CTRP3 attenuates cardiac dysfunction, inflammation, oxidative stress and cell death in diabetic cardiomyopathy in rats

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1 Dietologi (7) 6:6 7 DOI.7/s57 ARTICLE CTRP ttenutes ri ysfuntion, inflmmtion, oxitive stress n ell eth in ieti riomyopthy in rts ZhenGuo M,, & YuPei Yun,, & SiChi Xu,, & WenYing Wei,, & ChunRu Xu,, & Xin Zhng,, & QingQing Wu,, & HiHn Lio,, & Jin Ni,, & QiZhu Tng,,, Reeive: Deemer 6 /Aepte: Ferury 7 /Pulishe online: Mrh 7 # SpringerVerlg Berlin Heielerg 7 Astrt Aims/hypothesis Oxitive stress, inflmmtion n ell eth re losely involve in the evelopment of ieti riomyopthy (). Cq/tumour nerosis ftorrelte protein (CTRP) hs ntiinflmmtory properties ut its role in remins lrgely unknown. The ims of this stuy were to etermine whether CTRP oul ttenute n to lrify the unerlying mehnisms. Methos Streptozotoin (STZ) ws injete intrperitonelly to inue ietes in Sprgue Dwley rts. Criomyoytespeifi CTRP overexpression ws hieve using n enossoite virus system weeks fter STZ injetion. Results CTRP expression ws signifintly erese in ieti rt herts. Knokown of CTRP in riomyoytes t seline resulte in inrese oxitive injury, inflmmtion n poptosis in vitro. Criomyoytespeifi overexpression of CTRP erese oxitive stress n inflmmtion, ttenute myoyte eth n improve ri funtion in rts trete with STZ. CTRP signifintly tivte AMPtivte protein kinse α (AMPKα) n Akt (protein kinse B) in H9 ells. CTRP protete ginst highgluoseinue oxitive stress, inflmmtion n poptosis in vitro. AMPKα efiieny olishe the protetive effets of CTRP in vitro n in vivo. Furthermore, we foun tht CTRP tivte AMPKα vi the AMP exhnge protein iretly tivte y AMP (EPAC) mitogentivte protein kinse kinse (MEK) pthwy. lusions/interprettion CTRP protete ginst vi tivtion of the AMPKα pthwy. CTRP hs therpeuti potentil for the tretment of. Keywors AMPtivte protein kinse α. Apoptosis. Cq/ tumour nerosis ftorrelte protein. CTRP. Dieti riomyopthy. Inflmmtion. Oxitive stress Eletroni supplementry mteril The online version of this rtile (oi:.7/s57) ontins peerreviewe ut uneite supplementry mteril, whih is ville to uthorise users. QiZhu Tng qztng@whu.eu.n Deprtment of Criology, Renmin Hospitl of Wuhn University, Wuhn University, Jiefng Ro 8, Wuhn 6, People s Repuli of Chin Criovsulr Reserh Institute of Wuhn University, Wuhn, People s Repuli of Chin Huei Key Lortory of Criology, Wuhn, People s Repuli of Chin Deprtment of Criology, Renmin Hospitl of Wuhn University, Criovsulr Reserh Institute, Huei Key Lortory of Criology, Wuhn University, Jiefng Ro 8, Wuhn 6, People s Repuli of Chin Arevitions HNE Hyroxynenl AAV Aenossoite virus AMPKα AMPtivte protein kinse α BAX BCLssoite X protein BCL Bell lymphom CMKK Clium/lmoulinepenent protein kinse kinse CTRP Cq/tumour nerosis ftorrelte protein Dieti riomyopthy EPAC Exhnge protein iretly tivte y AMP FS Frtionl shortening GFP Green fluoresent protein High gluose LKB Liver kinse B LPS Lipopolyshrie

2 Dietologi (7) 6:6 7 7 LVID MEK MOI PKA PKC PPARα ROS SOD STZ Left ventriulr internl istoli imeter Mitogentivte protein kinse kinse Multipliity of infetion Protein kinse A Protein kinse C Peroxisome prolifertortivte reeptorα Retiveoxygenspeies Smll hirpin RNA Superoxie ismutse Streptozotoin Therefore, the ims of this stuy were to etermine whether CTRP oul protet ginst streptozotoin (STZ)inue ri injury n to eluite the potentil mehnisms. To the est of our knowlege, this is the first report of protetive role for CTRP in. Methos Mterils See eletroni supplementry mteril (ESM) Methos for full etils. Introution Dieti riomyopthy () is efine s ventriulr