsttin on totl linoleic cid (LA) conversion nd cholesterol synthesis revel tht enhncement of PUFA metbolism is lredy mximl t 0.5 p~ simvsttin, wheres cholesterol synthesis is further inhibited by concentrtions of simvsttin up to 5 p ~ The. effects of 5 p~ simvsttin on PUFA metbolism re prtilly prevented by mevlonte (1 mm) nd gernylgerniol (5 p ~ but ) not by frnesol (10 p~).il These dt indicte tht HMGCoA inhibitors hve profound effects on PUFA metbolism, nd tht the pthwys for cholesterol nd PUFA synthesis re mutully modulted.-rise, P., C. Colombo, nd C. Glli. Effects of simvsttin on the metbolism of polyunsturted ftty cids nd on glycerolipid, cholesterol, nd de novo lipid synthesis in THP-1 cells. J. Lipid Res : Supplementry key words THP-1 cells ftty cid desturtion nd elongtion simvsttin mevlonte isoprenoids lipid synthesis from glycerol nd cette Competitive inhibitors of 3-hydroxy-3methylglutrylcoenzyme A (HMGCoA) reductse (1) re widely used in the tretment of hypercholesterolemi. Although their ction is considered rther specific for the inhibition of cholesterol synthesis, nd ftty cid metbolism hs been generlly reported to be unffected (2, 3), recent studies hve shown tht HMGCoA reductse inhibitors hve dditionl effects. They enhnce ftty cid synthesis nd peroxisoml ctivity (4) nd induce elev- tion of rchidonic cid (AA) nd thromboxne production (5) in cultured cells. Furthermore, plsm nd erythrocyte long-chin polyunsturted ftty cids (LC- PUFA) re modified by the use of HMGCoA reductse inhibitors, in clinicl studies (6, 7). As it hs been shown tht the microsoml cholesterol content my influence the cyl-coa desturses (8), the rte-limiting enzymes of PUFA synthesis, this my be relevnt to the bove findings. In ddition, chnges in membrne lipids, resulting from complex modifictions of lipid metbolism, my be prtly involved in the reported effects of lovsttin, potent HMGCoA reductse inhibitor, on membrne fluidity nd ggregtion of pltelets in hypercholesterolemic ptients (9). It ppers, therefore, tht inhibition of HMG-CoA reductse results in wide spectrum of effects on lipid metbolism, but their reltionship to their primry mechnism of ction is still uncqrtin. Altkough HMGCoA reductse inhibitors ffect the formtion of the LGPUFA AA from its precursor linoleic cid (LA), in the n-6 series (5), it is not cler whether this effect pplies generlly to the desturtion of FA in other metbolic series, nd of endogenously synthesized FA. In ddition, the possible effects of HMGCoA reductse inhibitors on the formtion of 22- crbon PUFA, pthwy involving peroxisoml bxidtion (10) nd the concentrtion-effect reltionships Abbrevitions: AA, rchidonic cid; CE, cholesteryl esters; DAG, dicylglycerol; FA, ftty cids; FFA, free ftty cids; LA, linoleic cid; LGPUFA, long-chin polyunsturted ftty cids; MAG, monocylglycerol; MUFA, monounsturted ftty cids; MVA, mevlonic cid; PBS, phosphte-buffered sline; PL, phospholipids; PUFA, polyunsturted ftty cids; SAT, sturted ftty cids; TG, triglycerides; TL, totl lipids. HMGCoA, %hydroxy-3methylglutryl coenzyme A. 'To whom correspondence should be ddressed. Journl of Lipid Reserch Volume 38,

2 for the ctivity on FA nd cholesterol, s well s on other metbolic pthwys, hve not been explored in detil. We hve therefore investigted the ctivity of the potent HMGCoA reductse inhibitor simvsttin on the bove spects in the humn monocytic cell line THP-1. These cells re widely used in studies on lipid/lipoprotein metbolism (1 1) nd re ble to ctively desturte nd elongte exogenous n-6 ftty cids to long-chin derivtives, including the synthesis of 22 crbon PUFA (12). Simvsttin is chemicl derivtive of lovsttin (13) nd both re mong the most potent inhibitors of cholesterol synthesis in cultured cells (14). Our study confirms tht simvsttin enhnces the conversion of LA to AA, nd shows lso tht the synthesis of 22 : 6 (docoshexenoic cid, DHA) from 20: (eicospentenoic cid, EPA) nd the production of oleic cid (18: 1) from endogenously synthesized FA, using cette s substrte, re enhnced. Chnges in FA metbolism re ssocited with enhnced incorportion of glycerol in triglycerides. Finlly, mximl effects of simvsttin on PUFA metbolism re