rd owde* iegZag io .RdeadMcalJ .Wakelam O. J. Michael and Rudge A. is Simon cells Zhang, FFAR4 Qifeng Huh-7 Rohwedder*, receptor Arndt in acid acid fatty oleic the to by response mediated in formation droplet Lipid ARTICLE RESEARCH 3104 2014 April 26 Accepted 2013; November 6 Received ` Germany. Kiel, 24118 14, Garten Botanischen Christian-Albrechts-Universita address: *Present UK. Campus, 3AT, Research CB22 Babraham Cambridge Institute, Babraham Programme, Signalling HCV suppress can targeting droplet of – lipid lipid droplets of to lipid process at disturbance targeted assembling indeed, are particles capsid HCV (NS5A) the a in protein (HCV); resulting droplets, non-structural virus affects C are a type droplets and hepatitis lipid also (core) by addition, infection In droplets in 2008). important al., et Thiele lipid (Paul 2006; atherosclerosis Garg, of 2010), and Bostro Neuschwander-Tetri, formation (Agarwal 2008; diabetes Spandl, 2008; 2 (NASH) and type Borlak, steatohepatitis and these obesity non-alcoholic and of number a including generation (Anderson in implicated diseases, al., Extensive been et has 2006). of organelles (Schadinger storage al., types lipid et cell neutral are most Fujimoto droplets in Lipid 2005; 2009; droplets 2009). fatty long-chain Farese, generates al., of application acids and et the and Meex Walther organelles, dynamic 2010; highly 2009; Wu, (Goodman, and unique Hodges proteins a embedded of is which al., in set et 2006) 2009) (Tauchi-Sato Ohsaki, al., and phospholipids et Fujimoto of 2002; Cheng of monolayer 2000; up al., a made et by are (Zweytick surrounded lipids droplets Wan neutral Lipid of 2008). 1999; core al., al., a et et Fujimoto (Athenstaedt 2007; most al., organisms in et all found essentially organelles in storage cells intracellular are droplets Lipid INTRODUCTION receptor acid Fatty Phospholipase FFAR4, two PI3-kinase, D, acid, Oleic potential These droplets, Lipid distinct WORDS: uptake. KEY to lipid point upon formation points. droplet dependent intervention lipid is number lipid, of the droplets exogenous mechanisms in increase upon lipid sustained dependent of more not subsequent as the is initial This whereas known formation activities. (PLD) droplet (also D lipid phospholipase and AKT B) involving kinase (PI3-kinase), protein pathway 3-kinase a signalling through phosphoinositide signals which G-protein FFAR4, activating receptor by pertussis-toxin-sensitive acid formation we a fatty droplet Here, involves long-chain lipid uptake. are stimulates the this lipid number acid whether cellular oleic and by known that directed size show not is is droplet or it pathway lipid example, signalling in for changes – how regulated unclear is It ABSTRACT uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,30–15doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. me l,20) yrglto fthe of Dysregulation 2009). al., et ¨m ¨ uKe,MI-C Am MOIN-CC, Kiel, zu t rpe omto a bae nprusstxntetdcells pertussis-toxin-treated 7.95 in ablated was 1F). that was (Fig. condition with formation compared starved cell droplet per the droplets of of number the in 3.88 elevation min, (120 number 4.92 droplet lipid in increase , hrfr motn o el.Hwvr trmisunclear externally remains an through it about brought However, The is cells. 2010). regulation is al., this turnover for and whether et composition or important (Blouin generation, reduction caveolin-1 droplet therefore a of lipid with of sorting associated control of the 2 2009), loss in lipodystrophy al., a defect et congenital by (Boutet Berardinelli-Seip marked (BSCL2) is in is Lipodystrophy tissue tissue droplets, adipose 2008). adipose in lipid al., not but of syndrome, et tissues, (Fujimoto different constituent in Chanarin-Dorfman stored lipid and In deposited major a implications. triacylglycerol, Cle further 2008; have al., et Fujimoto 2006; 2011). al., al., et et (Sato production inligptwyadalne atn lipid-uptake-mediated lasting longer fatty-acid-receptor-mediated a acute pathway. and an to pathway used both signalling droplet be lipid paper through increase can this acids formation in fatty cells long-chain presented the that results evidence normal and provide The Under size, conditions. droplets was lipid hepatic uptake. average of Huh-7 represent number intermediate lipid high line an hepatic a contains cell of of line In hepatoma cell model unclear. this the conditions, is a uptake, this, as the nutritional investigate examined of in to indicator pathways an order exposure acids, signalling cell but, to fatty response 2006) of in to droplets al., lipid involvement of et formation the Andersson stimulated 2005; this, lipid ERK al., the been despite of and et has (PLD) growth (Nakamura D It and phospholipase pathway 2010). upon localisation dependent al., formation, is et droplets lipid the (Digel cellular that by uptake suggested directed thus, just and, is availability or pathway signalling activated bevbeb i fe laeadto tefl increase was (3.13 fold level min (the 30 starvation addition to oleate the 1.44 up was after control from starved min biphasic the 5 above increase a by was rapid addition observable lipid the oleate of in following increase process; number The of number dependent. addition the droplet concentration the was after lipid in and minutes 1) few increase (Fig. a oleate within This 100 could apparent 1). was of this (Fig. droplets cells; addition medium Huh-7 the in free by droplets reversed lipid an be overnight detectable by about of medium brought number culture cell withdrawal from serum acids fatty of Depletion RESULTS 0mn(3.23 min 60 h xesv cuuaino nrclua ii rpescan droplets lipid intracellular of accumulation excessive The 6 .9 Fg E.I oto nrae el,tefold the cells, untreated control In 1E). (Fig. 0.19) 6 .4,atrwihteefloe continuous a followed there which after 0.24), 6 .4,floe yapaeupaeto phase plateau a by followed 0.24), ` 6 0.17; m 6 , laet h serum- the to oleate M .7 Oleate-stimulated 0.37. 5 euto nthe in reduction 85% 6 ... n increased and s.e.m.) 6 .8 8 min, 180 0.18; ´ ment

