Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes

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1 Dietologi (216) 59: DOI 1.17/s ARTICLE Redundnt roles of the phosphtidte phosphtse fmily in triylglyerol synthesis in humn dipoytes An Temprno 1,2 & Hiroshi Semongi 3,4 & Gil-Soo Hn 5 & Dvid Sestián 6,7,8 & Jordi Cpelldes 8,9 & Cristól Moreno 1,8 & Jun Gurdiol 1 & Mrtin Witsh 11 & Cristól Rihrt 1,12 & Osr Ynes 8,9,13 & Antonio Zorzno 6,7,8 & George M. Crmn 5 & Symeon Siniossoglou 3 & Mere Mirnd 1,8 Reeived: 22 April 216 /Aepted: 23 My 216 /Pulished online: 25 June 216 # The Author(s) 216. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis In mmmls, the evolutionry onserved fmily of Mg 2+ -dependent phosphtidte phosphtses (PAP1), involved in phospholipid nd triylglyerol synthesis, onsists of lipin-1, lipin-2 nd lipin-3. While muttions in the murine Lpin1 gene use lipodystrophy nd its knokdown in mouse 3T3-L1 ells impirs dipogenesis, deleterious muttions of humn LPIN1 do not ffet dipose tissue distriution. However, redued LPIN1 nd PAP1 tivity hs een desried in prtiipnts with type 2 dietes. We imed to hrterise the roles of ll lipin fmily memers in humn dipose tissue nd dipogenesis. Methods The expression of the lipin fmily ws nlysed in dipose tissue in ross-setionl study. Moreover, the effets of lipin smll interfering RNA ()-medited depletion on in vitro humn dipogenesis were ssessed. Results Adipose tissue gene expression of the lipin fmily is ltered in type 2 dietes. Depletion of every lipin fmily memer in humn Simpson Goli Behmel syndrome (SGBS) pre-dipoyte ell line, lters expression levels of dipogeni trnsription ftors nd lipid iosynthesis genes Eletroni supplementry mteril The online version of this rtile (doi:1.17/s ) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. Symeon Siniossoglou ss56@m..uk Mere Mirnd mmg@meremirnd.net Jon XXIII University Hospitl, Pere Virgili Helth Reserh Institut (IISPV), Modulr Building, C/ Mllfre Gush, Trrgon 435, Spin Deprtment of Biohemistry nd Moleulr Biology, Rovir i Virgili University,Trrgon,Spin Cmridge Institute for Medil Reserh, University of Cmridge, Wellome Trust/Medil Reserh Counil Building, Hills Rod, Cmridge CB2 XY, UK Present ddress: Chesterford Reserh Prk, Little Chesterford, Sffron Wlden, UK Deprtment of Food Siene nd the Rutgers Center for Lipid Reserh, New Jersey Institute for Food, Nutrition nd Helth, Rutgers University, New Brunswik, NJ, USA Institute for Reserh in Biomediine (IRB Brelon), The Brelon Institute of Siene nd Tehnology, Brelon, Spin Deprtment of Biohemistry nd Moleulr Biology, Fulty of Biology, University of Brelon, Brelon, Spin Biomedil Reserh Networking Centre in Dietes nd Assoited Metoli Disorders (CIBERDEM), Instituto de Slud Crlos III, Mdrid, Spin Centre for Omi Sienes, Rovir i Virgili University, Reus, Spin Deprtment of Pulmonry, Critil Cre nd Sleep Mediine, University of Louisville, Louisville, KY, USA Division of Peditri Endorinology nd Dietes, Interdisiplinry Clini, University Clini for Child nd Adolesent Mediine, University of Ulm, Ulm, Germny GEMMAIR Reserh Group Applied Mediine, Deprtment of Mediine nd Surgery, Rovir i Virgili University (URV), Trrgon, Spin Deprtment of Eletroni Engineering, Rovir i Virgili University, Trrgon, Spin

2 1986 Dietologi (216) 59: in erly stges of differentition. Lipin-1 knokdown lone uses 95% depletion of PAP1 tivity. Despite the redued PAP1 tivity nd ltertions in erly dipogenesis, lipinsilened ells differentite nd umulte neutrl lipids. Even omintoril knokdown of lipins shows mild effets on triylglyerol umultion in mture dipoytes. Conlusions/interprettion Overll, our dt support the hypothesis of lterntive pthwys for triylglyerol synthesis in humn dipoytes under onditions of repressed lipin expression. We propose tht indution of lterntive lipid phosphte phosphtses, long with the inhiition of lipid hydrolysis, ontriutes to the mintenne of triylglyerol ontent to ner norml levels. Keywords Bsi siene. Cell lines. Humn. Lipid metolism Arevitions DAG Diylglyerol DNL De novo lipogenesis ER Endoplsmi retiulum FAO Ftty id oxidtion LPP Lipid phosphte phosphtses ORO Oil Red O stining PAP Phosphtidte phosphtse PPAR Peroxisome prolifertor tivted reeptor SGBS Simpson Goli Behmel syndrome Smll interfering RNA SAT Suutneous dipose tissue TAG