Adipose tissue NAPE-PLD controls fat mass development by altering the browning process and gut microbiota

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1 Reeived 11 Jul 1 Aepted Fe Pulished 11 Mr DOI: 1.138/nomms795 OPEN Adipose tissue NAPE-PLD ontrols ft mss development y ltering the rowning proess nd gut miroiot Luie Geurts 1, Amndine Everrd 1,, Mtthis Vn Hul 1,, Ahmed Essghir, Thiut Dupr 1, Séstien Mtmoros 1, Huert Plovier 1, Julien Cstel 3, Rphel G.P. Denis 3, Mrie Bergiers 1, Céline Drurt 1, Mireille Alhouyek, Nthlie M. Delzenne 1, Giulio G. Muioli, Jen-Bptiste Demoulin, Serge Luquet 3 & Ptrie D. Cni 1 Oesity is pndemi disese ssoited with mny metoli ltertions nd involves severl orgns nd systems. The endonninoid system (ECS) ppers to e key regultor of energy homeostsis nd metolism. Here we show tht speifi deletion of the ECS synthesizing enzyme, NAPE-PLD, in dipoytes indues oesity, gluose intolerne, dipose tissue inflmmtion nd ltered lipid metolism. We report tht Npepld-deleted mie present n ltered rowning progrmme nd re less responsive to old-indued rowning, highlighting the essentil role of NAPE-PLD in regulting energy homeostsis nd metolism in the physiologil stte. Our results indite tht these ltertions re medited y shift in gut miroiot omposition tht n prtilly trnsfer the phenotype to germ-free mie. Together, our findings unover role of dipose tissue NAPE-PLD on whole-ody metolism nd provide support for trgeting NAPE-PLD-derived iotive lipids to tret oesity nd relted metoli disorders. 1 Metolism nd Nutrition Reserh Group, WELBIO-Wlloon Exellene in Life Sienes nd BIOtehnology, Louvin Drug Reserh Institute, Université tholique de Louvin, Avenue E. Mounier, 73 B , 1 Brussels, Belgium. de Duve Institute, Université tholique de Louvin, Avenue Hipporte, 7 B1.7.5, 1 Brussels, Belgium. 3 Université Pris Diderot, Soronne Pris Cité, BFA, UMR851, CNRS, F-755 Pris, Frne. Bionlysis nd Phrmology of Biotive Lipids Reserh Group, Louvin Drug Reserh Institute, Université tholique de Louvin, Avenue E. Mounier, 7 B1.7.11, 1 Brussels, Belgium. These uthors ontriuted eqully to this work. Correspondene nd requests for mterils should e ddressed to P.D.C. (emil: ptrie.ni@ulouvin.e). NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.

2 NATURE COMMUNICATIONS DOI: 1.138/nomms795 Oesity hs rehed pndemi levels. In ddition to eing ssoited with mssive expnsion of dipose tissue, oesity is lso ssoited with luster of metoli ltertions, suh s type dietes nd rdiovsulr nd hepti diseses. Thus, it is of the utmost importne to unrvel the underlying mehnisms tht led to these metoli ltertions to disover new therpeuti strtegies. Oesity n e onsidered multi-system disese euse severl orgns nd systems prtiipte in this metoli ondition. Among those, the endonninoid system (ECS) ppers to e key regultor of energy homeostsis nd metolism. The ECS is omplex system omposed of severl iotive lipids interting with oth memrne-ound nd nuler reeptors, leding to rod rnge of physiologil effets. ECS tivity is minly ontrolled y key synthesis nd degrdtion enzymes 1. Anndmide (N-rhidonoylethnolmine, AEA) is one of the est hrterized endonninoids (ecbs) nd is involved in regultion of ppetite nd energy homeostsis,3.in ddition to AEA, other relted N-ylethnolmines (NAEs), suh s N-plmitoylethnolmine (PEA), N-steroylethnolmine (SEA) nd N-oleoylethnolmine (OEA) shre iosyntheti nd degrding pthwys with AEA. NAEs re typilly synthesized y the enzyme N-ylphosphtidylethnolmine phospholipse D (NAPE-PLD), lthough lterntive pthwys exist for AEA iosynthesis 1,. The ecbs re synthesized from ell memrne phospholipids nd re relesed to the extrellulr omprtment to trget their reeptors. ecbs t vi n utorine or prrine mehnism 5. The prinipl ecb reeptors re the G-oupled reeptors CB 1 nd CB, whih re minly trgeted y AEA nd -rhidonoylglyerol, the two mjor ecbs. Other NAEs, suh s OEA or PEA, tivte non-nninoid reeptors, inluding PPAR, GPR55 or GPR119 (refs 1, 8). The levels of ecbs re losely regulted y lne etween synthesis nd degrdtion. After relese, ecbs nd NAEs re rpidly degrded y luster of degrding enzymes suh s ftty id mide hydrolse (FAAH) or NAE hydrolyzing id midse (NAAA) 5. Beuse they re primrily synthesized on demnd, it is worthwhile to fous on ecb prodution. NAEs modulte food intke nd inflmmtory response 3,9,1 nd thus seem to t s importnt meditors of metoli homeostsis nd inflmmtion. Studies on totl Npepld knokout (KO) mie reveled no overt phenotype, highlighting lterntive synthesis pthwys for ertin long-hin NAEs suh s AEA nd suggesting role for NAPE-PLD in regulting lipid signlling systems,11. These studies foused on the entrl ECS, ut the ext role of NAPE-PLD per se in metolism in peripherl tissues hs yet to e investigted. Among the peripherl tissues involved in oesity, the dipose tissue plys entrl role. Besides storing exessive energy, the dipose tissue is n tive metoli orgn tht seretes mny meditors, suh s hormones nd ytokines (for exmple, dipokines) 1,13. We nd others hve previously underlined role for the ECS in dipogenesis nd dipose tissue funtion 1 17, therey designting the ECS s n importnt tor in dipose tissue metolism. We hypothesize tht NAEs produed y dipoytes re key meditors regulting whole-ody metolism nd energy homeostsis. To evlute the speifi role of NAEs produed in dipose tissue, we generted mouse model of dipoyte-speifi deletion of the Npepld gene nd investigted the physiologil role of dipose tissue NAPE-PLD under sl (ontrol diet (CT)) nd pthologil (diet-indued oesity (DIO)) onditions. We found in this study tht Npepld deletion in dipose tissue leds to development of oesity, impirment of gluose nd lipid homeostsis long with ltered dipose tissue metolism nd hnges in gut miroiot omposition. Results Npepld deletion is speifi of dipose tissue. To ssess the role of dipose tissue NAPE-PLD on metolism, Npepld lox/lox mie (onstrution in Supplementry Fig. 1) were rossed with Fp- Cre mie to generte mie with onditionl dipoyte-speifi KO () of NAPE-PLD. Fp-Cre-Npepld mie hve norml postntl development, in ontrst to other Fp-Cre mie strins tht n develop postntl lethlity 18. To onfirm the invlidtion of the Npepld gene in the dipose tissue of the mie, we ssessed the presene of the NAPE-PLD protein y Western lot nlysis in the white dipose tissue (WAT) of wildtype () nd mie (Fig. 1) nd found no detetle mounts of NAPE-PLD in the WAT of mie. In ontrst, we did not oserve redued NAPE-PLD levels in the rin, whih demonstrtes the speifiity of our model (Supplementry Fig. ). In ddition, the nlysis of messenger