Journal of Cell Science EERHARTICLE RESEARCH h el eeicbtdfr1hwt [ with h 1 for incubated lipids, cellular were in cells changes stimulated the examine To mechanisms. hstopaepoessget h novmn fdistinct of involvement the suggests process two-phase This 3 ]oet,adthe and H]-oleate, eetv clto fDGseis(i.2.Bcuelow Because 2). to (Fig. pointing [ species elevated, DAG of particularly of were incorporation acylation 54:2 and selective 52:3, 50:2, DAG 54:3 50:3, double 36:3 50:4, 48:2, carbon:total 52:2, and 48:3, acyl species 34:1 (total triglyceride 34:2, The ester bonds). extent, cholesterol lesser 18:1 a and species to particularly cells of and, serum-starved LD540 the 36:2 stimulation of elevated stimulation oleate that oleate the demonstrated min to species and 15 molecular a triglyceride the response confirmed of DAG, Analysis in cells. in starved content analysis not increase ester an cholesterol (data lipidomic showing no in also by pool experiments The was followed was There monoglyceride there starved content. shown). that oleate-containing oleate been demonstrated cellular detectable min had in 15 change that for minimal oleate cells with and treatment cells starved (0.06 biosynthesis. lipid acid in increase small a fatty an was suggesting there the in nevertheless, 1); into (Fig. increase time in this radiolabel increase at the droplets despite of lipid fractions, ester into incorporation cholesterol and oleate stimulated triglyceride of minimal incorporation 100 to was the exposed cells In in lipids. neutral increase 0.72 an demonstrating (phospholipid, oleate h 1 hshtdcai eeshddcesd implying decreased, had levels acid C17 and phosphatidic total the the min, C17 oleate. with 15 with at the stimulation that, loaded to showed prior analysis were h spectrometric Mass 2 for cells the acid of fatty serum-starved lipids heptadecanoate the oleate drops, into whether acids determine fatty lipid of To suggests incorporation droplets. rapid further lipid stimulated of the this underlies generation remodelling 1F), acute and/or (Fig. metabolism lipid detected stimulated that was ester cholesterol xrse stepooto fttlcut eetd and found detected) counts was total labelling of proportion cells, (TLC) (0.53 the then control phospholipids as chromatography was expressed the in classes thin-layer predominantly In lipid following between separation. radiolabel quantified of distribution oad hs ftecnrlfloiga2hepsr o100 to exposure h 2 a the following for and control into the starved incorporated of was been those label towards had (0.91 (MAG) the of that fraction proportion cells phospholipid greater In a monoacylglycerol h, 1G). 16 (Fig. ester (DAG), cholesterol (0.44 diacylglycerol triglycerides opeemdu r loson() cl as 30 bars: with Scale (D). incubation shown by also followed are was medium this complete green. and in (A), shown h have are 16 100 nuclei outlines for and Cell starved (red) (blue). were dye 33342 Cells LD540 Hoechst the with which with stained in stained been cells been Huh-7 of cells. have recordings Huh-7 droplets confocal in lipid 3D droplets of lipid projection Maximum in (A–D) increase Oleate-stimulated 1. Fig. 1 ,popoiis G icllcrl A at cd;T,tilcrds CE, triglycerides; TG, acids; ester. fatty ( cholesterol FA, fraction each diacylglycerol; as in DG, presented minute per phospholipids; are P, disintegrations Data of Methods. proportion was the and of the fraction Materials means to each in prior in described h radiolabel as 16 of determined for proportion serum-free The cultured (Starv). been experiments had cells the oleate; yperaigHh7clswt 0n/lprusstxn(T)fr1 h 16 mean for the [ (PTX) show of toxin Data distribution pertussis period. ng/ml starvation 10 the inhibited with during was cells formation Huh-7 (LD) pretreating droplet by lipid Data Oleate-stimulated imaging. (F) confocal cells. 3D starved the from mean cells the oleate-exposed represent in droplets lipid of m iioi assetoercaayi ftecnrlcells, control the of analysis spectrometric mass Lipidomic io [ of Ci m laefr1 i B r3h() oto u- el rw in grown cells Huh-7 Control (C). h 3 or (B) min 15 for oleate M ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal 3 ]oecai nDE o eoebigsiuae ih100 with stimulated being before h 1 for DMEM in acid H]-oleic 3 ]oet nlpd rmHh7clsta eeicbtdwith incubated were that cells Huh-7 from lipids in H]-oleate 6 ...adwr omlsdt eut rmserum- from results to normalised were and s.e.m. 3 6 ]oet noDG rgyeieand triglyceride DAG, into H]-oleate .6,wt mle mut in amounts smaller with 0.06), 6 6 .1.Teepootosreturned proportions These 0.01). 6 .5 rgyeie 0.26 triglyceride, 0.05; .1 n A (0.02 DAG and 0.01) m laefr1 i,there min, 15 for oleate M 6 6 ...()Relative (G) s.e.m. .6–dt are data – 0.06 m .()Quantification (E) m. 6 6 6 0.05), 3105 0.03) s.d.). m m M M

Journal of Cell Science EERHARTICLE RESEARCH 3106 ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal laefr1 i ntepeec of presence the in 10 min 15 100 for plus oleate cells starved bars, striped 100 plus cells starved cells; bars, starved horizontal-striped bars, filled cells; control bars, of Open means measurements. are duplicate data The (CE). cholesterol ester (C) (TG), triglyceride (DG), (B) Diacylglycerol (A) classes. lipid of separation HPLC-mediated Methods following and Materials as in out described carried was species lipid molecular individual of determination spectrometric Folch Mass the method. using extracted were and lipids added, the were standards lipid defined lipids. neutral analysis of spectrometric Mass 2. Fig. m m Y902(I-iaeinhibitor). (PI3-kinase LY294002 M laefr1 i;vertical- min; 15 for oleate M el eeisolated, were Cells m M