Triylglyerol VAT Viserl dipose tissue Introdution Triylglyerols (TAGs) re neutrl lipids tht t s the mjor energy storge moleules, repository for ftty ids, nd phospholipid preursors [1]. Adipoytes re the speilised ells for neutrl lipid storge nd one of their importnt physiologil funtions is to uffer the toxiity used y NEFAs. Exessive lorie intke or geneti disorders n led to lipid deposition in etopi tissues, impir their funtion nd led to dyslipidemi, insulin resistne nd type 2 dietes [2, 3]. Lipins re Mg 2+ -dependent phosphtidte phosphtses (PAP1) with entrl role in lipid metolism, nd tlyse the dephosphoryltion of phosphtidte to diylglyerol (DAG), whih n e (1) ylted to form TAG, or (2) used in phospholipids synthesis [4, 5]. A seond type of PAP tivityismeditedymg 2+ -independent trnsmemrne lipid phosphte phosphtses (LPPs, lso known s PAP2), whih re thought to regulte signlling properties of phosphtidte nd DAG [6]. Fungi, nemtodes nd insets express one lipin, wheres mmmls express three prlogues lled lipin-1, -2 nd -3 tht exhiit distint ut overlpping expression in mny mouse nd humn tissues [5]. Consistent with their key metoli role, loss of lipin funtion disrupts TAG prodution, memrne orgnistion nd phospholipid synthesis in severl model orgnisms [7]. Interestingly, esides their enzymti funtions, lipins lso regulte trnsription [8 13]. For instne, lipin-1 trnsriptionl o-regultion of the peroxisome prolifertor tivted reeptor (PPAR)A/PPAR otivtor 1α xis modultes ftty id oxidtion (FAO) in liver [9]. Lpin1 gene ws originlly identified s the defiient gene using lipodystrophy, insulin resistne, peripherl neuropthy nd neontl ftty liver in the fld (lso known s Lpin1) mouse model [14]. Loss of lipin-1 in mie loks dipogenesis t n erly stge preeding TAG umultion, suggesting distint role of lipin-1 in differentition [15, 16]. Consistently, smll interfering RNA ()-medited silening of lipin-1 in mouse 3T3-L1 ells potently inhiits dipogenesis [1, 17]. In ontrst, deleterious muttions in the LPIN1 gene in humns, whih led to reurrent rhdomyolysis in hildhood, do not ompromise dipose tissue [18]. The sis of this differene etween mie nd humns is unknown; it hs een hypothesised tht it is due to ompenstion y the other two lipins [18]. Nevertheless, geneti vrition in LPIN1 nd LPIN2, nd redued LPIN1 expression levels nd PAP1 tivity in humn dipose tissue hve een ssoited with type 2 dietes [19 25], whih suggests loss of their protetive role ginst lipotoxiity. While most of the studies hve een performed on mie, very little is known on the roles of lipin-1 in humn dipoyte physiology, while there is virtully no informtion on lipin-2 nd -3. This prompted us to investigte the funtions of the three lipins in type 2 dietes nd in humn dipoytes y studying the effets of loss-of-funtion. Methods Regents Unless otherwise stted, ll regents were supplied y Sigm-Aldrih Corportion (St. Louis, MO, USA). Humn insulin ws purhsed from NovoNordisk (Bgsværd, Denmrk); rosiglitzone from Cymn Chemil (Ann Aror, MI, USA); nd ell ulture medi from Gio (Thermo Fisher Sientifi, Wlthm, MA, USA). Humn dipose tissue iopsy olletion The ross-setionl study hs een previously desried [26, 27]. We used ohort of 71 prtiipnts for the gene expression nlysis, nd of 28 mles for protein nlysis. They were grouped s:

3 Dietologi (216) 59: (1) normoweight (BMI etween 18.5 nd 24.99), (2) oesity (BMI 25) nd (3) oesity with type 2 dietes (herefter type 2 dietes). Group hrteristis re desried in eletroni supplementry mteril (ESM) Tle 1A nd 1B, respetively. Smples were otined t the Jon XXIII University Hospitl (Trrgon, Spin). VAT nd SAT smples (gene expression) nd SAT smples (protein expression) were otined during dominl eletive surgil proedures for enign pthologies (holeystetomy or surgery for dominl herni). The Ethis Committee pproved the study nd informed onsent ws otined from ll prtiipnts. Prtiipnts hd no systemi disese other thn oesity [28] or type 2 dietes [29]. See ESM Methods for further detils. Cell ulture nd differentition Simpson Goli Behmel syndrome (SGBS) ells, well-estlished system for studies of humn dipoyte iology [3, 31], were differentited s desried [3], exept tht 1 nmol/l of insulin,.1 μmol/l ortisol, 2 μmol/l rosiglitzone nd 25 nmol/l dexmethsone were used. See ESM Methods for detiled informtion. Adipose-derived