RNA (mrna) expression from multiple tissues onfirms tht the deletion is speifi for different depots of WAT (suutneous, viserl nd epididyml) nd rown dipose tissue (BAT; Fig. 1), without ffeting Npepld expression in the liver, olon or musles, whih indites tht reomintion did not our in other tissues 19. During experiments, nd mie were fed either CT ( nd groups) or high ft diet (HFD; nd groups). Deletion of the Npepld gene ws verified in groups under oth diets (Fig. 1). Beuse we oserved residul expression of Npepld in the dipose tissue, we performed seprtion of the stroml vsulr frtion (SVF) nd dipoytes enrihed frtion in the WAT. This indited tht deresed expression of Npepld ours only in dipoytes frtion nd not in the SVF (Supplementry Fig. ). Some reports in the literture estlished Cre tivity medited y the Fp promoter in other ell types suh s mrophges.to verify Npepld expression in mrophges, we isolted peritonel mrophges from nd mie. We found tht mrophges from oth genotypes did not differ in Npepld expression (Supplementry Fig. ). Finlly, to ensure tht the deletion of Npepld is indeed reduing NAE levels we mesured the levels of NAEs produed y NAPE-PLD. Figure 1 illustrtes B% redution of PEA, OEA nd SEA levels in the dipose tissue of mie ompred with mie. In ontrst, we found no derese in NAEs levels in the rin when ompring oth genotypes (Supplementry Fig. ). The lk of signifint impt of Npepld deletion on AEA onfirms the existene of n lterntive synthesis pthwy for this NAE,11,1. Importntly, we determined tht HFD-treted mie exhiited similr levels of NAEs to mie, suggesting tht HFD tretment in itself hs NAE lowering effet tht ws only slightly intensified y the genotype. Moreover, we found tht Npepld deletion in dipose tissue leds to inresed NAE preursor levels (tht is, NAPEs) in dipose tissue, orroorting results of previous studies performed in Npepld / mie,11,1 (Supplementry Fig. ). Adipose tissue Npepld-deleted mie develop n oese phenotype. Surprisingly, under the CT diet, mie gined more weight thn mie, phenomenon exerted under the HFD (Fig. ). Body omposition mesured y NMR indited tht the mie umulte more ft mss, whih results in higher perentge of totl ody ft nd inresed ft mss gin (Fig.,) nd lower perentge of len mss when ompred with their ontrol ounterprts fter 8 weeks of CT diet (Supplementry Fig. 3). Food intke remins unhnged etween the nd mie ut is inresed in the HFD-treted groups ompred with the CT-treted groups (Fig. d). The inrese in ft mss gin oserved in mie is ssoited with n inrese in dipoyte size in oth mie on the CT diet nd HFD (Fig. e,f). NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI: 1.138/nomms795 ARTICLE This inrese is refleted y higher frequeny of lrger dipoytes in mie nd HFD mie ompred with mie (Fig. g). Moreover, the plsm levels of leptin, produed proportionlly to ft mss, re mrkedly inresed in mie ompred with mie nd re inresed even more in mie ompred with mie (Fig. h). We next ssessed whether dipose Npepld deletion hs n impt on whole-ody gluose metolism. We oserved tht mie re hyperglyemi in the fsted stte nd tht these mie develop gluose intolerne, s evidened y n orl gluose tolerne test (OGTT) (Fig. i). Importntly, this gluose intolerne is mintined throughout the durtion of the OGTT. In ddition, dipose Npepld deletion exerted HFD-indued gluose intolerne (Fig. i). The mie exhiit twofold higher level of plsm insulin in the fsted stte s well s fter the orl gluose lod, nd this ltter effet is lso present during HFD feeding (Fig. j). These oservtions re onfirmed y the inresed insulin resistne index oserved in during oth CT nd HFD diet feeding, the ltter eing worsened in mie ompred with mie (Fig. k). Adipose tissue Npepld deletion indues insulin resistne. Insulin resistne in mie is suggested y the inresed fsted glyemi, gluose intolerne, fsted nd fed hyperinsulinemi nd higher insulin resistne index (Fig. i k). To explore whih orgn my e responsile for insulin resistne, 55 kd 3 kd NAPE PLD 3 kd β-atin Npe pld mrna AEA (pmol g 1 ) Npe pld mrna SAT VAT EAT BAT Liver Colon Musle PEA (pmol g 1 ).... SAT VAT EAT BAT Liver Colon Musle OEA (pmol g 1 ) 1, SEA (pmol g 1 ) Figure 1 Speifi deletion of Npepld in dipose tissue. () Representtive dipose tissue immunolot of NAPE-PLD nd -tin in mie nd mie. () mrna reltive expression of Npepld in different dipose tissue deposits (suutneous, viserl, epididyml nd rown dipose tissue SAT, VAT, EAT nd BAT), s well s in liver, olon nd tiilis musle under CT diet nd HFD in mie nd mie (n ¼ 7). These dt (,) orrespond to the results of three independent experiments. Dt with indite signifint differene (Po.5) versus or ording to the unpired two-tiled Student s t-test. () SAT levels of AEA, PEA, OEA nd SEA (pmol per g tissue) mesured y HPLC-MS (n ¼ 1). Dt re presented s the men±s.e.m. Dt with different supersript letters re signifintly different (Po.5) ording to post-ho one-wy nlysis of vrine sttistil nlysis. NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.

4 NATURE COMMUNICATIONS DOI: 1.138/nomms795 Body-weight gin (g) d d Ft mss (% Totl ody weight) Ft mss gin (g) Food intke per dy (g) e f g h HFD CT Adipoyte men dimeter (μm) 5 3 1, Frequeny (%) Adipoyte dimeter (μm) Leptin (pg ml 1 ),, 1, 5, i j k Plsm gluose (mg dl 1 ) 75 7, 1,5 d -HED, 1, 5 5,, 75 3, 5 5, 5 1, Time (min) 9 1 Gluose AUC (mg l 1 min 1 ) Insulin (μg I 1 ) Insulin resistne index (AUC gluose x AUC insulin ) 1 Figure Adipose tissue Npepld deletion indues n oese-like phenotype. () Totl ody-weight gin (g) (n ¼ 7). () Totl ft mss (% of totl ody weight) mesured y TD-NMR (n ¼ 7). () Totl ft mss gin (g) mesured y TD-NMR (n ¼ 7). (d) Men dily food intke per mouse (g) (n ¼ 7). (e) Representtive hemtoxylin nd eosin-stined pitures of SATdeposits. Sle r, 1 mm. (n ¼ 1). (f) Men dipoyte dimeter (mm) determined y histologil nlysis (n ¼ 1). (g) Adipoyte distriution nd frequeny with respet to the men dimeter mesured y histologil nlysis (n ¼ 1). (h) Leptin plsm levels mesured in the ven v (mgml 1 ; n ¼ 1). (i) Plsm gluose (mg dl 1 ) profile nd the men re under the urve (AUC) mesured etween nd 1 min fter gluose loding (mg dl 1 min; n ¼ 7). (j) Plsm insulin levels t 3 min efore nd min fter gluose loding (n ¼ 7). (k) Insulin resistne index determined y multiplying the AUC of lood gluose y the AUC of insulin. These dt ( d nd i k) orrespond to the results of three independent experiments. Dt re presented s the men±s.e.m. Dt with different supersript letters re signifintly different (Po.5) ording to post-ho one-wy nlysis of vrine ( h nd