Macrophage mtorc1 disruption reduces inflammation and insulin resistance in obese mice

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1 Dietologi (1) 7:393 DOI 1.17/s ARTICLE Mrophge mtorc1 isruption reues inflmmtion n insulin resistne in oese mie Hongfeng Jing & Mrit Westerterp & Chunjiong Wng & Yi Zhu & Ding Ai Reeive: 1 April 1 /Aepte: 16 July 1 /Pulishe online: 1 August 1 # Springer-Verlg Berlin Heielerg 1 Astrt Aims/hypothesis Inflmmtory ftors serete y mrophges ply n importnt role in oesity-relte insulin resistne. Being t the rossros of nutrient hormonl signlling network, the mmmlin trget of rpmyin omplex 1 (mtorc1) ontrols importnt funtions in the regultion of energy lne n peripherl metolism. However, the role of mrophge mtorc1 in insulin resistne is still unler. In the urrent stuy, we investigte the physiologil role of mrophge mtorc1 in regulting inflmmtion n insulin sensitivity. Methos We generte mie efiient in the regultory ssoite protein of mtor (Rptor) in mrophges, y rossing Rptor (lso known s Rptor) floxe mie (Rptor flox/flox ) with mie expressing Cre reominse uner the ontrol of the Lysm-Cre promoter (M- ). We fe mie how or high-ft iet (HFD) n ssesse insulin sensitivity in liver, musle n ipose tissue. Susequently, we mesure inflmmtory gene expression in liver n ipose tissue n investigte the role of Rptor efiieny in the regultion of inflmmtory responses in peritonel mrophges from Eletroni supplementry mteril The online version of this rtile (oi:1.17/s-1-33-) ontins peer-reviewe ut uneite supplementry mteril, whih is ville to uthorise users. H. Jing: M. Westerterp : D. Ai Division of Moleulr Meiine, Deprtment of Meiine, Columi University, New York, NY, USA M. Westerterp Deprtment of Meil Biohemistry, Aemi Meil Center, University of Amsterm, Amsterm, The Netherlns C. Wng: Y. Zhu : D. Ai () Deprtment of Physiology n Pthophysiology, Tinjin Meil University, 37 Tinjin, People s Repuli of Chin e-mil: einn@gmil.om HFD-fe mie or in plmiti i-stimulte one mrrowerive mrophges (BMDMs). Results M- mie fe HFD h improve systemi insulin sensitivity ompre with Rptor flox/flox mie. Mrophge Rptor efiieny reue inflmmtory gene expression in liver n ipose tissue, ftty liver n ipose tissue mrophge ontent in response to HFD. In peritonel mrophges from mie fe with n HFD for 1 weeks, mrophge Rptor efiieny erese inflmmtory gene expression, through ttenution of the intivtion of Akt n susequent inhiition of the inositol-requiring element 1α/lun NH -terminl kinse nuler ftor kpp-lighthin-enhner of tivte B ells (IRE1α/JNK/NFκB) pthwys. Similrly, mtor inhiition s result of Rptor efiieny or rpmyin tretment erese plmiti iinue inflmmtory gene expression in BMDMs in vitro. Conlusions/interprettion The isruption of mtorc1 signlling in mrophges protets mie ginst inflmmtion n insulin resistne potentilly y inhiiting HFD- n plmiti i-inue IRE1α/JNK/NFκB pthwy tivtion. Keywors Inflmmtion. Insulin resistne. mtorc1 Arevitions ATF6 Ativting trnsription ftor 6 ATM Aipose tissue mrophge BMDM Bone mrrow-erive mrophge CD Chow iet ConA Connvlin A DIO Diet-inue oesity eifα Eukryoti trnsltion initition ftor α ER Enoplsmi retiulum HFD High-ft iet IPGTT Intrperitonel gluose tolerne test IPITT Intrperitonel insulin tolerne test IRE1α Inositol-requiring element 1α