stem ells were isolted from dipose tissue (n = 3 femle donors; ge (yers) 37.4 ± 6.4, BMI (kg/m 2 ) 25.9 ± 3.) from ptients undergoing eletive liposution surgery. See ESM Methods for the isoltion, prolifertion nd dipogeni proedure detils. Quntifition of expression levels Protein ontent in ell lystes (lysis uffer: 5 mmol/l HEPES ph 7.4, 15 mmol/l NCl, 4 mmol/l MgCl 2, 1% Triton X-1 nd protese inhiitors) ws quntified y using the iinhonini id ssy (Piere, Rokford, IL, USA). See ESM for western lot proedures. Antiodies ginst lipin fmily memers were previously desried [17, 32]. Quntittive PCR ws performed s previously desried [27], nd expressed reltive to ylophilin A nd to ontrol. See ESM for detiled informtion nd ESM Tle 2A nd 2B for ommeril regents. Cell Frtiontion For ell frtiontion, SGBS ells were grown nd differentited until dy 1. Suellulr frtions were otined y using hypotoni lysis followed y high slt extrtion of nulei. See ESM Methods for further detils. Gene silening Trnsfetions of SGBS ells with oligonuleotides were rried out y using Lipofetmine RNAiMAX trnsfetion regent (Thermo Fisher Sientifi). Pre-dipoyte knokdowns were performed s two-shots trnsfetion of mix of two duplexes per gene (1 nmol/l of eh duplex) (see ESM Tle 2C): reverse trnsfetion t the strt of the experiment nd forwrd trnsfetion the dy efore onfluene. The non-trgeting ontrol onentrtion depended on totl mount of the single nd multiple knokdowns. Neutrl lipid umultion nd metolism Dt were otined from differentited SGBS ells nd normlised y protein ontent. For TAG nlysis, ells were proessed s previously desried [32], exept tht the superntnt frtion ws nlysed with the Serum Triglyeride Determintion kit (Sigm). Glyerol relese to ell ulture medi ws quntified y using the Free Glyerol Determintion Kit (Sigm). Ftty id nd gluose inorportion into TAGs, nd FAO nlysis were performed s previously desried [33], with slight modifitions (see ESM Methods for detils). Enzyme ssy Cell lystes (lysis uffer: 5 mmol/l Tris-HCl ph 7.5,.25 mol/l surose, 1 mmol/l 2-merptoethnol, protese inhiitors) were sujeted to entrifugtion t 1 g for 1 min t 4 C to remove ell deris. Protein onentrtion ws determined y the method of Brdford [34] using BSA s referene protein. Preprtion of the sustrte nd mesure of PAP tivity ws s previously desried [35 37]. See ESM Methods for detils. The Mg 2+ -independent LPP tivity ws mesured in the sme retion mixture exept tht 2 mmol/l EDTA ws sustituted for.5 mmol/l MgCl 2.TheMg 2+ -dependent PAP1 tivity ws determined y sutrtion of LPPs tivity from PAP tivity. A unit of PAP tivity (expressed s units/mg protein) ws defined s the mount of enzyme tlysing the formtion of 1 nmol of produt/min. Metolomi nlysis SGBS ells were grown, trnsfeted with s explined ove, nd differentited to dy 4. In rief, lipids were extrted from lyophilised smples y using dihloromethne/methnol nd wter. The orgni phse (lipidi) ws olleted, dried under strem of nitrogen, nd resuspended in etonitrile/isopropnol/wter for untrgeted LC-MS nlysis. Differentilly regulted lipids (p vlue <.5 nd fold >2) were retined for ompound identifition y MS/MS nlyses. See ESM Methods for detiled proedures. Sttistil nlysis Sttistil nlysis ws performed y using the SPSS softwre version 15 (Chigo, IL, USA). ANOVA, Kruskl Wllis, Person χ 2,Spermnorreltion nd Liner Stepwise Regression tests were performed for the humn ohort nlysis, nd the Generl Liner Model Univrite test for in vitro experiments. Sttistil power in the ohort nlysis ws 8%. The level of signifine ws set t α=.5. Results Adipose tissue expression levels of the lipin fmily is ltered in type 2 dietes To investigte the roles of lipin prlogues in dipose tissue, we strted y exmining their gene expression in pired dominl suutneous (SAT) nd viserl (VAT) dipose tissue iopsies. As shown in