ysfuntion in ieti iniviuls tht nnot e srie to oronry rtery isese n hypertension []. is hrterise y the loss of myoytes, myofiril epletion n umultion of ollgen [] n hs een ientifie s mjor preisposing ftor for hert filure in ieti ptients [, ]. The Frminghm stuy reporte tht the hert filure rte in men n women with ietes is, respetively, twie n five times tht in people without ietes [5]. The prevlene of istoli ysfuntion is s high s 6% in ieti iniviuls without oronry rtery isese [, 6]. Moreover, the pthologil hnges of riomyopthy in ptients with ietes oul not e effetively reverse fter intensive loo gluose ontrol [7]. Therefore, moleules tht seletively inhiit the progression of woul e of gret therpeuti interest. Dieti ri ysfuntion is preisely oorinte y n orhestrte network omprise of numerous pthogeni ftors. Emerging t hve ientifie key role for inrese oxitive stress, inflmmtion n susequent ell eth [8 ]. Hyperglyemi n inflmmtion ll result in the overproution of retive oxygen speies (ROS) [], whih inue lipi peroxition n erese ntioxint pity, eventully leing to the loss of myoytes n ri ysfuntion []. Aitionlly, ROS n myoyte eth promote the trnsition from firolst to myofirolst n interstitil firosis []. Therefore, eveloping n effetive strtegy to suppress the overproution of ROS, inflmmtion n ell eth in the hert is urgently neee for ieti ptients. Cq/tumour nerosis ftorrelte proteins (CTRPs), ientifie se on sequene homology with the gloulr omin of iponetin, re reognise s novel serete metoli regultors []. CTRP, expresse preominntly y ipose tissue, is lso foun in the hert n liver [5]. CTRP regultes hepti gluose output [6], ttenutes hepti stetosis [7] n inhiits inflmmtory responses [8, 9]. Although it hs een shown to suppress poptosis n reue interstitil firosis in ishemi mouse herts [5, ], CTRP hs not een further investigte for itionl iologil tivity in the hert, espeilly in riomyoytes. Animls n niml moel All niml experimentl proeures followe Ntionl Institutes of Helth guielines n the guielines of our hospitl. Mle Sprgue Dwley rts (8 9 weeks ol, 8 g) were otine from the Institute of Lortory Animl Siene, Chinese Aemy of Meil Sienes (Beijing, Chin). The rts were llowe free ess to foo n wter n were house t ontrolle temperture ( 5 C) n humiity (5 ± 5%) on h light rk yle. After eing fste for h, the rts were given single intrperitonel injetion of STZ (65 mg/kg). Rts with fsting loo gluose >.9 mmol/l in three inepenent mesurements were efine s hving ietes n were use for further stuies. After weeks of ietes, the rts were given single tilvein injetion of enossoite virus (AAV) Ctrp (lso known s Cqtnf) or AAV green fluoresent protein (Gfp) t ose of 5 prtiles per rt. The rts were lso infuse suutneously with the reominnt humn gloulr omin of CTRP (. μg g y )vin Alzet osmoti minipump (Duret, Cupertino, CA, USA) for weeks eginning weeks post STZ injetion. Dieti rts were groupe y rnom numer tle. The tretment n susequent nlyses were performe in lin fshion for ll groups. See ESM Methos for further etils. Ehoriogrphi n hemoynmi mesurements Ehoriogrphy ws performe s previously esrie [ ]. Hemoynmi vriles were nlyse using mirotip trnsuer theter (SPR89; Millr Instruments, Houston, TX, USA) n Millr Pressure Volume System (MPVS; Millr Instruments). Histologil nlysis, immunofluoresene, immunohistohemistry n TUNEL stining The hemtoxylin eosin n Pirosirius Re stining were performe using stnr proeures [ ]. To etet the protein level of CTRP in herts, setions were inute with rit ntirt CTRP ntioy (:) n were oserve uner n Olympus DX5 fluoresene mirosope (Olympus, Tokyo, Jpn). For immunohistohemil nlysis, setions were inute with ntihyroxynonenl (HNE, :) or ntitnfα (:). TUNEL stining ws performe using ommeril