Journal of Cell Science uniiaino ii pce napriua ii ls htcnanaC1-at-cldrvdgroup C-17-fatty-acyl-derived a contain that class ( lipid lipid particular C-17 cells a in heptadecanoate-loaded species in lipid lipids of of Quantification measurement spectrometric Mass 1 Table ARTICLE RESEARCH i.3 eedneo otne ii rpe omto pnetaellroleate. extracellular upon formation droplet lipid continued of Dependence 3. Fig. the support data These increased. declined was ester cholesterol incorporation increase C17 no it oleate of was level there as the makes but – min incorporation and 15 C17 lipids in after DAG total increased MAG, Additionally, DAG other 1). from and Table MAG lipids (see to triglyceride in to shift acid relative phosphatidic the quantify triglyceride to impossible of amounts increased E1 383 14 201 8385 0.25 8 17 8310 170 0.99 13 21 8245 1.63 180 14 44 8600 170 1.8 CE PA TG DAG MAG C26 3028 2331 0.1 2082 0.13 2390 0.13 2060 0.12 class lipid particular ( a Lipid in present lipids all of Quantification PC FACoA E212120110 13.2 379 52,100 4010 154 15,428 210 13.5 471 44,845 3494 11,545 117 211 51,005 13.9 494 10,895 3502 135.5 45,905 271 12.5 734 11,805 2771 29.5 phosphati PC, acylCoA; fatty FACoA, ester; cholesterol sing CE, a acid; from measurements phosphatidic duplicate PA, of triglyceride; means 3 are TG, data diacylglycerol; of spectrometry. The addition DAG, mass monoacylglycerol; by the quantified MAG, and indicated by times followed the BSA, at extracted DMEM+1% were lipids in The oleate. h 18 for incubated were Cells PC FACoA CE PA TG DAG MAG 0mn usqety ihrti a anand(lc as rtemdu a eoe n,floigaPSws,teclswr nuae nfresh quantif mean in and the incubated staining as were LD540 expressed cells by are identified the and were wash, (starvation) droplets PBS cells lipid a fatty-acid-starved and following from time-points, and, results stated removed to the was at normalised P medium fixed were the were data or cells The bars) The microscopy. (black bars). maintained (open was medium this fatty-acid-free either Subsequently, min. 10 5 .;3 min, 30 0.9; 6 10 6 6 g -i lae1-i lae3-i lae10mnoleate 120-min oleate 30-min oleate 15-min oleate 0-min ng) enovo de 10 6 P g -i lae1-i lae3-i lae10mnoleate 120-min oleate 30-min oleate 15-min oleate 0-min ng) 5 .;10min, 120 0.5; rgyeiesnhss h xrml high extremely The synthesis. triglyceride P , 10 2 10 8 min 180 ; P , 10 2 10 (Student’s t -test). m etdcnae olwn ute ,teclswr tmltdwt 100 with stimulated were cells the h, 2 further a Following heptadecanoate. M a eoe n h el eewse ihwre PBS, warmed with washed were cells the and removed was 100 containing the external time-points of incorporation min) the 15 than to oleate. rather from synthesis lipids, (up increased cellular from comes early existing droplets in the lipids in at increase that proposition nclsta eesiuae yteadto fmedium of addition the by stimulated were that cells In tre u- el eeepsdt 100 to exposed were cells Huh-7 Starved ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal m laefr1 i,atrwihteoleate the which after min, 10 for oleate M 6 ...The s.e.m. m lae(A for (OA) oleate M P vle r:1 min, 15 are: -values dylcholine. eexperiment. le e yusing by ied 3107 m M

Journal of Cell Science EERHARTICLE RESEARCH ii rpe ubrosre fe i laestimulation 3108 oleate min 5 a after observed (1.45 number 3.52 droplet followed was lipid exposure absence) oleate its min in 1.6 10 starved min the (i.e. 20 fold of min had The that by 30 above 3). oleate after droplets (Fig. cells lipid min the of control 20 number further though the a in least as increase at continued, for present, formation remained droplet lipid 6 .6fl fe ,wihwssmlrt h odcag in change fold the 6 to similar was which h, 2 after 0.16-fold 0.17). 6 .8 u hsdcie to declined this but 0.28, P3kns)ptwyi rca o ii rpe eeainin generation droplet lipid for droplet crucial lipid is 3-kinase for phosphoinositide pathway the important (PI3-kinase) that be demonstrates 4 to Fig. a proposed formation. of been signalling involvement of have the number pathways indicating A pathway. further signalling shown), fatty-acid-stimulated capric not fatty (e.g. acids long-chain (data fatty other short-chain acid) not of but addition acid), linoleic the (e.g. to acids response in cells starved nices nlpddoltgnrto a loosre in observed also was generation droplet lipid in increase An ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal ro osiuainwt 100 with stimulation to prior (10 LY294002 inhibitor PI3- kinase the with cells preincubated Huh-7 were (C) acid. capric short- chain the with stimulation following observed was membrane plasma the No to (B) translocation (arrows). GPR1–EGFP min 1 for oleate with stimulation following was observed membrane plasma the to EGFP–Grp1-PH of Translocation (A) protein. fusion the stably expressing cells of recording disk confocal spinning by examined was EGFP–Grp1-PH of localisation The activity. PI3-kinase droplet by lipid formation of Regulation 4. Fig. PtdIns(3,4,5)P (Student’s min, 15 The (starvation). cells fatty-acid-starved lipid in of droplets number the to normalised mean the as are expressed Data cells. untreated bars, closed min. cells; 180 LY294002-treated or bars, min Open 15 for (OA) oleate el fe tmlto ih100 with stimulation after cells ncdw el seFg 8). Fig. (see cells FFAR4- knockdown bars, open control cells; bars, Huh-7 Closed test). U Whitney P the as mean expressed are and (Starv) cells fatty-acid-starved to from normalised results were data The oleate. 5 .;10min, 180 0.2; 6 ..The s.d. P 5 t ts) D h ai of ratio The (D) -test). .0;10min, 180 0.005; 3 :PtdInsP P P P vle r:1 min, 15 are: -values vle are: -values 5 .9(Mann- 0.09 2 6 nHuh-7 in s.e.m., P m , M m m 0.001 M) M

