FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells

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1 FATP1 chnnels exogenous FA into 1,2,3-tricyl-sn-glycerol nd down-regultes sphingomyelin nd cholesterol metbolism in growing 293 cells Grnt M. Htch,*, Anne J. Smith, Fred Y. Xu,*, Angel M. Hll, nd Dvid A. Bernlohr 1, Deprtments of Phrmcology* nd Therpeutics, University of Mnitob, Winnipeg, Mnitob, Cnd; nd Deprtment of Biochemistry, Moleculr Biology nd Biophysics, The University of Minnesot, Minnepolis, MN Abstrct Biosynthesis of lipids ws investigted in growing 293 cells stbly expressing ftty cid (FA) trnsport protein 1 (FATP1), bifunctionl polypeptide with FA trnsport s well s ftty cyl-coa synthetse ctivity. In short-term (30 s) incubtions, FA uptke ws incresed in FATP1 expressing cells (C8 cells) compred with the vector (s determined by BODIPY 3823 stining nd rdioctive FA uptke). In long-term (4 h) incubtions, incorportion of [ 14 C]cette, [3H]oleic cid, or [ 14 C]lignoceric cid into 1,2,3-tricyl-snglycerol (TG) ws elevted in C8 cells compred with vector, wheres incorportion of rdiolbel into glycerophospholipids ws unltered. The increse in TG biosynthesis correlted with n increse in 1,2-dicyl-sn-glycerol cyltrnsferse ctivity in C8 cells compred with vector. In contrst, incorportion of [ 14 C]cette into sphingomyelin (SM) nd cholesterol, nd [3H]oleic cid or [ 14 C]lignoceric cid into SM ws reduced due to reduction in de novo biosynthesis of these lipids in C8 cells compred with vector. The results indicte tht exogenously supplied FAs, nd their subsequently produced cyl-coas, re preferentilly chnneled by n FATP1 linked mechnism into the TG biosynthetic pthwy nd tht such internlized lipids downregulte de novo SM nd cholesterol metbolism in ctively growing 293 cells. Htch, G. M., A. J. Smith, F. Y. Xu, A. M. Hll, nd D. A. Bernlohr. Ftty cid trnsport protein 1 chnnels exogenous ftty cid into 1,2,3-tricyl-sn-glycerol nd down-regultes sphingomyelin nd cholesterol metbolism in growing 293 cells. J. Lipid Res : Supplementry key words trnsport CoA synthetse esterifiction ftty cid trnsport protein 1 Ftty cids (FAs) re key components of membrne glycerolipids, re involved in signl trnsduction mechnisms, nd serve s n importnt source of metbolic fuel though -oxidtion (1). Circulting FAs re tken up into cells either by diffusion due to their solubility in the membrne or by one or more proteins tht fcilitte their trnsport into the Mnuscript received 19 Mrch 2002 nd in revised form 2 My DOI /jlr.M JLR200 cell (2, 3). To dte, t lest three seprte fmilies of proteins hve been described: FA trnslocse/cd 36 (FAT/CD 36), plsm membrne (PM) FA binding protein (FABPpm), nd FA trnsport protein (FATP). Five members of the FATP fmily hve been described in murine cells nd sixth identified from nlysis of the humn genome (lbeled ftty cid trnsport protein 1 (FATP1)-6) (4). The N-terminl 51 mino cids of FATP re vrible nd my represent specific trgeting of the protein to different subcellulr comprtments. One of the members of this fmily, FATP1, is expressed on the PM s well s intrcellulr membrnes (5, 6). FAs enter into glycerolipids by de novo biosynthesis by successive cyl-coa dependent cyltion of sn-glycerol- 3-phosphte to form lysophosphtidic cid followed by phosphtidic cid (PA) (7). PA is then converted to 1,2- dicyl-sn-glycerol (DG) by PA phosphohydrolse (8). DG is then condensed with nother molecule of cyl-coa to form 1,2,3-tricyl-sn-glycerol (TG) nd free CoA in rection ctlyzed by DG:cyl-CoA cyltrnsferse. In ddition to TG, DG produced from PA phosphohydrolse my be utilized for de novo phosphtidylcholine (PC) nd phosphtidylethnolmine (PE) biosynthesis by their respective cytidine diphosphte (CDP)-choline nd CDP-ethnolmine pthwys (7). PA my lso serve s precursor for phosphtidylinositol (PI) synthesis vi CDP-DG production ctlyzed by PA:CTP cytidylyltrnsferse. In ddition to glycerolipid de novo biosynthesis, ftty cyl-coas my enter into glycerophospholipids vi decyltion followed by recyltion pthwys (9). Ftty cyl-coa enters into sphingolipids vi two routes: condenstion of serine with plmitoyl-coa ctlyzed by serine plmitoyltrnsferse Abbrevitions: ACS, cyl-coa synthetse; DG, 1,2-dicyl-sn-glycerol; DGAT, 1,2-dicyl-sn-glycerol cyltrnsferse; ER, endoplsmic reticulum; FA, ftty cid; FATP1, ftty cid trnsport protein 1; GPAT, sn-glycerol-3-phosphte cyltrnsferse; MHC, myosin hevy chin; PA, phosphtidic cid; PC, phosphtidylcholine; PE, phosphtidylethnolmine; PI, phosphtidylinositol; PM, plsm membrne; PS, phosphtidylserine; SM, sphingomyelin; TG, 1,2,3-tricyl-sn-glycerol. 1 To whom correspondence should be ddressed. e-mil: bernl001@umn.edu Copyright 2002 by Lipid Reserch, Inc Journl of Lipid Reserch Volume 43, 2002 This rticle is vilble online t