3 8 Dietologi (7) 6:6 7 kit (Millipore, Billeri, MA, USA), following the mnufturer s instrutions.see ESM Methos for etils. Aenovirl vetor onstrution The soure of enovirl (A)α enoviruses ws esrie in our previous rtiles [, ]. Knokown of CTRP ws rrie out using enovirl vetors rrying Ctrp smll hirpin RNAs (s), whih were generte y Hnio (Shnghi, Chin). Srmle ws use s the ontrol. Cell ulture n tretment Neontl rt riomyoytes were isolte s previously esrie []. To etet expression of CTRP, the myoytes were stine with ntiαtinin (:) n ntictrp (:). H9 ells were purhse from the Cell Bnk of the Chinese Aemy of Sienes (Shnghi, Chin), where myoplsm etetion, DNAfingerprinting n isozyme nlyses were performe. Myoplsm ontmintion ws not etete in H9 ells. To investigte the protetion of CTRP, H9 ells were pretrete with reominnt humn gloulr omin of CTRP ( μg/ml) for min followe y norml gluose (5.5 mmol/l) or high gluose (, mmol/l) tretment. To knok own AMPtivte protein kinse α (AMPKα), H9 ells were infete with Aα (multipliity of infetion [MOI] = 5) or A for h [, ]. To knok own CTRP, H9 ells were infete with AshCtrp (MOI = 5) for h. For inhiition of Akt, H9 ells were inute with Akt/ kinse inhiitor ( μmol/l). To explore the mehnism y whih CTRP tivte AMPKα, H9 ells were inute with H89 ( protein kinse A [PKA] inhiitor, μmol/l), 5 Ao (n enylte ylse inhiitor, μmol/l), U6 ( mitogentivte protein kinse [MEK] inhiitor, μmol/l) or STO69 ( lium/ lmoulinepenent protein kinse kinse [CMKK] inhiitor, 8 nmol/l). To knok own exhnge protein iretly tivte y AMP (EPAC), smll interfering RNA (siep) ws use. To inue ell eth, H9 ells were trete with TNFα (5 ng/ml for h) or H O ( μmol for h). See ESM Methos for further etils. Western lot n reversetrnsription n reltime PCR Western lot n reltime PCR were performe s previously esrie [ ]. See ESM Methos n ESM Tles, for informtion out the soure, hrteristis n vlition of the ntioies. Detetion of totl superoxie ismutse tivity, NADPH oxise tivity, lipi peroxition n AMP The fresh rt herts (8 mg) were homogenise n then entrifuge ( g, min) to ollet the superntnt frtions. The tivity of totl superoxie ismutse (SOD) n NADPH oxise, s well s the level of lipi peroxition, ws mesure y the ommeril kits. The H9 ells were lyse to etet the levels of AMP fter CTRP tretment. AMP ws etete using ommeril kit. Knokown of AMPKα in the ieti herts To knok own AMPKα in the ieti herts, n intrmyoril injetion of Aα ws ministere, s esrie previously for AAVCtrp n AAVGfp []. The level of Ampkα (lso known s Prk) mrna ws exmine t ifferent time points post infetion. After 6 weeks, Doppler nlysis n hemoynmi mesurements were performe n the rts were euthnise. Detetion of ROS n ell viility H9 ells were ulture in sixwell pltes n were expose to n CTRP for h. To etet ROS, the ells were inute with,7 ihloroihyrofluoresein iette (DCFHDA) for min t 7 C. The proution of ROS y the ells ws oserve uner n Olympus IX5 fluoresene mirosope. To quntify the proution of ROS, the fluoresene intensity ws mesure using flow ytometry. Cell viility ws etermine using the ell ounting ssy (CCK8; Dojino Moleulr Tehnologies, Rokville, MD, USA) following the mnufturer s protool. Sttistil nlyses The results in our stuy re presente s the men ± SEM. When ompring multiple groups, onewy ANOVA ws use, followe y post ho Tukey s test.an unpire, twosie Stuent s t test ws use to ompre ifferenes etween two groups. A repetemesures ANOVAws lso use. Smple size ws etermine s esrie in our previous stuy []. No smples hve een exlue from nlysis. Sttistil signifine ws