Journal of Cell Science uniidb h PtdIns(3,4,5)P the by quantified lyarl nlpddoltfrain(aauae l,2005, al., et with cells (Nakamura Huh-7 of formation incubation droplet However, 2006). lipid al., et in Andersson role a play droplets. lipid the and domain nlssdmntae htteicesdPtdIns(3,4,5)P increased the that spectrometric demonstrated mass Additional analysis 4D). Fig. (see ratio 4,5-bisphosphate) nw spoenkns ) hswsdtcal y1 min 10 that cells by in detectable inhibited was was but min, This 30 10 to B). with preincubated (also were continued blotting AKT kinase and western of 5A) protein Second, (Fig. phosphorylation shown). as the (not stimulated known oleate species that 38:3 showed or 38:4 the EERHARTICLE RESEARCH inligi laesiuain he taeiswr adopted. (3,4,5)-trisphosphate were phosphatidylinositol strategies PI3-kinase- [PtdIns(3,4,5)P of three measurement upon stimulation, PI3-kinase First, oleate of generation involvement in a the droplet signalling indicates confirm This To of signalling. 2). dependent (Fig. dependence ester LY294002 cholesterol by strong and inhibited triglyceride were DAG, species in oleate- the increases that showed stimulated also analysis spectrometric Mass 4C). 5.10 (Fig. (control, min droplet lipid 180 time- in at stimulation min increase number fold 15 10 oleate the the suppressed significantly at to and generation with point droplet prior lipid inhibitor) Preincubation inhibited completely PI3-kinase cells. (a LY294002 starved oleate-stimulated cells. 100 oleate-stimulated in with AKT treated of Phosphorylation 5. Fig. noet-tmltdices nclua PtdIns(3,4,5)P cellular in increase oleate-stimulated an roet-tmltdcls ewe h PtdIns(3,4,5)P the between cells, domain oleate-stimulated control Grp1-PH either in colocalisation, the or no of was there localisation that demonstrated the also translocation the of membrane Examination plasma stimulated 4B). induce (Fig. to oleate able 4A). not that (Fig. was formation, membrane plasma show the 100 contrast, to to By domain PH GFP the of with translocation tagged domain h rsneo %BAdrn tmlto ihoet i o alter not did oleate with stimulation during response. PI3- BSA 100 the the 1% with with of stimulation incubated presence to were The prior that (LY) cells LY294002 using inhibitor above (phospho)-AKT.kinase as phosphorylated blotting and AKT Western for (B) blotting western and PAGE u- el tbyepesn PtdIns(3,4,5)P a expressing stably cells Huh-7 rvosrprshv ugse htPDadEKsignalling ERK and PLD that suggested have reports Previous m 3 ocnrto yms pcrmtydemonstrated spectrometry mass by concentration ] lae(A n eletat eesbetdt SDS- to subjected were extracts cell and (OA) oleate M m arcai,wihddntidc ii droplet lipid induce not did which acid, capric M m Y902(i.5) hr,w used we Third, 5B). (Fig. LY294002 M 6 3 :PtdInsP .9 L240,1.79 +LY294002, 0.19; 2 (phosphatidylinositol 3 bnigGRP1-PH -binding m oleate. M A el were Cells (A) 3 a either was 3 -binding 6 3 0.21) ,as m M ciaino FR,cagsi h PtdIns(3,4,5)P oleate the to in linked changes To pathways min. FFAR4, 15 signalling of at FFAR4- primarily the activation with apparent transfected characterise was phosphorylation transiently further this cells AKT 8C,D); (Fig. of pEGFP reduced that with demonstrated compared cells knockdown blo western formation, droplet lipid ihtefntoal ialdcntutFA4HpGP(5min, (15 FFAR4RH-pEGFP 0.76 construct disabled functionally the with laefie oices h hshrlto fEK(aanot (data (10 ERK inhibitor ERK of the phosphorylation of inclusion the the and increase shown), to failed oleate hN ol ervre ytasetytasetn h el with 2.26 cells min, the transfecting (15 transiently FFAR4-pEGFP by reversed be could shRNA 2.37 3.64 both (pLKO, to FFAR4-knockdown, 8B) response (Fig. in the reduction 1.37 min from a 15 showed numbers line a droplet cell with control FFAR4- compared the pLKO in line droplets cell lipid of knockdown Quantification 8A). (Fig. of Western expression control. FFAR4 the suppressed a shRNA as the used that line demonstrated be blotting empty- to cell line an cell pLKO-vector-based with Huh-7 along vector-containing established, stable was shRNA a FFAR4 expressing formation, droplet To lipid oleate-stimulated regulating 8A). in (Fig. FFAR4 cells of importance HEK293 the in determine not responsible but cells receptor HeLa fatty in was the detectable FFAR4 also FFAR1 8A). as (Fig. not formation former droplet lipid the but oleate-stimulated for to FFAR4 the pointing the receptor, of acid induced presence the GW9508 demonstrated formation, droplet 7B). (Fig. AKT lipid of phosphorylation in increase rvddb nuaigclsoengtwt etsi oi,which toxin, pertussis with overnight cells incubating was receptor- by proposition this provided for a evidence Further stimulates increase formation. droplet an lipid to oleate leads in that that cells Huh-7 in suggest pathway signalling data activated lipid phospholipase These the in of 6E). the activity (Fig. of the involved regulate one to is ARF1, known GTPases with 6A–D) PLD1 small colocalised (Fig. also that PLD1 detection suggesting processing. droplet droplets, droplet PLD1 of lipid colocalization lipid exclusive with not for but extensive LD540 demonstrated with stained inhibiting in effective 3.19 was inhibitor, PLD1 (PLD1 5.1 formation not droplet droplet but lipid PLD1 lipid of in inhibition change (fold 1.06 1.26 PLD inhibition, effective either PLD1 of following lipid equally inhibition number prevented min, In was 15 6D). 2009) for (Fig. isoform oleate al., stimulation with oleate et stimulated nM to cells 100 response (Scott in either inhibitor formation with droplet PLD2 Preincubation By or activities. both formation. PLD1 PLD2 upon dependent droplet and be to lipid PLD1 found was oleate-stimulated generation droplet affect contrast, to failed tmlto 10mn i o ute nraelpddoltnumber droplet 5.1 lipid droplet increase (oleate, further not in did min) increase (180 stimulation fold 2.69 the 2.88 (oleate, oleate when GW9508, by induced that min, to similar 15 was This formation after 7A). (Fig. apparent acids fatty exogenous was of absence the in formation in droplet cells lipid Huh-7 induced FFAR4, 1F). and thereby primarily FFAR1 (Fig. receptor, upon acid active pathway fatty formation, long-chain this the of agonist in an droplet GW9508, receptor Gi/Go-coupled lipid a implicating oleate-stimulated inhibited etr ltigo ebaepeaain rmHh7cells Huh-7 from preparations membrane of blotting Western xmnn u- el tbyepesn EF-L1adco- and pEGFP-PLD1 expressing stably cells Huh-7 Examining 6 6 6 6 .9 L2ihbtr 4.23 inhibitor, PLD2 0.19; .7 8 i,3.08 min, 180 0.17; .4 n 8 i laesiuain(LO 7.18 (pLKO, stimulation oleate min 180 a and 0.14) .1 nrae,3.12 untreated, 0.31; ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal 6 .9 W58 2.71 GW9508, 0.19; 6 .2;hwvr ncnrs ooet,alonger a oleate, to contrast in however, 0.72); 6 6 .1.I aallwt h euto in reduction the with parallel In 0.31). .4.Teihbtr feto the of effect inhibitory The 0.34). 