ttined t concentrtions lower thn those required for mximl effects on cholesterol synthesis. Chemicls MATERIAL AND METHODS Linoleic cid, essentilly ftty cid-free bovine serum lbumin, RPMI 1640 medium, penicillin/streptomycin, mevlonic cid, ll-trns gernylgerniol, nd frnesol were from Sigm (St. Louis, MO); fetl clf serum (FCS), Pmercptoethnol were from Msci Brunelli S.p.A. (V.le Monz 272, Milno, Itly); solvents nd silic-gel 60R were from E. Merck (D-6100 Drmstdt, Germny). [ 1-14C]LA (specific ctivity 53 pci/pmol), [ 1 (3)-3H]glycerol (specific ctivity 3.1 Ci/mmol) nd [1-I4C]cetic cid sodium slt (specific ctivity 60 mci/ mmol) were purchsed from Amershm; [ 1-14C]EPA (specific ctivity 52 mci/ mmol) from DuPont-NEN. Simvsttin in lctone form ws from Merck, Shrp & Dohme Reserch Lbortories (Woodbridge, NJ). Cell culture THP-1 cells were grown in RPMI 1640 medium with 10% FCS, 100 pg/ml penicillin, 100 IU/ml streptomycin, nd 1 % Pmercptoethnol t 37 C in humidified tmosphere of 5% COL, nd 95% ir. Cells tht were not induced by incubtion with phorbol esters were used in the vrious experiments. Experimentl design The content of severl flsks of THP-1 cells ws centrifuged t 200 gfor 10 min; the pellet ws resuspended in RPMI medium without FCS nd the cells were counted in Cell Counter R Model 2 M (Coulter Instrument, Miln, Itly). The concentrtion of cells ws djusted to lo6 cells/ml. Simvsttin, used in opened form t different (0.5-5 p ~ concentrtions ) (15), ws dded t this time. After 24 h, rdioctive substrtes, [ l-"c]la 0.1 pci/ml or [I4C]EPA 0.08 pci/ml or [3H]glycer~1 0.5 pci/ml or [ I4C]cetic cid sodium slt 1 pci/ml, were dded for dditionl 24 h. Time course experiments for the conversion of LA were crried out by incubting THP-1 cells with [1-I4C]LA 0.1 pci/ml, for 4, 8, 16, nd 24 h. In ll experiments FA nd glycerol were dded dissolved in 0.5% ethnol (finl volume). At the end of the incubtion period cells were centrifuged nd resuspended in PBS; before the second nd lst wshing the pellet ws resuspended in given volume of PBS nd cells were counted. Cell tretment with cholesterol precursors Mevlonic cid ( p ~, ll-trns ) gernylgerniol (5 p~), nd frnesol (10 p ~ were ) dded, t the indicted concentrtions, t beginning of the experiment, without or with simvsttin. Lipid extrction Lipid extrction ws crried out ccording to the method of Folch, Lees, nd Slone Stnley (16). The lipid concentrtion of the smples, dissolved in chloroform-methnol 2: 1, ws determined by using microblnce (C-31, Chn Instruments, Cerritos, CA) (17). Determintion of cell TG content Levels of TG in THP-1 cells were determined on lipid extrcts by the use of commercil kit (F. Hoffmnn- L Roche Ltd., Bsel) nd vlues were expressed s percent of totl lipids. Anlysis of FA-ssocited rdioctivity The determintion of the rdioctivity incorported in individul FA ws crried out by HPLC. The FA methyl esters, prepred by trnsesterifiction with 3 N methnolic-hc1 (Supelco, Bellefonte, PA), were seprted by two-solvent system t flow rte of l ml/min: solvent A cetonitrile nd solvent B wter, in ccord with Moore, Yoder, nd Spector (18). The column used ws LiChrospher 100, RP-18 (5 pm) (E. Merck, D-6100 Drmstdt, Germny); the HPLC used ws Jsco Model 880-PU (Jpn Spectroscopic, Tokyo 192, Jpn). The detection of rdioctivity ssocited to FA methyl esters ws chieved by using n on-line rdiodetector (Flo-one bet A 200, Rdiomtic Instruments nd Chemicls, A Cmberr Compny, Tmp, FL) equipped with 500 pl flow cell, using Flo-Scint A s scintilltion fluid Journl of Lipid Reserch Volume 38, 1997