ssigne t p<.5. Results CTRP ws ownregulte in the ieti herts in vivo CTRP expression ws ssesse in riomyoytes isolte from neontl rts. We foun tht CTRP expression in riomyoytes ws not s low s previously esrie (Fig. ) [5]. The level of CTRP protein in the hert ws out.7 times higher thn tht in plsm n pproximtely hlf tht in ipose tissue (Fig. ). Immunofluoresene nlyses revele tht CTRP protein levels were rmtilly reue in herts of ieti rts (Fig. ). This ws onfirme y western lot (Fig. ). These results inite role for CTRP in. CTRP eletion inrese inflmmtion, oxitive stress n myoyte eth t seline Next, we investigte whether the reue CTRP level ffete riomyoytes. We knoke own CTRP in H9 ells. Downregultion of CTRP inrese the mrna levels of Tnfα (lso known s

4 Dietologi (7) 6:6 7 9 Positive Negtive CTRP/ (fol) CTRP 8 6 αatinin αatinin Ig G CTRP Plsm Hert Aipose 6 kd 7 kd Plsm Hert Aipose DAPI DAPI CTRP/DAPI Merge Merge Tnf) n Mp (Cl) t seline without ffeting Il6 (Il6) (Fig. ). CTRP efiieny lso inrese the mrna level of P67phox (Nf) n ROS proution in sl onitions without ffeting Pphox (Cy) (Fig. e g). Moreover, knokown of CTRP erese the viility of H9 ells (Fig. h). CTRP CTRP/ (fol) kd 7 kd Fig. CTRP expression ws erese in the ieti rt herts. () Representtive imges of immunofluoresene of CTRP in riomyoytes from neontl rts (n =6). () Protein level of CTRP (n =6).() Immunofluoresene of CTRP in the ieti herts (n =5). () Representtive western lot nlysis of CTRP in the herts (n = 6). Sle r, μm. p <.5 for inite omprisons., ontrol (no ietes) CTRP ttenute STZinue ri injury n improve ri funtion Rts trete with STZ exhiite typil symptoms of ietes, inluing polyipsi n polyuri, long with inrese loo gluose (ESM Tle ). STZ injetion reue oy weight (ESM Tle ). Overexpression of CTRP in the herts (Fig. ) i not ffet loo gluose n oy weight (ESM Tle, ). Rts trete with STZ injetion for weeks evelope erement in ri funtion with mrke erese in frtionl shortening (FS) n left ventriulr internl istoli imeter (LVID); overexpression of CTRP in the herts improve the erese ri funtion (Fig., ). Compre with ontrol rts (no overexpression of CTRP), the rts trete with STZ isplye systoli ysfuntion (P/t mx 7%) n istoli ysfuntion (P/t min 5%) (Fig., e). However, these ietesrelte ri funtionl ltertions were ttenute fter CTRP overexpression. Rts with ietes h erese hert weighttooy weight rtio n inrese ell re n overexpression of CTRP protete ginst these pthologil hnges (Fig. f, g). Next, we etermine whether infusion of exogenous reominnt humn CTRP woul ttenute ietesrelte ri ysfuntion. Aministrtion of CTRP slightly erese loo gluose n signifintly inrese P/t mx n P/t min, initing the therpeuti potentil of CTRP in (ESM Fig. ). Cri firosis in the ieti rts ws lso evlute. The ollgen volume ws inrese in the ieti herts n ws not ffete y CTRP (ESM Fig., ). This ws further onfirme y exmining the mrna levels of firoti mrkers (ESM Fig., ). CTRP CTRP/ (fol) Pphox mrna (fol) shctrp 6 kd 7 kd Tnfα mrna (fol) e f g h shctrp shctrp P67phox mrna (fol) shctrp shctrp Fig. CTRP efiieny inrese inflmmtion, oxitive stress n ell eth t seline. () Protein level of CTRP in riomyoytes from neontl rts (n =6). ( ) mrna level of inflmmtory ftors fter knokown of CTRP in neontl rt riomyoytes (n = 6). (e, f) mrna level of Pphox (e) np67phox (f) in neontl rt Il6 mrna (fol) shctrp shctrp Mp mrna (fol) Cell viility (fol)..8. shctrp shctrp riomyoytes. (g) Representtive fluoresene mirosopy imges of DCFDA fluoresene in neontl rt riomyoytes. Sle r, μm. (h) Cell viility fter knokown of CTRP in neontl rt riomyoytes (n =6). p <.5 for inite omprisons. refers to srmle ontrol.