6 6 .5 8 i,5.64 min, 180 0.35; .4;wees fe 8 min, 180 after whereas, 0.24); 6 tn foet-tmltdFFAR4- oleate-stimulated of tting 0.68). 6 6 .9.I aallwt the with parallel In 0.89). .1 FFAR4-knockdown, 0.41; 6 .9 L2inhibition, PLD2 0.19; 6 .1 untreated, 0.21; 6 m PD98059) M .8 u not but 0.58) 3 :PtdInsP 6 6 3109 1.02; 0.14; 2

Journal of Cell Science EERHARTICLE RESEARCH 3110 6. Fig. e etpg o legend. for page next See ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal

Journal of Cell Science dt o hw) niaigta h aesgaln aha is this receptors. pathway acid signalling and fatty same both the cells, by that used indicating pLKO LY294002 shown), with not mock-transfected preincubation (data by ablated in was droplets response observed lipid recovered of number was the in as increase similar a generated oleate FR.Siuaino h FR-rnfce el ih100 for with substitute FFAR4-knockdown cells also FFAR1-transfected could the in of FFAR1 Stimulation receptor formation FFAR4. alterative droplet the but lipid cells, of stimulation the in FFAR4-knockdown detected the in was observed min not (0.84 15 was cells this for but cells, stimulation pLKO oleate to response L2ihbtr h aawr omlsdt h ubro ii rpesin mean droplets the lipid as of expressed number are the and to (starvation) normalised cells were bars, fatty-acid-starved horizontal-striped data inhibitor; The 100 PLD1 inhibitor. with bars, PLD2 PLD2 open stimulation or cells; to PLD1 control prior the bars, nM) with Closed 100 cells lipid at Huh-7 with (each of PLD1 Preincubation inhibitors of (D) the association white). – strong (C, ImageJ a droplets pixels using showed colocalised determined plugin isolated was colocalisation The images RGB red). LD540 (B, and LD540 GFP–PLD1 with from stained and is and green) Huh-7-cells formation. (A, in droplet droplets lipid lipid for with colocalises necessary partially PLD 6. Fig. ARTICLE RESEARCH eimrpdyidcsteapaac fdtcal lipid detectable serum-free of to oleate appearance of [ the addition The induces the droplets. that rapidly LD540. clear as medium such they is dyes whether it lipid-specific structures using or However, lipid-free detected starvation become be whether or to cannot detectable state that be response to to in possible small too fatty not lost become is exogenous are It of droplets 1). subsequent addition the (Fig. the oleate the and notably following starvation acids, number to droplet response in in recovery lipid reduction of their by process regulated increased of a consequence a uptake. to solely lipid being it point than rather paper generation, presented results this 2012; the droplet Hussain, nevertheless, in and 2012); Lipid (Sturley Wolins, and organelle formation. Brasaemle the and their in modification uptake, storage of lipid subsequent known a cellular is regulation requires little as clearly that acute Thus, formation surprising are operate is synthesis. the it they viral and also about crucial, and instead, is can signalling regulation structures; their intracellular that for storage organelles platform simple dynamic than specialised droplets a more lipid in However, lipids droplets. store are lipid cells in been that manner essential has regulated thus is highly resistance tissues It insulin failure. and as heart even cells such and disorders, in pathological lipid with excess associated of accumulation The DISCUSSION pLKO- in FFAR4- changes 1.74 in A the spectrometry 4D). with (Fig. mass cells compared by expressing cells determined knockdown were ratio The diint h eimteewslmtdicroaino fatty of incorporation limited was 1) there oleate following (Table medium min) the experiments 30 to and spectrometry (15 addition times mass short at acid that demonstrated fatty C-17 and ncnrlsavdcls(o shown). 100 (not observed with cells was min (yellow) starved 180 colocalisation control Some or in imaging. min and 15 fixing to for prior Arf1–mCherry stimulated oleate and were left) Cells cells. (green, middle). Huh-7 GFP–PLD1 (red, transfected with transiently transfected of were recordings Cells confocal 3D of projection inhibitor, inhibitor, o nycudtaseto fFA4pG eceteoleate the rescue FFAR4-pEGP of transfection could only Not h yai aueo ellrlpddolt sdemonstrated is droplets lipid cellular of nature dynamic The P vle r:1 i L1inhibitor, PLD1 min 15 are: -values P P 5 5 6 .02 8 i L1inhibitor, PLD1 min 180 0.0002; .9(Student’s 0.09 0.19). 