Chronic inflammation is a pathogenic factor in

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1 ORIGINAL ARTICLE Dynmi, M2-Like Remodeling Phenotypes of CD11 Adipose Tissue Mrophges During High-Ft Diet Indued Oesity in Mie Merv E. Shul, Gre Bennett, Ktherine J. Strissel, Andrew S. Greenerg, nd Mrtin S. Oin OBJECTIVE To identify, lolize, nd determine M1/M2 polriztion of epidydiml dipose tissue (eat) mrophges ( s) during high-ft diet ()-indued oesity. RESEARCH DESIGN AND METHODS Mle C7BL/6 mie were fed n (6% ft kl) or low-ft diet (LFD) (1% ft kl) for 8 or 12 weeks. eatm s (F4/8 ells) were hrterized y in vivo fluoresent leling, immunohistohemistry, fluoresene-tivted ell sorting, nd quntittive PCR. RESULTS Reruited interstitil mrophge gltose-type C- type letin (MGL)1 /CD11 nd rown-like struture ssoited /CD11 nd med /CD11 eatm s were identified fter 8 weeks of. med /CD11 ells omprised 6% of CD11 eatm s. CD11 eatm s expressed mixed M1/M2 profile, with some M1 trnsripts upregulted (IL-12p4 nd IL-1 ), others downregulted (inos, spse-1, MCP-1, nd CD86), nd multiple M2 nd mtrix remodeling trnsripts upregulted (rginse-1, IL-1R, MMP-12, ADAM8, VEGF, nd Cle-7). At week 12, eh eatm sutype displyed n enhned M2 phenotype s ompred with week 8. CD11 sutypes downregulted IL-1 nd genes mediting ntigen presenttion (I-, CD8) nd upregulted the M2 hllmrk Ym-1 nd genes promoting oxidtive metolism (PGC- 1 ) nd dipogenesis (MMP-2). med /CD11 eatm s upregulted dditionl M2 genes (IL-13, SPHK1, CD163, LYVE-1, nd PPAR- ). med /CD11 ATM s expressing elevted PGC-1, PPAR-, nd Ym-1 trnsripts were seletively enrihed in eat of oese mie fed pioglitzone for 6 dys, onfirming the M2 fetures of the med /CD11 eatm trnsriptionl profile nd impliting PPAR tivtion in its eliittion. CONCLUSIONS These results 1) redefine the phenotypi potentil of CD11 eatm s nd 2) suggest previously unppreited phenotypi nd funtionl ommonlity etween murine nd humn ATM s in the development of oesity nd its omplitions. Dietes 9: , 21 From the Oesity nd Metolism Lortory, Jen Myer U.S. Deprtment of Agriulture Humn Nutrition Reserh Center on Aging, Tufts University, Boston, Msshusetts. Corresponding uthors: Mrtin S. Oin, mrtin.oin@tufts.edu, or Andrew S. Greenerg, ndrew.greenerg@tufts.edu. Reeived 21 Septemer 29 nd epted 14 Ferury 21. Pulished hed of print t on 2 Ferury 21. DOI: /d y the Amerin Dietes Assoition. Reders my use this rtile s long s the work is properly ited, the use is edutionl nd not for profit, nd the work is not ltered. See -n-nd/3./ for detils. The osts of pulition of this rtile were defryed in prt y the pyment of pge hrges. This rtile must therefore e herey mrked dvertisement in ordne with 18 U.S.C. Setion 1734 solely to indite this ft. Chroni inflmmtion is pthogeni ftor in oesity omplitions, in prtiulr insulin resistne (1,2). A signifint dvne in our understnding of oesity-ssoited inflmmtion nd insulin resistne hs een reognition of the underlying role of dipose tissue mrophges (ATM s) (1 4). Tissue M s re phenotypilly heterogeneous nd re rodly hrterized ording to tivtion (polriztion) stte y the M1/M2 lssifition system (,6): Clssil M1 tivtion indued y interferon- nd lipopolyshride (LPS) defines proinflmmtory, miroiidl M s. M1 M s re the first line of defense ginst intrellulr pthogens, nd they prime towrd Th1 dptive immune response y produing interleukin (IL)-12 (7). In ontrst, M s expressing one of severl overlpping M2 polriztion sttes re tivted y IL-4/ IL-13 (M2 or lterntive tivtion), diverse stimuli (e.g., poptoti ells) in onert with LPS (M2), or IL-1, trnsforming growth ftor (TGF)-, or gluoortioids (M2). M2-polrized M funtion in tissue remodeling during development nd in response to injury, nd they promote the resolution of ute inflmmtion. Although M2 polrized M s re lso involved in ntiprsite defense nd Th2 priming, it hs een suggested tht the predominnt role of M2-polrized M s is the regultion of tissue homeostsis (8,9). Thus, M2-polrized M s typilly upregulte rginse (ARG)-1 nd downregulte induile nitri oxide sythse (inos) (therey promoting ollgen prodution t the expense of miroiidl NO); elevte the expression of inflmmtion-suppressive ftors (IL-1 nd IL-1R), mtrix metlloproteinses (MMPs), nd prongiogeni meditors; nd express n overll ttenuted proinflmmtory nd Th1-priming gene expression profile. Our urrent understnding of how ATM s promote oesity-ssoited inflmmtion nd insulin resistne is sed in lrge prt on the phenotype swith model of Lumeng et l. (1). In this model, oesity promotes the reruitment