5 Dietologi (7) 6:6 7 CTRP CTRP/ (fol) P/t mx (mmhg/s) 8 6 GFP Dys 6 Weeks P/t min (mmhg/s) 6 kd 7 kd 6 8 LVID (mm) 9 6 Fig. CTRP improve ietesinue ri ysfuntion. () Western lot nlysis of CTRP in rt herts fter infetion with n AAV (n =5).(, ) Altertion of LVID () n FS () fter overexpression of CTRP (n = 6). White squres, ontrolgfp; lk squres, ontrolctrp; white tringles, GFP; lk tringles, e Time (weeks) f HW/TL (mg/m) 5 5 FS (%) g Cell re (µm ) 6 5 Time (weeks) CTRP. (, e) Effet of CTRP on hemoynmi mesurements (n =8). (f) The rtio of hert weight (HW) to tii length (TL) (n =). (g) Setionl re of myoytes (n = 6). White rs, GFP; lk rs, CTRP. p <.5 for inite omprisons., ontrol (no ietes) Overexpression of CTRP in the ieti rt herts reue oxitive mge As oxitive stress is key feture of, the effet of CTRP on oxitive injury ws evlute. Western lotting showe tht overexpression of CTRP signifintly erese the NADPH oxise suunit 67phox n inrese SOD expression in ieti rt herts (Fig. ). Our results lso onfirme tht the upregultion of P67phox n Gp9phox (lso known s Cy) mrna inue y STZ injetion ws reue in ieti rts with CTRP overexpression (Fig., ). CTRP overexpression in the ieti rts lso reue the norml tivity of NADPH oxise (Fig. ). Compre with rts in the ontrol groups, totl SOD tivity in the hert ws signifintly reue in ieti rts n ws signifintly inrese y overexpression of CTRP in the herts (Fig. e). CTRP overexpression lso reue the proution of myoril lipi peroxition in the rts with ietes (Fig. f). Immunohistohemil nlyses of HNE in the herts lso onfirme the ttenution of lipi peroxition in the CTRP group ompre with the GFP group (Fig. g). CTRP prevente ietesinue inflmmtion in the herts A previous stuy foun tht lower irulting levels of TNFα were oserve in CTRP trnsgeni mie [8]. In the present stuy, CTRP lso h signifint effets on myoril Tnfα n Mp in rts with ietes (Fig. 5 ). However, CTRP h no effet on Ilβ (lso known s Il) nil6 mrna in ieti rt herts (Fig. 5 ). Immunohistohemistry n western lot nlysis lso onfirme tht TNFα levels were signifintly inrese in the GFP group ut not in the CTRP group (Fig. 5e, f). CTRP erese ietesinue ell eth in the herts As shown in Fig. 5g, n inrese proportion of poptoti ells ws oserve in ieti herts n this ws reue signifintly following CTRP overexpression. The inhiitory effets of CTRP on poptosis were further onfirme y western lot results showing tht CTRP upregulte the expression of Bell lymphom (BCL) n reue the expression of BAX (Fig. 5g, h). CTRP tivte AMPKα n Akt in the ieti herts Next, we investigte the preise mehnisms y whih CTRP protete ginst ietesrelte ri injury. Compre with the ontrol groups, rts with ietes isplye erese phosphoryltion of Akt n AMPK in the herts, n CTRP signifintly inrese the phosphoryltion of AMPKα n Akt (Fig. 6). We lso exmine the phosphoryltion of etylcoa roxylse, whih reflets the tivity of AMPKα. As expete, CTRP promote AMPKα tivity (Fig. 6) in ieti herts. CTRP i not ffet protein kinse C (PKC), nuler ftore p5relte ftor or peroxisome prolifertortivte reeptorα (PPARα) in ieti herts lthough it inrese the level of PPARα in norml herts (Fig. 6). CTRP tivte AMPKα n inhiite inue ROS genertion n inflmmtion in H9 ells Given the oservtion tht CTRP tivte AMPKα n Akt in vivo, we exmine the effets of CTRP on AMPKα n Akt in vitro n foun tht reominnt CTRP ( μg/ml) signifintly inrese the phosphoryltion of AMPKα n Akt (Fig. 7). CTRP lso inrese the tivity of etyl