3 ]oet noprto xeiet Fg 1G) (Fig. experiments incorporation H]-oleate t ts) .,nnsgiiat E Maximum (E) non-significant. N.S, -test). u- el xrsigGFP–PLD1 expressing cells Huh-7 P 5 6 P 9.4 .2fl tmlto in stimulation 0.32-fold 5 6 1 6 10 10 2 2 5 5mnPLD2 min 15 ; 8 8 i PLD2 min 180 ; m lae(OA). oleate M 6 m s.e.m. M m M oaiaino PtdIns(3,4,5)P of localisation oterl fteercposi laesiuae ii droplet lipid oleate-stimulated in receptors these pointing of FFAR1, not role but the FFAR4, express cells to study Huh-7 acid The this formation. fatty in not droplet examined but FFAR4 lipid short-term in and the increase long-term stimulating FFAR1 the in the oleate replace of can is agonist receptors, formation molecule GW9508, small that droplet and a the can lipids oleate monounsaturated that of long-chain pulse for formation, a specific data that droplet show the which lipid by 7, and supported stimulate 3 is 2, This Figs is in process. response presented signalling acute a dependent more of the undoubtedly incorporation, suggestive and is uptake oleate Whereas increase upon formation. their longer-time-dependent of mechanisms the distinct to points oleate content. increase triglyceride significant total a also in and lipids droplet-associated into oleate hsi eetbea nices nPtdIns(3,4,5)P in increase an as detectable Gi. is pertussis-toxin-sensitive This the activates through receptor In FFAR4 presumably inhibitors. to the ERK PI3-kinase, ligating due specific signalling, is the ERK more PLD of result for to generation in in addition newer evidence difference a no differences of this use that found our apparent possible we is PLD2, with it and involvement; formation, PLD1 a of plays droplet functions activity PLD in that been proposal role the inhibitor our for has PLD Although support 2006). provide requires It al., studies et formation (Andersson cascade. droplet activity ERK signalling lipid and that a suggested through previously formation droplet 8). (Fig. formation min shRNA droplet 120 suppressed an after significantly and using incubation a min FFAR4, following 15 formation the droplet oleate-stimulated of ablated approach, depletion indeed, formation; h tmlto fPDatvt yoet cigtruhafatty a droplet. through lipid acting the oleate at by regulator activity major PLD a of as stimulation a 2004), The PLD1 is al., to PLD1, PLD2 pointing effect. et of thus minimal term (McDermott inhibition a isoform has the longer PLD2 plasma-membrane-localised by of contrast, oleate- inhibited inhibition the by acute also whereas is upon formation; formation effect droplet and droplet inhibitory PLD1 lipid Both an droplet. stimulated lipid have might the inhibitors it upon that effect PLD2 direct and more cascade, a oleate-stimulated have downstream the is in more signalling PI3-kinase PLD be of that small to suggests regulatory This its appears ARF1. with along GTPase, contrast, droplet, the by stimulates with associated that PLD, closely process formation. signalling droplet receptor-linked lipid the is this that ihoet eosrtsacerricroaino de [ added of incorporation in clearer increases a incubation longer demonstrates variable the oleate or contrast, 1; with either By fusion (Table droplets. size droplet particular droplet into analysis stimulated lipid uptake in to spectrometric increase due non-regular the being the mass with stimulates as 2), the droplets, Fig. addition lipid by into oleate lipid demonstrated cellular that (see endogenous droplets implies of lipid incorporation of This size and a 1A–C). number being Fig. the there in despite increase ester, cholesterol significant and triglycerides into acid epnet laebtntcpi cdsiuain n eddnot did we PtdIns(3,4,5)P and stimulation, plasma of acid accumulation the capric detect not in but in oleate increase GFP–Grp1-PH-domain to an transfected by response a observed mimicked of be localisation we membrane could addition, and In LY294002 to GW9508. inhibitor sensitive in was PI3-kinase increase which an the by phosphorylation, and AKT cells FFAR4-knockdown oleate-stimulated stimulation not oleate but to control response in in spectrometry, mass by measured hscerybpai eeaino ii rpesi epneto response in droplets lipid of generation biphasic clearly This cuaino h FR at cdrcpo tmltslipid stimulates receptor acid fatty FFAR4 the of Occupation ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal 3 ttepam ebaeimplies membrane plasma the at 3 ttelpddolt The droplet. lipid the at 3 as as mass, 3111 3 H]-

Journal of Cell Science oeua pce ssmlrt htgnrtdb L (Pettitt PLD by the generated stimulated that that early to the similar provided only is phosphatidic-acid-dependent, species that can molecular or is pathways times, suggesting pathway phosphatidic-acid-generating these inhibition, at partial other compensate only that is there either time-points pathway. receptor-stimulated later the to At it suggested linking time-points, been early formation droplet at of oleate-stimulated also Inhibition ablates 2008). fully only al., has activity et PLD signalling Bell 2006; Wilson, insulin and different (Vereshchagina in through pool droplet types lipid the cell upon AKT of effect regulatory 2012). (LPS) the al., A et lipopolysaccharide (Khatchadourian microglia, LY294002 following to sensitive In is droplets stimulation regulating suggested. lipid in previously of signalling been PI3-kinase generation has for droplets role as lipid A activity, remains that determined. PLD1 signalling known AKT be control for (officially to function thereby separate a Arno and be might ARF as there activate such that CYTH2) (GEFs) factors exchange EERHARTICLE RESEARCH 3112 regulatory fundamental PtdIns(3,4,5)P a implies Although oleate min to role. its exposure 15 PI3- that after min fact both How 180 number the and droplet resolved. although in be increase unclear, the to is ablates inhibition process remains lipid the 2012) of regulates fission al., kinase or et 2009) (Long al., droplets the be et formation, from (Robenek budding might droplet reticulum by droplets, enzyme endoplasmic lipid existing using this oleate-stimulated happens this that whether in suggests involved events directly fission and species. PLD fusion unidentified thus an treatment implicate oleate to following need receptor-mediated PLD2 the – and removes identified PLD1 been al., never of et has stimulation (Kasai that enzyme cells long-standing PLD in oleate-stimulated the 1998) an for is explanation there that an issue provides also receptor acid h eonsdrl fPDi euaigmmrn trafficking, membrane regulating in PLD of role recognised The 3 a euaegaienucleotide guanine regulate can eetr ncnrs otemr eeeiu fet observed this effects absence. by deleterious its more activated in uptake the