3 Seprtion of lipid clsses The lipid clsses (PL, TG, CE, DAG, FFA) were seprted by monodimensionl TLC using silic-gel 60R pltes nd s mobile phse hexne-diethyl ether-cetic cid 70: 30: 1.5; DAG, free cholesterol, nd MAG were seprted by monodimensionl TLC using silic-gel 60R pltes nd chloroform-methnol 98: 2. Lipids were detected on pltes dried under N2 by exposure to iodine vpors; the spots were scrped from pltes nd the rdioctivity ws detected fter ddition of 1 ml methnol-wter 1 : 1 nd 10 ml of scintilltion fluid. Seprtion of FA ccording to unsturtion levels Seprtion of sturted, mono, nd polyunsturted FA methyl esters ws crried out by monodimensionl TLC on silic-gel 60R pltes impregnted with 15% AgNO,, using hexne-diethyl ether 95: 5 s developing solvent. Spots, loclized using rdio scnner LB Dunnschicht-scnner 2 (Berthold, D 7547 Wildbd 1, Germny), were scrped into vils nd the rdioctivity ws detected fter ddition of 1 ml methnol-wter 1 : 1 nd 10 ml of scintilltion fluid. Smples used for further HPLC seprtions were extrcted from silic-gel (14). After solvent evportion, smples were redissolved in solvents for chromtogrphic nlysis, nd liquots were injected in HPLC. FA seprtion in GC FA methyl esters, prepred from TL extrcts, were nlyzed on GC (Dni 8610, Monz, Itly) using the cpillry column Supelco Omegwx TM 320 (Supelco, Bellefonte, PA), 30 m, 0.32 I.D., 0.25 pm film, nd the temperture ws progrmmed from 120 C to 220 C. Peks were identified by using pure reference compounds nd quntified by the use of n internl stndrd (19 : 0), dded to the smples. sttisticl nlysis Significnce of differences, when compring vlues in control nd simvsttin-treted cells, ws ssessed by the use of Student's t-test. RESULTS Effects of simvsttin tretment on the FA composition of TI- -1 cell lipids Cells incubted in the presence of 5 ~ L simvsttin M showed significnt, lthough quntittively modest, chnges in the FA profiles of TL (Tble 1). In generl, TABLE 1. Mjor polyunsturted ftty cids nd rtios in control nd simvsttin-treted THP-1 cells Ftty Acids 18: 1 20: 3 n-9 20:3 n-6 20: 4 n-6 22 : 4 n-6 22 : 5 n-6 20 : 5 n-3 22 : 5 n-3 22:6 n-3 SAT MUFA PUFA n-6 n-3 UI Control ? t ? % weight 33.45? 0.98" " " " " " " " " " * 0.53" " SAT, sturted ftty cids; MUFA, monounsturted ftty cids; PUFA, polyunsturted ftty cids; UI, unsturtion index = sum of percentge levels X number of double bonds. Vlues re weight percentge levels nd represent the verge? SE of four independent determintions. "Significntly different from controls t P < sturted FA tended to be reduced by simvsttin, but the difference ws significnt only for the totl SAT. PUFA of the n-6 nd n-3 series were ll rther low in these cells, when compred to blood monocytes (12); neverless, in the treted cells there ws mrked increment of the mjor products in both series, nmely 20: 4 n-6 nd 22:6 n-3. Elevtion of n-9 ftty cids, in ddition to 18 : 1, included 20 : 3, product of desturtion nd elongtion of oleic cid nd mrker of essentil ftty cid deficiency in vivo. As consequence of the elevtion of most PUFA, the unsturtion index (UI) of cell lipids ws significntly incresed. Effects of different concentrtions of LA on its conversion nd esterifiction In order to explore the metbolic chnges responsible for the observed modifictions in FA composition, we hve investigted the synthesis of LCPUFA from lbeled precursors. We hve first studied the conversion of LA to AA nd the FA esterifiction into lipid clsses t two very different substrte concentrtions: 2 nd 50 ~LM. We found tht while the proportion of the substrte being converted, t 24 h incubtion, ws not ffected by its concentrtion ( % with 2 p~ vs Ir 0.6% with 50 ~LM LA), the reltive esterifiction into different lipid clsses ws mrkedly modified. At 2 p~ LA, the reltive proportions of incorportion were Risk, Colombo, nd Glli Sivstijn nd lipid metbolism in monocytic THP-1 cells 1301

4 401 T TABLE 2. Product-precursor rtios t the desturtion nd rlongtion steps in the conversion of LA to AA 18:3/18:2 (A6 desturse) :4/20:3 (A5 desturse) 0.86 t " 20:3/18:3 (C18 elongse) 3.57 t :4/20:4 ((220 elongse) t t , linoleic cid; AA, rchidonic cid. Vlues re the verge t- SE of 4 experiments. "Significntly different from controls t P < :3 20:3 20:4 22:4 Fig. 1. Rdioctivity recovered in individul ftty cids of'n-6 series s percentge of totl rdioctivity, in cells incubted with ["GILA. Vlues re the verge 2 SE of dt from four different experiments; control; H simvsttin, 5 PM;, significntly different from control t P > % in PL, 6.6% in TG, with less thn 0.5% in DAG, FFA nd CE, wheres t 50 p~ the proportions were 36.3% in PL, 55% in TG, 4.3 in DAG, 2.8% in FFA, nd 1.4% in CE. These dt prompted us to use the 2 p~ LA concentrtion for studies on the effects of simvsttin s the mjor esterifiction occurred in PL nd smll incorportion occurred in the most lbile lipid pools (DAG nd FFA). Effect of simvsttin on the conversion of LA The concentrtion of the drug nd the time period of incubtion were chosen on the bsis of vilble dt (15), nd the initil set of experiments ws crried out with 5 p~ simvsttin. The incubtion of cells with lbeled LA, in the presence of this concentrtion of the drug did not modify the totl incorportion of the substrte in cell lipids (2,854? 