2 39 Dietologi (1) 7:393 JNK Jun NH -terminl kinse mtorc1/ Mmmlin trget of rpmyin omplex 1/ NF-κB Nuler ftor κb NMR Nuler mgneti resonne PERK RNA-epenent protein kinse-like ER kinse p7s6k p7 S6 kinse Rptor Regultory ssoite protein of mtor SFA Sturte ftty i TG Triylglyerol TLR Toll-like reeptor UPR Unfole protein response WAT White ipose tissue XBP1 X ox ining protein 1 Introution Over reent ees, it hs eome ler tht oesity is ssoite with the tivtion of enoplsmi retiulum (ER) stress signlling n inflmmtory pthwys, whih ontriute to oesity-relte riovsulr isese, metoli synrome n type ietes [1 3]. ER stress signlling, whih is referre to s the unfole protein response (UPR), is triggere y three ownstrem proteins, inositol-requiring element 1α (IRE1α), tivting trnsription ftor 6 (ATF6) n RNA-epenent protein kinse-like ER kinse (PERK). Among these three pthwys, the IRE1α X ox ining protein 1 (XBP1) rnh hs een implite in oesity-inue insulin resistne n type ietes [3 ]. In oesity, tissue mrophges, whih umulte in ipose tissue n liver, re in n inflmmtory stte, n re the mjor soure of lol inflmmtion n insulin resistne [6 8]. In mrophges, the Jun NH -terminl kinse (JNK) signlling pthwymeitesinflmmtoryftorproutionnplys key role in estlishing oesity-inue insulin resistne [9, 1]. Sturte ftty is (SFA) re systemilly elevte in iet-inue oesity (DIO) [11, 1] n trigger inflmmtion n poptosis in mrophges through ER stress [13, 1]. This stimultion is meite, t lest in prt, y signlling through Toll-like reeptor (TLR) n TLR n the ssoite tivtion of JNK [1]. Mmmlin trget of rpmyin omplex 1 (mtorc1) ts s hu, whih integrtes severl environmentl ues suh s nutritionl stimuli n regultes mny ellulr proesses inluing utophgy, protein trnsltion n riosoml iogenesis []. The mtorc1 is ompose of mtor, regultory ssoite protein of mtor (Rptor) n mlst8, n is sensitive to rpmyin. mtorc1 is one of the key regultors for ell growth n metolism through mtorc1-meite iret phosphoryltion of riosoml p7 S6 kinse (p7s6k) n eukryoti initition ftor E (eife)- ining protein. Rpmyin hs een suggeste for the tretment of mtorc1-relte iseses, inluing ner, riovsulr iseses n metoli isorers [16]. Interestingly, these iseses re lso onsiere to e ER stress-relte isorers. In the liver, geneti n iet-inue long-term tivtion of mtorc1 uses ER stress [17] n results in metoli isorers [18]. ER stress intersets with mny ifferent inflmmtory signlling pthwys [19]. However, the funtion of mrophge mtorc1 in inflmmtory response n insulin resistne use y oesity-relte ER stress remins unknown. In this stuy, we set out to exmine the role of mrophge mtorc1 in insulin resistne n the mehnisms involve. For our stuies, we rosse Rptor (lso known s Rptor) floxe(rptor flox/flox )mie with Lysm-Cre mie, generting Lysm-CreRptor flox/flox mie, whih we will refer to s M- mie. We foun tht M- mie h erese mtorc1 tivity in mrophges. M- n ontrol Rptor flox/flox mie were fe high-ft iet (HFD) to inue insulin resistne. On n HFD, M- mie isplye improve insulin sensitivity n reue inflmmtory ftor expression in liver n ipose tissue. Mehnistil stuies showe tht this my e ue to erese mrophge inflmmtory gene expression vi ttenution of IRE1α/JNK/nuler ftor κb (NF-κB) pthwy tivtion. Methos Animls n iet Rptor flox/flox mie (stok No ; The Jkson Lortory, Br Hror, ME, USA) were mte with trnsgeni mie expressing Cre reominse uner the ontrol of the Lysm promoter (Lysm-Cre; stok No. 781; Jkson Lortories) to generte mie with or without Rptor (M- ) expression in myeloi ells. Rptor flox/flox littermtes without the Cre reominse trnsgene were use s ontrols throughout the stuy. Mle mie (8 weeks ol) were fe n HFD (6% of energy from ft; D19i; Reserh Diets, New Brunswik, NJ, USA) or how iet (CD; iet 3; Purin Mills, Gry Summit, MO, USA) for 1 weeks. Mie were kille 1 min fter i.p. injetion of PBS or insulin (.7 U/kg oy weight). Boy omposition mesurements were performe with the minispe TD NMR nlyzer (Bruker, Billeri, MA, USA). All protools were pprove y the Institutionl Animl Cre n Use Committee of Columi University. Priniples of Lortory Animl Cre (NIH pulition no. 8 3, revise 198; gov/grnts/olw/referenes/phspol.htm, esse 1 Jnury 1) were followe.