of M1-polrized ATM s tht shift the noninflmmtory milieu mintined y M2 polrized, resident ATM s towrd proinflmmtory stte. In epididyml AT (eat), these M1 ATM s (eatm s) re distinguished y 1) loliztion to rown-like strutures (CLS) nd ell lusters of tive remodeling round ded dipoytes (7,11,12), 2) elevted expression of the CC hemokine reeptor CCR2 nd the dendriti ell mrker CD11 in onjuntion with downregultion of the signture M2 letin mrophge gltose-type C-type letin (MGL)1 (11), nd 3) y n M1 gene expression profile feturing upregulted inos nd downregulted ARG-1 (1,11,13). The M1 polriztion of eatm s is in ertin respets ounterintuitive. Adipose tissue expnsion is proess of dietes.dietesjournls.org DIABETES, VOL. 9, MAY

2 REMODELING PHENOTYPES OF CD11 MACROPHAGES oordinted tissue remodeling nd repir involving removl of poptoti ells (dipoytes), extrellulr mtrix remodeling, ngiogenesis, nd new dipogenesis (12,14 16). In other tissues, these types of remodeling nd reprtive proesses typilly involve M2-polrized M s (,6). Moreover, these fetures of dipose tissue remodeling re mnifest in or ner the lusters to whih CCR2 nd/or CD11 eatm s seletively lolize, nd roles for lusterssoited eatm s in dipoyte lerne nd ngiogenesis hve een proposed (7,12,14). In humns, ft mss expnsion is ssoited with the umultion of ntiinflmmtory or mixed M1/M2-polrized ATM s (17). These ATM s exhiit remodeling phenotypes (18) hrterized y inresed MMP tivities nd elevted expression of lymphti vessel endothelil hyluronn reeptor (LYVE)-1, meditor of dipose tissue ngiogenesis (19). Considered together, these oservtions suggest tht one or more popultions of reruited eatm s re likely to express fetures of n M2-like remodeling phenotype during the development of murine oesity nd insulin resistne. Here we identify, lolize, nd trnsriptionlly hrterize eatm sutypes in mie fed high-ft diet () for 8 or 12 weeks, period of progressively inresing ody weight, eat remodeling, eatm reruitment, nd whole-ody insulin resistne (12). Our oservtions redefine the phenotypi potentil of CD11 eatm s nd suggest tht oesity nd insulin resistne develop in this model in ssoition with oinident M2 nd M1 phenotypi progression. RESEARCH DESIGN AND METHODS Animls nd diets. Six-week-old mle C7BL/6j mie were fed low-ft diet (LFD) (1% energy from ft) or n (6% energy from ft) (12) for 8, 1, or 12 weeks t The Jkson Lortory (Br Hror, ME). Following overnight shipment, mie were fed the sme diets in virl pthogen free fility for 2 3 dys efore use. Mie fed n for 1 weeks were mintined for n dditionl 6 dys t the Humn Nutrition Reserh Center on Aging on either n or n ontining (.1%, wt/wt) pioglitzone (PIO) ( PIO). Six-week-old stndrd diet fed mie were mintined for n dditionl 4 weeks on stndrd diet or following in vivo phgoyte leling with PKH26 (see elow). Mie were killed y CO 2 /ervil dislotion, nd eat ws proessed s desried elow. All proedures dhered to Humn Nutrition Reserh Center on Aging Institutionl Animl Cre nd Use Committee Guidelines. Intrperitonel insulin tolerne test (ITT). Whole-ody insulin resistne ws determined in mie s desried (12). Immunohistohemistry nd immunofluoresene. Prffin setions were proed with got nti-mouse (R&D Systems, Minnepolis, MN) or with IgG isotype ontrol followed y horserdish peroxidse onjugted seond ntiody (12). For immunofluoresene, eat ws fixed in zin-prformldehyde (Zn-PFA) (2), mined into mm piees, loked with horse serum, nd inuted with ntiody in PBS/horse serum (o/n, 4 C) followed y DyLight488-onjugted seondry ntiody nd Hoehst regent. Stroml vsulr ell isoltion. eat ws pled into Kres-Henseleit uffer (21) supplemented with 4% ftty id free BSA, mmol/l D-gluose, nd 2 nmol/l PIA; mined; entrifuged (g, min, room temperture); digested (3 4 min, 37 C) with endotoxin-free Lierse 3 (.3 units/ml, Rohe Applied Siene, Indinpolis, IN) ontining units of DNse I (Sigm-Aldrih); pssed through sterile 1- m striner (Fisher Sientifi, Frnklin, MA); nd entrifuged (g, min, room temperture). Following inution with erythroyte lysis uffer, stroml vsulr ells (SVCs) were resuspended (1 1 6 ells/1 l) in old fluoresene-tivted ell sorting (FACS) uffer (PBS ontining 1 mmol/l EDTA, 2 mmol/l HEPES, nd 1% [wt/vol] ftty id free BSA) until immunoleling. Flow ytometry nd SVC sorting. SVCs were inuted on ie (1 min) with FBlok ( g/ml) (BD Phrmingen, Sn Jose, CA), followed y inution with fluorophore-onjugted ntiodies or isotype ontrols. Antiodies inluded phyoerythrin-cy onjugted F4/8 (ebiosiene, Sn Diego, CA), phyoerythrin-onjugted CD11 (BD