117 X lo3 cpm/mg TL in control vs. 2, X 10' cmp/mg TL in simvsttin-treted cells), but tended to shift the reltive incorportion of LA nd metbolites in TG versus PL from TG/PL rtio of to vlue of % (nonsignificnt difference). Mesurements of the conversion of LA to its metbolites showed tht tretment with simvsttin, while it did not enhnce the totl rdioctivity incorported into the cells, resulted in significnt increment of the totl conversion of LA to its derivtives, from 29.5? 1.5% to 53.1? 3.2%, nd in mrkedly incresed incorportion of rdioctivity in 20: 4 (Fig. 1). Mesurements of product/precursor rtios for the desturtion nd elongtion rections (Tble 2) indicte tht simvsttin enhnced exclusively the A5 desturtion step, with no involvement of the other steps. The clculted net AA synthesis in control nd simvsttin treted cells ws 3.3 nd 9.2 nmol/mg cell TL, respectively. The reltive lbeling of individul FA in cell PL nd TG (Tble 3) indictes tht greter increments of AA, in treted cells, occurred in PL rther thn in TG. Lbeling of 22:4 ws lso mrkedly incresed in PL, by simvsttin, wheres lbeling of this FA ws not detectble in TG in both control nd treted cells. In order to explore whether the chnges of lbeling of LA-derived products were relted to possible chnges in their turnover rtes rther thn in their biosynthesis, we hve lso crried out time course experiment in which the formtion of products ws monitored between 4 nd 24 h incubtion of the substrte, in control nd simvsttin-treted cells. Figure 2 reports the vlues only for the incorportion of rdioctivity in 20 : 3 nd 20 : 4 s these FA underwent the most prominent modifictions in control nd treted cells. In controls mximl totl conversion of LA occurred lredy t 4 11 (37.2% conversion in simvsttin vs. 20.5% in controls), with limited chnges lter on. In ddition, in the simvsttin cells, the lbeling of 20:4 exceeded tht of 20:3 lredy t 4 h, nd there ws n dditionl increment of 20:4 up to 16 h, without significnt chnges in 20: 8 lbeling. In the simvsttin-treted cells there ws lso TABLE 3. Percentge of the totl rdioctivity incorported in ftty cids derived from linoleic cid Ftty Acids In phospholipid 18:s 20 : 4 20:s 22:4 20:2 In triglyceride 18:s 20:4 20:3 20:2 Control 4.13 r+_ t f t t t 1.18 Vlues re the verge -t SE of 4 experiments. "Significntly different from control t P < Simvbttin 5 flht t 5.38" t '' Journl of Lipid Reserch Volume 38, 1997

5 C.e c 0 m 0.- U s Lipid Clsses O* I I I I I Time (h) TABLE 5. Incorportion of ['4C]cette in totl lipids nd lipid clsses Control cpm/mgtl X i 9.41 % of Incorported rdioctivity CE 2.66 i 1.42 TG i 4.79 FFA MAG Cho 9.27 i 0.60 DAG 2.71 i 0.04 PL i 4.54 Simvsttin 5 FM i 184' t IT 0.23' 0.38 t 0.01" 1.36 i 0.20' CE, cholesteryl esters; TG, triglycerides; FFA, free ftty cids; MAG, monocylglycerol; Cho, cholesterol; DAG, dicylglycerol, PL, phospholipids. Vlues re the verge t SE of 4 experiments. "Significntly different from controls t P < 'Significntly different from controls t P < Fig. 2. Timecourse of the lbeling of 20 : 3 nd 20 : 4 fter incubtion with ["GILA, in control nd simvsttin-treted cells. Vlues re the verge of duplicte nlyses crried out on smples mde up by lest three pooled cell preprtions for ech time point; (0) 20:3 control; (0) 20:4 control; (0) 20:3 simvsttin 5 PM; (W) 20:4 simvsttin 5 PM. formtion of 22:4, mounting to bout 1.5% of the rdioctivity, versus undetectble levels in controls. In controls, lbeling of 20: 4 remined somewht lower thn tht of 20:3 throughout the whole time period. Subsequent experiments were devoted to investigte the effects of simvsttin on the conversion of lbeled 20 : 5 n-3 to its derivtives (Tble 4). Totl conversion ws enhnced of bout 