3 Dietologi (1) 7: Metoli nlyses We mesure loo gluose onentrtion y gluose meter (OneTouh, Milpits, CA, USA) n plsm insulin onentrtion y ELISA (Millipore, Temeul, CA, USA) for intrperitonel gluose tolerne (IPGTT), intrperitonel insulin tolerne (IPITT) n fsting/refeeing tests. We performe the IPGTT fter 16 h fst (18: hours to 1: hours) using n i.p. injetion of g per kg oy weight gluose. For the IPITT, fsting loo gluose ws mesure ( h fst, loo tken from the til vein). Insulin ws then injete intrperitonelly (.7 U/kg) n loo gluose ws mesure gin t time points of 3, 6, 9 n 1 min, post injetion. For fsting/re-feeing stuies, mie were fste for 16 h n loo ws ollete. Mie were then re-fe for the inite time. A smll piee of liver ws tken, weighe n homogenise. Next, we extrte hepti lipis, mesure triglyerol (TG) levels, n normlise them to tissue weight. To mesure TG n holesterol ontent, olorimetri ssys from Thermo (Wlthm, MA, USA) n Wko (Rihmon, VA, USA), respetively, were use. Mrophge ulture See eletroni supplementry mteril (ESM) Methos for etils. Rel-time quntittive PCR nlysis See ESM Methos for etils. Western lot nlysis See ESM Methos for etils. Immunohistohemistry See ESM Methos for etils. Sttistis All t re presente s mens ± SEM (n is inite in the figures n/or legens). The t test ws use to efine ifferenes etween two tsets. To efine ifferenes etween multiple tsets, two-wy ANOVA ws use with Bonferroni multiple omprison post test. The riterion for loxp FRT NEO FRT loxp Trgeting vetor Primer Rptor NEO Exon 6 Exon 7 + FLP eleter Initilly trgete llele Exon 6 + Lysm-Cre Floxe llele Primer LysmCre LysmCre WT M- Rptor mtor β-atin Rptor protein 1. mtor protein 1... e P-S6 T-S6 Insulin f P-S6/T-S6 protein 3 1 Insulin Fig. 1 Genertion of M- mie. () A trgeting vetor, ontining NEO seletion ssette flnke y flippse-speifi FRT sites, n hving exon of the Rptor gene flnke y loxp site, ws use to generte mie with floxe Rptor lleles (Rptor flox/flox mie). To generte M- ( )mie,rptor flox/flox ( )miewere rosse with Lysm-Cre +/ mie. () The effiieny of genomi reomintion in M- mie ws nlyse y PCR. WT, wil type. () ConA-eliite peritonel mrophges were isolte from Rptor flox/flox n M- mie. Protein levels of Rptor n totl mtor were mesure y western lot. () Rptor n mtor were quntifie n normlise to Rptor flox/flox mrophges. (e) BMDMs were isolte from Rptor flox/flox n M- mie. The BMDMs were serum strve for 6 h n trete with serum-free DMEM or with insulin (1 nmol/l) in serum-free DMEM for 3 min. Phospho-S6 riosoml protein (P-S6) n totl S6 (T-S6) were nlyse y western lot. (f) The rtio of phospho-s6 to totl S6 ws quntifie n normlise to Rptor flox/flox mrophges trete with PBS. All t re presente s mens ± SEM; n=3. p<., vs or inite omprison

4 396 Dietologi (1) 7:393 signifine ws set t p<.. Sttistil nlyses were performe using GrphP Prism version.1 (GrphP, Sn Diego, CA, USA). Results Genertion of M- mie To ress the role of mrophge mtorc1 in oesity-relte insulin resistne n inflmmtion in vivo, we generte mouse moel in whih the Rptor gene ws isrupte y homologous reomintion in mrophges. Mie with Rptor floxe lleles (Rptor flox/flox mie) were rosse with trnsgeni mie expressing Cre reominse uner the ontrol of the Lysm promoter (Fig. 1), whih is highly expresse in mrophges n neutrophils []. We employe Rptor flox/flox LysmCre +/ mie s M- mie n Rptor flox/flox s ontrol mie (Fig. 1). Next, we ssesse the effiieny of Rptor eletion in M- mrophges. Rptor protein expression ws reue y >9% in onnvlin A (ConA)- eliite peritonel mrophges, while the totl mtor level ws similr ompre with levels in Rptor flox/flox ontrols (Fig. 1, ). Moreover, mrophge Rptor efiieny olishe the insulin-inue mtorc1 tivity in one mrrow-erive mrophges (BMDMs), s shown y erese S6 phosphoryltion (Fig. 1e, f ). Mrophge Rptor efiieny results in improve systemi insulin sensitivity espite similr oy mss n iposity mtorc1 tivity is inrese in mouse moels of oesity n type ietes, ontriuting to insulin resistne in numer of ell types [1, ]. Consistent with previous reports, mtorc1 tivity ws inrese in ConA-eliite peritonel mrophges of o/o mie n DIO mie (ESM Fig. 1). To ssess the role of mrophge mtorc1 in Fig. Mrophge Rptor efiieny i not ffet oy omposition n plsm lipi levels. Rptor flox/flox ( ) n M- ( ) mie were fe CD or HFD for 1 weeks. () Boyweightws mesure: re she line, +CD;reunroken line, +HFD;lk she line, +CD; lk unroken line, + HFD; p<., CD vs HFD. ( f) After 1 weeks of HFD, oy weight (), oy ft () n len oy mss () were mesure y NMR n ft (e)n len oy mss (f) were mesure s perentge of oy weight (BW). (g i) After 1 weeks of HFD, plsm TG (g), holesterol (Chol) (h) n HDL-holesterol (HDL hol) (i) levels were ssesse. All t re presente s mens ± SEM; n= Boy weight (g) Boy weight (g) e Ft (% of BW) Time (weeks) 1 Boy ft (g) f Len (% of BW) 8 6 Len oy mss (g) 1 g h i Plsm TG (mmol/l) Plsm Chol (mmol/l) 3 1 Plsm HDL hol (mmol/l).. 1..