pathway lipid to unregulated signalling following contrast key the a and in to points for receptor, This epidemiological liver. role fatty a was regulatory including combined there a obesity, absence in its of by in increase importance a – an shown The demonstrated was 2012). receptor using al., FFAR4 been functioning et (Ichimura al., have approach reducing The knockout dysfunction diet. et in high-fat FFAR4 a role Ichimura to a of response in play effects formation droplet could lipid deposition. lipid inhibitors acute reducing in PLD effective and be Alternatively, drugs, to prove anti-insulin also as might considered PI3- they as been occurs. such formation have diseases, thus, droplet inhibitors excess in kinase droplets diabetes, and, whereas, lipid 2 lipids, type the formation and for obesity states, devices in droplet healthy storage switch In as lipid serve disease. important and of an elevated health be amount to between could exposure the acids of regulating length fatty could and of This magnitude the concentrations uptake. the in following that results lipid suggest exposure exogenous longer primarily of cells whereas incorporation in lipid, exposure droplets endogenous for of short-term growth from crucial formation; or is generation droplet the acid lipid stimulates fatty of of mechanism concentrations the elevated to exposure storage. lipid cellular – promote formation mTOR to droplet known lipid in activate is increase to mTOR means indeed, an a into has be might feeding PLD this this both activity are although if activities 2001), Nevertheless, PI3-kinase An suggestion. al., contentious and formation. et a be (Fang droplet to mTOR which proved lipid phosphatidic regulate activity, that for can reports PI3-kinase the acid essential to by suggested contrast fully is possibility in additional be is to This appears 2001). al., et h aai hspprsgetamdli hc h eghof length the which in model a suggest paper this in data The ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal tmltdcells. stimulated GW9508- the in (pAKT) phosphorylation show AKT to blots Western (B) non-significant. N.S., test). P P min, 15 are: values mean the as expressed are and (starvation) cells fatty-acid-starved from results to normalised were data The stimulation. (OA) oleate after bars) (closed cells control with compared bars) (open GW9508 agonist FFAR4 the with stimulated cells in recordings 3D confocal from droplets lipid of agonist. FFAR4 with cells of Stimulation 7. Fig. 5 5 .03(Student’s 0.0003 .;10min, 180 0.8; 6 ...The s.e.m. A Quantification (A) P - t -

Journal of Cell Science EERHARTICLE RESEARCH ora fCl cec 21)17 1431 doi:10.1242/jcs.145854 3104–3115 127, (2014) Science Cell of Journal el.()Rltv uniiaino h data the of quantification Relative (D) cells. fatty-acid-starved shRNA- FA-Starv, the cells. in treated AKT of Phosphorylation (C) P knockdown+FFAR4RH, shRNA FFAR4 niio LY294002. PI3-kinase LY, inhibitor 8C). Fig. in shown blot (representative in phosphorylation changes AKT determining blots multiple from knockdown+FFAR4RH, shRNA FFAR4 min 15 knockdown+FFAR4–GFP, knockdown, P shRNA FFAR4 min 180 knockdown+FFAR4–GFP, shRNA FFAR4 min 15 knockdown, shRNA FFAR4 the as expressed mean are Data oleate. OA, (FFAR4RH). mutant dominant-negative non-functional a with transfection additional bars, horizontal-striped FFAR4– GFP; with transfection additional vertical-striped bars, vector; pLKO with empty transfected an cells bars, closed FFAR4 shRNA; expressing stably cells Open Huh-7 cells. bars, Huh-7 of in effects FFAR4 The of (B) knockdown type. wild Hela wt, and cells. cells Huh-7 in (FFA4) FFAR4 signalling. stimulated oleate- mediates acid FFAR4 fatty receptor long-chain The 8. Fig. 5 5 .9 Mn-hte test). U (Mann-Whitney 0.391 .7;10mnFA4shRNA FFAR4 min 180 0.375; 6 ...The s.e.m. P vle r:1 min 15 are: -values A eeto of Detection (A) P 5 P P .7;10min 180 0.078; 5 5 P 5 0.531; 0.093; 0.014; 3113

Journal of Cell Science niios h L1adPD niioswr sda described as or used agonists were acids, inhibitors fatty for PLD2 with free) stimulation and endotoxin to PLD1 free, prior The acid h fatty inhibitors. 16 was (BSA; for medium albumin starvation (PAA) serum The and DMEM bovine (VWR). penicillin-streptomycin 1% in 1% with coverslips supplemented 50% DMEM round with penicillin- of replaced 1% 22-mm and confluency (PAA) on (FCS) a serum streptomycin calf to fetal 10% grown with supplemented were cells Huh-7 stimulation and culture Cell METHODS AND MATERIALS ARTICLE RESEARCH 3114 (6 pellets Cell spectrometry Mass PtdIns(3,4,5)P stimulation, After analysis Lipid ECL Hyperfilm (Amersham). using film detection chemiluminescence anti- ECL performance by high rabbit (HRP-coupled followed antibody and was secondary the anti-rabbit-IgG) (MBL) the with Incubation anti-FFAR4 with (Abcam). FFAR1 rabbit blotting (Cell Technologies), anti-phospho-AKT preceded anti-AKT, Signaling rabbit transfer antibodies: primary Semi-dry following kaleidoscope standards. with gels acrylamide pre-stained 10% using performed was SDS-PAGE blotting Western ms. 350 Olympus an using recording during were PBS 0.265 cells of The resolution of 800 a min. at with ml 10 FV1000 1 Bonn, for stained FLUOVIEW Thiele, (1:10,000) under Christophe were 33342 times by viewed Samples three Hoechst provided washing PBS; PBS. and then in Germany) ice-cold and 1:1000 temperature (diluted of room LD540 ml at 1 min 45 with for PBS in 800 with incubating by fixed were Cells 10 of concentration microscopy final Confocal a at was stimulation DMSO before in min (Calbiochem) 20 LY294002 applied 2011). al., et (Norton previously ta. 03.Hh7clswr nuae ih1 with incubated were cells Huh-7 Gaunt 2013; 2013). al., al., et et (Griffiths previously described as spectrometry determined mass were lipids by neutral and 2011), al., et (Clark previously described otxdfr2 tro eprtr n oiae na ice-cold an in was sonicated mixture 1000 at and