30% nd this ws reflected in enhnced lbeling of both 22 : 5 nd 22 : 6. We could not detect, in our conditions, formtion of peks corresponding to the intermedite products 24:5 nd 24: 6 (lo), possibly becuse of the reltively low rtes of conversion of EPA to DHA in THP-1 cells, resulting in lbeling of intermedites below the detection limit. The product/precursor rtios for the elongtion nd the desturtion t position 4 (Tble 4), were both enhnced by simvsttin. TABLE 4. Conversion of 20:5 n-3 (EPA) Simvsttin Control 5 CIM % of Totl conversion i 1.36" % Rdioctivity 22:5 n t 0.95" 22:6 n ? ' Product-precursor rtio 22:5/20:5 (C20 elongse) t 0.02" 22:6/22:5 (desturse) 0.04 i i 0.01' Vlues re the verge 2 SE of 3 experiments. "Significntly different from controls t P < "Significntly different from controls t P < Lipid synthesis from cette Acette incorportion into TL ws substntilly the sme in both control nd treted cells. The mjor difference, concerning the incorportion of cette into individul lipid clsses (Tble 5), ws the lmost complete inhibition of cholesterol synthesis, s expected, in simvsttin-treted cells, nd this ws ssocited with significnt reduction lso of MAG nd DAG lbeling. Lbeling of CE ws not ffected, in spite of the drstic reduction of cholesterol synthesis. Alkline hydrolysis of TL nd preprtion of methyl esters reveled tht over 90% of the rdioctivity in control cells nd more thn 98% in simvsttin-treted cells ws ssocited with the FA moiety. This prompted us to nlyze the distribution of the rdioctivity in the FA, seprted s methyl esters by AgN03-impregnted TLC nd by HPLC coupled with rdiodetector. In simvsttin treted cells (Fig. 3), there ws mrkedly greter incorportion into MUFA, nd correspondingly lower incorportion into SAT (pnel A), when compred to control cells, with n increment of the MUFA/SAT rtio from 0.42 to The incorportion into individul FA (pnel B) showed reduction of 16:O nd increment of 18: 1 in simvsttin-treted versus control cells. Effect of mevlonte nd isoprenoids on LA conversion In order to ssess whether the effects of simvsttin on PUFA metbolism were relted to inhibition of the formtion of intermedites in cholesterol synthesis, we hve supplemented mevlonte t the concentrtions of 100,500, nd 1000 PM (5, 15), frnesol (10 p ~ nd ) gernylgerniol (5 p ~ (15) ) together with the dministrtion of simvsttin. There ws dose-dependent reduction (Tble 6) of the effects of simvsttin on LA conversion nd AA synthesis by mevlonte (MVA), l- Risi, Colombo, nd GuUi Sivsttin nd lipid metbolism in monocytic THP-1 cells 1303

6 1-1 A TABLE 6. Effect of mevlonte nd isoprenoids on LA conversion Totl LA Conversion AA Synthesis % rdioctivity Control 23.9 If: r Simvsttin 5 pm 47.3 t pm MVA 100 pm " 27.4 t 0.lb 5 p~ + MVA 500 p~ 39.0 t 0.lh 22.8 c 0.1' 5 pm MVA 1000 pm 32.8 t 0.1' 16.4 t 0.3'.5 p~ frnesol 10 p~ 45.5 c t 0.3.i p~ + GG 5 p~ 36.4 t 0.4' 20.0 c 0.1' LA, linoleic cid; AA, rchidonic cid; MVA, mevlonic cid; GG, gernylgerniol. Vlues re the verge 2 SE of different experiments. "Significntly different from simvsttin t P < 'Significntly different from simvsttin t P < 'Significntly different from simvsttin t P < :O SAT T MUFA B T 16:O 18:O 16:l 18:l Fig. 3. FA lbeling in cells incubted with [14C]cette. Pnel A percentge of rdioctivity incorported in sturted nd monounsturted FA in control nd simvsttin-treted cells. SAT, sturted; MUFA, monounsturted ftty cids. Pnel B: percentge of rdioctivity incorported in individul ftty cids in control nd simvsttintreted cells. Vlues re the verge 5SE of dt from three experiments; 0 control; simvsttin 5 p ~,. significntly different from control t P < though even t 1000 PM the effects were reduced only bout 50%. Administrtion of gernylgerniol, but not of frnesol, ws lso ble to mrkedly reduce the effects of simvsttin on PUFA metbolism. Lipid synthesis from glycerol As FA metbolism my be coupled to lipid synthesis, we then explored the effects of simvsttin on the production of glycerolipids from lbeled glycerol. More thn 95% of the rdioctivity ws incorported into TG nd PL, in both control nd simvsttin treted cells. The percentge of incorportion of glycerol in TG ws enhnced, wheres tht in PL ws reduced by the drug (Fig. 4) with significntly greter reltive incorportion into TG. In order to explore whether chnges in the synthesis of TG from different precursors (glycerol nd cette) were lso ssocited with enhnced TG levels, we hve then mesured the endogenous concentrtion of this lipid clss in control nd treted cells nd found significnt elevtion of TG concentrtion fter simvsttin 80.