2 nd N-cyltion of sphingnine (sphingosine) ctlyzed by sphingnine (sphingosine) N-cyltrnsferse (10). Numerous experimentl models nd humn metbolic disorders hve shown tht sphingomyelin (SM) biosynthesis is regulted in concert with cholesterol biosynthesis (11). Acyl-CoA synthetses (ACSs) re fmily of proteins tht ligte FA to CoA with concomitnt pyrophosphorylmedited hydrolysis of ATP (12). Recent studies hve indicted tht there my be selectivity of cyl-coa pool for vrious glycerolipid biosynthetic pthwys (13, 14). For exmple, incubtion of heptocytes, prepred from fsted or fed rts, with tricsin C inhibited TG biosynthesis but not FA oxidtion (13). FATP1 exhibits FA ligse ctivity towrd long chin nd very long chin FAs suggesting tht FA trnsport my be linked to CoA esterifiction (15). The bove studies prompted us to exmine whether or not the CoA synthetse component of FATP1 ctivity mechnisticlly linked FA uptke to selection of cyl-coa pools for vrious lipid biosynthetic pthwys. Our surprising results indicte tht FATP1 upregultes de novo TG biosynthesis while down regulting de novo SM nd cholesterol metbolism in growing 293 cells. MATERIALS AND METHODS [ 3 H]oleic cid, l-[ 3 H]serine, [methyl- 3 H]choline, [1-14 C]cette, [ 3 H]ethnolmine, nd [myo- 3 H]inositol were obtined from Amershm. [1-14 C]lignoceric cid ws obtined from Americn Rdiochemicls Compny. Thin lyer pltes (silic gel G 0.25 mm) were obtined from Fisher Scientific. Lipid stndrds were obtined from Avnti Polr Lipids Inc. Tricsin C ws obtined from BIOMOL. Cell culture regents were obtined from GIBCO. Oleic cid nd lignoceric cid were obtined from Nu- Chek Prep., Inc., Elysin, MN. Phloretin ws obtined from ICN Biomedicls, Inc. Fluorescein-conjugted secondry ntibody ws obtined from Orgnon Teknik. Polyclonl nti-cveolin-1 (C13630) ntibody ws obtined from Trnsducin Lbortories. All other regents were of nlyticl grde nd obtined from Sigm Chemicl Co., St. Louis, MO. The murine FATP1 cdna ws generous gift of Dr. Jen Schffer, St. Louis, MO. Genertion of stble cells expressing FATP1 FATP1 subcloned into pcdna3.0 (InVitrogen) or vector control ws linerized nd introduced into 293 cells by electroportion (BTX Corp., Sn Diego, CA) ccording to the mnufcturers instructions. 293 cells re n excellent model system to evlute FATP1 function in lipid metbolism for they re well defined cell system, grow esily in culture, nd hve been shown to tolerte FATP expression (5). Dilutions of the electroported cells were plted in DMEM contining 10% FBS nd incubted t 37 C, 5% CO 2, nd fter 48 h selection ws initited with the ddition of 400 g/ml geneticin (GIBCO/BRL Life Technologies). After 8 dys colonies were selected using cloning rings nd individul lines developed. Once estblished, severl lines were nlyzed for FATP1 expression using commercilly generted (bioworld, Dublin OH) ffinity-purified polyclonl nti-peptide ntibody mde in rbbits nd directed towrd murine FATP1 mino cid sequences A cell line stbly expressing high level of mfatp1 (C8) ws selected for further study long with vector-trnsformed line (vector). C8 nd vector cells were mintined on 400 g/ml geneticin. Locliztion of FATP1 in C8 cells Vector nd C8 cells were plted onto 13 mm coverslips nd fixed with 3% formldehyde nd 0.1% glutrldehyde in the presence or bsence of 0.01% Triton X-100 t 37 C. After fixtion, cells were incubted for 2 h t 37 C with nti-fatp1, wshed with PBS, nd incubted with fluorescein-conjugted secondry ntibody for 1 h t 25 C. Cells were viewed using Nikon Eclipse E800 photomicroscope equipped with brightfield, DIC, phse nd fluorescence optics including 100 W mercury lmp epifluoresence illumintion with stndrd fluorescein/gfp (excittion filter nm, brrier nm) filter sets. Digitl imges were collected using CoolCm liquid-cooled, threechip color CCD cmer (Cool Cmer Co., Dectur, GA) nd cptured to personl computer using Imge Pro Plus version 4.1. softwre (Medi Cybernetics, Silver Springs, MD). FA uptke experiments C8 or vector cells (200,000 per well) were plted on 13 mm coverslips in 12-well pltes pre-treted with polylysine for enhnced cell dherence nd incubted for 48 h in DMEM contining 10% FBS. Uptke experiments were performed on cells t 50 90% confluence. Cells were incubted for 2 h with serumfree DMEM, wshed twice with PBS, nd then incubted for min t 37 C in Krebs-Ringer Hepes contining 2 mm glucose, ph 7.4, before inititing uptke studies. Stock solutions (5 mm) of non-rdioctive oleic cid or lignoceric cid were prepred s described (16), nd rdioctive lipids were dded to chieve the indicted specific rdioctivity. To initite uptke, cells were incubted t 37 C with KRH, 2 mm glucose, ph 7.4, contining 50 M [ 3 H]oleic cid (8 mc i /mmol) or 50 M [ 14 C]lignoceric (3 mc i /mmol) cid bound to FA free BSA (1:1 molr rtio). After 30 s, the cells were rpidly wshed three times in ice cold PBS contining 200 M phloretin, incubted t room temperture in 0.5% SDS for 30 min, nd the rdioctivity determined by liquid scintilltion counting. A duplicte set of coverslips treted similrly were utilized for phosphorus determintions. FA uptke ws expressed s nmol FA internlized/30 s/nmol lipid phosphorus. The concentrtion of phosphorus did not differ between vector nd C8 cells. For experiments using BODIPY 3823, cells were plted onto coverslips t 250,000 cells/well nd fter 48 h cells were incubted with 20 M BODIPY 3823 in BSA (1:1 molr rtio) nd uptke determined s described (5) using Nikon E800 Fluorescence microscope. Rdiolbeling of phospholipid nd tricylglycerol pools Cells were plted on 10 cm pltes nd incubted in DMEM contining 10% FBS. Subconfluent cells were incubted for 4 h with serum-free DMEM contining 0.1 mm [ 3 H]oleic cid (2 Ci / dish) or 0.1 mm [ 14 C]lignoceric cid (2 Ci /dish) bound to lbumin (1:1 molr rtio). For other lbeling, cells were incubted for 4 h with either 0.1 M [ 14 C]cette (2 Ci/dish), 0.5 mm l-[ 3 H]serine (10 Ci /dish), 28 M [methyl- 3 H]choline (2 Ci / dish), 28 M [ 3 H]ethnolmine (2 Ci /dish), or 0.4 mm [myo- 3 H]inositol (2 Ci /dish). For ll lbeling, the medium ws removed nd the cells wshed twice with ice-cold PBS nd then hrvested from the dish with 2 ml of methnol-wter (1:1, v/v) using rubber policemn. A 25 l liquot ws tken for the determintion of totl rdioctivity ssocited with the cells nd nother for protein determintion. To fcilitte phse seprtion, chloroform nd wter were dded until finl rtio of chloroform-methnolwter of 4:2:3 (v/v/v) ws chieved. The mixture ws centrifuged nd the queous phse removed. The orgnic phse ws wshed once with theoreticl queous phse (chloroform-methnol-wter, 3:48:47, v/v/v). The queous phse ws removed nd combined with the queous phse from the first centrifugtion nd 0.5 ml liquot of the combined queous frction ws tken for the deter- Htch et l. FATP1 chnnels ftty cid into TG 1381

3 mintion of rdioctivity. The orgnic phse ws dried under nitrogen nd resuspended in 125 l of chloroform-methnol (2:1, v/v) nd 50 l liquots were plced on thin-lyer pltes for the seprtion of neutrl lipids nd phospholipids. Phospholipids were seprted by scending TLC in solvent system contining chloroform-methnol-cetic cid-wter (70:30:8:3, v/v/v/v). The TG, DG, nd FA frctions were seprted by TLC in solvent system contining benzene-diethylether-bsolute ethnol-cetic cid (50:45:2:0.2, v/v/v/v). Cholesterol ws seprted by TLC in solvent system contining hexnes-diethylether-cetic cid (70:30:2, v/v/v). Silic gel corresponding to the lipids of interest ws removed nd rdioctivity in these determined by liquid scintilltion counting s described (17). Enzymtic ssys nd other determintions Phospholipid nd cholesterol pool sizes were determined s described (18, 19). FA nd TG pool sizes were mesured by gs chromtogrphy using C19 s internl stndrd s described (20). For Oil Red O stining 0.5% solution ws prepred in isopropnol then diluted with 1.5 ml wter, filtered, nd used immeditely s described (21). Preprtion of cveolin-1 contining membrne frctions nd seprtion using Optiprep were exctly s described (22). Microsoml frctions were prepred from vector nd C8 cells nd 1,2-dicyl-sn-glycerol cyltrnsferse (DGAT) ctivity determined in these frctions s described (23). Microsoml nd mitochondril frctions were prepred from vector nd C8 cells nd sn-glycerol-3-phosphte cyltrnsferse (GPAT) ctivity determined in these frctions s described (24). FATP1 dependent FA CoA ligse ctivity ws mesured s described by Coe et l., (15). Protein ws mesured by the method of Lowry (25). Student s t-test or Dunnet s t-test for multiple comprisons with single men were used for determintion of sttisticl significnce. The level of significnce ws defined s P RESULTS FATP1 is loclized to non-cveolin-1 contining PMs nd internl membrnes in C8 cells The subcellulr locliztion of FATP1 ws investigted in vector nd in C8 cells over-expressing FATP1. As shown in Fig. 1, the level of FATP1 ws mrkedly elevted in C8 cells compred with vector. In ddition, FATP1 ws clerly loclized to the PM in vector nd C8 cells. Consistent with the results of Schffer nd collegues (5, 6), dditionl fluorescence ws detected on internl membrnes nd suggested tht some FATP1 my reside within the ER. In greement with the immunofluorescence studies, semi quntittive nlysis of Western blots of FATP1 expression suggested pproximtely 20 more FATP1 in C8 cells s compred with vector cells (Fig. 1, inset). Although FATP1 is expressed to high levels, the bundnce of the protein in 293 cells is less thn in cultured 3T3-L1 dipocytes (results not shown). As such, while overexpressed, the mount of FATP1 is not in excess of tht normlly found. Frctiontion of membrnes by grdient density centrifugtion in the presence of Optiprep (22) nd subsequent Western blotting indicted tht FATP1 did not co-loclize with cveolin-1 (Fig. 2). Hence, heterologously expressed FATP1 is loclized in 293 cells to non-cveolin-1 contining regions of the plsm nd internl membrnes. Fig. 1. Locliztion of ftty cid trnsport protein 1 (FATP1) protein in vector nd C8 cells. Vector nd C8 cells were glutrldehyde/ formldehyde fixed nd incubted with nti-fatp1 ntibody followed by fluorescein-tgged got nti-rbbit secondry ntibody nd nlyzed by immunofluorescence microscopy. A nd C: Represent vector cells. B nd D: C8 cells. Fluorescence of monolyers treted identiclly but lcking primry ntibody is depicted in C nd D. The inset in D represents Western blotting of totl cell membrne from C8 nd vector cells with nti-fatp1 ntibody Journl of Lipid Reserch Volume 43, 2002

4 Fig. 2. FATP1 does not co-loclize with cveolin-1 in C8 cells. Detergent-solubilized C8 cell extrct ws nlyzed exctly s described by Oliferenko et l. (22) using flottion grdient system in the presence of vrying concentrtions of Optiprep. Following centrifugtion, frctions were recovered nd proteins nlyzed by SDS- PAGE. The presence of FATP1 nd cveolin-1 in ech frction ws ssessed using immunoblotting nd detected using enhnced chemiluminescence. Numbers represent moleculr mss in kd. Fig. 3. Ftty cid (FA) CoA ligse ctivity of FATP1 nd its sensitivity to tricsin C. Vector or C8 cells were lysed by repeted freeze-thw cycles nd totl protein ssyed for FA-CoA ligse ctivity using either oleic cid (18:1) or lignoceric cid (24:0) in the presence or bsence of the indicted concentrtion of tricsin C. Control ctivity (white