Phrmingen, Frnklin Lkes, NJ), got nti-mouse (R&D Systems), nd donkey nti-got DyLight488 (Jkson ImmunoReserh Lortories, West Grove, PA). Cells were nlyzed using FACSliur flow ytometer equipped with CellQuest softwre (Beton Dikinson, Sn Jose, CA) or sorted on MoFlo multilser system sorter (Bekmn Coulter, Bre, CA) using Summit softwre (Bekmn Coulter). Gting nd ompenstion strtegies re depited in Fig. 1 nd supplementl Fig. 1 (ville t Sorted ATM s were olleted in uffer ontining RNse inhiitor nd stored t 7 C. PKH26 leling of dipose tissue phgoytes. Six-week-old stndrd diet fed mie were injeted (intrperitonelly) with 2 l of. mol/l PKH26 (Sigm-Aldrih, St. Louis, MO). Four dys lter, mie were ssigned to either stndrd diet or ohorts for 4 weeks, fter whih ATM s ineat were nlyzed y FACS. Quntittive PCR. RNA ws extrted using RNesy Mini nd RNesy MinElute Clenup kits (Qigen) nd reverse trnsried nd mplified using the WT-Ovtion RNA Amplifition System (NuGEN Tehnologies, Sn Crlos, CA). Quntittive PCR ws onduted using SYBR Green (Applied Biosystems) (12). Fold differenes in gene expression were lulted s 2 Ct using ylophilin A s the housekeeping gene. Primer sequenes re in supplementl Tle 1. Sttistis. Dt re expressed s mens SE. Mens were ompred y t test or y ANOVA or GLM proedures in onjuntion with Tukey honestly signifint differene test (SAS version 9.1). Signifine ws set t P.. RESULTS CD11 ATM s with differentil expression levels re reruited to nd umulte in eat of mie fed n. As reported (12) feeding the for 8 weeks inresed ody nd eat weights nd indued whole-ody insulin resistne (Tle 1). Although totl SVCs inresed in response to, SVCs per grm eat did not (Tle 1). Flow ytometry (Fig. 1A) onfirmed tht the preponderne ( 9%) of eatm s (F4/8 ells) in mie fed LFD did not express CD11 nd tht the indued the umultion of CD11 eatm s (1). Overll, there ppered to e ontinuum of expression rther thn distint popultions of -expressing ells (Fig. 1A nd C). When gted for, CD11 eatm s unexpetedly exhiited rod distriution of stining intensity tht prtilly overlpped tht of / CD11 ells (Fig. 1A, ottom pnel). The overlpping nd nonoverlpping ells were defined s med /CD11 nd /CD11, respetively, sed on stining intensity (Fig. 1A nd 1B) nd trnsript levels (Fig. 1C nd supplementl Tle 2). When only CD11 ells were onsidered, ells designted s med /CD11 expressed three times the level of mrna thn ells designted /CD11 (P.1). Surprisingly, med /CD11 eatm s omprised the mjority of CD11 ells (Fig. 1D). In vivo pulse experiments with the phgoyte-leling dye PKH26 (Fig. 1E) suggest tht similr to /CD11 ells (Fig. 1E nd 11), med / CD11 ATM s re reruited to eat (i.e., re not derived from resident /CD11 ells present t the initition of ). PKH26 leling (Fig. 1E) lso reveled -indued reruitment of new (PKH26-negtive) /CD11 eatm s. CD11 eatm s re the predominnt omponent of CLS nd ell lusters surrounding ded dipoytes (11). Immunofluoresent stining of whole eat leled numerous -expressing ells within suh lusters (Fig. 2A, ottom left pnel), onsistent with the presene of med /CD11 eatm s (Fig. 1). As previously reported (11), some ell lusters were omposed predominntly of eatm s (Fig. 2A, ottom right pnel). Immunohistohemistry reveled -expressing ells within CLS (Fig. 2B, left pnel) nd in res of tive remodeling round ded dipoytes (Fig. 2B, right pnel). These results suggest tht med /CD11 eatm s re 1172 DIABETES, VOL. 9, MAY 21 dietes.dietesjournls.org

3 M.E. SHAUL AND ASSOCIATES A D ATMΦs (x1 4 )/g eat % of ATMΦs CD11 CD Counts 8wks LFD 8wks LFD LFD 26% CD11 F4/8 F4/8 1% 2% - CD11 + CD11 8wks 12 wks LFD 12wks 3% 13% Whole F4/8 popultion 93% med CD11 + 8wks 12wks LFD 12wks 29% % 12% (R1) (R2) 72% (R3) CD B - CD11 + (R1) med CD11 + (R2) + CD11 - (R3) C Reltive gene expression (ritrry units) E + CD11 - PKH26 + med CD PKH CD PKH26 leling (%) F4/8 merge F4/ CHOW CHOW CHOW + CD LFD + CD med CD11 - CD CD11 + CD11 med CD CD11 + FIG. 1. CD11 eatm s exhiiting differentil expression ( med /CD11 nd /CD11 ) umulte in the eat of mie fed n. SVCs were otined y ollgense digestion from eat of mie fed n or LFD for 8 weeks, leled with F4/8,, nd CD11 ntiodies nd nlyzed y flow ytometry. A: -ssoited inrese in F4/8 /CD11 eatm s reflets inreses in two ATM sutypes, designted med /CD11 (R2) nd /CD11 (R1) ording to their stining intensity. B: Confirmtion of protein expression in /CD11 nd med /CD11 eatm s y fluoresene mirosopy of sorted eatm s (s in A). C: Gene expression for F4/8,, nd CD11 in sorted eatm sutypes. D: Quntifition of eatm sutypes in response to 8 nd 12 weeks of demonstrting the solute nd proportionl inrese in CD11 nd CD11 sutypes during the time ourse. P <.; P <.1, ANOVA nd Tukey test. E: Evidene tht med /CD11 eatm s re reruited rther thn derived y phenotypi progression from resident /CD11 ells. Len mie were pulsed with the phgoyte-speifi dye PKH26 nd susequently fed 2 stndrd diet or n for 1 month followed y FACS of eatm s. Only ATM s present in eat efore lerne of the dye (24 h) express the lel. (A high-qulity digitl representtion of this figure is ville in the online issue.) dietes.dietesjournls.org DIABETES, VOL. 9, MAY