- E 60 > +-' 0 CFI Y- O $? *O 0 TG PL Fig. 4. Incorportion of [3H]glycerol in lipids of control nd simvsttin-treted cells. PL, phospholipids; TG, triglycerides. Vlues re the verge 5SE of dt from three experiments; 0 control, simvsttin 5 p ~,. sttisticlly different from control t P < 0.01; b, sttisticlly different from control t P < Journl of Lipid Reserch Volume 38, 1997

7 pm Simvsttin Fig. 5. Dose-response curves for the effects of simvsttin on LA metbolism nd cholesterol synthesis. Left xis: totl [14C]LA conversion in control nd simvsttin-treted cells. Vlues re the verge +SE of dt from three experiments., Sttisticlly different from control t P < 0.05; Right xis: cholesterol lbeling from ['%]cette s percentge of the incorportion in totl lipids. Vlues t ech time point re different from ech other, t the specified levels: 6, Sttisticlly different from control t P < 0.01; c, sttisticlly different from 0.5 JIM simvsttin t P < 0.005; d, sttisticlly different from 0.5 JIM simvsttin t P< 0.01; e, sttisticlly different from 0.5 JIM simvsttin t P < 0.02;f; sttisticlly different from 1 JIM simvsttin t P< 0.01; g, sttisticlly different from 1 JIM simvsttin t P< 0.02; h, sttisticlly different from 2 JIM simvsttin t P < (0) linoleic cid, (A) cholesterol. ( SE s % of TL in treted cells vs _+ 2.0 SE in controls, P = 0.046). After nlyzing the effects of simvsttin on vrious pthwys of lipid metbolism, t the concentrtion of 5 p~ simvsttin, we proceeded to evlute the comprtive dose-response curves for the effects of the drug on cholesterol synthesis nd on FA metbolism. We hve therefore mesured the conversion of lbeled LA to LC- PUFA nd of cette to cholesterol in the presence of concentrtions from 0.5 to 5 p~ simvsttin (Fig. 5). Totl conversion of LA ws mximlly elevted lredy t 0.5 PM simvsttin, with miniml dditionl chnges t greter concentrtions, wheres the inhibition of incorportion of cette into cholesterol ws further reduced from 9.3% cette incorported in controls to 2.1% t 0.5 VM nd 0.35% t 5 PM simvsttin, tht is with further sttisticlly significnt reduction between 0.5 nd 5 PM simvsttin. DISCUSSION In the monocytic cell line THP-1, inhibition of cholesterol synthesis, lmost complete in the presence of 5 p~ simvsttin, ws ssocited with enhnced lbeling of n-6 LC PUFA from LA, s previously described (5), nd with enhnced conversion of 20:5 n-3 to 22:s nd 22: 6. The effects of simvsttin on AA lbeling ppered to be minly the result of enhnced synthesis nd to be selective for the A5 desturtion, on the bsis of clcultions of the product/precursor rtios. An initil nd selected ctivtion of the A5 desturtion step ws lso supported by timecourse studies indicting tht in simvsttin-treted cells, lbeling of 20: 4 ws mrkedly greter thn tht of 20:3 lredy t 4 h of incubtion with LA, with further increment exclusively for 20:4, in contrst to the sitution in control cells. It is lso unlikely tht the enhnced AA lbeling ws the result of reduced degrdtion, s in control cells, i.e., in the bsence of simvsttin, lbeling of this ftty cid remined constnt between 4 nd 24 h of incubtion, without significnt losses of rdioctivity. The elevtion of 22:5 nd 22 : 6 lbeling from 20 : 5, nd the enhnced formtion of MUFA from cette further support generlized stimultion of FA desturtion by simvsttin. Totl lipid synthesis from cette, however, with the excep tion of cholesterol lbeling, ws not ffected by simvsttin. It should be pointed out, however, tht uncertinties exist in quntittive interprettions of ftty cid nd cholesterol synthesis from rdiolbeled cette due to possible differences in the specific ctivity of cellulr cetyl CoA substrte pools. The use of tritited wter would hve llowed more relible ssessment of lipid synthesis. Formtion of CE from cette ws lso mrginlly