5 Dietologi (1) 7: regulting systemi insulin sensitivity, we fe Rptor flox/flox n M- mie CD or HFD for 1 weeks. HFD feeing inue oy weight gin in mie of oth genotypes, without ifferenes etween the genotypes (Fig. ). Boy omposition ws mesure y nuler mgneti resonne (NMR). Len weight, oy ft n oy omposition were similr in Rptor flox/flox n M- mie on HFD (Fig. f ). In ition, plsm TG, totl holesterol n HDL-holesterol levels were similr in the two groups on the HFD (Fig. g i). Sustine HFD feeing les to insulin resistne To evlute the funtion of mrophge Rptor efiieny in insulin resistne, IPGTTs n ITTs were performe. In the IPGTT plsm gluose n insulin levels were omprle etween Rptor flox/flox n M- mie on CD (Fig. 3 ). After 1 weeks of HFD, M- mie showe less of n inrese in serum gluose t 3 n 6 min fter gluose injetion ompre with their Rptor flox/flox ontrols (Fig. 3). The AUC for gluose uring the IPGTT ws reue y 13%, wheres insulin levels were reue y 3% t 1 min fter gluose injetion in HFD-fe M- ompre with Rptor flox/flox mie (Fig. 3, e). Injetion of insulin in HFD-fe, ut not CD-fe M- mie, le to n enhne hypoglyemi response (p<.) ompre with Rptor flox/flox mie, ompnie y reution of % in the AUC for gluose (Fig. 3f h). We next mesure plsm gluose n insulin levels in response to fsting/re-feeing. After h of re-feeing fter mie h een fste overnight, plsm gluose n insulin levels were omprle etween Rptor flox/flox n M- mie on the CD (Fig., ). In ontrst, lthough plsm gluose levels were unhnge in HFD-fe M- ompre with Rptor flox/flox mie (Fig. ), HFD-fe M- mie isplye reue plsm insulin level fter n h of re-feeing (Fig. ). These t suggeste tht reution of mtorc1 in mrophges le to improve systemi insulin resistne inue y the HFD. To unerstn the mehnisms y whih mrophge Rptor ltion improve insulin sensitivity, we ssesse insulin signlling in the liver, white ipose tissue (WAT) n skeletl musle. Mie were fe HFD for 1 weeks, n fste overnight efore eing ministere sline or insulin. We foun tht the insulin-inue tyrosine phosphoryltion of Akt t Ser73 ws enhne in WAT, liver n skeletl musle of mie (Fig. ), ompre with Rptor flox/flox mie, onsistent with systemilly improve insulin sensitivity. Mrophge Rptor ltion inhiits HFD-inue ipose tissue mrophge umultion, hepti stetosis n inflmmtion The HFD moel is known to e ssoite with hepti stetosis. Mrophge Rptor efiieny erese Gluose AUC (mmol/l x min) Gluose AUC (mmol/l x min) g Gluose AUC (mmol/l x min) Gluose (mmol/l) f Gluose (mmol/l) 3 1, 1, 1,, 3,, 1, IPGTT Time (min) ITT Insulin (pmol/l) e Insulin (pmol/l) h Time (min) Gluose AUC (mmol/l x min) 6 1, 1, min 1 min min 1 min Fig. 3 Insulin resistne ws ttenute y mrophge Rptor efiieny. ( e) IPGTT ws performe fter 1 weeks of CD or HFD feeing in Rptor flox/flox ( )nm- ( ) mie. () Gluose levels were mesure: re she line, + CD; re unroken line, + HFD; lk she line, + CD; lk unroken line, +HFD.p<., vs.() AUC of gluose levels in CD-fe mie. () Plsm insulin of CD-fe mie ws mesure t the inite time points. () AUC nlysis of gluose levels in HFD-fe mie. p<.. (e) Plsm insulin of HFD-fe mie ws mesure t the inite time points. p<. (f h) ITT ws performe fter 1 weeks of CD or HFD feeing in n M- mie. (f) Gluose levels were mesure: re she line, + CD; re unroken line, + HFD; lk she line, + CD; lk unroken line, + HFD. p<., vs.(g, h) AUC of gluose levels in CD-fe mie (g) orhfd-femie(h). All t re presente s mens ± SEM; n=