br), 1 M tricsin C (light gry br), 10 M tricsin C (gry br), 100 M tricsin C (drk gry br), DMSO control (blck br). FA-CoA ligse ctivity nd the uptke of FAs re elevted in 293 cells over-expressing FATP1 The FA-CoA ligse ctivity of cellulr extrcts expressing FATP1 ws compred with tht from vector cells using both oleic cid nd lignoceric cid. Consistent with previous reports for FATP1 (15) nd FATP4 (26), FATP1 contining frctions hd elevted esterifiction ctivity using both long chin (18:1) nd very long chin (24:0) FAs. The mount of esterifiction ctivity ttributble to FATP1 ws similr for long chin nd very long chin FA (Fig. 3). This indicted tht FATP1 is brod substrte rnge enzyme s opposed to more fmilir ACS fmily of CoA ligses tht exhibit much more specificity for long chin over very long chin FAs (27). Since the ACS fmily of CoA ligses re sensitive to inhibition by tricsin C (28, 29), we evluted if FATP1 ws similrly inhibited. As shown in Fig. 3, extrcts from both vector nd FATP1 expressing cells were sensitive to incresing tricsin C levels using oleic cid s substrte but were mrkedly resistnt to tricsin C inhibition using lignoceric cid s substrte. Moreover, the frction of ctivity inhibited by tricsin C in vector extrcts ws greter thn from C8 cells. These results suggest tht FATP1 is brod rnge CoA ligse tht is reltively insensitive to tricsin C inhibition nd tht tricsin C sensitivity cn be used s tool to evlute lipid trfficking through either the ACS or FATP fmily of enzymes. To exmine if lipid uptke ws elevted in C8 cells overexpressing FATP1, cells were incubted for 30 s with FA nlog BODIPY 3823 nd the incorportion evluted by fluorescence microscopy. C8 cells exhibited elevted BODIPY 3823 stining compred with vector suggesting tht FA uptke ws elevted in cells over-expressing FATP1 (Fig. 4). This ws nticipted since FATP1 ws isolted by Schffer nd Lodish (5) s hving BODIPY 3823 uptke ctivity. To exmine if long chin nd very long chin FA uptke were elevted in cells over-expressing FATP1, 293 vector nd C8 cells were incubted for 30 s t 37 C with KRH, 2 mm glucose, ph 7.4, contining 50 M [ 3 H]oleic cid or 50 M [ 14 C]lignoceric cid bound to BSA (1:1 molr rtio) nd rdioctivity incorported into cells determined. [ 3 H]oleic cid incorportion into vector ws nmol/30 s/nmol Pi nd incresed 1.8-fold to nmol/30 s/nmol P i in C8 cells. [ 14 C]lignoceric cid incorportion into vector ws nmol/30 s/nmol Pi nd incresed 2.6-fold to nmol/30 s/nmol Pi in C8 cells (Fig. 5). These dt indicte tht C8 cells over-expressing FATP1 exhibit functionl elevtion in FA uptke compred with vector nd tht uptke ctivity is, generlly, proportionl to FATP1 enzymtic ctivity. It should be noted tht the rdiolbeling methodology used in this study simply mesures cellssocited rdioctivity nd does not mechnisticlly distinguish between bilyer trnsit (trnsport) vs. intercltion nd lterl diffusion in the PM. As such, the term uptke hs been used to signify such. TG synthesis is incresed nd SM synthesis decresed in 293 C8 cells over-expressing FATP1 Since some of FATP1 ws loclized to internl membrnes in 293 cells, nd the endoplsmic reticulum (ER) is the principl site of lipid synthesis in mmmlin cells, we investigted if over-expression of FATP1 ffected de novo biosynthesis of lipids from long chin FA. To evlute Htch et l. FATP1 chnnels ftty cid into TG 1383

5 Fig. 4. FA uptke in vector nd C8 cells s ssessed by BODIPY 3823 incorportion. Vector nd C8 cells were incubted for 30 s with BODIPY 3823 nd nlyzed by fluorescence microscopy. this possibility, 293 vector nd C8 cells were cultured for 7 dys, stined with Oil Red O (21), nd nlyzed by light microscopy. Oil Red O stining of C8 cells ws enhnced compred with vector indicting incresed ccumultion of neutrl lipid in 293 cells over-expressing FATP1 (Fig. 6) nd suggestive of effects on de novo biosynthesis of lipids. A similr ccumultion of TG hs been reported by Souz et l., (30) who co-expressed ACS1 nd FATP1. To evlute de novo lipid biosynthesis in FATP1 expressing cells, 293 vector nd C8 cells were incubted for 4 h with 0.1 mm [ 3 H]oleic cid bound to lbumin (1:1 molr rtio) nd rdioctivity incorported into lipids determined. Oleic cid in the quntity of 0.1 mm ws chosen to pproximte the physiologicl level of plsm FA (31). Totl rdioctivity incorported into 293 C8 cells ws elevted 30% (P 0.05) compred with vector (Tble 1). Rdioctivity incorported into TG in 293 C8 cells ws incresed 72% (P 0.05) compred with vector, indicting n pprent elevtion in TG synthesis from oleic cid. Rdioctivity observed s free FA ccounted for less thn 1% of the totl rdioctivity ssocited with the cells nd ws unltered in 293 C8 cells compred with vector. In the phospholipid frction, rdioctivity incorported into SM ws reduced 34% (P 0.05) in 293 C8 cells compred with vector indicting n pprent reduction in SM biosynthesis. In contrst, rdioctivity incorported into DG nd the glycerophospholipids PC, PE, phosphtidylserine (PS), nd PI/PA were unltered in 293 C8 cells compred with vector. Thus, in 293 C8 cells over-expressing FATP1, de novo TG biosynthesis from oleic cid ws elevted nd SM biosynthesis from oleic cid reduced. Long chin sphingoid bses re synthesized de novo from serine nd plmitte (32). To exmine if the reduction in oleic cid incorportion into SM in 293 cells ws truly due to reduction in de novo SM biosynthesis, 