k). indites signifint differene versus (Po.5) (i) nd t oth time points ( 3 nd ) versus (Po.5) (j); indites signifint differene (Po.5) versus nd ; nd indites signifint differene versus s determined y two-wy sttistil nlysis (i,j). we nlyzed insulin-indued phosphoryltion of the insulin reeptor (p-ir). We found tht following insulin stimultion, phosphoryltion of IR ws strongly redued in the liver nd in the musles of mie, wheres the redued phosphoryltion of IR in dipose tissue ws not sttistilly signifint (Fig. 3 ). To further nlyze insulin resistne in the liver, we next nlyzed insulin-indued phosphoryltion of Akt (p-akt), downstrem meditor in the insulin signlling pthwy. Phosphoryltion of Akt on the serine site fter insulin stimultion ws redued in the liver (Fig. 3d), onfirming insulin resistne in this orgn, wheres p-akt levels were not ffeted in musle or dipose tissue (dt not shown). Finlly, we found tht gluose-- phosphtse tivity ws inresed nd glyogen ontent deresed in the liver, onfirming insulin resistne in this orgn (Fig. 3e,f). Npepld deletion impts dipose nd whole-ody lipid profiles. Npepld deletion leds to deresed synthesis of iotive meditors (tht is, NAE) whih ould e involved in the regultion of lipid synthesis nd relese y the dipose tissue. To exmine this possiility, we performed n nlysis of irulting lipids nd dipose tissue lipidomis. We oserved inresed irulting triglyerides (TAG) nd holesterol levels (Fig.,) in mie, wheres irulting non-esterified ftty ids levels were not ffeted y the deletion (Fig. ). In ddition, NAEs shre similr iosyntheti pthwys with eiosnoids nd their derivtives (nmely prostglndins (PG)), whih re lso importnt lipid meditors involved in metolism nd inflmmtion. As we nnot exlude the possiility tht dipose tissue Npepld deletion lso ffets these lipid meditors nd tht this intertion ould e n dditionl mehnism involved in hnges in energy homeostsis nd gluose metolism, we quntified the dipose levels of eiosnoids, PG, ermides nd phospholipids using lipidomi pproh. We oserved tht the deletion of Npepld in dipose tissue dereses PG nd eiosnoid onentrtions in dipose tissue to the sme extent s the HFD NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI: 1.138/nomms795 ARTICLE pirβ β-tin Insulin kd 3 kd p-ir/β-tin (.u.) d pakt Thr pakt Ser β-tin Insulin kd pirβ β-tuulin Insulin kd 55 kd p-ir/β-tuulin (.u.) p-akt Thr /β-tin (.u.) p-akt Ser /β-tin (.u.) e f pirβ 95 kd.5 β-tin 3 kd 1. Insulin + + p-ir/β-tin (.u.) GPse tivity (U per g 1 liver) Glyogen (μg per mg 1 protein), 1,5 1, 5 Figure 3 Adipose tissue Npepld deletion indues insulin resistne in the liver. () Representtive liver immunolots for p-ir nd -Atin in nd mie with or without insulin stimultion. Rtio of the insulin-stimulted p-ir in the liver on the loding ontrol s mesured y densitometry in nd mie (n ¼ ). () Representtive skeletl musle immunolots for p-ir nd -tuulin in nd mie with or without insulin stimultion. Rtio of the insulin-stimulted p-ir in the musle on the loding ontrol s mesured y densitometry in nd mie (n ¼ 3 ). () Representtive dipose tissue immunolots, for p-ir nd -Atin in nd mie with or without insulin stimultion. Rtio of the insulin-stimulted p-ir in the dipose tissue on the loding ontrol s mesured y densitometry in nd mie (n ¼ 3). (d) Representtive liver immunolots for p-akt Thr38, p-akt Ser73 nd -Atin in nd mie with or without insulin stimultion. Rtio of the insulin-stimulted p-akt Thr38 nd p-akt Ser73 in the liver on the loding ontrol s mesured y densitometry in nd mie (n ¼ ). (e) Liver GPse tivity (U per g liver) (n ¼ 1). (f) Liver glyogen ontent (mg per mg proteins; n ¼ 1). Dt re presented s men±s.e.m. Dt with re signifintly different (Po.5), dt with re signifintly different (Po.1), dt with tend to e signifintly different (P ¼.) ording to the unpired two-tiled Student t-test. tretment (Fig. d,e). We found in mie signifintly inresed levels of very long-hin ermides (Fig. f) ompred with mie. Interestingly, we oserved tht HFD tretment does not ffet long-hin ermide levels, s there is no exertion of the ondition under HFD. Furthermore, mie exhiit drop in the ontent of severl phospholipids in the WAT ompred with mie (Supplementry Tle 1). Altogether, these dt onfirm strong impt of Npepld deletion on lipid metolism in the sl stte. Npepld deletion modifies inflmmtion nd rowning proess. To etter explin the inresed ft mss nd ltered lipid profile oserved following the deletion of Npepld in dipose tissue, we nlyzed the gene expression profiles in the WAT y mirorry. These reveled n inresed expression of lrge numer of genes involved in inflmmtory pthwys nd deresed expression of numerous genes involved in dipose tissue metolism in, nd mie ompred with mie (Fig. 5). Genes were ssigned to relevnt pthwys y nlyzing gene ontology using the ioinformtis tool DAVID (Supplementry Fig. ). Figure 5 represents reltive rtios in our experimentl groups of seleted genes tht were down or upregulted y t lest 1.5-fold in versus mie. The entire list of genes is ville in Supplementry Dt 1. To onfirm the results otined from the mirorry nlyses, we performed quntittive PCR (qpcr) mesurements on key inflmmtory mrkers s well s on lipid metolism nd rowning mrkers. All of the qpcr results supported the results from the mirorrys nd onfirmed tht mie exhiit mrkedly inresed inflmmtory tone in dipose tissue (Fig. 5). It is worth noting tht the levels of inflmmtory mrkers in mie re similr to those of nd mie or re even more pronouned thn under HFD onditions, emphsizing the effet of Npepld deletion on dipose tissue inflmmtion under physiologil onditions. We hve onfirmed the presene of inflmmtion in mie y histologil stining for the F/8 mrker in WAT setions (Fig. 5, quntifition in Supplementry Fig. 5). With regrd to lipid metolism, we oserved deresed expression of severl genes involved in lipogenesis (Fsn nd A), -oxidtion (Aox1 nd Asl1) or key regultors of lipid metolism (Ppr nd Pprg1) in mie (Fig. 5d). Seeking mehnisti explntion for the inresed ft umultion, we identified y mirorry mrked downregultion of key mrkers of rowning, nmely Pprg1, Up1, Cide nd Elovl3 in mie (Fig. 5). This downregultion ws onfirmed y qpcr (Fig. 5d) nd y immunologil stining for UCP1 (Fig. 5e, quntifition in Supplementry Fig. 5). Beuse we oserved inresed ft mss ut no inrese in food intke, these results suggest the impt of dipose Npepld deletion on the rowning proess. These different mrkers were similr etween nd (Fig. 5 e). We nd others hve linked the inresed expression NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.