4 1988 Dietologi (216) 59: LPIN1 mrna level (reltive to ylophilin A) d Lipin-1 protein level (reltive to tin) LPIN2 mrna level (reltive to ylophilin A) e Lipin-2 protein level (reltive to tin) Fig. 1 nd ESM Fig. 1, LPIN1 expression ws redued in oth SAT (p <.1)ndVAT(p =.21) in the oesity nd type 2 dietes groups ompred with normoweight. LPIN2 expression ws similr mong groups. In ontrst, SAT LPIN3 expression ws signifintly inresed in the type 2 dietes (p =.18). At the protein level, SAT showed only lipin-1 ws downregulted in oesity nd in type 2 dietes ompred with normoweight (p =.34)(Fig.1d f). Correltion nlysis showed negtive ssoition of LPIN1 expression levels with BMI, HOMA-IR nd plsm TAG levels. In ontrst, expression of LPIN3 positively orreltes with fsting gluose (SAT) nd NEFA (VAT) (Tle 1). Regression nlysis ws performed, ssessing ge LPIN3 mrna level (reltive to ylophilin A) f Lipin-3 protein level (reltive to tin) Fig. 1 Altered expression of the lipin fmily in type 2 dietes dipose tissue. Prtiipnts were grouped y BMI nd type 2 dietes (). ( ) mrna expression reltive to ylophilin A nd to lirtor tht onsisted of mix of mrna smples (n = 17, 43 nd 11 smples for the normoweight [], oesity nd groups, respetively), nd (d f) protein expression normlised to tin (n =9,1 nd 9)were quntified in humn dominl suutneous dipose tissue. Dt represent men ± SD; p <.5, p <.1 vs normoweight; ANOVA nd Kruskl Wllis tests nd sex s onfusing nd interting vriles, nd showed tht (1) SAT LPIN1 expression depends negtively on HOMA-IR (R =.466, p =.5, exluded vriles: BMI, TAG, ge nd sex; LPIN1 SAT =.37 log 1 HOMA +.984), nd (2) SAT LPIN3 expression depends positively on plsm gluose levels (R =.414, p =.2, exluded vriles: ge ½ nd sex; LPIN3 SAT ¼ 1 1 1:22 log 1Gluose :961 ). The oserved hnges of the lipin fmily expression in type 2 dietes prtiipnts my ount for the ltered PAP1 tivity in dipoytes from these ptients. Moreover, LPIN3 expression is ssoited with fsting gluose levels. Altertions in lipin-3 protein levels in SAT of prtiipnts with type 2 dietes my e msked y its presene lso in the stroml vsulr frtion (dt not shown). The three lipin fmily memers hve role in erly humn dipogenesis Next, we exmined the expression of lipins during dipogenesis. Lipin-1 ws indued, nd lipin-2 nd lipin-3 levels were present long SGBS dipogenesis with slight vrition (Fig. 2, ). Thus, SGBS dipoytes, similr to differentited dipose-derived stem ells (ESM Fig. 1d), express the three lipin prlogues. Finlly, under seline onditions, the three lipins prtitioned etween the ytosoli, intrnuler nd memrne-ound forms in SGBS dipoytes (ESM Fig. 1e). To ddress the effets of deresed PAP1 tivity on dipogenesis, we depleted eh lipin memer prior to the indution of dipogenesis in SGBS pre-dipoytes (y using ; see Methods nd Fig. 2). Cells were nlysed t dy 4 fter differentition to ssess dipogeni erly events. Protein expression nlysis ws used to onfirm knokdowns nd evlute possile ompenstory mehnisms mong lipins. Lipin-1 depleted ells responded y ompenstory upregultion of lipin-2 protein (Fig. 2d nd ESM Fig. 1f), ut not LPIN2 trnsript levels (dt not shown). Conversely, single lipin-2 nd lipin-3 knokdowns led to lower protein levels of the other fmily memers (Fig. 2d). Tle 1 Correltion nlysis in dominl SAT nd VAT dipose tissue iopsies from 71 prtiipnts Vrile SAT VAT BMI HOMA-IR Gluose Insulin Triylglyerol NEFA Glyerol Spermn oeffiient of the orreltion nlysis is shown. p <.5,p <.1, p <.1 Where sttistil power is lower thn 8%: p <.5

5 Dietologi (216) 59: Fig. 2 Single knokdowns of lipins in SGBS pre-dipoytes. SGBS pre-dipoytes were indued to differentite, nd mrna () nd protein () levels were nlysed during dipogenesis t the given time points (n = 3). Dt represent men ± SD of fold indution over dy. () Knokdowns of single lipin memers were performed in pre-dipoytes, dipogenesis ws indued (dy ) nd ells were olleted t dy 4. (d) Lipin protein levels (n =7),(e) PAP1 tivity (ontrol: 23.1 ± 5.12 nmol min 1 mg 1 )(n =3), (f) ftty id (FA) nd gluose inorportion into TAGs (ontrol, ftty ids: 22.8 ± ritrry units TAG/μg protein, gluose: 3.47 ± 6.3 ritrry units TAG/μg protein) (n =3),ndmRNAlevels of (g) erly dipogeni trnsription ftors (n =3),nd (h) lipogeni genes (n =3)were nlysed. Gene expression is expressed reltive to ylophilin A nd to non-trgeting ontrol. Protein expression is normlised to tin levels. Dt represent men ± SD of fold inrese over non-trgeting ontrols (set s 1). p <.5,p <.1, p <.1,GenerlLinel Model Univrite test. (, ) White squres, LPIN1; grey squres, LPIN2; lk squres, LPIN3; (d h) white rs, LPIN1 knokdown; grey rs, LPIN2 knokdown; lk rs, LPIN3 knokdown mrna level (fold over dy ) Medi supplements: MIX Dy: d Protein level (fold) g mrna level (fold) Lipin-1 CEBPA Dy of differentition Anlysis Lipin-2 CEBPB CEBPD Lipin-3 PPARG