293 vector nd C8 cells were incubted for 4 h with 0.5 mm l-[ 3 H]serine nd rdioctivity incorported into SM determined. Totl rdioctivity incorported into 293 C8 cells ws unltered compred with vector (Tble 2). In contrst, rdioctivity incorported into SM ws reduced 26% (P 0.05) in 293 C8 cells compred with vector. Rdioctivity incorported into PS nd PE ws unltered compred with the vector, indicting tht PS biosynthesis nd its subsequent decrboxyltion to PE ws unffected in 293 C8 cells. Thus, in 293 C8 cells over-expressing FATP1, de novo biosynthesis of SM ws reduced. Since PC is the mjor phospholipid found in mmmlin cells (7), we exmined if elevtion in expression of FATP1 ltered de novo biosynthesis of PC or of other phospholipids. C8 cells were incubted with [methyl- 3 H]choline for 4 h nd rdioctivity incorported into PC, SM, nd LPC determined. Totl rdioctivity incorported into 293 C8 cells ws unltered compred with vector (Tble 3). In ddition, rdioctivity incorported into PC nd LPC ws unltered in C8 cells compred with vector. In contrst, rdioctivity incorported into SM ws reduced 55% (P 0.05) in C8 cells compred with controls confirming the Fig. 5. FA uptke in vector nd C8 cells. FA uptke using 50 M totl lipid in 1:1 molr complex with BSA for 30 s. ws performed s described in Mterils nd Methods. A: Represents oleic cid uptke while B represents lignoceric cid. Error brs re SD. P Journl of Lipid Reserch Volume 43, 2002

6 Fig. 6. Oil Red O stining for neutrl lipids in vector nd C8 cells. Vector nd C8 cells were formldehyde fixed nd incubted with Oil Red O nd visulized by light microscopy. decrese in de novo SM synthesis. In ddition, FATP1 overexpression in C8 cells did not ffect [myo- 3 H]inositol uptke or incorportion into PI nor [ 3 H]ethnolmine incorportion into PE (dt not shown). Thus, the reduction in de novo SM biosynthesis from [ 3 H]serine, [ 3 H]oleic cid, nd [methyl- 3 H]choline in C8 cells ws specific for this phospholipid. PC nd PE were the mjor phospholipids in 293 cells nd ccounted for 56% nd 24%, respectively, of the totl phospholipid mss. Over-expression of FATP1 in 293 C8 cells did not ffect the PC nd PE pool sizes compred with the vector (dt not shown). Elevted TG biosynthesis is correlted with decresed cholesterol nd SM biosynthesis in 293 cells over-expressing FATP1 A previous study hd indicted negtive correltion between TG nd SM-cholesterol levels in the surfctntccommodted epidermis (33). To explore if this reltionship ws observed in 293 cells over-expressing FATP1, we simultneously exmined TG, SM, nd cholesterol biosynthesis in 293 C8 nd vector cells. 293 vector nd C8 cells were incubted for 4 h with [ 14 C]cette nd rdioctivity TABLE 1. Incorportion of [ 3 H]oleic cid into lipids of 293 vector nd C8 cells incorported into TG, SM, nd cholesterol determined. Totl incorportion of rdioctivity into 293 C8 cells ws unltered compred with the vector (Tble 4). Rdioctivity incorported into TG ws incresed 69% (P 0.05) in 293 C8 cells compred with the vector. Since totl uptke of [ 14 C]cette into the 293 C8 cells ws unltered but incorportion into TG elevted compred with vector, de novo TG biosynthesis ws truly stimulted in C8 cells. In contrst, rdioctivity incorported into SM nd cholesterol ws reduced 33% (P 0.05) nd 57% (P 0.05), respectively, in 293 C8 cells compred with vector. Rdioctivity incorported into ll other phospholipids ws unffected (dt not shown). These dt indicte tht negtive correltion between de novo TG nd SM-cholesterol biosynthesis exists in 293 cells over-expressing FATP1. We exmined the pool sizes of FA, TG, SM, nd cholesterol in these cells. As seen in Tble 5, the pool sizes of FA nd TG were elevted 60% (P 0.05) nd 2.2-fold (P 0.05), respectively, in 293 C8 cells compred with vector. In contrst, SM nd cholesterol pool sizes were unltered. Since the cholesterol nd SM pool sizes were unltered but cholesterol nd SM de novo biosynthesis were decresed in 293 C8 cells, we interpret this dt to suggest tht SM nd cholesterol metbolism in C8 cells ws lower thn tht of the vector. The mechnism for the elevtion Vector C8 Totl incorportion (dpm 10 6 /mg protein) Cellulr lipid clss TG (dpm 10 5 /mg protein) DG (dpm 10 4 /mg protein) PC (dpm 10 5 /mg protein) PE (dpm 10 4 /mg protein) PI/PA (dpm 10 4 /mg protein) FA (dpm 10 3 /mg protein) PS (dpm 10 3 /mg protein) SM (dpm 10 3 /mg protein) 293 vector nd C8 cells were incubted with [ 3 H]oleic cid bound to lbumin for 4 h nd the rdioctivity incorported into cells nd lipids determined. Dt represent the men SD of three determintions. P 0.05 for comprison of C8 with vector. TABLE 2. Incorportion of l-[ 3 H]serine into SM, PE, nd PS of 293 vector nd C8 cells Vector Totl incorportion (dpm 10 5 /mg protein) Cellulr lipid clss PE (dpm 10 3 /mg protein) PS (dpm 10 3 /mg protein) SM (dpm 10 3 /mg protein) 293 vector nd C8 cells were incubted with l-[ 3 H]serine for 4 h nd the rdioctivity incorported into cells, SM, PE, nd PS determined. Dt represent the men SD of three determintions. P 0.05 for comprison of C8 with vector. C8 Htch et l. FATP1 chnnels ftty cid into TG 1385