6 NATURE COMMUNICATIONS DOI: 1.138/nomms795 TAG (mg dl 1 ) Prostglndins (fmol mg 1 ) 3 1 PGD PGE TXB Cholesterol (mg dl 1 ) 1 5 Eiosnoids (fmol mg 1 ) S-HETE 13S-HODE S-HETE -keto-pgf1 9S-HODE NEFAs (mm).8... Cermides (pmol mg 1 ) N-C19:-Cer N-C1:-Cer N-C:-Cer N-C3:-Cer N-C:-Cer Figure Adipose tissue Npepld deletion lters dipose nd whole-ody lipid metolism. () Plsm triglyeride ontent (mg dl 1 ; n ¼ 7). () Plsm holesterol ontent (mg dl 1 ; n ¼ 7). () Plsm non-esterified ftty id (NEFA) levels (mm; n ¼ 7). These dt ( ) orrespond to the results of three independent experiments. (d) SAT prostglndin ontent mesured y LC-MS/MS in fmol per mg tissue (n ¼ 1). (e) SAT eiosnoid ontent mesured y LC-MS/MS in fmol per mg tissue (n ¼ 1). (f) SAT ermide ontent mesured y ESI-MS/MS in pmol per mg tissue (n ¼ 1). See lso Supplementry Tle 1. Dt re presented s men±s.e.m. Dt with different supersript letters or symols re signifintly different (Po.5) ording to post-ho one-wy nlysis of vrine. indites signifint differene (Po.5) versus, nd indites signifint differene (Po.5) versus nd ording to post-ho one-wy nlysis of vrine. of lipopolyshride (LPS) inding protein (LBP) in the dipose tissue to inflmmtion nd plsm LPS levels 3,.AsLp mrna were drstilly inresed in the dipose tissue of mie ording to oth mirorry nd qpcr dt, we mesured plsm LPS levels, nd found tht the inresed Lp mrna expression nd the higher inflmmtion in mie were ssoited with signifint inrese in metoli endotoxemi (tht is, inresed plsm LPS levels 5 ) (Fig. 5f). Npepld deletion impirs dpttion to old exposure. To investigte whether redued WAT rowning is usl ftor for, or metoli onsequene of, the phenotype oserved, we sumitted ody weight-mthed nd mie (Fig. ) to old exposure for 7 h. Under norml onditions, this effetively upregultes Up1 nd onsequent het-prodution ( rowning ) in mie, in n ttempt to ounter the drop in environmentl temperture. mie mintined lower ody temperture throughout old exposure nd hd signifintly lower men NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

7 NATURE COMMUNICATIONS DOI: 1.138/nomms795 ARTICLE Seleted genes from 1.5-fold regulted in log-rtio Lp PAI1 Lep Pltp Fp1 Cd8 Wisp Cd11 Cl11 Lyz1 C5r1 Hmgs Ple1 Emr Cd Alox5p Bl F/8 Plg5 Gdpd1 Cd8 C1q Cr5 Tlr13 Npy1r C Il Strd Vpre Ant Def5 Aot3 Vpre1 Tnfrsf Def Str Cxl Cd79 Pgk1 At Gpd1 Otop1 Pprg1 Asl5 Ass1 Ldh Hsd171 Pnliprp1 Pprg1 Me1 Pnlip Hdh Pfkl Advl Ge1 Mtfp1 Cox8 Fp3 Hdh Cox71 Aot Ppr Cpt1 Pdh Gpd Mpzl Asm3 Sl7 Pnk1 Aot11 Cide Aly A Elovl Up1 Elovl3.CT.HFD.HFD Conditions Adipose tissue mrna levels (reltive expression) HFD CT Serum LPS (EU ml 1 ) F/8 Cd11 Cd8 MCP1 F/8 II1 Cd Cd3g PAI1 Lp Negtive ontrol BAT HFD CT Adipose tissue mrna levels (reltive expression) Positive ontrol Fsn A Aox Asl1 Ppr Pprg1 UCP-1 Cide Up1 Elovl3 Prdm1 Negtive ontrol Figure 5 Adipose tissue Npepld deletion indues inflmmtion nd ltered metolism in SAT. () Het mp from mirorry nlysis representing seleted genes involved in inflmmtion or lipid metolism, sed on the list of genes down or upregulted 1.5-fold in versus mie. See lso Supplementry Fig. nd Supplementry Dt 1. () mrna expression of dipose tissue inflmmtion mrkers mesured y RT qpcr (n ¼ 7 for F/8, Cd11, MCP1 nd Il1 nd n ¼ 1 for the other mrkers). The lk-dotted line represents the levels for mrna expression. Dt in regrding inflmmtory mrkers orrespond to the results of three independent experiments. () Representtive pitures of stining for F/8 in SAT. Sle r, 1 mm. (n ¼ 1). See lso Supplementry Fig. 5. (d) mrna expression of dipose tissue metolism mrkers mesured y RT qpcr (n ¼ 1). The lk-dotted line represents the levels for mrna expression. (e) Representtive imges of stining for UCP1 in SAT. Sle r, 1 mm. (n ¼ 1). See lso Supplementry Fig. 5. (f) Plsm LPS levels (EU per ml) mesured in the portl vein (n ¼ 1). Dt re presented s the men±s.e.m. Dt with different supersript letters re signifintly different (Po.5) ording to post-ho one-wy nlysis of vrine. indites signifint differene (Po.5) versus, nd indites signifint differene (Po.5) versus nd ording to post-ho one-wy nlysis of vrine. ody temperture fter 7 h of old exposure (Fig.,). qpcr mrkers of the rowning progrmme in WAT (Up1, Elovl3, Cide nd Pprg1) indited tht old exposure resulted in norml rowning of the WAT in mie, ut tht this indution ws impired in mie (Fig. d g). Browning phenotype is minly onserved in ex vivo explnts. Beuse drenergi stimultion of WAT indues the rowning proess, this phenomenon in vivo is onsidered s symptheti event 7. Indeed, mny tions of so-lled rowning gents my e tred k to indiret mehnisms tht led to tivtion of the symptheti nervous system (SNS) nd susequent indution of rowning 7. However, some gents my hve n impt on rowning in diret nd ell-utonomous mnner. To further understnd the impt of dipose tissue-speifi Npepld deletion on the rowning proess, we wondered if this phenotype ws NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.

8 NATURE COMMUNICATIONS DOI: 1.138/nomms795 Body weight (g) 3 1 -CE -CE Body temperture ( C) Alimtiztion AUC 3 1 AUC 3 1 Dy 1 Dy Dy 3 Dy Cold exposure (hours) Dissetion (RT) Men t during old exposure ( C) CE -CE Up1 mrna 1 8 -RT -RT -CE -RE Elovl3 mrna 1 5, -RT -RT -CE -RE Cide mrna, -RT -RT -CE -RE Pprg1 mrna,, -RT -RT -CE -RE Figure Adipose tissue Npepld deletion indues n ltered response to old exposure. () Totl ody weight (g; n ¼ 7). () Body temperture evolution ( C) during 7-h old exposure (n ¼ 7). Inserts represent the re under the urve (AUC) of dy nd 3, with seline set t 3 C. () Men ody temperture (t ) mesured in C during old exposure (n ¼ 7). (d) Up1 mrna levels, (e) Elovl3 mrna levels, (f) Cide mrna levels nd (g) Pprg1 mrna levels (reltive expression) in SAT of room temperture (RT) nd old-exposed (CE) nd mie (n ¼ 7). Dt re presented s men±s.e.m. Two-wy repeted-mesure nlysis of vrine (ANOVA; genotype time) reveled signifint effet of genotype (P ¼.), signifint effet of time (Po.1), nd non-signifint intertion etween ftors (P ¼.). Post-ho pirwise omprisons with Bonferroni orretion did not show signifint differenes t ny of the time points in. Dt with re signifintly different (Po.5) ording to the unpired two-tiled Student t- test. Dt with different supersript letters re signifintly different (Po.5) ording to post-ho one-wy nlysis of vrine. 3 1 regulted y ell-utonomous funtions or ws linked to hnges in the symptheti tone in vivo. To ddress this question, we isolted suutneous ft pds (explnts) nd ultured them ex vivo for h to eliminte peripherl drenergi stimultions. Interestingly we found tht fter h inution in ulture medium, dipose tissue explnts from mie tend to reprodue the in vivo phenotype on rowning mrkers, lthough this derese did not reh sttistil signifine (Supplementry Fig. ). These results suggest tht the effet of Npepld deletion on rowning nnot entirely e ttriuted to hnge in the symptheti drive. Furthermore, we did not find ny hnges in mrna expression of the 3 reeptor in vivo, whih ould hve een expeted s 3 reeptors re losely regulted t the expression level y symptheti inputs (Supplementry Fig. ). Adipose tissue Npepld deletion hnges gut miroiot. The metoli endotoxemi oserved in mie indites puttive shift in gut miroiot omposition. Beuse we previously demonstrted strong ssoition etween dipose tissue, ecb ontent nd gut miroiot 1,, we nlyzed the gut miroiot using high-throughput sequening. Consistent with previous studies,5,8, we oserved signifint hnge in gut miroiot omposition under HFD ompred with the