4 REMODELING PHENOTYPES OF CD11 MACROPHAGES TABLE 1 Body nd eat weights, SVC numers in eat, nd whole-ody insulin resistne (ITT) in mie fed either the LFD or for 8 (n 6) or 12 (n 7 11) weeks Body weight (g) eat weight (g) signifint omponent of some ut not ll ell lusters surrounding moriund dipoytes. eatm s express mixed M1/M2 trnsriptionl profiles fter 8 weeks of. As expeted, interstitil /CD11 eatm s in mie fed n LFD displyed elevted levels of nonil M2 trnsripts (IL-13, IL-1, Ym-1, sphingosine kinse 1 [SPHK1], signl trnsduer nd tivtor of trnsription [STAT]-6, CD26 [mnnose reeptor], nd CD163 [hemogloin svenger reeptor]) nd reltively redued levels of M1 trnsripts (IL-12p4 nd IL-1 ) (Tle 2; quntittive dt summrized in Tle 2 re presented in supplementl Fig. 2). In ontrst, / CD11 eatm s t week 8 expressed elevted levels of CCR2 nd severl hllmrk M1 trnsripts (inos nd IL-12p4), oinident with redued expression of some M2 trnsripts (IL-13, SPHK1, CD26, nd CD163) nd inreses in M2 genes with inflmmtion-suppressive funtions (IL-1, IL-1R, nd STAT-6) (Tle 2). These results indite tht the reruits new interstitil / CD11 eatm s (Fig. 1E) tht express enhned M1 nd ltered M2 trnsriptionl profiles. Notly, neither CD11 eatm sutype expressed n overll M1-polrized phenotype fter 8 weeks of. Most surprisingly, ARG-1 expression ws upregulted nd inos expression ws drmtilly ttenuted in CD11 eatms s ompred with interstitil /CD11 eatm s (Tle 2). CD11 eatm s lso upregulted genes promoting tissue remodeling, inluding MMP-12 (22), nd proteinse with disintegrin nd metlloproteinse domin (ADAM)-8, meditor of Th2-dependent irwy remodeling in sthm (23). CD11 eatm s lso expressed reltively high levels of vsulr endothelil growth ftor (VEGF), nd the /CD11 sutype expressed more TGF -1 mrna (Tle 2). These remodeling signtures were ssoited with n M2-like expression pttern of severl genes, inluding the upregultion of IL-1R interleukin-1 reeptor ntgonist nd the downregultion of the M1-ssoited genes MCP-1 monoyte hemotrtnt protein, spse-1, nd toll-like reeptor (TLR)-4. Both CD11 sutypes lso expressed reltively greter mounts of M1 trnsripts with proinflmmtory (IL-1 ) nd Th1-priming (IL-12p4) funtions, nd they downregulted severl inflmmtion-suppressive (M2) genes (IL-1, IL-13, nd STAT6) nd the prototypi M2 mrkers Ym-1 CD26 nd CD163 (Tle 2). These dt re onsistent with the reported M1 polriztion of nd CD11 eatm s (1,11,24). However, expression of tumor nerosis ftor- nd IL-6 were omprle in CD11 nd CD11 eatm s (dt not shown), perhps refleting the indution of these genes in CD11 ATM s y the ollgense proedure (2). In summry, CD11 eatm s in mie fed n for 8 weeks express mixed M1 (proinflmmtory) nd M2 (remodeling) trnsriptionl profiles. Totl numer of SVCs ( 1 6 ) Are under urve (ITT) 8 weeks LFD , weeks , weeks LFD weeks , Dt re mens SE. Mens identified y different symols re signifintly different (P., ANOVA nd Tukey proedure). Levels of M1 nd M2 trnsripts in med /CD11 eatm s were intermedite etween those mesured in /CD11 nd /CD11 eatm s, respetively (Tle 2 nd supplementl Fig. 2). When only gene expression dt for -fed mie were ompred, trnsript levels of 17 of 34 genes were signifintly different in med /CD11 eatm s s ompred with / CD11 eatm s (supplementl Tle 2). These oservtions support the viewpoint tht med /CD11 eatm s re distint sutype with n intermedite phenotype onsistent with the expression of oth nd CD11. Prolonged feeding promotes distintive ptterns of ltered gene expression in -expressing nd CD11-expressing eatm s. As reported (12), 12 weeks of indued weight gin, eat remodeling (mnifest s redued eat mss oinident with inresed SVCs), nd exerted whole-ody insulin resistne (Tle 1). Eh of the three eatm sutypes inresed in numer (per grm eat) (Fig. 1D, upper pnel), ut the proportion of eatm s tht were /CD11 tully deresed (Fig. 1D, ottom pnel). Of note, med / CD11 eatm s remined the predominnt CD11 eatm sutype (Fig. 