ffected, suggesting tht the endogenous cholesterol pool, not modified by tretment (not shown), served s substrte for esterifiction of the newly synthesized FA. In cells exposed to cetylted LDL, insted, simvsttin ws shown to inhibit the incorportion of [ 1-I4C]olete in cholesteryl esters (19). The enhnced lbeling of TG from glycerol in simvsttin-treted cells, ssocited with the lredy reported (4) elevtion of cellulr TG, suggests tht the production of the unsturted FA exceeded the rtes of esterifiction into PL, shifting the process towrds TG synthesis. The effects of simvsttin on FA metbolism were dependent upon inhibition of mevlonte formtion, s supplementtion of this metbolic intermedite normlized the rtes of PUFA formtion, enhnced by simvsttin (5). The reversl of the effect of simvsttin on FA metbolism by mevlonte ws concentrtiondependent, lthough t high concentrtions, nd mrked reversl ws obtined lso with gernylgerniol t much lower concentrtions (5 PM), frnesol (10 PM) being, insted, inctive. The bove re the highest nontoxic concentrtions of the isoprenoids reported in the literture (15, 20). Although the discrepncy between gernylgerniol nd frnesol demnds further investigtion, it my reflect different fte of the two isoprenoids. &si, Colombo, nd GUi Simvsttin nd lipid metbolism in monocytic THP-1 cells 1305

8 The Clj isoprenoid frnesyl, in its pyrophosphte form functions poorly s substrte for elongtion to gernylgernyl pyrophosphte (21) nd, t lest in the liver, seprte enzyme ppers to ctlyze the synthesis of gernylgernyl pyrophosphte from gernyl-pyrophosphte (22). Furthermore, in mmmlin cells incubted with lbeled fmesol or gernylgerniol, completely different sets of protein become lbeled (23). In ddition, s the mjor clss of lbeled preriylted proteins produced in eukryotic cells is derived from gernylgernyl-pyrophosphte (20). Inhibition of protein gernylgernyltion my be mjor component of the effect of simvsttin on FA metbolism. Our dt confirm the complexity of the interctions between cholesterol nd FA metbolism described in studies on somewht different spects, phrmcologiclly nd/or nutritionlly pproched. Enhnced ctivity of the heptic mitochondril crnitine plmitoyltrnsferse hs been observed, for instnce, fter inhibition of the HMGCoA reductse (24), wheres the dministrtion of exogenous cholesterol resulted in enhnced TG synthesis while reducing FA oxidtion (25). Conversely, the dministrtion of FA stimulted cholesterol synthesis (26, 27). In summry, simvsttin in the monocytic cell line THP-1 enhnces the unsturtion of exogenously supplemented PUFA nd of FA formed from endogenous precursors, thus resulting in incresed unsturtion of cell lipids; TG synthesis is lso enhnced. The mchinery for unsturted FA formtion is quite sensitive to the ctivity of simvsttin, s mximl effects occur lredy t the lowest concentrtion tested. The observed chnges in FA nd lipid metbolism my be responsible for the reported effects of HMGCoA reductse inhibitors on plsm FA in hypercholesterolemic subjects (6, 7) nd possibly for the modifictions of pltelet function (9). The mechnisms re not cler but it would pper tht chnges in the lipid environment surrounding the FA metbolizing enzymes, induced by the drug, my ply role in the described processes (28). Inhibition of protein gernylgernyltion my lso be involved. IJ The uthors wsh to thnk Dr. M. Som for kindly providing us with simvsttin. Mnuscnpt recezwd 8 August 1996, zn rmrdfm 23 llecrmbw 1996, nd zn rereutsed form I8 Mrch 1997 REFERENCES 1. Endo, A., Y. Isujit, M. Kurod, nd K. Tnzw Inhibition of cholesterol synthesis in vivo nd in vitro by ML236A nd ML236B, competitive inhibitors of shydroxy-3-methylglutryl-coenzyme A reductse. Eur. J. Biorhem. 77: Kneko, I., Y. Hzm-Shimd, nd A. Endo Inhib itory effects on lipid metbolism in cultured cells of ML- 236B, potent inhibitor of 3-hydroxy-3-methylglutry-coenzyme A reductse. Eur. J. Biochem. 87: Brown, M. S., J. R. Fust, nd J. L. Goldstein Induction of.%hydroxy-.