e PAP1 tivity (fold) SREBF1 Protein level (fold over dy ) h mrna level (fold) PCK1 ACACA f Inorportion into TAGs (fold) SCD1 GPAT3 FA AGPAT Dy of differentition Gluose DGAT1 DGAT2 Anlysis of PAP1 tivity showed tht lipin-1 ounted for lmost ll PAP1 tivity (lipin-1-depleted ells showed 5% of PAP1 tivity in the ontrol), with lipin-2 nd lipin-3 single knokdowns reduing it to 49% nd 61%, respetively (Fig. 2e). Next, we nlysed inorportion of ftty ids nd gluose into TAGs. While esterifition of ftty ids ws downregulted in lipin-1- nd lipin-2-depleted ells, gluose inorportion into TAGs deresed in ll three lipin-depleted ells (Fig. 2f). Finlly, we nlysed the expression levels of trnsription ftors tht promote erly dipogenesis. CEBPA ws downregulted in ells depleted of ny lipin fmily memer, nd CEBPB ws deresed upon lipin-2 defiieny (Fig. 2g). Under these onditions, gene expression of two key trnsription ftors regulted y CCAAT/enhner inding proteins, PPARG nd SREBP1 were downregulted (Fig. 2g), nd lso t the protein level (ESM Fig. 2, ). CEBPD showed signifint upregultion in ells depleted of ny lipin fmily memer (Fig. 2g), proly due to the triggering of ompenstion mehnism. Given the role of lipins in neutrl lipid iosynthesis, we next explored expression of lipogeni genes. The expression of these genes ws signifintly downregulted in ells depleted of ny lipin fmily memer (Fig. 2h). In ontrst, glyerol-3-phosphte yltrnsferse ws signifintly

6 199 Dietologi (216) 59: upregulted in the lipin-1 nd lipin-2 knokdowns (Fig. 2h). Lipid quntifition y mss spetrometry showed tht, mong the lipid speies tht were ltered, the levels of most DAG (ESM Fig. 2) nd TAG (ESM Fig. 2d) speies were downregulted in ells depleted of ny lipin ompred with ontrols. Interestingly, phosphtidte levels did not hnge (dt not shown). Overll, despite the rosstlk etween lipin fmily memers, lipin-1 silening leds to 95% depletion of PAP1 tivity. However, our results point to role of ll three memers on erly stges of humn dipogenesis. Single lipin silening does not lok TAG umultion in fully differentited dipoytes Next, we nlysed the effets of lipin silening in SGBS pre-dipoytes on lte dipogenesis. Similrly to the ove experiments, we depleted eh lipin memer prior to indution of dipogenesis in SGBS pre-dipoytes, nd ells were nlysed t dy 1 fter differentition (Fig. 3), where protein downregultion of lipins still persisted (Fig. 3 nd ESM Fig. 3). At dy 1, lipin-1 depleted ells still responded y ompenstory upregultion of lipin-2 protein (Fig. 3 nd ESM Fig. 3). Additionlly, oth single lipin-2 nd lipin-3 knokdowns led to lower protein levels of the other lipins. PAP1 tivity nlysis showed tht lipin-1 ounted for lmost ll PAP1 tivity (lipin-1-depleted ells showed 3% of the tivity in the ontrol). Lipin-2 nd lipin-3 single knokdowns redued PAP1 tivity only slightly (84% nd 74%, respetively) (Fig. 3). Next, we ssessed neutrl lipid umultion. Depletion of ny lipin triggered slight redution (15% to 34%) in TAG umultion (Fig. 3d). Expression of key trnsription ftors for lipogenesis (ESM Fig. 3, ) nd genes of lipid iosynthesis (ESM Fig. 3) were ltered to lesser extent ompred with the effets oserved t dy 4. Finlly, ftty id esterifition into TAGs nd α-glyerophosphte synthesis/de novo lipogenesis (DNL) were downregulted in ells depleted of ny lipin (Fig. 3e). Compred with dy 4, lipin-3 depletion lso showed redued ftty id esterifition into TAGs t dy 1. Overll, the effets of lipin knokdown were mild on fully differentited dipoytes suggesting tht ells overme the initil impirment of lipid gene expression. This my e due to the existene of enough remnnt protein mounts of lipins, lthough PAP1 tivity t dy 1 ws still lmost sent in the lipin-1 knokdowns. Medi supplements: MIX Insulin Dy: Protein level (fold) d TAG level (fold) Lipin-1 Lipin-2 Lipin-3 PAP1 tivity (fold) Inorportion into TAGs (fold) Comintoril silening of lipins does not further lter the phenotype of fully differentited dipoytes We then sked whether ompenstion y other lipin fmily memers might help ells to reover the initil ltertions shown t dy 4 fter dipogenesis. We used omintoril knokdown to deplete omintions of two of the three lipins in SGBS predipoytes (see Methods), nd nlysed ells t dy 1 of differentition (s in Fig. 3). Doule lipin-1 nd lipin-3 knokdowns led to n