1D). At week 12 eatm s exhiited glol hnges in the expression of genes involved in inflmmtion, ntigen presenttion, tissue remodeling, nd metolism onsistent with inresed M2 polriztion, espeilly in the two -expressing sutypes (supplementl Fig. 3). The diretion nd mgnitude of these hnges re summrized in Fig. 3. / CD11 nd med /CD11 eatm s upregulted hllmrk M2 genes (IL-13, Ym-1, SPHK1, nd TGF -1), downregulted inos, nd upregulted the dipogeni metlloproteinse MMP-2 (26) (Fig. 3). med /CD11 eatm s seletively upregulted LYVE-1 (19,27) while mintining reltively high levels of VEGF nd MMP-12 (Fig. 3). When ompred with enhmrk M2-polrized ( /CD11 ) eatm s from mie fed n LFD (Tle 3), -expressing eatm s in mie fed n for 12 weeks hd omprle or more pronouned M2-like expression profile of genes regulting inflmmtion, lipid metolism, tissue remodeling, nd ntigen presenttion. However, they ontinued to express M1-like levels of CD163, CD26, SPHK1 (redued), nd IL-12p4 (inresed) s ompred with M2-polrized eatm s from mie fed n LFD (Tle 3). Feeding the for 12 weeks roustly upregulted the trnsriptionl otivtor peroxisome prolifertor tivted reeptor (PPAR)- otivtor (PGC)-1 in oth CD11 eatm sutypes (Fig. 3), suggesting enhned oxidtive (i.e., M2-ssoited) metolism (28). At this time, mrna levels of oth PGC-1 nd PPAR- (ut neither PPAR- nor PPAR- ) were three- to fivefold greter 1174 DIABETES, VOL. 9, MAY 21 dietes.dietesjournls.org

5 M.E. SHAUL AND ASSOCIATES A Isotype ontrol Isotype ontrol + Hoehst A + Hoehst A + Hoehst B 4 mm mm FIG. 2. -expressing eatms re ssoited with CLS nd ontiguous remodeling res in eat. A: immunostining ws performed on eat whole mounts from mie fed n nd exmined y fluoresene mirosopy. Lower left pnel: -expressing ells (green) were lolized to lusters (rrow) nd interstitium (rrowheds). Right lower pnel: An exmple of n -negtive luster. Blue olor (Hoehst s regent) identifies nulei of ells. B: immunostining of prffin-emedded setions from mie fed n. ells re seen ssoited with ells in CLS (left pnel) nd in ssoited remodeling res (right pnel, rrowheds). (A high-qulity digitl representtion of this figure is ville in the online issue.) in med /CD11 eatm s thn in the other two eatm sutypes (P.) (supplementl Fig. 4). CD11 eatm s expressed reltively less IL-1 nd genes involved in ntigen presenttion (I-, CD8, nd CD86) (Fig. 3). Overll, these results demonstrte M2-like hnges in lipid/oxidtive metolism nd inflmmtory gene expression in CD11 eatm s etween weeks 8 nd 12 of. Importntly, when ompred with enhmrk M2-polrized eatm s (Tle 3), CD11 eatm sofmie fed n for 12 weeks expressed omprle or greter levels of multiple M2-ssoited trnsripts (STAT6, IL1- R, C type letin [Cle]-7, Arg-1, PGC-1, TGF, MMP-2, MMP-12, VEGF, nd ADAM-8) nd equivlent or redued levels of severl M1-ssoited trnsripts (inos, CD8, nd CD86). These oservtions undersore the M2/remodeling fetures of the CD11 eatm trnsriptionl profile during -indued oesity. med /CD11 ATM s preferentilly umulte in eat of oese mie fed PIO. As oth PGC-1 nd PPAR- re trgets of PPAR-, it ws plusile tht the M2-like gene expression hnges oserved in med / CD11 eatm s etween weeks 8 nd 12 of (Fig. 3) refleted inresed PPAR- tivtion (29). Aordingly, we phenotyped eatm s from mie mintined on for 1 weeks nd then fed either or ontining the PPAR- /- gonist PIO ( PIO) for n dditionl 6 dys. PIO tretment signifintly meliorted hyperinsulinemi nd upregulted unoupling protein-1 in eat (Fig. 4), onfirming PPAR- tivtion. Coinidentlly, mrna levels of oth F4/8 nd were inresed in mie fed dietes.dietesjournls.org DIABETES, VOL. 9, MAY

6 REMODELING PHENOTYPES OF CD11 MACROPHAGES TABLE 2 Mixed M1/M2 remodeling trnsriptionl profiles in CD11 eatm s t week 8 Gene LFD / / med / / CD11 CD11 CD11 CD11 M2 ARG IL-1R TGF PGC ADAM MMP VEGF Cle7 1 1 IL IL Ym STAT-6 1 SPHK CD CD MMP M1 inos IL IL12p I CCR MCP Cspse TLR4 2 2 CD CD8 STAT-1 2 nd 1 rrows indite redued or inresed gene expression, respetively, s ompred with /CD11 eatm s from LFDfed mie (n 6) (see online ppendix Fig. 2 for quntittive dt summrized in this tle). Trnsript levels designted y different symols re signifintly different (P., ANOVA nd Tukey proedure). PIO (Fig. 4), onsistent with the reruitment of M2-polrized eatm s. Unexpetedly, CD11 mrna levels lso inresed (Fig. 4). Flow ytometry reveled seletive inrese in med /CD11 eatm s in mie fed PIO, resulting in signifint inrese in the proportion of CD11 ells expressing (Fig. 4). These results suggest tht med /CD11 ATM s re preferentilly reruited nd/or umulte in eat of -fed mie in response to short-term systemi PPAR- gonism. Importntly, med /CD11 eatm s from mie fed PIO displyed elevted levels of Ym-1, PGC-1, nd PPAR- (ut not PPAR- ) mrnas (Fig. 4). This pttern of M2-ssoited gene expressions mirrors in prt tht oserved in med /CD11 eatm s from mie fed for 12 weeks (Fig. 3 nd supplementl Fig. 4). Mixed M1/M2 remodeling trnsriptionl profile in whole eat during -indued oesity. Finlly, we determined mixed M1/M2 nd remodeling trnsriptionl profile t the tissue level during -indued oesity. Quntittive PCR of seleted eat trnsripts indited tht the expression of M2/remodeling genes, IL-1, IL-13, TGF -1, ADAM-8, nd MMP-2 inresed progressively in eat during the ourse of (Fig. ). MMP12 mrna (indued 3-fold in CD11 eatm s) (Tle 3) ws upregulted 1- nd 1-fold in eat t weeks 8 nd 12, respetively (dt not shown). As expeted, trnsript levels of M1 ytokines (IL-12p4, IL-1, nd tumor nerosis ftor- ) lso inresed during the time ourse, ut inos mrna levels deresed t week 12 (Fig. ), onsistent with downregultion in eatm s (Tle 3 nd supplementl Fig. 3). Overll, these dt demonstrte progressive, oordinte inreses in the expression of oth M2/remodeling nd M1-ssoited genes in eat during the ourse of -indued oesity nd insulin resistne (Tle 1). DISCUSSION Employing n estlished model of -indued oesity (12), we demonstrte tht eatm s reruited in response to express mixed M1/M2 nd remodeling trnsriptionl profiles nd tht these profiles eome more M2-like with extended feeding. Humn ATM s hve reently een shown to express mixed M1/M2 remodeling phenotypes (17,18), thus distinguishing them from the M1- polrized eatm s reported for oese mie (11,24). By demonstrting the pleiotropi trnsriptionl profiles of eatm s in murine oesity, the present study suggests previously unppreited phenotypi nd funtionl ommonlity etween murine nd humn ATM s in the development of oesity nd its omplitions. We identified three sutypes of reruited eatm s in mie fed n, inluding the /CD11 nd /CD11 eatm s tht were previously reported to e M2 nd M1 polrized, respetively (11). In ontrst to reent report (3), our sorting strtegy did not identify CD11 nd CD11 eatm s s F4/8 lo nd F4/8 hi, respetively (Fig. 1A nd C). As expeted, interstitil /CD11 eatm s were M2 polrized in mie fed n LFD (Tle 2). indued the reruitment of / CD11 eatm s expressing n ltered M2 trnsriptionl profile nd elevted levels of severl M1 trnsripts (Tle 2). These reruitment dt, otined in young, len PKH26-injeted mie susequently fed n differ from results of prior study (11) tht reported little or no reruitment of interstitil ( ) eatm s in older, oese -fed mie injeted with PKH26. did not eliit lssil M1 polriztion in / CD11 eatm s ut rther mixed M1/M2-like pttern of gene expression hrterized y enhned expression of IL-12p4 nd IL-1 (M1) oinident with downregulted expression of inos nd spse-1 nd upregulted IL-1R (M2). Moreover, /CD11 eatm s upregulted trnsripts involved in mtrix remodeling nd ngiogenesis (ARG-1, ADAM-8, MMP-12, VEGF, nd TGF -1). During the preprtion of this mnusript, Fujisk et l. (31) reported high levels of ARG-1 gene expression in CD11 eatm s in mie fed n for 17 weeks. Our results extend this oservtion nd suggest tht /CD11 eatm s in the present study express mixed M1/M2 remodeling phenotype. Surprisingly, luster-ssoited eatm s expressing oth nd CD11 (i.e., med /CD11 ) onstituted the mjority (6 7%) of CD11 eatm s (nd % of CD11 eatm s fter 2 weeks of [not shown]). While this mnusript ws in review, Westott et l. (32) reported the unntiipted oservtion of sustntilly redued numers of CD11 eatm s in / mie fed n, therey supporting our oservtion of the 1176 DIABETES, VOL. 9, MAY 21 dietes.dietesjournls.org

7 M.E. SHAUL AND ASSOCIATES 1 8 Pro/nti-inflmmtory mrkers Arg/lipid metolism Remodeling Antigen presenttion Fold Chnge in gene expressions CD11 - med CD CD IL-13 Ym-1 STAT 6 SPHK1 IL-1 IL1R CD163 CD26 IL1 IL-12p4 TLR4 ARG inos PGC1 PGC1 ADRP LYVE-1 MMP2 TGF1 MMP12 MCP-1 I- CD8 CD86 FIG. 3. Prolonged (12-week) feeding promotes M2-ssoited gene expression differentilly in -expressing nd CD11-expressing eatm s. mrna levels of inflmmtion-, metolism-, remodeling-, nd ntigen presenttion relted genes were nlyzed y quntittive PCR fter 12 weeks of in eh eatm sutype (supplementl Fig. 3). mrna levels were ompred with levels determined fter 8 weeks of (set s 1 nd indited y the horizontl line). Dt for eh eatm sutype re from 6 mie (week 8) nd 9 11 mie (week 12), respetively. P <.; P <.1, ANOVA nd Tukey proedure. preponderne of -expressing