6 398 Dietologi (1) 7:393 Gluose (mmol/l) Gluose (mmol/l) Fst Fst Refeh Refeh Refeh Insulin (pmol/l) Insulin (pmol/l) Oil Re O stining in the liver, refleting erese lipi umultion (Fig. 6), whih potentilly ontriutes to the reue liver to oy weight rtio (Fig. 6). Lipi nlysis showe tht the hepti TG levels were signifintly erese in M- mie (Fig. 6). The inhiition of hepti stetosis in M- mie ws ssoite with 1 1, 8 6 Fst Refeh Fst Refeh Refeh Fig. Plsm insulin level ws reue in M- mie fter refeeing. Plsm gluose (, ) n insulin (, ) levels were mesure fter mie h een fste overnight (Fst) n fter re-feeing for h (Refeh) or h (Refeh) in Rptor flox/flox ( )nm- ( ) mie fter 11 weeks of CD (, ) orhfd(, ). All t re presente s mens ± SEM; n=. White rs, ; lk rs,.p<., vs reue mrna expression of the mrophge mrker C68 n the inflmmtory ytokines Tnf n Cl (ut not Il1), in the liver (Fig. 6 g). When WAT ws exmine, we foun tht mrophge Rptor efiieny erese the F/8- positive stining (Fig. 6h), refleting the ontent of ipose tissue mrophges (ATMs) n the mrna expression of mrophge mrker C68 (% n % reution, respetively) (Fig. 6i, j), suggesting erese ATM umultion. ATMs re reporte to onsist of t lest two types, referre to s lssilly tivte M1 n lterntively tivte M mrophges [3]. The expressions of ll M1 mrkers mesure were ownregulte y mrophge Rptor efiieny. For exmple, the mrna level of inflmmtory ytokines Tnf, Cl n inos (lso known s Nos) ws reue y 63%, 7% n 7%, respetively (Fig. 6k, l, o). On the other hn, the M mrker Arg1 ws inrese y mrophge Rptor efiieny (Fig. 6n) n Il1 ws unhnge (Fig. 6m). The rtio of inos/arg1 ws reue y 8% (Fig. 6p), suggesting M polristion, s esrie previously []. Rptor eletion in mrophges inhiits inflmmtory gene expression vi the IRE1α/JNK/NFκB pthwy in the HFD moel Our fining tht mrophge Rptor efiieny resulte in erese inflmmtion in the liver n ipose tissue le us to hypothesise tht mtorc1 my ply role in ontrolling mrophge inflmmtion in response to HFD hllenge. To investigte the role of mtorc1 in mrophge Fig. Insulin sensitivity ws improve y mrophge Rptor efiieny. Rptor flox/flox ( )nm- ( )miefehfdfor 1 weeks were injete with PBS or insulin (.7 U/kg oy weight). At 1 min fter injetion, the levels of phospho-aktser73 (P-Akt) n totl-akt (T-Akt) in WAT (), liver () nmusle () were mesure y western lot n the rtio of P-Akt to T-Akt ws quntifie. All t re presente s mens ± SEM; n=. White rs, ; lk rs,.p<. for inite omprisons P-Akt T-Akt P-Akt T-Akt PBS PBS Insulin Insulin PBS PBS Insulin Insulin WAT P-Akt/T-Akt Insulin Liver P-Akt/T-Akt Insulin P-Akt T-Akt PBS Musle P-Akt/T-Akt Insulin PBS Insulin Insulin

7 Dietologi (1) 7: Fig. 6 Mrophge Rptor efiieny results in reue mrophge inflmmtory tivtion in ipose tissue n liver. () Oil re O (ORO) stining ws performe in liver (mgnifition ). ()The rtio of liver weight n oy weight (LW/BW). () Liver TG ontent ws mesure n normlise to liver weight. ( g) mrna expression of mrophge mrkers C68 n inflmmtory ftors Tnf, Cl n Il1 in liver of HFD-fe Rptor flox/flox ( )nm- ( ) mie. (h) Hemtoxylin n eosin (HE) n F/8 stining of ipose tissue (mgnifition 1). (i) ATMs s perentge of totl ells epite in (h). (j p) mrna levels of mrophge mrkers C68, n M1 n M mrkers in iposetissueofhfd-fe n M- mie were mesure y quntittive PCR. Dt re presente s mens ± SEM; n=. p<., vs C68 mrna h ORO HE F/8 ATM ontent (% of totl ells) e f g Tnf mrna j k l LW/BW (mg/g) Cl mrna i Liver TG ontent (mg/g tissue) Il1 mrna m C68 mrna 1.. Tnf mrna 1.. Cl mrna 1.. Il1 mrna 1.. n o p Arg1 mrna. 1.. inos mrna 1.. inos/arg1 mrna 1.. inflmmtion in HFD-fe mie, we isolte ConA-eliite peritonel mrophges from Rptor flox/flox n M- mie fe CD or HFD for 1 weeks, n nlyse signlling pthwys ownstrem of mtorc1 immeitely fter mrophge isoltion. HFD inrese mtorc1 tivity, ssesse y phosphoryltion of S6, in Rptor flox/flox, ut not in M- mrophges (Fig. 7). Our previous stuy showe tht ER stress ws tivte y long-term mtorc1 tivtion in liver [18]. We thus investigte the role of mrophge Rptor in ER stress. HFD tivte the ER