CT diet. Interestingly, the deletion of Npepld profoundly ffeted gut miroiot under CT diet onditions, s oserved in the Prinipl oordintes nlysis (Fig. 7). More speifilly, the undne of opertionl txonomi units is signifintly different in mie ompred with mie (Fig. 7). At the txonomi level, two phyl, six fmilies nd eight gener were signifintly modified in mie ompred with mie (Fig. 7 d nd Supplementry Tles 3 5). Interestingly, gut miroiot from mie differed from tht of the mie ut lso from tht of the HFD-treted mie (Fig. 7,d), suggesting tht the effets of Npepld deletion on gut miroiot my e different thn those indued y the HFD tretment. This finding strongly suggests tht the deletion of Npepld in dipose tissue hs profound influene on gut miroiot omposition in physiologil onditions nd therey suggests for the first time the existene of n dipose tissue to gut miroiot xis. Long-term ntiioti tretment improves gluose homeostsis. To investigte if gut miroiot my influene the phenotype oserved following Npepld deletion, we treted mie with ntiiotis for 1 weeks. Interestingly we found tht long-term ntiioti tretment in mie under CT (-Ax) redued ody-weight gin nd ft mss development (Fig. 8,). Antiiotis lso improved gluose tolerne nd insulin resistne index in mie (Fig. 8 f), suggesting diret impt of gut miroiot on energy nd gluose homeostsis. Gut miroiot trnsfer prtilly replites the phenotype. To see whether the ltered gut miroiot omposition in mie is usl ftor for, or metoli onsequene of, the phenotype oserved nd to further eluidte whether the profound shift in gut miroiot omposition oserved in mie my ontriute to the phenotype, we trnsferred gut miroiot from ody-weight-mthed or mie into germ-free (GF) reipient mie. Both donors nd reipients were kept on CT diet. After weeks, we found tht gut miroiot trnsfer from 8 NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

9 NATURE COMMUNICATIONS DOI: 1.138/nomms795 ARTICLE. PCoA-PC1 vs PC Bteroidetes PC-Perent vrition explined.9% Proteoteri Firmiutes Deferriteres Atinoteri PC1-Perent vrition explined.35%.. PCoA-PC3 vs PC PC-Perent vrition explined.9% d 1S rrna sequenes (%) PC3-Perent vrition explined 1.7%.8.1 Adlerreutzi Alloulum Anerotrunus Bilophil Clostridium Dehloterium Delfti Dore Enteroous Esherihi Ltoillus Ltoous Muispirillum Odoriter Olsenell Osillospir Prteroides Prevotell Pseudomons Ruminoous Sutterell Turiiter Unlssified Unlssified Atinoteri Unlssified Alphproteoteri Unlssified Arhe Unlssified Bteri Unlssified Bteroidee Unlssified Bteroidles Unlssified Bteroidles other Unlssified Bteroidetes Unlssified Betproteoteri Unlssified Burkholderiles Unlssified Clostridi Unlssified Clostridiee Unlssified Clostridiles Unlssified Corioteriee Unlssified Cynoteri Unlssified Deferriterles Unlssified Deltprototeri Unlssified Desulfovirionee Unlssified Desulfovirionles Unlssified Enteroteriee Unlssified Erysipelotrihee Unlssified F1 Fmilies Alligenee Bteroidee Clostridiee Corioteriee Erysipelotrihee Lhnospiree Rikenellee Ruminooee S-7 Non-identified Unlssified Firmiutes Unlssified Lhnospiree Unlssified Ltoillee Unlssified Ltoillee other Unlssified Peptooee Unlssified Peptostre ptooee Unlssified Porphyromondee Unlssified Proteoteri Unlssified Rikettsiles Unlssified Rikenellee Unlssified Rikenellee other Unlssified Ruminooee Unlssified S-7 Unlssified Steptooee Unlssified Steptophyt Figure 7 Adipose tissue Npepld deletion ffets gut miroiot omposition. () PCoA sed on the weighted UniFr nlysis on opertionl txonomi units (OTUs; n ¼ 1). Eh symol representing single smple is oloured ording to the group. () Composition of undnt teril phyl identified in the gut miroiot of nd mie (n ¼ 1). () The different OTUs signifintly ffeted y dipose tissue Npepld deletion under CT diet. A representtive 1S rrna gene from eh of the differentilly expressed OTUs in versus mie ws ligned nd used to infer the phylogeneti tree presented in this figures (n ¼ 1). The olour in front of the OTU indites the fmily of the OTU. (d) Reltive undnes (perentge of 1S rrna sequenes) of the vrious teril gener in eh smple mong eh group of mie (n ¼ 1). In d, the different phyl, fmilies nd gener re represented y different olour odes. See lso Supplementry Tles 3 5. donors signifintly inresed ft mss gin nd the diposity index (sum of the weights of the different dipose tissue depots) (Fig. 9,). There ws trend towrds inresed totl ody-weight gin ut this did not reh sttistil signifine (Fig. 9). We found tht gut miroes from mie inrese dipose tissue inflmmtion (inresed Cd11 mrna levels Fig. 9d) nd mrkedly derese mrkers of -oxidtion nd rowning (Aox, Pprg1, Cide, Elovl3, Up1 Fig. 9d). To investigte if gut miroiot trnsfer my lso ffet insulin sensitivity, we nlyzed Sl (GLUT) expression in suutneous dipose tissue (SAT) nd musle nd gluose-- Phosphtse (Gp) expression in the liver nd found tht the NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.

10 NATURE COMMUNICATIONS DOI: 1.138/nomms795 Body-weight gin (g) Plsm gluose (mg dl 1 ) Insulin (μg I 1 ) Ax Time (min) -Ax 1 Gluose AUC (mg I 1 min 1 ) Adiposity index (% BW) Insulin resistne index (AUC gluose xauc insulin ) 1, 3,, 1, miroiot trnsfer from mie did not ffet these prmeters. Furthermore, miroiot trnsfer did neither lter liver glyogen levels nor GPse tivity (Fig. 9e,f). Thus, these results suggest tht gut miroiot trnsfer is suffiient to rpidly reprodue the phenotype oserved in dipose tissue of Npeplddeleted mie (tht is, ft gin nd rowning), wheres the impt on gluose metolism is not yet oserved. Together, the results otined following ntiioti exposure nd miroiot trnsfer onfirm the key role plyed y gut miroiot in shping the host phenotype nd demonstrte the ritil influene of dipose tissue NAPE-PLD on gut miroiot. Disussion The ECS nd its relted iotive lipids suh s NAEs ply key roles in the regultion of energy homeostsis. In this pper, we disovered the essentil role of the ecb synthesizing enzyme NAPE-PLD in dipose tissue (summrized in Fig. 1). Mie Ax -Ax -Ax Figure 8 Antiioti tretment improves metolism in dipose tissue Npepld-deleted mie. () Totl ody-weight gin (g; n ¼ 5). () Adiposity index (% Totl ody weight) fter long-term ntiioti tretment (n ¼ 5). () Plsm gluose (mg dl 1 ) profile nd (d) the men re under the urve (AUC) mesured etween nd 1 min fter gluose loding (mg l 1 min 1 ; n ¼ 5). (e) Plsm insulin levels t 3 min efore nd min fter gluose loding (mgl 1 )(n ¼ 5). (f) Insulin resistne index determined y multiplying the AUC of lood gluose y the AUC of insulin. Dt re presented s men±s.e.m. Dt with re signifintly different (Po.5) ording to the unpired two-tiled Student t-test, or following two-wy sttistil nlysis in. Dt with indites signifint differene (Po.1) versus time point 3 min in mie in e. lking the Npepld gene in their dipose tissue re prone to oesity nd ssoited metoli disorders. Remrkly, the phenotype of mie develops in physiologil stte (tht is, CT diet). This indites tht NAPE-PLD plys n importnt role in the regultion of sl metolism, energy homeostsis nd inflmmtion. Surprisingly, Npepld-deleted mie under HFD hve higher ody-weight gin nd insulin resistne index, wheres other metoli prmeters re not exerted under this pthologil ondition. Sine the levels of dipose tissue NAEs re similr etween nd mie under HFD, this my explin why we do not oserve inresed inflmmtion nd ltered lipid metolism under HFD. Nevertheless, Npepld deletion is effetive when differentition of dipose tissue is omplete, thus efore the eginning of HFD tretment. Therefore