upregultion of lipin-2 protein levels (Fig. 4 nd ESM Fig. 4). This ompenstory pttern hindered the effiieny of lipin-2 knokdown in the doule (lipin-1 nd -2) nd triple knokdown ells. Nevertheless, PAP1 tivity in the triple knokdown ws residul ompred with the ontrol (.18 ±.31 vs ± 3.42 nmol min 1 mg 1 ). Despite the lmost omplete lk of PAP1 tivity, neutrl lipid umultion ws not totlly ompromised in thetripleknokdown(fig.4). This is in greement with the ft tht deleterious muttions in LPIN1 do not ffet dipose tissue development in humns, nd it suggests tht it is unlikely to e due to ompenstory upregultion of lipin-2 (Fig. 2d), sine the doule lipin-1 nd -2 knokdown shows similr redution in the ontent of neutrl lipids (Figs 3d, 4). e FA Gluose Anlysis Fig. 3 Lipin-depleted SGBS pre-dipoytes n differentite into dipoytes. () Knokdowns of single lipin memers were performed in pre-dipoytes, dipogenesis ws indued (dy ), nd ells were olleted t dy 1. () Lipin protein levels (n =3),() PAP1 tivity (ontrol: ± 1.38 nmol min 1 mg 1 )(n =3), (d) totl TAG ontent (ontrol: ± mmol l 1 mg 1 )(n = 3), nd (e) ftty id (FA) nd gluose inorportion into TAGs (ontrol, ftty ids: 74.6 ± ritrry units TAG/μg protein, gluose: 3.73 ± 2.6 ritrry units TAG/μg protein) (n = 3) were nlysed. Dt represent men ± SD of fold inrese over non-trgeting ontrols (set s 1); p <.5, p <.1, p <.1; Generl Linel Model Univrite test. White rs, LPIN1 knokdown; grey rs, LPIN2 knokdown; lk rs, LPIN3 knokdown

7 Dietologi (216) 59: Protein level (fold) Lipin-1 Lipin-2 Lipin-3 Fig. 4 Comintoril depletion of lipins in SGBS pre-dipoytes fter full differentition. Multiple knokdowns of lipin memers were performed in pre-dipoytes, dipogenesis ws indued (dy ) nd ells were olleted t dy 1. () Lipin protein levels (n =3 6), () totl TAG ontent (ontrols: ± 36.4 nd ± mmol l 1 mg 1 for doule nd triple knokdowns, respetively) (n = 5), nd () ftty id (FA) nd gluose inorportion into TAGs in the triple knokdown (ontrol, ftty TAG level (fold) LPIN1,-2 LPIN1,-3 LPIN2,-3 LPIN1,-2,-3 Inorportion into TAGs (fold) FA Gluose ids: ± ritrry units TAG/μg protein, gluose: 1 ± 2.26 ritrry units TAG/μg protein) (n = 3) were nlysed. Dt represent men ± SD of fold inrese over non-trgeting ontrols (set s 1); p <.5, p <.1, p <.1; Generl Linel Model Univrite test. White rs, LPIN1 nd LPIN2 knokdown; light grey rs, LPIN1 nd LPIN3 knokdown; drk grey rs, LPIN2 nd LPIN3 knokdown; lk rs, triple knokdown Moreover, depletion of the three lipins did not signifintly ffet ftty id esterifition into TAGs nd only slightly downregulted α-glyerophosphte synthesis/dnl (Fig. 4), pointing to n improvement ompred with dy 4 (dy 4 inorportion of ftty ids:.52 ±.35, p =.6, nd of gluose:.32 ±.15, p <.1, fold over the ontrol level). Consumption of TAGs is redued in lipin-depleted SGBS dipoytes Lipid droplets umulte neutrl lipids in dynmi mnner, with lne mong lipid iosynthesis, lipid hydrolysis nd FAO in mitohondri. To test whether ells protet intrellulr lipids y reduing its onsumption, we nlysed the rtes of plmitte oxidtion. The results show similr FAO rtes in the ontrol, single (Fig. 5) nd triple knokdown (Fig. 5). In ontrst, levels of glyerol relese were downregulted ompred with ontrols in the single (Fig. 5), nd in ll omintoril knokdowns (Fig. 5d). Thus, redued onsumption my ontriute to the umultion of neutrl lipids in lipin-depleted dipoytes. The LPP fmily is upregulted under onditions of repressed lipin expression Next, we tested whether the Mg 2+ -independent LPPs ould provide n lterntive pthwy for DAG synthesis. LPP tivity ws highly indued in the lipin-1 single knokdown ( ± 1.18-fold inrese over ontrol levels) (Fig. 6), nd in the triple knokdown (2.74 ±.97-fold inrese) t dy 1 of differentition (Fig. 6). Moreover, the expression of LPP3 (lso known s Plpp3/ PPAP2B) ws upregulted in the single (Fig. 6) nd in the omintoril lipin knokdowns t dy 1 (Fig. 6d) nd, similrly to lipogeni genes, upregultion ws higher t dy four (ESM Fig. 5). This ws onfirmed y using other ommeril soures of lipin-1 (ESM Fig. 5) nd nlysing LPP expression t dy four of differentition (ESM Fig. 5). Proprnolol, nonspeifi ß-loker, is n effetive PAP1 tivity inhiitor nd modestly effetive s LPP inhiitor, with the LPP3 isoform eing more sensitive to inhiition [36]. Proprnolol tretment during dipogenesis (Fig. 