stress rnhes PERK n ATF6 in oth Rptor flox/flox n M-Rptor flox/flox mrophges, reflete y inrese phosphoryltion of eukryoti trnsltion initition ftor α (eifα) n nuler ATF6, respetively (Fig. 7 ). Interestingly, the HFD-inue tivtion of IRE1α rnh, reflete y XBP1 spliing, ws muh more rmti in Rptor flox/flox thn in M- mrophges (Fig. 7), suggesting tht this ER stress rnh my e moulte y mtorc1 tivity. mtorc1 exerts negtive feek regultion on Akt tivtion [1, ]. To test whether Akt ws involve in mtorc1-triggere IRE1α tivtion, we mesure Akt tivity. Phospho-Akt levels were erese in mrophges from Rptor flox/flox mie on HFD ompre with those on CD, while this suppression of Akt ws resue y mrophge Rptor efiieny (Fig. 7e, f). In Rptor flox/flox

8 Dietologi (1) 7:393 mrophges, the suppression of Akt ws inversely orrelte with phosphoryltion of JNK n NF-κB (P6), n reovery of Akt phosphoryltion y Rptor efiieny ws ssoite with lunte JNK n P6 tivtion (Fig. 7g, h). Prlleling the erese level of phospho-jnk n phospho-p6, the mrna levels of inflmmtory genes in HFD-fe mie were reue y mrophge Rptor efiieny (Fig. 7i, j). Rptor ltion or mtorc1 inhiition y rpmyin ereses the inflmmtory response to plmiti i vi the IRE1α/JNK-NFκB pthwy Reent eviene points to the importne of SFAs, suh s plmiti i, whih re systemilly elevte in DIO n inue insulin resistne y tivtion of intrellulr inflmmtory signlling pthwys [6, 7]. We thus investigte whether Rptor efiieny ffete the inflmmtory response to plmiti i in mrophges. Sine rpmyin lso inhiits mtorc1, we investigte the effets of rpmyin on inflmmtory gene expression in mrophges onomitntly. BMDMs were isolte from M- n Rptor flox/flox mie n hllenge with plmiti i. Conomitntly, BMDMs from Rptor flox/flox mie were pre-trete with rpmyin followe y plmiti i hllenge. Plmiti i inue mtorc1 tivity, ssesse y phosphoryltion of S6 in Rptor flox/flox mrophges, n this response ws lunte y rpmyin (Fig. 8, ) n y mrophge Rptor efiieny Fig. 7 Mrophge Rptor efiieny suppresse the HFDinue IRE1α/JNK/NF-κB pthwy y inresing Akt phosphoryltion. ConA-eliite mrophges were isolte from Rptor flox/flox ( )n M- ( ) mie fe CD or HFD for 1 weeks. () Western lot of phosphorylte (P-) n totl (T-) S6 n eifα. () Rtioof inite phosphorylte to totl protein (P/T) in (), normlise to mie on CD. () Western lot of nuler (N-) ATF6 n lmin A/C. () mrna expression of splie n totl Xp1 ws mesure y quntittive PCR. The rtio of splie (s) n totl (t) Xp1 mrna ws lulte. (e h) Western lot ws performe to mesure P- n T- Akt (e), JNK n P6 (g). The rtio of phosphorylte to totl protein (P/T) for (e) n(g) ws normlise to on CD (f n h). (i, j) mrna expression of Tnf n Cl ws mesure y quntittive PCR. Dt re presente s mens ± SEM; n=. White rs, ; lk rs,.p<. for inite omprisons P-eIFα T-eIFα N-ATF6 Lmin A/C CD HFD CD HFD e P-Akt T-Akt g P-JNK P-JNK1 T-JNK T-JNK1 i P-S6 T-S6 P-P6 T-P6 Tnf mrna 1 CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD P/T-S6 protein s/t Xp1 mrna f P/T-Akt protein h P/T JNK protein j Cl mrna CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD CD HFD P/T-eIFα protein P/T-P6 protein 1 CD HFD CD HFD CD HFD CD HFD

9 Dietologi (1) 7:393 1 Fig. 8 Rpmyin suppresse the plmiti i-inue IRE1α/ JNK/NF-κB pthwy y inresing Akt phosphoryltion. BMDMs were isolte from Rptor flox/flox ( ) mie, pre-inute with or without rpmyin (R, 1 nmol/l) for 3 min n trete with BSA or BSA-onjugte plmiti i (PA,. mmol/l) for h. () Western lot of phosphorylte (P-) n totl (T-) S6 n eifα. () Rtioof inite phosphorylte n totl protein (P/T) in (), n normlise to BMDMs trete with BSA. () Western lot of nuler (N-) ATF6 n lmin A/C. () mrna expression of splie (s) n totl (t) Xp1. The rtio of splie n totl Xp1 ws lulte. (e h) WesternlotofP-ntotlT-Akt (e), JNK n P6 (g). The rtio of inite phosphorylte n totl protein (P/T) in (e) n (g) ws normlise to trete with BSA (f, h). (i, j) mrna expression of Tnf n Cl. Dt re presente s mens ± SEM; n=3. White rs, BSA; lk rs, plmiti i. p<. for the inite omprisons P-S6 T-S6 P-eIFα T-eIFα N-ATF6 Lmin A/C e g P-Akt T-Akt P-JNK P-JNK1 T-JNK T-JNK1 P-P6 T-P6 PA PA PA PA P/T-S6 protein s/t Xp1 mrna f P/T-Akt protein h P/T JNK protein P/T-eIFα protein P/T-P6 protein i Tnf mrna (fol vsctrl) j Cl mrna (ESM Fig., ). Plmiti i inue the tivtion of ll three ER stress pthwys, inluing PERK, ATF6 n IRE1α in Rptor flox/flox mrophges, reflete y inrese phosphoeifα, nuler ATF6 n XBP-1 spliing, respetively (Fig. 8 n ESM Fig. ). Consistent with the in vivo results, only the tivtion of the IRE1α pthwy ws erese y the mtorc1 inhiitor rpmyin (Fig. 8, ) n y Rptor efiieny (ESM Fig., ). Moreover, the repression of phospho-akt y plmiti i ws ttenute y rpmyin (Fig. 8e, f) n y Rptor efiieny (ESM Fig. e, f), inversely orrelte with the hnges in levels of phospho-jnk, phospho-p6, Tnf mrna n Cl mrna (Fig. 8g j n ESM Fig. g l). These oservtions thus onfirme our in vivo finings, initing tht Akt-meite IRE1α tivtion plys ruil role in mtorc1-triggere inflmmtion.

10 Dietologi (1) 7:393 Disussion In reent ees, lol inflmmtion ws foun to e importnt in the suppression of insulin signlling pthwys n insulin resistne [8]. The mtorc1 pthwy, tivte in oesity, is responsive to type ietes n insulin resistne prtly vi ER stress pthwys [18, 9]. It hs een shown tht inhiition of mtor y rpmyin promotes proution of proinflmmtory ytokines n loks the relese of IL-1 in humn monoytes n mouse mrophges in response to enotoxin, suggesting tht mtor is nti-inflmmtory [3 33]. However, it is unler whether mrophge mtorc1 is involve in the evelopment of insulin resistne. Surprisingly, in the present report, we foun tht mrophge mtorc1 hs proinflmmtory role in iet-inue insulin resistne. Our stuies proue two min finings: (1) mrophge Rptor efiieny improve systemi insulin sensitivity, lthough oy omposition ws unhnge; () mrophge Rptor efiieny suppresse the tivtion of JNK n NF-κB pthwys y resuing Akt n seletively repressing IRE1α in response to HFD n plmiti i (shemtilly shown in Fig. 9). A novel fining of this stuy is tht mrophge Rptor efiieny erese insulin resistne y mrkely suppressing the lol inflmmtory response to HFD. We showe tht the HFD-impire insulin sensitivity ws improve y mrophge Rptor efiieny. In ition, mrophge Rptor efiieny rmtilly loke the HFD-inue tivtion of mtorc1 tivity. Moreover, this protetion lso inlue signifint reution in hepti stetosis n ipose tissue mrophge umultion. Sine the polristion of mrophges in ipose tissue is importnt for oesity n insulin resistne [3], we stuie M1/M mrkers. Consistent with finings from stuies y Byles et l [], mrophge Rptor Akt XBP-1 spliing mtorc1 IRE1α JNK Inflmmtion SFA ER stress ATF6 NF-κB PERK eifα Fig. 9 The proinflmmtory role of mtorc1 in SFA-inue inflmmtion. mtorc1 is tivte y plmiti i, leing to suppression of Akt phosphoryltion. As Akt suppresses tivtion of IRE1α, mtorc1- meite inhiition of Akt uses seletive tivtion of the IRE1α/JNK/ NF-κB pthwy n inues inflmmtion efiieny erese the mrna levels of severl M1 inflmmtory genes, suh s Tnf n Cl, in WAT, n inrese the M polristion mrker Arg1. Although the preise role of mrophge polristion in meiting insulin resistne remins inompletely efine, ATMs were foun to e responsile for lmost ll ipose tissue TNF-α expression, n neutrlistion of TNF-α in oese rts use improve peripherl uptke of gluose [1, 8]. Our stuy suggeste tht mrophge Rptor efiieny erese insulin resistne y ttenuting mrophge inflmmtion. Our results seem to e in onflit with those from Weihrt et