CD11 eatm s in the present study (Fig. 1). Osionl eatm s expressing oth nd CD11 were previously identified next to lusters (11), ut the phenotype of these eatm s ws undetermined. med /CD11 eatm s express mixed M1/M2 phenotype with trnsript levels intermedite etween /CD11 nd /CD11 eatm s. This intermedite phenotype rised the possiility tht med /CD11 eatm s were derived from interstitil /CD11 ells. Lipid svenging, key funtion of eatm s in oesity (7), promotes CD11 expression in M s (33) nd ould oneivly promote the phenotypi progression of CD11 eatm s to CD11 phenotype. However, the lmost totl sene of PKH26 dye mong med /CD11 eatm s rgues ginst their phenotypi progression from resident /CD11 eatm s (Fig. 1E). Irrespetive of origins, the phenotype of med /CD11 eatm s is likely to reflet exposure to two sets of dipose tissue miroenvironmentl ues (e.g., ytokines, lipid, nd hypoxi) tht individully eliit the disrete phenotypes of / CD11 nd /CD11 eatm s, respetively. dietes.dietesjournls.org DIABETES, VOL. 9, MAY

8 REMODELING PHENOTYPES OF CD11 MACROPHAGES TABLE 3 Fold differene in mrna levels of genes regulting inflmmtion, metolism, tissue remodeling, nd ntigen presenttion in eatm sutypes fter 12 weeks of reltive to enhmrk M2-polrized /CD11 eatm s from mie fed n LFD for 8 weeks 8 weeks LFD 12 weeks CD11 CD11 med CD11 CD11 Pro-/nti- Inflmmtory mrkers Gene IL Ym STAT SPHK IL IL-1R CD CD Cle IL Cspse IL-12p TLR MCP Metolism Genes ARG inos PGC PGC ADRP Remodeling Genes MMP MMP TGF VEGF ADAM LYVE Antigen presenttion Genes I CD CD Dt re mens SE. Mens designted y different symols re signifintly different (P., ANOVA nd Tukey proedure). Continued (12-week) promoted M2-like trnsriptionl modultion in eh of the three eatm sutypes (Fig. 3). -expressing eatm s upregulted hllmrk M2 genes nd downregulted inos (Fig. 3). The med / CD11 sutype dditionlly upregulted MMP-2, CD163, nd LYVE-1 (Fig. 3) nd ws three times more likely thn other eatm sutypes to stin for the IB4 isoletin (dt not shown), mrker of dipogeni nd ngiogeni tivities in eat of oese mie (14). The inreses in LYVE-1 nd MMP-2 mrnas re intriguing in light of proposed role of LYVE-1 eatm s in new vessel development during eat expnsion (19), nd the demonstrtion tht MMP-2 is required for diet-indued dipoyte hypertrophy (26). These oservtions re onsistent with the loliztion of med /CD11 eatm s to remodeling lusters (Fig. 2) nd suggest funtionl role in ngiogenesis nd/or dipogenesis. An unexpeted feture of CD11 eatm gene expression t week 12 ws the roust upregultion of PGC-1 (Fig. 3) (rev. in 34). Although little is known onerning the role of PGC-1 in M polriztion (3), its role in promoting mitohondril iogenesis nd oxidtive metolism suggests tht, similr to PGC-1 (28), PGC- 1 medited gene expression promotes/mintins the M2 stte. med /CD11 eatm s dditionlly expressed high levels of PPAR- mrna, suggesting inresed ftty id metolism nd lunted proinflmmtory responses to Th1 ytokines (36). At week 12, oth CD11 eatm sutypes expressed redued levels of M1-ssoited trnsripts (inos, IL-1, I-, nd CD8) s well s ARG-1 mrna. These redutions suggest reltively detivted M2 phenotype, onsistent with the elevted IL-1 gene expression oserved in eat t this time (Fig. ) (12). Similr downregultion of M1 ytokines, inos nd ARG-1 is oserved in M2-polrized M s during the reprtive phse of murine musulr dystrophy (37). Systemi PPAR- tivtion y thizolidinediones (TZDs) promotes ATM reruitment, M2-ssoited gene expression, nd tissue remodeling in eat of oese rodents (29,3,38,39). Surprisingly, ute (1-week) exposure to TZDs lso inreses CD11 mrna in eat (38) (Fig. 4). The seemingly nomlous oservtion of inresed CD11 gene expression in response to M2-polrizing tretment is explined y our demonstrtion tht med /CD DIABETES, VOL. 9, MAY 21 dietes.dietesjournls.org

9 M.E. SHAUL AND ASSOCIATES A B C % ells (of F4/8+ ells) Fsting gluose (mmol/l) Body weight (gr) Reltive gene expressions (ritrry units) Ym PPARα eat weight (gr) Fsting insulin (µu/ml) PIO +PIO +PIO + CD11 - med CD CD PPARγ PGC1 +PIO eat gene expression med CD11 + /-CD PIO +PIO +PIO F4/8 CD11 UCP-1 +PIO + CD11 - med CD CD11 + FIG. 4. Pioglitzone tretment preferentilly enhnes umultion of med /CD11 eatm s nd promotes their expression of M2- ssoited genes. Mie fed n for 1 weeks were fed n or ontining PIO (.1% [wt/wt]) ( PIO) for 6 dditionl dys. A: Body nd eat weights (top pnels), fsting gluose nd insulin levels (ottom pnels), nd gene expression reltive to mie fed n lone (right pnel), n 4. B: Flow-ytometri nlysis of eat inditing seletive inrese in med /CD11 eatm s (left pnel) nd enrihment in the proportion of CD11 ells expressing (right pnel), n 6. C: Gene expression inditing PIO-ssoited inrese in Ym-1, PGC-1, nd PPAR- trnsripts in the med /CD11 sutype, n 6. Similr ptterns of gene expression were oserved t week 12 (Fig. 3 nd supplementl Fig. 4). P <., t test, or ANOVA with Tukey proedure. dietes.dietesjournls.org DIABETES, VOL. 9, MAY