6e) ltered lipogeni gene expression (Fig. 6f) nd loked lipid droplet formtion in SGBS dipoytes (ESM Fig. 5d). Disussion Herein we hve ddressed the ontriution of the lipin prlogues to humn dipogenesis. Deresed lipin expression levels nd PAP1 tivity in dipose tissue hve een linked with insulin resistne [23 25]. In our ohort, dipose tissue expression onfirmed tht lipin-1 is ltered in type 2 dietes nd tht LPIN1 is negtively ssoited with insulin resistne. Moreover, we show tht LPIN3 trnsript levels re positively relted with fsting gluose levels. This prompted us to nlyse seprtely the effets of gene silening of eh lipin prlogue in dipogenesis. Previous studies hve estlished tht lipin-1 plys mjor role in ft metolism in rodents, with lipin-1 defiieny using lipodystrophy fetures in mie nd rts [14, 38]. The role of lipins in humn dipogenesis is still undefined: muttions in the LPIN1 gene hve not yet een deteted in humn lipodystrophy [39, 4]. Moreover, deleterious LPIN1 muttions use peditri rhdomyolysis while ft distriution, verge weight nd plsm iohemil vriles re norml [18]. Our dt suggest tht the lk of n essentil role for PAP1 tivity in humn dipogenesis is not due to

8 1992 Dietologi (216) 59: Plmitte oxidtion rte (fold) Relesed glyerol (fold) ompenstion y lipin-2 or lipin-3. We find tht the triple lipin silening in SGBS pre-dipoytes mintins the ility to inorporte ftty ids into TAG nd umulte neutrl lipids despite loss of nerly ll PAP1 tivity. SGBS pre-dipoytes re differentited in the sene of serum nd, therefore, neutrl lipids should e otined from DNL. Besides, lthough glyeroneogenesis is the mjor pthwy for glyerol synthesis in the mture dipoyte [41], during dipogenesis glyolysis my ount for n importnt soure of α-glyerophosphte. Thus, gluose inorportion into TAGs my ount for DNL, ut lso for α-glyerophosphte synthesis vi glyeroneogenesis nd glyolysis. Downregulted α-glyerophosphte synthesis/ DNL points to roder effets of lipin silening other thn TAG synthesis. Moreover, the puttive intrnuler roles of lipin-1, -2 nd -3 in SGBS dipoytes dd some omplexity to the ove. d Relesed glyerol (fold) LPIN1,-2, LPIN1,-2 LPIN1,-3 LPIN2,-3 LPIN1,-2,-3 Fig. 5 Consumption of neutrl lipids in fully differentited lipin-depleted SGBS pre-dipoytes. Knokdown of single, doule nd triple lipin fmily memers ws performed in pre-dipoytes, dipogenesis ws indued (dy ) nd ells were olleted t dy 1. Plmitte oxidtion rtes in () single (ontrol:.77 ±.37 nmol h 1 mg 1 )(n =3), nd () triple knokdowns (ontrol:.63 ±.28 nmol h 1 mg 1 )(n = 3); glyerol relese in () single (ontrol:.42 ±.12 mmol l 1 mg 1 )(n =3),nd(d) omintoril knokdowns (ontrols, doule:.36 ±.21, nd triple:.21 ±.7 mmol l 1 mg 1 )(n = 5) were nlysed. Dt represent men ± SD of fold inrese over non-trgeting ontrols (set s 1); p <.1, p <.1; Generl Linel Model Univrite test. (, ) White rs, LPIN1 knokdown; grey rs, LPIN2 knokdown; lk rs, LPIN3 knokdown; (, d) white rs, LPIN1 nd LPIN2 doule knokdown; light grey rs, LPIN1 nd LPIN3 doule knokdown; drk grey rs, LPIN2 nd LPIN3 doule knokdown; lk rs, triple knokdown Plmitte oxidtion rte (fold) LPP tivity (fold) mrna level (fold) e f mrna level (fold) LPP PPARG SREBP1 LPP2 LPP3 AGPAT2 DGAT1 DGAT2 LPP tivity (fold) d mrna level (fold) LPIN1,-2,-3 The lipin fmily memers ooperte for optiml PAP tivity in mouse dipose tissue [42], liver [43, 44], rin [43] nd humn primry myolsts [45]. Our dt show tht humn lipin-1 ounts for most PAP1 tivity in SGBS dipoytes nd tht upregultion of lipin-2 in lipin-1- depleted ells does not ompenste for PAP1 tivity. By ontrst, downregultion of PAP1 tivity in lipin-2 nd lipin-3 single knokdowns nnot e fully ttriuted to their own depletion sine they lso downregulte lipin LPP1 LIPIN1 LIPIN2 LIPIN3 LPP1 LPP2 LPP3 LPP2 PrPol Medi supplements: MIX Insulin Dy: LPP3 Anlysis Fig. 6 Indution of the LPP fmily under onditions of repressed lipin expression. Knokdowns of single, doule nd triple lipin fmily memers were performed in pre-dipoytes, dipogenesis ws indued (dy ) nd ells were olleted t dy 1. LPP tivity in () the single (ontrol: 6.1 ±.6 nmol min 1 mg 1 )(n = 3) nd () triple knokdowns (ontrol: 4.82 ±.53 nmol min 1 mg 1 )(n = 3), nd expression levels of the LPP fmily in () the single (n =7)nd(d) omintoril knokdowns (n =4 7) were nlysed. (e) SGBS pre-dipoytes were indued to differentite in presene of 1 μmol/l of proprnolol (PrPol). (f) mrna levels of lipogeni genes were nlysed (n = 3). Dt represent men ± SD of fold inrese over non-trgeting ontrols ( d) or non-treted ontrol (f); p <.5, p <.1, p <.1; Generl Linel Model Univrite test. (, ) White rs, LPIN1 knokdown; grey rs, LPIN2 knokdown; lk rs, LPIN3 knokdown; (, d) white rs, LPIN1 nd LPIN2 doule knokdown; light grey rs, LPIN1 nd LPIN3 doule knokdown; drk grey rs, LPIN2 nd LPIN3 doule knokdown; lk rs, triple knokdown; (f) white rs, proprnolol-treted ells