7 TABLE 3. Incorportion of [methyl- 3 H]choline into SM, PC, nd LPC of vector nd C8 cells TABLE 5. Pool size of FA, cholesterol, TG, nd SM of 293 vector nd C8 cells Vector C8 Vector C8 Totl incorportion (dpm 10 5 /mg protein) Cellulr lipid clss PC (dpm 10 4 /mg protein) LPC (dpm 10 2 /mg protein) SM (dpm 10 2 /mg protein) 293 vector nd C8 cells were incubted with [methyl- 3 H]choline for 4 h nd the rdioctivity incorported into cells, SM, PC, nd LPC determined. Dt represent the men SD of three determintions. P 0.05 for comprison of C8 with vector. Cellulr lipid clss TG (nmol/mg protein) FA (nmol/mg protein) SM (nmol P i /mg protein) Cholesterol ( g/mg protein) Lipids were extrcted from 293 vector nd C8 cells nd the pool size of FA, cholesterol, TG, nd SM determined. Dt represent the men SD of three determintions. P 0.05 for comprison of C8 with vector. in TG pool size ws exmined. Mitochondril nd microsoml GPAT ctivities were unltered between vector nd C8 cells (Tble 6). In contrst, DGAT ctivity ws incresed 40% (P 0.05) in C8 cells compred with vector. Thus, the increse in TG formtion in C8 cells ws due to n increse in DGAT ctivity. We next exmined whether or not the inverse correltion between de novo TG nd SM biosynthesis in 293 cells over-expressing FATP1 held true for very long chin FAs. 293 vector nd C8 cells were incubted for 4 h with 0.1 mm [ 14 C]lignoceric cid bound to lbumin (1:1 molr rtio) nd rdioctivity incorported into lipids determined. Incorportion of [ 14 C]lignoceric cid into TG ws incresed 35% (P 0.05) from dpm 10 2 /mg protein in vector to dpm 10 2 /mg protein in C8 cells (verge of three seprte dishes). In contrst, incorportion of [ 14 C]lignoceric cid into SM ws reduced 53% (P 0.05) from dpm 10 2 /mg protein in vector to dpm 10 2 /mg protein in C8 cells (verge of three seprte dishes). Incorportion of [ 14 C]lignoceric cid into other phospholipids ws unffected (dt not shown). Thus, in 293 C8 cells over-expressing FATP1 de novo TG biosynthesis from very long chin FA ws elevted nd SM biosynthesis from very long chin FA reduced. FATP1-mdedited uptke of oleic cid is insensitive to tricsin C Since FATP1 CoA synthetse ctivity ws found to be insensitive to tricsin C inhibition wheres ACS forms 1 nd 4 re sensitive to the inhibitor [28, 29], we exmined how the presence of tricsin C would ffect FA incorportion nd its subsequent conversion into TG in C8 cells over-expressing FATP1. Cells were incubted for 4 h with [ 3 H]oleic cid bound to lbumin (1:1 molr rtio) nd totl rdioctivity incorported into cells nd TG determined. Incorportion of [ 3 H]oleic cid into 293 C8 cells ws elevted 41% (P 0.05) compred with vector (Tble 7). The presence of tricsin C (10 M) resulted in 47% reduction (P 0.05) in incorportion of [ 3 H]oleic cid into 293 vector cells compred with untreted vector cells. In ddition, the presence of tricsin C resulted in 33% reduction (P 0.05) in incorportion of [ 3 H]oleic cid into C8 cells over-expressing FATP1 compred with untreted C8 cells. Incorportion of [ 3 H]oleic cid into untreted 293 vector nd tricsin C-treted C8 cells ws similr. Incorportion of [ 3 H]oleic cid into TG in 293 C8 cells ws elevted 83% (P 0.05) compred with vector. The presence of tricsin C resulted in 86% reduction (P 0.05) in incorportion of [ 3 H]oleic cid into TG in 293 vector cells compred with untreted vector cells. In ddition, the presence of tricsin C resulted in 41% reduction (P 0.05) in incorportion of [ 3 H]oleic cid into TG in C8 cells compred with untreted C8 cells. Incorportion of [ 3 H]oleic cid into TG of untreted 293 vector nd tricsin C-treted C8 cells ws similr. Consistent with tricsin C not ffecting FATP1 enzymtic ctivity, tricsin C did not ffect the FATP1 medited elevtion in [ 3 H]oleic cid incorportion into C8 cells nd its subsequent conversion into TG. DISCUSSION The objective of this study ws to exmine the regultion of lipid metbolism from long chin nd very long chin FAs nd to explore metbolic chnnelling of metbolites derived from FATP1 ction. Although some FATP1 TABLE 4. Incorportion of [ 14 C]cette into cholesterol, TG, nd SM of 293 vector nd C8 cells TABLE 6. GPAT nd DGAT ctivities of 293 vector nd C8 cells Vector Totl incorportion (dpm 10 5 /mg protein) Cellulr lipid clss TG (dpm 10 3 /mg protein) SM (dpm 10 3 /mg protein) Cholesterol (dpm 10 2 /mg protein) 293 vector nd C8 cells were incubted with [ 14 C]cette for 4 h nd the rdioctivity incorported into cells, cholesterol, TG, nd SM determined. Dt represent the men SD of three determintions. P 0.05 for comprison of C8 with vector. C8 Enzyme ctivity Vector C8 nmol/min mg protein GPAT microsoml Mitochondril DGAT Mitochondril nd microsoml frctions were prepred from 293 vector nd C8 cells nd mitochondril nd microsoml GPAT nd microsoml DGAT ctivities determined. Dt represent the men SD of three determintions. P 0.05 compring C8 cells to vector cells Journl of Lipid Reserch Volume 43, 2002