The centrifuged then temperature of added. and min, room ml were 2 1.5 for at chloroform bath standards, water s of sonication 20 internal ml d7-cholesterol as for 3 of ester vortexed and ng 100 17:0-cholesterol NaCl acid, of 0.88% fatty ng ng 50 17:0-free 100 12:0/12:0-DAG, of and of ng 200 ng 12:0-MAG, 100 ice. acid, of 12:0/12:012:0-triglyceride, of dry of 12:0/12:0-phosphatidic ng on 400 ng of tubes 100 17:0/20:4-phosphatidylinositol, ng glass of 100 silanised ng 400 12:0/12:0-phosphatidylcholine, in with methanol spiking of After ml 1.5 in resuspended aiatvt a eemndb cnilto onig u- el were cells Huh-7 the 3 counting. and with scintillation plate loaded 60 the by from gel determined scraped was silica vapour, radioactivity iodine activated with on identified (80:20:1). were TLC acid Lipids hexane:diethylether:acetic by using developed separated plates were (Merck) lipids and 1 standard, of the using total pellets cell A from extracted method. were Folch Lipids stimulation. to prior h 1 for BSA fatty-acid-free 1% and penicillin-streptomycin 1% containing DMEM ih3m fsnhtclwrpaeadtersligetatoswere extractions resulting the and extracted phase was lower phase synthetic upper The of collected. ml was 3 phase with lower The min. 15 F ectto 8 m emission for nm, settings 488 filter with (excitation used Huh7- was GFP microscope GPR1–GFP-transfected confocal disc of spinning a imaging cells, live-cell Volocity lipid For with 33342; of filter (PerkinElmer). performed quantification Hoechst the (3D) and was for Three-dimensional LD540, nm droplets LD540. for 425–420 for nm SDM490, nm 488 were 500–600 and recording 33342 for Hoechst settings for nm 405 were The eiilnsrpoyi n %ftyai-reBAfr2hbfr oleate before h 2 for stimulation. 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Journal of Cell Science ag . iel-ah . ahan . lngn .adCe,J. Chen, and A. Flanigan, R., Bachmann, M., Vilella-Bach, Y., Fang, ie,M,Eeat .adFu and R. Ehehalt, M., Digel, Cle lr,J,Adro,K . ui,V,Sih .S,Kre . aea,M .O., J. M. Wakelam, F., Karpe, S., T. Smith, V., Juvin, T. E., K. Fujimoto, Anderson, and J., Y. Clark, Shinohara, M., Suzuki, Y., Ohsaki, A., Fujita, J., Cheng, E. N. Wolins, and L. D. Brasaemle, ott . lMuai,H,Po,M,Nmn,M,Kalu,E,Clr,O., Colard, E., Khallouf, M., Nemani, M., Prot, H., Mourabit, El E., Boutet, Bostro Ko A., Eberl, S., Lay, Le M., C. Blouin, el . ag . hn . ceihn .C,Gn,D-. ag .Z,Yu, R.-Z., Yang, D.-W., Gong, C., J. McLenithan, H., Chen, H., Wang, M., Bell, tesad,K,Zetc,D,Jnrst,A,Khwi,S .adDu,G. Daum, and D. S. Kohlwein, A., Jandrositz, D., Zweytick, K., Athenstaedt, Bostro L., Andersson, J. Borlak, and N. Anderson, an,E,Zag . hug . aea,M .O,Lvr .and A. Lever, O., J. M. Wakelam, W., Cheung, Q., Zhang, E., Y. Shinohara, and Gaunt, M. Suzuki, J., Cheng, Y., Ohsaki, T., Fujimoto, lndinto cloned ARTICLE RESEARCH gra,A .adGr,A. Garg, and K. A. Agarwal, (UK). References and Council project; Research LipidomicNet Sciences FP7 Biological Union and European Biotechnology the the by by supported was work This manuscript. Funding the wrote S.A.R. mass and the A.R. M.J.O.W., performed performed analysis. Q.Z. also lipid and S.A.R. biology, spectrometric molecular experiments, the the experimental and of the majority experiments designed the and project performed the A.R. strategy. conceived A.R. and S.A.R. M.J.O.W., contributions Author interests. competing no declare authors The interests Competing uioo . ndk,J,Hma .J,Ymgci . oi . iah,Y., Higashi, M., Mori, S., Yamaguchi, J., K. Homma, J., Onoduka, Y., Fujimoto, Y. Ohsaki, and T. Fujimoto, CGTCCAT-3 3 5 GFP, CGTGTCCCAGCAG-3 5 CTCC-3 was pEGFP-C1 GACACGTGGATCTGGTGGCT-3 5 the using performed 5 3 was pEGFP-C1 9 9 nrdcino h 20 mutatio R270H the of Introduction . hshtdcai-eitdmtgncatvto fmO signaling. mTOR of activation 294 mitogenic acid-mediated Phosphatidic rmlv microscopy. live from iu oe3 nhptctstigr h omto flrelpddroplets. lipid large of formation the triggers hepatocytes in L. 3a Hepatology Rubbia-Brandt, core S., virus Pascarella, C K., Guilloux, al. M., et Vinciguerra, D., Suter, 272 tpes .R n akn,P T. P. spectrometry. mass by Hawkins, tissues and and cells in species R. of molecular L. incorporation Stephens, uniform shows droplets. lipid microscopy existing into electron triglycerides Quantitative (2009). expansion. and assembly droplet lipid of epndfcec lesftyai et9dstrto n ii rpe formation droplet lipid lipodystrophy. and congenital desaturation Berardinelli-Seip Delta9 in acid fatty alters Chre deficiency A.-M., Seipin Durand-Schneider, M., Maurice, phospholipid surface in defects. alterations maturation adipocytes: and al. composition et caveolin-deficient J.-F. Gautier, in O., analysis Bourron, F., Lasnier, X., Liepvre, nui sensitivity. insulin S.-O. Olofsson, droplet lipid abnormal cells: liver in resistance. insulin of downregulation al. induction et and C. protein metabolism Londos, J., coat M. Quon, droplet K., S. Fried, D., 19) dniiainadcaatrzto fmjrlpdpril rtiso the of proteins particle lipid cerevisiae. major Saccharomyces of yeast characterization and Identification (1999). al. et A. formation. M. droplet Frohman, P., lipid cytosolic Huang, regulate L., Asp, M., Ruiz, D., steatohepatitis. and steatosis in targets eslegr U. Desselberger, ifrnito,addeath. differentiation, and rpes lsi rael ihnwoutfits. new with organelle classic a droplets: manner a in al. hepatocyte human synthetase. et cultured Acyl-CoA H. a of in Itabe, dependent formation J., droplet lipid Noda, induce T., acids Kinoshita, M., Makita, platform. unique a as structure old ;3 mn,S,Pyo,M,SnhzPrj,A,Bugi,L,Rmdr,P., Ramadori, L., Bourgoin, A., Sanchez-Pareja, M., Peyrou, S., ´ment, 9 1942-1945. , ,P,Adrsn . i . ekn,R,Hjud . Bore K., Højlund, R., Perkins, L., Li, L., Andersson, P., m, ¨ 9 21) onrglto fpopaaeadtni ooo yhepatitis by homolog tensin and phosphatase of Down-regulation (2011). 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