9 Dietologi (216) 59: Ativtion of non-lipin ompenstory pthwys my lso msk the lipin phenotypes. Nevertheless, (1) we found no evidene, t the mrna level, of upregultion of the monoylglyerol O-yltrnsferse pthwy tht n generte DAG from monoylglyerol [46, 47] (dtnot shown); nd (2) holesteryl esters levels re likely not upregulted sine the effet of lipin knokdown on neutrl lipid ontent looks similr y Oil Red O stining (ORO) nd TAG ontent (ORO:.76 ±.6, nd TAG:.85 ±.13 in the lipin-1 knokdown, nd ORO:.74 ±.12, nd TAG:.54 ±.33 in the triple lipin knokdown, ompred with the ontrol set s 1). In ontrst, LPP tivity ws indued in the lipin knokdowns, possily due to n erlier upregultion of their trnsript levels (oserved y dy 4). Moreover, inhiition of oth lipins nd LPPs with proprnolol ompletely loked lipid droplet formtion, lthough this ft must e tken with ution euse of the non-speifiity of this ompound. LPPs hydrolyse phosphtidte s well s different sustrtes, nd t on the outer surfe of plsm memrne nd in the luminl surfe of endoplsmi retiulum (ER) nd Golgi memrnes [6] nd thus, it is not ler if they n hve ess to the phosphtidte formed from glyerol phosphte nd yl-coa. However, in yest, TAG synthesised oth in the ytosoli nd luminl leflets of the ER memrnes re effiiently pked into lipid droplets [48]. Another strtegy to protet the lipid storge when glyerolipid synthesis is ompromised might e downregultion of lipid hydrolysis nd FAO. Lipin-1 regultes sl lipolysis [49], nd modultes FAO trnsript expression levels [9]. We show tht ll omintoril knokdowns of lipins downregulte sl lipolysis to greter extent thn redution of neutrl lipid ontent. In summry, we onfirm tht dipose tissue expression of the lipin fmily is ltered in type 2 dietes. Furthermore, loss of nerly ll PAP1 tivity, due to omined lipin-1, -2 nd -3 knokdown, hs only mild effets on finl dipogenesis nd lipid umultion in SGBS ells. Conversely, lipin-2 nd lipin-3 my ontriute little to totl PAP tivity, ut still ply role in erly dipogenesis. Our results suggest ompenstion strtegy to umulte ner norml neutrl lipid levels, tivting other pthwys (suh s LPPs) nd inhiiting TAG hydrolysis. More work is lerly required to deipher whether LPPs or other unknown pthwys my ompenste lk of PAP1 tivity. Aknowledgements We re grteful to I. Mylonis (Lortory of Biohemistry, Fulty of Mediine, University of Thessly, Greee) for dvie with the ell frtiontion protool. Funding This study ws supported y reserh grnts from the Instituto de Slud Crlos III (ISCIII, Spnish Ministry of Eonomy nd Competitiveness) (PI1/967 nd CP11/21 to MM); the R. Brri Privte Foundtion (PV12142S to MM); the Medil Reserh Counil (G71446 to SS); nd Ntionl Institutes of Helth Grnt (GM2814 to GMC). CIBER de Dietes y Enfermeddes Metólis soids (CB778/12) is n inititive of the ISCIII. MM knowledges support from the Miguel Servet tenure trk progrmme (CP11/21), from the Fondo de Investigión Snitri (FIS) o-finned y the Europen Regionl Development Fund (ERDF), nd supported y Slvdor de Mdrig Moility fellowship from the Spnish Ministry of Edution (PR ). AT is the reipient of FI-DGR fellowship ( /212) from the Agèni de Gestió d Ajuts Universitris i de Reer (AGAUR). The funders hd no role in study design, dt olletion nd nlysis, deision to pulish, or preprtion of the mnusript. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Author ontriutions SS, AZ nd MM were the min ontriutors in the oneption, design nd interprettion of the dt, nd in writing the mnusript. AT, HS, G-SH, DS, JC, CM nd MM performed the experiments nd dt nlysis, nd revised the mnusript. JG, MW, CR, OY nd GMC were responsile for quisition of dt nd nlysis, nd revised the mnusript. All uthors hd finl pprovl of the sumitted nd pulished versions. 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