inresed ody-weight gin nd insulin resistne my e diretly ttriuted to Npepld deletion wheres other metoli ltertions re proly due to long-term NAEs redution. Using different pprohes (tht is, mirorry, qpcr nd histology), we determined tht mie develop mrked inflmmtory tone. This phenotype my e explined y different mehnisms, inluding the ft tht Npepld deletion deresed the levels of the nti-inflmmtory PEA. Indeed, PEA hs een identified previously s iotive lipid with nti-inflmmtory properties 9,1,9. Moreover, the ltered regultion of severl PGs, phospholipids, ermides nd eiosnoids my ontriute to ltering the regultion of inflmmtory pthwys 3. Interestingly, it hs een shown tht n inflmmtory stimulus suh s LPS redues Npepld expression nd PEA prodution in RAW.7 mrophges 31. These in vitro dt, together with our in vivo findings, support role for NAPE-PLD in regulting the norml inflmmtory response. The ECS plys n importnt role in regulting gluose homeostsis 3 3. Aordingly, our findings highlight the impt of dipose tissue Npepld on gluose metolism. Perturtions of gluose homeostsis hve een linked to dipose tissue inflmmtion 35,3, suggesting tht the inflmmtory tone developed in our model ontriutes to the oserved gluose intolerne nd insulin resistne. We lso found tht insulin resistne ours minly in the liver rther thn skeletl musle or dipose tissue. Nevertheless, we nnot exlude tht insulin resistne ould ffet those orgns t lter time sine phosphoryltion of IR is lso signifintly ffeted in skeletl musle nd there is n importnt trend towrds redued insulin-indued phosphoryltion of IR in dipose tissue. Thus, the speifi kineti of development of insulin resistne wrrnts further investigtions. In ddition, we found tht Npepld deletion in dipose tissue leds to n inrese in irulting TAG nd holesterol levels, underlying perturtions in lipid metolism. To further eluidte the mehnisms linking iotive lipids produed y NAPE-PLD nd other lipid ndidtes known to ply mjor role in inflmmtion nd insulin resistne, we performed thorough lipidomi nlysis, inluding ermides, eiosnoids nd PGs. Cermides link inflmmtion to insulin resistne 37. Moreover, reent study reported diret link mong long-hin ermides, ecb nd insulin tion in the liver 38. Interestingly we oserved tht mie exhiit inresed levels of long-hin ermides in their dipose tissue, suggesting tht ltered NAE prodution in dipose tissue my impt ermide levels nd susequently led to other metoli disturnes. The ecb nd eiosnoid metolisms re losely relted 17,39. Eiosnoids nd PG re synthesized from rhidoni id (AA) vi the COX pthwy, whih n lso metolize ecb 39. Moreover, in mie, we found n upregultion for severl genes involved in the regultion of inflmmtion nd immunity s well s iotive lipid metolism (Alox5p, Plg5 nd Ple1; Fig. 5). In ontrst, 1 NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

11 ARTICLE NATURE COMMUNICATIONS DOI: 1.138/nomms795 F- W FG T W T FG W FG KO Pp r g 1 U p 1 C id e El ov l3 KO T KO Liver GPse tivity (U g 1 liver) KO 5 A ox C d1 1 1 F- G F- T 1 G W F- FG G Liver glyogen ( g mg 1 proteins) GF-CT GF-KO 1 KO T W FG 8 SAT mrna levels 5 F- 1 G G 3 Adiposity index (g) Ft mss (% BW) Body-weight gin (g) Figure 9 Gut miroiot trnsfer to germ-free (GF) mie reprodues prt of the phenotype. () Body-weight gin (g) fter gut miroiot trnsfer to germ-free mie (n ¼ 7). () Ft mss weight in perentge of ody weight fter gut miroiot trnsfer to germ-free mie (%; n ¼ 7). () Adiposity index (g) fter gut miroiot trnsfer to germ-free mie (n ¼ 7). (d) mrna expression of Cd11, Aox, Pprg1, Up1, Cide nd Elovl3 in SAT (n ¼ 7). (e) Liver glyogen ontent (mg per mg proteins; n ¼ 7). (f) Liver GPse tivity (U per g liver; n ¼ 7). Dt re presented s men±s.e.m. Dt with Po.5, Po.1 nd Po.1 re signifintly different ording to the unpired two-tiled Student s t-test. Crosstlk O N H «Len» gut miroiot OH NAPE-PLD NAE Norml rowning Norml gluose homeostsis Norml lipid flow Altered gluose nd lipid homeostsis Inflmmtion LPS O «Altered» gut miroiot N H OH NAPE-PLD NAE Deresed rowning Modified Crosstlk Figure 1 Shemti overview of the phenotype oserved following Npepld deletion in dipoytes. When NAPE-PLD is present nd funtionl, dipose tissue homeostsis is mintined nd this prtiiptes in norml rosstlk etween dipose tissue nd gut miroiot. Moreover the presene of NAPE-PLD prtiiptes in norml gluose homeostsis nd lipid flows. When NAPE-PLD is sent, dipose tissue metolism is deregulted, with n inrese in inflmmtion, derese in the rowning pity nd exessive ft mss development. Npepld deletion lso indues ltertions in gluose homeostsis nd lters norml lipid flows nd lipid homeostsis in irultion nd in dipose tissue. Altered dipose metolism following Npepld deletion indues dysiosis of the gut miroiot, whih in turn prtiiptes in the metoli ltertions oserved in the dipose tissue. we oserved derese in eiosnoids nd PG in the dipose tissue of mie s well s in the dipose tissue of HFD-treted mie, where n inrese in these pro-inflmmtory lipid meditors ould hve een expeted. However, reent dt hve suggested tht these lipids my exhiit oth pro- nd ntiinflmmtory properties ording to the pthologil sitution. On the other hnd, PG derivtives n e synthesized y the metolism of ecb vi the COX pthwy, leding to the formtion of PG-glyerol esters or PG-ethnolmides, whih exert nti-inflmmtory effets,1. In ddition, these iotive lipids my t s resolvins, therey ontriuting to the omplex resolution of inflmmtion3. Whether the derese in PGD nd PGE oserved here ontriutes to the modultion of these omplex intertions requires further investigtion. Finlly, NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved. 11

12 NATURE COMMUNICATIONS DOI: 1.138/nomms795 AA levels re unhnged in mie nd re deresed under HFD ompred with mie (Supplementry Fig. 7). The glol ltered lipid profile oserved in our model suggests tht the deresed level of the sustrte (AA) my explin why PGs re not inresed. Tken together, these dt shed light on the influene of NAPE-PLD on iotive lipid levels in dipose tissue nd the pronouned ltered dipose tissue lipid metolism in our mie model. Furthermore, we found inresed levels of NAPEs (NAEs preursors, Supplementry Fig. ) in dipose tissue of mie ompred with mie, onfirming dt otined in previous studies,11,1. These lipids ould lso e potentil ontriutors to the phenotype oserved sine some NAPEs hve een shown to present metoli properties, nmely on energy homeostsis. Nevertheless, this sttement wrrnts further investigtion in our model. mie lso exhiited deresed mrna levels of Up1 nd other rown ft ell-enrihed genes, suh s Cide nd Pprg1, in their WAT, suggesting tht their dipose tissue loses thermogeni potentil. This finding ws further supported y the mirorry nlysis, whih indited redution in the expression of severl genes generlly ssoited with rown/eige dipoytes, suh s Ev1 (lso known s myelin protein zero-like, Mpzl) nd the mitohondril genes Cox71 nd Cox8. It hs reently een suggested tht suset of dipoytes in the SAT deposits n tivte thermogeni progrmme, nmely eige or rite ells 3,. Animls defiient in funtionl eige ells develop oesity, in onjuntion with huge inrese in SAT deposits 5. Remrkly, we oserved tht more thn 1 genes involved in the regultion of these metoli proesses were signifintly ffeted in the sene of NAPE-PLD. Bsed on these oservtions, we propose tht the inresed ft mss development is ssoited with n ltered rowning proess in the dipose tissue nd redued pity to develop norml thermogeni progrmme. Interestingly, the mirorry