8 TABLE 7. Effect of tricsin C on incorportion of [ 3 H]oleic cid into 293 vector nd C8 cells Vector C8 Vector C8 ( Tricsin C) ( 10 M Tricsin C) Totl incorportion (dpm 10 6 /mg protein) b c Cellulr lipid clss (dpm 10 5 /mg protein) TG b c 293 vector nd C8 cells were incubted with [ 3 H]oleic cid for 4 h nd the rdioctivity incorported into cells nd TG determined. Dt represent the men SD of three determintions. P 0.05 vector versus C8, b P 0.05 vector versus vector tricsin C, c P 0.05 C8 versus C8 tricsin C. is found in internl membrnes, the protein is lrgely loclized to the PM in 293 cells (5). Our findings confirm this observtion nd re consistent with recent report demonstrting the presence of FATP1 on internl membrnes in NIH-3T3 cells (6). The results presented herein clerly indicte the surprising finding tht FATP1 chnnels exogenous FA towrd TG biosynthesis while down regulting SM nd cholesterol metbolism in growing 293 cells. The elevtion in DGAT ctivity nd TG biosynthesis from oleic cid or lignoceric cid in growing 293 C8 cells over-expressing FATP1 nd lck of ltered GPAT ctivity nd incorportion of these FAs into DG nd glycerophospholipids indicte tht FATP1 my ply role in TG synthesis t the ER. These observtions support the hypothesis of functionlly independent cyl-coa pools within mmmlin cells which my be chnnelled towrd specific ftes rther thn being freely vilble for ll possible enzymtic rections (27, 28, 29). The question might be sked does PM FATP1 regulte TG metbolism? PM FATP4 hs been shown to regulte FA bsorption in intestinl mucosl cells (34). In these cells, the PM is in close ssocition with the ER. As reviewed by Stremmel et l. (3), it is possible tht FATP4 my ply role in ctivtion of FAs for TG synthesis t the ER in these cells. The sme mechnism of ctivtion of FAs might hold true for FATP1 in 293 cells since the immunofluorescence microgrphs indicted some res of PM FATP1 ssocition with ER. The ACSs referred to s the ACS fmily re fmily of enzymes tht ctlyze the condenstion of long chin FA with CoA to produce cyl-coa (12). The cyl-coas formed re utilized for glycerolipid synthesis, esterifiction of cholesterol, nd -oxidtion, s well s signl trnsduction (35). At lest five different isoforms of ACS exist in mmmlin tissue. Both FATP1 nd FATP4 hve been shown to exhibit brod rnge FA CoA synthetse ctivity (4, 15, 26) nd re members of relted, five-member gene fmily in mice (six members in the humn FATP fmily). However, the specific ctivity of the ACS fmily ppers to be t lest n order of mgnitude greter thn tht of the FATP enzymes (Fig. 3). Tricsin C is non-specific competitive inhibitor of the ACS clss of enzymes nd ws shown to be potently effective ginst ACS1 nd ACS4 isoforms (28, 29). Recently the existence of tricsin C-insensitive ACS (ACS 5) loclized to the mitochondril membrne ws demonstrted (28). In the current study, tretment of C8 293 cells with tricsin C did not ffect the enzymtic ctivity of FATP1 or the FATP1-medited contribution of [ 3 H]oleic cid incorportion into cells nor [ 3 H]oleic cid incorportion into TG. These dt in sum suggest tht FATP1 is tricsin C-insensitive CoA synthetse in growing 293 cells. The results presented herein should be contrsted with the findings of Chui et l., (36) who developed trnsgenic mouse line overexpressing ACS1 specificlly in the crdiomyocyte under control of myosin hevy chin (MHC) promoter. Such MHC-ACS1 trnsgenic mice developed crdic hypertrophy nd ccumulted 12-fold more TG thn did wild-type nimls. In contrst to our findings, MHC-ACS1 crdiomyocytes lso exhibited 50% incresed ccumultion of choline glycerophospholipids nd 15% increse in ethnolmine glycerophospholipids, but no increse in free FA. 293 cells expressing FATP1 similrly ccumulted TG hd n expnded free FA pool, but did not exhibit ny incresed ccumultion of glycerophospholipids nor ltertions in mitochondril nd microsoml GPAT ctivity. Since overexpression of mitochondril GPAT ctivity ws ssocited with incresed TG biosynthesis nd reduction in phospholipid biosynthesis in CHO nd HEK293 cells (37), it would be of interest to determine in MHC-ACS1 crdiomyocytes if there is n upregultion of DGAT nd downregultion of GPAT to ccount for the bifurcted lipid trfficking. It is not cler if the difference in lipid metbolism reflects differences in cell types, or in specific lipid trfficking events linked to either ACS1 or FATP1. Clerly, NIH-3T3 cells overexpressing both ACS1 nd FATP1 exhibit elevted FA uptke nd TG ccumultion (30, 38). However, the effect of overexpression of both ACS1 nd FATP1 on phospholipid metbolism in these cells is unknown. Since cyl-coas hve been implicted s potentil nturl lignds for nucler trnscription fctors, one ttrctive hypothesis is tht cells overexpressing FATP1 nd/or ACS1 ffect cyl-coa pools sufficiently to lter expression of genes whose protein products re linked to complex lipid synthesis. Mny studies hve indicted coordinted link between SM nd cholesterol biosynthesis (11). In ddition, PUFA regultion of SM nd cholesterol biosynthesis is well documented. For exmple, ddition of exogenous oleic cid or other PUFAs to heptocytes resulted in reduced de novo SM synthesis vi inhibition of serine plmitoyltrnsferse Htch et l. FATP1 chnnels ftty cid into TG 1387