l, who reporte tht mtorc1 inhiition y rpmyin inrese the proution of proinflmmtory ytokines in monoytes n mrophges [3]. However, in ontrst to Rptor efiieny, long-term rpmyin tretment lso inhiits mtorc ssemly n Akt signlling, s hs een shown in severl ell lines [3], whih my explin the isrepny etween our stuy n tht of Weihrt et l. In ition, our stuy inite the existene of n mtorc1 Akt feek loop. Mrophge Rptor efiieny reverse the loss of Akt-meite IRE1α inhiition n, onsequently, JNK n NF-κB tivtion. The possile mehnism of the seletive inhiition of IRE1α y Akt might e through TRAF n Bl- fmily memers. As Hu et l n Kto et l hve shown, tivtion of Akt ownregultes expression of TRAF, whih is n ptor protein tht ouples IRE1α to JNK n NF-κB tivtion[3], wheres inhiition of Akt inrese TRAF [36]. In ition to TRAF, Bl- fmily memers, suh s Bx n Bl- homology omin 3 (BH3)-only proteins, whih were inhiite y Akt, iretly moifie IRE1α tivity [37, 38]. Moreover, the erese expression of ownstrem trget of IRE1α, splie Xp1, my lso ontriute to the nti-inflmmtory effet of mrophge Rptor efiieny, s it hs een shown to e require for the TLR signlling-relte proution of proinflmmtory ytokines suh s TNF-α n hemokine (C-C motif) lign [39, ]. In summry, our results emonstrte tht mrophge mtorc1 is importnt in regulting HFD-inue insulin resistne y moultion of the inflmmtory response. Tretment with the immunosuppressive rug rpmyin effetively ttenute inflmmtion [1]. However, long-term ministrtion of rpmyin inue insulin resistne, whih ws meite y mtorc loss [] n hyperlipiemi [3]. Different from long-term rpmyin tretment, our stuy showe tht geneti isruption of mtorc1 in mrophges ttenute the inflmmtory response n further improve insulin sensitivity, whih oul potentilly hve enefiil effet in insulin resistne. Aknowlegements The uthors woul like to thnk A. Tll, Columi University, New York, NY, USA, for his vie n help on experimentl esign n mnusript writing.

11 Dietologi (1) 7:393 3 Funing DA is supporte y the Ntionl Nturl Siene Fountion of Chin (Nos 8136 n ). YZ is supporte y the Mjor Ntionl Bsi Reserh Grnt of Chin (No. 1CB9). MW is supporte y The Netherlns Orgniztion of Sientifi Reserh (NWO VENI grnt ). Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. Contriution sttement HJ ontriute to the onept n esign, t quisition, nlysis n interprettion n rfting of the rtile. MW ontriute to the onept n esign, t quisition n revision of the rtile. CW n YZ ontriute to the t quisition n nlysis n revision of the rtile. DA ontriute to the onept n esign, t quisition, nlysis n interprettion of t n revision of the rtile. DA is the gurntor of the work. All uthors pprove the finl version. Referenes 1. Hotmisligil GS, Shrgill NS, Spiegelmn BM (1993) Aipose expression of tumor nerosis ftor-lph: iret role in oesity-linke insulin resistne. 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12 Dietologi (1) 7: Gri SJ, Hilemn DA, Frnkel SK et l () Phosphoryltion of Bx Ser18 y Akt regultes its tivity n poptosis in neutrophils. J Biol Chem 79: Zeng L, Liu YP, Sh H, Chen H, Qi L, Smith JA (1) XBP-1 ouples enoplsmi retiulum stress to ugmente IFN-et inution vi is-ting enhner in mrophges. J Immunol 18:3 33. Mrtinon F, Chen X, Lee AH, Glimher LH (1) TLR tivtion of the trnsription ftor XBP1 regultes innte immune responses in mrophges. Nt Immunol 11: Chen WQ, Zhong L, Zhng L et l (9) Orl rpmyin ttenutes inflmmtion n enhnes stility of therosleroti plques in rits inepenent of serum lipi levels. Br J Phrmol 6: Lmming DW, Ye L, Ktjisto P et l (1) Rpmyin-inue insulin resistne is meite y mtorc loss n unouple from longevity. Siene 33: Ai D, Chen C, Hn S et l (1) Regultion of hepti LDL reeptors y mtorc1 n PCSK9 in mie. J Clin Invest 1: 16 17