nlysis lso reveled the downregultion of Fp3 nd mitohondril ftty id oxidtion genes, suh s Ass1, Asl5 nd Cpt1. These enzymes hve een found to e upregulted fter old exposure or -drenergi stimultion nd re linked to rown-like trnsformtion of the WAT. Their downregultion might reflet redued -oxidtion due to the further loss of the BAT-hrteristis of the WAT. This defet in -oxidtion my potentilly ontriute to the elevted levels of irulting lipids oserved. Moreover, phrmologil lokde of the CB 1 reeptor indues the tivtion of BAT thermogenesis ssoited with enhned gluose nd lipid utiliztion 7,8, while OEA hs een reently implited in the enhnement of -drenergimedited thermogenesis in rts 9, linking ECS nd thermogenesis. Altogether, the ltered lipid metolism oserved in our model ould e linked to the ltered rowning proess. For exmple, PGE hs reently een identified s key regultor of white-to-rown dipogenesis 5 nd the redued PGE levels oserved in the WAT my therefore prtiipte in deresed rowning. To further explore the effets of Npepld deletion on the rowning proess, we exposed nd mie to old exposure nd found tht Npepld-deleted mie lerly present n ltered indution of old-indued rowning proess. This proess seems to e due to Npepld deletion independently of symptheti drive sine dipose tissue explnts from donors tend to develop the sme phenotype. Overll, these dt lerly indite tht dipose tissue NAPE-PLD is key enzyme involved in the regultion of energy homeostsis y regulting the rowning proess. Nevertheless, the usl impt of Npepld deletion on rowning proess merits further investigtions to sertin the diret effets on rowning, independently of het-loss mehnism or symptheti nervous system tivtion 7. Using 5-pyrosequening, we disovered tht ltering the NAE synthesis in dipose tissue profoundly lters the gut miroiot omposition. For instne, the gener Ltoillus nd Alloulum were deresed in mie ompred with mie (Supplementry Tle 5). We hve previously reported tht HFD feeding redues the undne of Alloulum, wheres preiotis inrese this genus; redue ft mss, metoli inflmmtion nd Lp mrna expression; nd inrese insulin sensitivity. Similrly, the undne of Alloulum is inresed in rts tht were fed with ererine, whih prevents oesity nd insulin resistne on HFD tretment 51. Severl strins of Ltoillus re ommonly used proiotis 5,53. It is therefore not unoneivle tht the effet of Npepld deletion in dipose tissue indues metoli ltertions ssoited with metoli funtions ssumed y gut miroes. We lso oserved signifint impt of HFD tretment on the gut miroiot omposition, onfirming previously pulished dt. Furthermore, Npepld deletion leds to n inrese in portl LPS, suggesting n ltered intestinl rrier funtion 5. We thus postulte tht the deresed prodution of NAEs in the dipose tissue hs n impt on the gut miroiot nd the gut rrier funtion 1. By treting mie with ntiiotis nd y trnsferring gut miroes from nd mie to GF reipients, we ould demonstrte the ontriution of the gut miroiot on the phenotype oserved in mie. These results knowledge n importnt role for the gut miroiot, whih seems to e independent of ody weight, s dipoyte-speifi Npepld deletion results in distint gut miroiot omposition from tht otined under HFD onditions nd s trnsfer of gut miroiot from ody-weight-mthed donors is suffiient to effetively trnsfer prt of the phenotype. However, we my not ompletely rule out the omplementry ssoition of ody weight nd gut miroiot modultion over the long term sine ntiioti tretment redues ody weight nd improves gluose homeostsis in mie. To note, only weeks follow-up fter gut miroiot trnsfer (ompred with 8 weeks in the other in vivo studies) were enough to prtly reprodue the phenotype. Conversely, insulin sensitivity nd gluose tolerne my not yet e ffeted weeks fter gut miroiot trnsfer in our mie model, ut rther oserved fter long-term modultion of gut miroiot (tht is, ntiioti tretment). Also, whether one or severl speifi gut miroes ontriute to this phenotype requires further investigtions. In onlusion, our study highlights the essentil ontriution of the dipose tissue NAE synthesis pthwy, to whole-ody energy metolism nd physiology. In the sene of this funtionl synthesis pthwy in the sl stte, mie develop oesity, dipose tissue inflmmtion, insulin resistne, gluose intolerne nd perturtion of the lipid metolism. This phenotype is prtly medited y the ltertion of gut miroiot omposition nd y n ltered rowning progrmme (Fig. 1). Tken together, these dt underlie the importne of tissue-speifi differenes in ECS regultion, with speil emphsis on dipose tissue. Methods Mie. Genertion of dipose tissue Npepld mie. Adipose tissue-speifi Npepld-deleted mie ( mie) were generted y rossing mie ering the Cre reominse expressed under the ontrol of the Fp promoter (Fp-Cre) (C57BL/ kground, Jkson-Lortory, Br Hror, ME, USA) with mie hrouring loxp-flnked Npepld llele. Npepld loxed mie were generted s previously desried 55. Deletion ws effetive when dipose tissue rehes mturity nd mie were orn t norml Mendelin rtios. All mouse experiments were pproved y nd performed in ordne with the guidelines of the lol ethis ommittee for niml re of the Helth Setor of the UCL under the supervision of Prof. F. Lemigre nd Prof. JP Dehoux nd under the speifi numer 1/UCL/MD/. Housing onditions were speified y the 1 NATURE COMMUNICATIONS :95 DOI: 1.138/nomms795 & Mmilln Pulishers Limited. All rights reserved.

13 NATURE COMMUNICATIONS DOI: 1.138/nomms795 ARTICLE Belgin Lw of 9 My 13 regrding the protetion of lortory nimls (greement numer LA1331). experiments. Cohorts of 8-week-old mle mie nd littermtes were housed in groups of two mie per ge (filter-top ges) with free ess to food nd wter. The mie were fed CT (AIN93Mi, Reserh Diet) or HFD (% ft, D19, Reserh Diet). Tretment ontinued for 8 weeks. This experiment ws replited independently three times. The ontrol mie were littermtes hrouring the Npepld loxp-flnked llele ut not the Cre reominse. Body weight, food intke nd wter onsumption were reorded one week. Body omposition ws ssessed one week using 7.5-MHz time-domin NMR (TD-NMR) (LF5 Minispe, Bruker, Rheinstetten, Germny). After 7 weeks of tretment, n OGTT ws performed s previously desried in freely moving mie 5,3. To nlyze the insulin signlling pthwy, mie reeived 5 U insulin (Atrpid; Novo Nordisk A/S, Denmrk) under nesthesi (isoflurne, Forene, Aott, Queenorough, Kent, Englnd), or n equl volume of PBS into the portl vein to nlyze signlling response to insulin. Three minutes fter injetion, mie were killed nd liver, SAT nd gstronemius skeletl musle were rpidly disseted. At the end of the tretments, the mie were nesthetized with isoflurne fter -h fsting period. Portl nd v vein lood smples were olleted for further nlysis. After exsnguintion, mie were killed y ervil dislotion. Tissues were preisely disseted, weighed nd immeditely snp-frozen in N nd stored t 8 C for further nlysis. Cold exposure experiment. Cohorts of 8-week-old mle mie nd littermtes were rered t room temperture under stndrd housing onditions (filter-top ges) with free ess to food nd wter for 1 weeks. The mie were fed CT diet (AIN93Mi, Reserh Diet, New Brunswik, NJ, USA). Body weight, food intke nd wter onsumption were reorded one week. Body omposition ws ssessed one week using 7.5-MHz TD-NMR. After 1 weeks follow-up, two groups of ge- nd ody weight-mthed nd mie were seprted nd individully housed. One hlf of the nimls from eh genotype ws trnsferred to old room t 8 C, wheres the other hlf remined t room temperture (n ¼ 7 