9 ctivity (39). Moreover, ddition of exogenous oleic cid nd other PUFAs to vrious cell lines reduced the nucler content of sterol responsive element binding protein-1 nd trnscription of vrious sterol responsive element regulted genes including hydroxymethylglutryl-coa reductse, which would result in reduction in cholesterol biosynthesis (40, 41). In C8 cells over-expressing FATP1, the pool size of FA ws elevted nd incorportion of [ 3 H] serine or [methyl- 3 H]choline into SM nd [ 14 C]cette into both SM nd cholesterol were reduced compred with vector cells. This indictes concerted regultion of these lipids t the biosynthetic level in growing 293 cells. The current study is the first to show coordintion between cholesterol nd SM biosynthesis in growing 293 cells. Since the pool sizes of cholesterol nd SM were not ltered in 293 C8 cells over-expressing FATP1, but de novo biosynthesis from vrious metbolic precursors ws reduced, cholesterol nd SM metbolism in these cells must be lower. Hence, FATP1 my ply role in the regultion of cholesterol nd SM metbolism through provision of exogenous FAs to down-regulte the biosynthesis of these lipids. In summry, the dt present in this pper clerly indicte tht in these ctively growing 293 cells overexpressing FATP1, there is medited metbolic chnnelling of exogenous FA towrd TG formtion nd down-regultion of cholesterol nd SM metbolism. This work ws supported by grnts from the Ntionl Science Foundtion (D.A.B.) nd the Cndin Institutes of Helth Reserch (G.M.H.). G.M.H. ws Hert nd Stroke Foundtion of Cnd visiting scientist. REFERENCES 1. Vn der Vusse, G. J., M. Vn Bilsen, nd J. F. Gltz Crdic ftty cid uptke nd trnsport in helth nd disese. Crdiovsc. Res. 45: Higgins, C. F Flip-flop: The trnsmembrne trnsloction of lipids. Cell. 79: Stremmel, W., J. Pohl, A. Ring, nd T. Herrmnn A new concept of cellulr uptke nd intrcellulr trfficking of long chin ftty cids. Lipids. 36: Hirsch, D., A. Sthl, nd H. F. Lodish A fmily of ftty cid trnsporters conserved from mycobcterium to mn. Proc. Ntl. Acd. Sci. USA. 95: Schffer, J. E., nd H. F. Lodish Expression cloning nd chrcteriztion of novel dipocyte long chin ftty cid trnsport protein. Cell. 79: Lewis, S. E., L. L. Listenberger, D. S. Ory, nd J. E. Schffer Membrne topology of the murine ftty cid trnsport protein 1. J. Biol. Chem. 276: Vnce, D. E Glycerolipid biosynthesis in eukryotes. In Biochemistry of Lipid, Lipoproteins nd Membrnes. D. E. Vnce nd J. E. Vnce, editors. Elsevier, Amsterdm Brindley, D. N., nd D. W. Wggoner Mmmlin lipid phosphte phosphohydrolses. J. Biol. Chem. 273: Lnds, W. E Stories bout cyl chins. Biochim. Biophys. Act. 1483: Merrill, A. H., Jr., D. D. Jones An updte on the enzymology nd regultion of sphingomyelin metbolism. Biochim. Biophys. Act. 1044: Ridgwy, N. D Interctions between metbolism nd intrcellulr distribution of cholesterol nd sphingomyelin. Biochim. Biophys. Act. 1484: Kornberg, A., nd W. E. 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Fleischer Phospholipid composition of humn, bovine nd frog myelin isolted on lrge scle from brin nd spinl cord. Lipids. 5: Zltkis, A., nd B. Zk Study of new cholesterol regent. Anl. Biochem. 56: Trdi, P. G., J. J. Mukherjee, nd P. C. Choy The quntittion of long chin cyl-coa in mmmlin tissue. Lipids. 27: Fried, B., nd I. L. Shpiro Thin lyer chromtogrphic nlysis of phospholipids in Echinostom revolutum (Tremtod) dults. J. Prsitol. 61: Oliferenko, S., K. Pih, T. Hrder, V. Gerke, C. Schwrzler, H. Schwrtz, H. Beug, U. Gunthert, nd L. A. Huber Anlysis of CD-44 contining lipid rfts: Recruitment of nnexin II nd stbiliztion by the ctin skeleton. J. Cell Biol. 146: Colemn, R. A Dicylglycerol cyltyrnsferse nd monocylglycerol cyltrnsferse from liver nd intestine. Methods Enzymol. 209: Hldr, D., nd A. Vncur Glycerophosphte cyltrnsferse from liver. Methods Enzymol. 209: Lowry, O. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. 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Greenberg Modultion of hormone-sensitive lipse nd protein kinse A-medited lipolysis by perilipin A in n denovirl reconstituted system. J. Biol. Chem. 277: Robinson, J., nd E. A. Newsholme The effect of dietry conditions nd glycerol concentrtions on glycerol uptke by rt liver nd kidney cortext slices. Biochem. J. 112: Merrill, A. H., Jr., E. Wng, nd R. E. Mullins Kinetics of long chin (sphingoid) bse biosynthesis in intct LM cells: effects of vrying the extrcellulr concentrtions of serine nd ftty cid precursors of this pthwy. Biochemistry. 27: Ando, H. Y., G. G. Gzdick, E. T. Sugit, nd R. L. Schunre Predicting coordinted lipid biosynthesis: ppliction to the surfctnt-ccommodted epidermis. Lipids. 23: Sthl, A., D. J. Hirsch, R. E. Gimeno, S. Punreddy, P. Ge, N. Wtson, S. Ptel, M. Kotler, A. Rimondi, L. A. Trtgli, nd H. F. Lodish Identifiction of the mjor intestinl ftty cid trnsport protein. Mol. Cell. 4: Colemn, R. A., T. M. Lewin, nd D. M. 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10 nd nutritionl regultion of enzymes of tricylglycerol synthesis. Annu. Rev. Nutr. 20: Chui, H-C., A. Kovcs, D. A. Ford, F-F. Hsu, R. Grci, P. Herrero, J. E. Sffitz, nd J. E. Schffer A novel model of lipotoxic crdiomyopthy. J. Clin. Invest. 107: Igl, R. A., S. Wng, M. Gonzlez-Bro, nd R. A. Colemn Mitochondril glycerol phosphte cyltrnsferse directs the incorportion of exogenous ftty cids into tricylglycerol. J. Biol. Chem. 276: Grgiulo, C., S. Stuhlstz-Krouper, nd J. E. Schffer Locliztion of dipocyte long-chin ftty cyl-coa synthetse t the plsm membrne. J. Lipid Res. 40: Messmer, T. O., E. Wng, V. Stevens, nd A. H. Merrill, Jr Sphingolipid biosynthesis in rt liver cells: effects of serine, ftty cids nd lipoproteins. J. Nutr. 119: Worgll, T. S., S. L. Sturley, T. Seo, T. F. Osborne, nd R. J. Deckelbum Unsturted ftty-cid medited decreses in sterol regultory element-medited gene trnscription re linked to cellulr sphingolipid metbolism. J. Biol. Chem. 273: Hnnh, V. C., J. Ou, A. Luong, J. L. Goldstein, nd M. S. Brown Unsturted ftty cids down regulte srebp isoforms 1 nd 1c by two mechnisms in HEK-293 cells. J. Biol. Chem. 276: Htch et l. FATP1 chnnels ftty cid into TG 1389