%methylglutryl-coenzyme A reductse ctivity in humn fibroblsts incubted with compctin (ML-23613), competitive inhibitor of the reductse. J. Biol. Chpm. 253: Willims, M. L., G. K Menon, rid K. P. Hnley HMGCoA reductse inhibitors perturb ftty cid metbolism nd induce peroxisomes in kertinocytes../. Lifiid. Res. 33: Hrboticky, N., L. Tng, B. Zimmer, I. Lux, rid P. C:. Weber Lovsttin increses rchidonic cid levels nd stiniultes thromboxne synthesis in humn liver nd monocytic cell lines. J. Clin. Znvesl 93: Doorml vn, J. J., W. J. W. Bos, F. A.J. Muskiet, nd H. Doorenbos Simvsttin influences linoleic cid metbolism. Phm. Weekbl. Sci. Ed. 11: Agheli, N., nd B. Jcotot Effect of simvsttin nd fenofibrte on the ftty cid composition of hypercholesterolemic ptients. Br. J. Clin. Phmncol. 32: Leikin, A. T., nd R. R. Brenner In vivo cholesterol removl from liver microsomes induces chnges in ftty cid desturse ctivity. Biochim. Biophys. Act. 963: Hochgrf, E., Y. Levy, M. Avirm,J. G. Brook, nd U. Cogin Lovsttin decreses plsm nd pltelet cholesterol levels nd normlizes elevted pltelet fluidity nd ggregtion in hypercholesterolemic ptients. Met& olism. 43: Voss, A., M. S. Reinhrt, S. Snkrpp, nd H. Sprechcr The metbolism of 7,10,13,16,19-docoshexenoic cid is independent of 4desturse. J. Biol. Chm. 266: Auwerx, J The humn leukemi cell line, THP-1: multifcetted model for the study of monocyte-mcrophge differentition. Expm mti. 47: Glell, G., F. Mrngoni, P. Risk, C. Colombo, G. Cklli, nd C. Clli n-6 nd n-3 ftty cid ccumultion in THP-1 phospholipids. Biochim. Biuphys. Act. 1169: Alberts, A. W Discovery, biochemistry nd biology of lovsttin. Am.J. Crdiol. 62: Cohen, I,. H., A. Vn Vliet, L. Roodenburg, L. C. M. Jnsen, nd M. Griffioen Prvsttin inhibited the cholesterol synthesis in humn heptom cell line HepG2 less thn simvsttin nd lovsttin, which is reflected in the upregultion of the 3-hydroxy-3-me thylglutryl coenzime A reductse nd squlene synthse. Biochem. I hnnnrol. 45: Corsini, A., M. Mzzotti, M. Riteri, M. R. Som, G. Gbbini, R. Funiglli, nd R. Poletti Reltionship between nievlonte pthwy nd rteril myocyte prolifertion: in vitro studies with inhibitors of HMGCoA reductse. Alherosrhosis. 101: Folch, J., M. Lees, nd G. H. Slone Stnley A simple method for the isoltion nd purifiction of totl lipids from niml tissue. J. Bid. Chem. 226: Kouser, G., G. Kritchevsky, A. Ymnioto, G. Simon, C. Glli, nd A. J. Bumn Diethylminoethyl nd triethylminoethyl-celluse column chromtogrphy procedures for phospholipids, glycolipids nd pigments. Meth- O ~ Enqmol. S 14: Journl of Lipid Reserch Volume 38, 1997

9 18. Moore, S. A., E. Yoder, nd A. A. Spector Role of the blood-brin brrier in the formtion of long-chin n-3 nd n-6 ftty cids from essentil ftty cid precursor. J. Neurochem Kempen, H. J. M., M. Vermeer, E. de Wit, nd L. M. Hvekes Vsttins inhibit cholesterol ester ccumultion in humn monocytederived mcrophges. Arte- ~osch. Thromb. 11: Frnsworth, C. C., M. H. Gelb, nd J. A. Glomset Identifiction of gernylgernyl-modified proteins in HeL cells. Science. 247: Sito, A., nd H. C. Rilling, Prenyltrnsferse: product binding nd rection termintion. J. Biol. Chem. 254: Sgmi, H., K Ishi, nd K Ogur Occurrence nd unusul properties of' gernylgernyl pyrophosphte synthetse of pig liver. Biochem. Znt. 3: Crick, D. C., D. A. Andres, nd C. J. Wechter Frnesol is utilized for protein isoprenyltion nd the bio- synthesis of cholesterol in mmmlin cells. Bioch. Biophys. Res. Commun. 211: Cook, G. A., B. Khn, nd M. Heimberg Feeding of lovsttin to rts increses the ctivity of the heptic mithocondril outer crnitine plmitoyltrnsferse. Biochem. Biophys. Res. Commun. 150: Fungwe, T. V., L. M. Cgen, G. A. Cook, H. G. Wilcox, nd M. Heimberg Dietry cholesterol stimultes heptic biosynthesis of triglyceride nd reduces oxidtion of ftty cids in the rt. J. Lipid Res Goh, E. H., nd M. Heimberg Stimultion of heptic cholesterol biosynthesis by oleic cid. Biochem. Biophys. Res. Commun. 55: Fungwe, T. V., J. E. Fox, L. M. Cgen, H. G. Wilcox, nd M. Heimberg Stimultion of ftty cid hiosynthes by dietry cholesterol nd of cholesterol synthesis hy dietry ftty cid.j. Lipid Res Leikin, A., nd M. Shinitzky Chrcteriztion of the lipid surrounding the Adesturse of rt fiver microsomes. Biochim. Biophys. Act. 1256: Rist?, Colombo, nd GUi Sivsttin nd lipid metbolism in monocytic THP-1 cells 1307