10 REMODELING PHENOTYPES OF CD11 MACROPHAGES Gene expression (ritrry units) 8wks LFD 8wks 4 12wks F4/8 CD11 IL-1 IL-13 TGF1, inos IL-12p4, IL-1 TNF MMP2 ADAM-8 FIG.. Coordinte expression of M1 nd M2/remodeling genes in whole eat during the time ourse. Quntittive PCR ws performed on eat from mie fed the LFD (set s 1 ) or the for either 8 or 12 weeks (n 6 8 mie per group). Mens designted y different letters re signifintly different (P <., ANOVA nd Tukey proedure). eatm s seletively umulte in eat of oese mie fed the TZD PIO, PPAR- /- gonist (4). med /CD11 eatm s in mie fed PIO upregulted the M2- ssoited trnsripts Ym-1, PGC-1, nd PPAR- (Fig. 4) similr to med /CD11 eatm s in mie fed n for 12 weeks (Fig. 3 nd supplementl Fig. 4). These oservtions support the notion tht med /CD11 eatm s eome more M2 polrized t week 12 nd suggest tht PPAR- (nd/or PPAR- ) tivtion y endogenous lignds ontriutes to this polriztion. TZDs promote insulin sensitivity, in prt, y promoting ft oxidtion nd the remodeling of dipose tissue with dditionl, smll dipoytes (29,39,41,42). Our dt suggest tht these tions my e promoted in eat with miniml inflmmtory impt y the seletive reruitment of med /CD11 eatm s expressing high levels of IL-13, PGC-1, MMP-2, nd LYVE-1 nd reltively redued levels of IL-1 nd IL-12p4 s ompred with / CD11 eatm s (Tle 3). However, the whole-ody insulin-sensitizing effets of PIO lso reflet its enefiil tions in multiple ells nd tissues, inluding suutneous dipose tissue (39,41,42). Thus, despite inreses in med /CD11 t week 12, inresed insulin resistne (Tle 1) is not totlly unexpeted t this time given the oinident inrese in /CD11 eatm s (Fig. 1D) in the sene of the pleiotropi meliortive tions of PIO. In losing, we note tht the mixed M1/M2 eatm phenotypes desried ove were ssoited with progressively inresing nd oordinte expression of M1 nd M2/remodeling genes in whole eat (Fig. ). Multiple eat ell types, in ddition to eatm s, undoutedly ontriute to this mixed inflmmtory profile. Nevertheless, our dt demonstrte tht -indued whole-ody insulin resistne (Tle 1) develops in this model oinident with oth n M2 s well s n M1 progression in eat. This onlusion my reflet our use of 6% (kl), ontining 33% more energy from ft thn the diet used y Lumeng nd ollegues (1,11) to eluidte the M1 eatm swith. Although qulittively identil, the higher ft ontent my promote more eat remodeling nd my oinidentlly ttenute M proinflmmtory signling (43). In prtiulr, lipid svenging y CD11 eatm s t sites of dipoyte deth (7) my render them prtiulrly prone to the M1-inhiiting nd/or M2-promoting effets of prtiulr ftty ids (43,44). Future studies will ddress mehnisms y whih dietry ft nd/or dipose tissue miroenvironments shpe ATM polriztion nd its inflmmtory nd metoli sequele. ACKNOWLEDGMENTS This work ws supported y Ntionl Institute of Helth Grnts DK74979 (to M.S.O.) nd TH32HL69772 (to G.B.), Amerin Dietes Assoition Grnts 1-6-RA-96 (to M.S.O.) nd 7-8-RA-7 (to A.S.G.), Tked Phrmeutils (to M.S.O.), nd U.S. Deprtment of Agriulture Contrt no (to A.S.G.). No potentil onflits of interest relevnt to this rtile were reported. Prts of this rtile were presented t the Keystone Symposi, The Mrophge: Intersetion of Pthogeni nd Protetive Inflmmtion, Bnff, Alert, Cnd, Ferury 21. We thnk Allen Prmelee nd Stephen Kwok from the Tufts University flow ytometry ore fility for ssistne. REFERENCES 1. Gutierrez DA, Puglisi MJ, Hsty AH. Impt of inresed dipose tissue mss on inflmmtion, insulin resistne, nd dyslipidemi. Curr Di Rep 29;9: Shoelson SE, Herrero L, Nz A. Oesity, inflmmtion, nd insulin resistne. Gstroenterology 27;132: Weiserg SP, MCnn D, Desi M, Rosenum M, Leiel RL, Ferrnte AW Jr. Oesity is ssoited with mrophge umultion in dipose tissue. J Clin Invest 23;112: Odegrd JI, Chwl A. Mehnisms of mrophge tivtion in oesityindued insulin resistne. Nt Clin Prt Endorinol Met 28;4: Mrtinez FO, Helming L, Gordon S. Alterntive tivtion of mrophges: n immunologi funtionl perspetive. Annu Rev Immunol 29;27: DIABETES, VOL. 9, MAY 21 dietes.dietesjournls.org

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