9/group). Mie were fsted during the dy period nd fed d liitum during the night. Mie were kept in the old room for 7 h nd fter 18 h limtion in the old room, ody temperture ws monitored t different intervls during two dys with retl proe (RET-3, World Preision Instruments, Aston Stevenge, UK). After 7 h, ll mie were srified s desried ove. Antiioti tretment experiment. Cohorts of 8-week-old C57/Bl femle mie were housed in groups of or 3 mie/ge (filter-top ges) with free ess to food nd wter. The mie were fed CT diet for 1 weeks. Hlf of the mie (n ¼ 5 per group) reeived ntiiotis (1. g l 1 mpiillin (Sigm, St Louis, MO) nd.5 g l 1 neomyin (Sigm)) in their drinking wter during the experimentl period 1. Body weight ws reorded one week. Body omposition ws ssessed one week using 7.5-MHz TD-NMR. After 11 weeks, n OGTT ws performed s desried ove. After 1 weeks follow-up, mie were killed s desried ove. Gut miroiot trnsplnttion experiments. The el ontents of 3 mie nd 3 littermtes (ody weight mthed) were trnsplnted into 1 GF mie (7-week-old Swiss-Wester mles, Toni, Hudson, NY, USA) s previously desried 5. Eh donor ws used to trnsplnt two or three GF reipients. Eh el smple ( mg) ws smpled in n neroi hmer nd suspended in PBS (1.5 ml per eum). Cel ontents were then immeditely dministered (. ml per mouse) to the GF mie. GF mie were housed individully in ventilted ges (IVC AERO GM5, Tenil-BMI, Someren, The Netherlnds) nd fed CT diet for weeks. Body weight, food intke nd wter onsumption were reorded one week. Body omposition ws ssessed one week using 7.5-MHz TD-NMR. After weeks follow-up, mie were killed s desried ove. Insulin resistne index. The plsm insulin onentrtions were mesured in plsm olleted from til lood during OGTT using n ELISA Kit (Merodi, Uppsl, Sweden) ording to the mnufturer s instrutions. The insulin resistne index ws determined y multiplying the re under the urve of oth the lood gluose ( 3 to 1 min) nd the plsm insulin ( 3 to min) otined from the OGTT. RNA extrtion nd rel-time qpcr nlysis. Totl RNA ws prepred from tissues using the TriPure regent (Rohe). The quntifition nd integrity nlysis of the totl RNA were performed y running 1 ml of eh smple on n Agilent 1 Bionlyzer (Agilent RNA Nno Kit, Agilent). The omplementry DNA ws prepred y reverse trnsription, nd rel-time qpcr ws performed s previously desried. RPL19 RNA ws hosen s the housekeeping gene. Primer sequenes re provided in the Supplementry Tle. Mirorry nlysis. Equl mounts of RNA from five mie per group were pooled within eh group. Mirorrys were performed s previously desried 57. Mouse gene ST mirorry hips were used for hyridiztion (MoGene 1. ST, Affymetrix). The expression kit (Amion) ws used for omplementry RNA preprtion from the totl RNA. The hyridiztion, wsh nd sn were done ording to the Affymetrix kits nd proedures speifi to the mouse gene ST hips. After the sn, the qulity ontrols of the hyridiztion were heked using the Affymetrix Gene Expression Console softwre. Using the Affymetrix APT suite tools, we normlized the dt y the RMA-Sketh proedure nd omputed the signl detetion P vlues using the DABG lgorithm. All the proe sets tht hve the DABG P vlue.5 in ll onditions were disrded from the nlysis. The rest of proes sets were kept for fold-hnge nlysis. Funtionl nnottion nd pthwy nlysis ws done using the DAVID we tool 58. Both tools were fed with the list of seleted offiil gene nmes s input, nd the threshold of signifine ws set y defult to P vlues o.5. DNA isoltion from mouse el smples nd qpcr nd sequening. Cel ontents were olleted nd kept frozen t 8 C until use. Metgenomi DNA ws extrted from the el ontent using the QIAmp DNA Stool mini-kit (Qigen, Hilden, Germny) ording to the mnufturer s instrutions. The V1-V3 region of the teril 1S rrna gene ws mplified using roded primers 7f nd 53r (ref. 59), nd the high-throughput sequening results of the purified mplions were nlyzed on Rohe FLX Genome Sequener using titnium hemistry (DNAVision, Gosselies, Belgium). The resulting reds were proessed through the QIIME v1.7. pipeline. The undne of identified nd unlssified tx ws trnsformed using the Hellinger method fter removing tx representing o.1% of the totl undne. Prinipl oordintes nlysis ws lulted using the weighted UniFr distne. Opertionl txonomi units were identified using the ulust onsensus txonomy lssifier with.97 threshold ginst the Greengenes dtse. Phylogeneti trees were generted using QIIME 1.7. nd visulized using itol v... SDS PAGE nd immunolotting. For the totl lystes, tissues were homogenized with TissueLyser II (Qigen) in RIPA uffer 1 supplemented with oktil of protese inhiitors (Sigm) nd phosphtse inhiitors. Equl mounts of proteins were seprted y SDS PAGE nd trnsferred to nitroellulose memrnes. For detetion of proteins of the insulin pthwy, tissues were homogenized in ERK uffer (Triton X-1.1%, HEPES 5 mm, NCl 5 M, Glyerol 1%, MgCl 1.5 mm nd DTT 1 mm) supplemented with oktil of protese inhiitors nd phosphtse inhiitors. Memrnes were inuted overnight t C with the following ntiodies diluted in Tris-uffered sline tween- ontining 1% non-ft dry milk: NAPE-PLD (1:; 95397, Am, Cmridge, MA, USA), p-ir (1:1,; s-513, Snt Cruz, CA, USA), p-akt Thr38 (1:1,; 95L, Cell Signling, Dnvers, MA, USA) nd p-akt Ser73 (1:1,; L, Cell Signling). Quntifition of phospho-proteins ws performed on six nimls with insulin injetion per group. The loding ontrol ws -tin (1:1,; 7) or -tuulin for skeletl musle (1:8; s-91). Full unedited lots re ville in Supplementry Fig. 8. Histologil nlysis nd immunohistohemistry. The tissues were fixed in % formldehyde. Hemtoxylin nd eosin stining ws performed using stndrd protools on 5-mm dipose tissue setions. Adipoyte size (hemtoxylin nd eosin-stined setions), mrophge infiltrtion (F/8:, Am) nd UCP1 stining (381, Am) were determined using ImgeJ (version 1.8r, Ntionl Institutes of Helth, Bethesd, Mrylnd, USA). Seprtion of dipoytes nd the SVF. Aout 3 mg of SAT deposit were disseted nd ut in smll piees nd digested with ollgense A (Rohe) for min t 37 C. Digested tissue ws filtered nd entrifuged t g for 1 min. The infrntnt ontining the SVF nd the superntnt ontining dipoytes were wshed three times in Kres-BSA1% solution nd stored t 8 C in Tripure Regent (Rohe) for further RNA extrtion. Primry peritonel mrophge isoltion. Murine peritonel mrophges were otined y eliiting n ute peripherl inflmmtory retion with n i.p. injetion of thioglyolte. Isolted primry mrophges were inuted t 37 C for h, wshed with PBS nd then frozen in Tripure Regent for further RNA extrtion. Adipose tissue explnts ulture. Suutneous dipose depots from mie (1 mie nd 1 mie) were preisely disseted, nd ll visile vessels, prtiles nd onjuntive tissues were removed. The ft tissue ws then ut into smll piees ( mm 3 ), pooled per genotype nd pled into Kres uffer (ph 7.) ontining.5% (w/v) ftty id-free BSA, peniillin/streptomyin (1:1) nd fungizone (1:1) (Invitrogen). A totl of mg dipose tissue ws rinsed in PBS nd inuted in 1-mm Petri dishes ontining 1 ml MEM (Invitrogen) supplemented with.5% (w/v) ftty id-free BSA, peniillin/streptomyin (1:1) nd fungizone (1:1). All onditions were repeted in six different dishes. Dishes were ultured for h t 37 C in5%co tmosphere. The sl onentrtion of gluose in fresh medi ws 5 mmol l 1. At the end of the experiment, dipose mteril ws olleted nd immeditely frozen in liquid nitrogen, nd stored t 8 C until susequent mrna nlysis. NATURE COMMUNICATIONS :95 DOI: 1.138/nomms & Mmilln Pulishers Limited. All rights reserved.