6 Dietologi (7) 6:6 7 Fig. CTRP ttenute ietesinue oxitive injury. () Western lot nlysis of p67phox n SOD in ieti rt herts (n =6).(, ) PCR nlysis of P67phox ()n Gp9phox () in ieti herts (n =6).() NADPH oxise tivity in ieti herts y CTRP overexpression (n =7).(e) Totl SOD tivity in ieti herts fter CTRP overexpression (n =7).(f) Lipi peroxition in ieti herts (n = 7).(g) Immunohistohemil nlysis of HNE in ieti herts. Representtive imges n quntifition (n = 6) re shown. Sle r, μm. White rs, GFP; lk rs, CTRP. p <.5 for inite omprisons., ontrol (no ietes). MDA, mlonilehye p67phox/ (fol) NADPH oxise tivity (fol) p67phox SOD 5 GFP CTRP GFP CTRP SOD/ (fol) 67 kd 5 kd 7 kd P67phox mrna (fol).8 e SOD tivity (U/mg) f MDA (nmol/mg) Gp9phox mrna (fol) g GFP CTRP HNE expression (fol) CoA roxylse (ESM Fig. ). Intriguingly, ministrtion of CTRP inue sustntil reution of ROS in H9 ells expose to for h (Fig. 7 ). siering the tivtion of AMPKα n Akt fter CTRP exposure, we further ssesse whether AMPKα or Akt ws involve in the CTRPmeite protetion. H9 ells were infete with enovirus to knok own AMPKα or were trete with n Akt inhiitor. Surprisingly, AMPKα knokown ut not the inhiition of Akt loke the protetion ffore y CTRP ginst ROS proution (Fig. 7 ). Fluoresene mirosopi oservtions of DCFHDAlelle ells n etetion of p67phox onfirme tht CTRP ttenute inue ROS y tivting AMPKα (Fig. 7, e). Given the inhiitory role of CTRP on inflmmtion in vivo, we then ssesse whether CTRP oul ffet inue inflmmtion in vitro. H9 ells trete with CTRP ( μg/ml for h) h lower mrna levels of Tnfα t seline when ompre with untrete ells (Fig. 7f, g). Moreover, CTRP ttenute inue inflmmtion in H9 ells, s shown y the ltere mrna levels of Tnfα n Mp (Fig. 7f i). The hnges in these levels were lunte y AMPKα efiieny n were unffete y Akt inhiition (Fig. 7f i). CTRP inhiite inue ell eth y tivting AMPKα in H9 ells Next, we etermine whether CTRP protete ginst inue eth of myoytes. H9 ells expose to for 7 h h erese ell viility n fter CTRP tretment the ell viility ws inrese (Fig. 8, ). AMPKα knokown ut not the inhiition of Akt olishe the protetion of CTRP ginst inue ell eth (Fig. 8, ). lso ownregulte BCL n upregulte BAX in H9 ells (ESM Fig. ); however, these ltertions were loke y CTRP tretment. The effets of CTRP were olishe y AMPKα efiieny (ESM Fig. ).GiventheinreseTNFα uring the proess of, the H9 ells were trete with TNFα (5 ng/ml). CTRP signifintly inhiite TNFαinue poptosis, n effet whih ws reverse y AMPKα efiieny ut not y Akt inhiition (Fig. 8, e). firmtion ws provie y western lot nlysis of BCL n BAX (ESM Fig. ). H O ws use to inue myoyte poptosis. AMPKα efiieny ompletely loke the protetion provie y CTRP ginst H O inue poptosis (Fig. 8f). This ws further onfirme y TUNEL stining (ESM Fig. ).

7 Dietologi (7) 6:6 7 Fig. 5 CTRP ttenute ietesinue inflmmtion. ( ) PCR nlysis of myoril Tnfα, Ilβ, Il6 n Mp mrna in rts with ietes (n =6).(e) Immunohistohemil nlysis of TNFα in ieti herts. Representtive imges n quntifition (n = 6) re shown. Sle r, μm. (f) Western lot nlysis of TNFα in ieti herts (n =6).(g) TUNEL stining in ieti herts (n =6). Arrows inite poptoti ells. (h) Western lot nlysis of BCL n BAX in ieti herts (n = 6). Sle r, μm. White rs, GFP; lk rs, CTRP. p <.5 for inite omprisons., ontrol (no ietes) Tnfα mrna (fol) e g GFP Il β mrna (fol) CTRP CTRP TNFα expression (fol) h BCL BAX Il6 mrna (fol) GFP CTRP f TNFα TNFα/ (fol) GFP CTRP GFP CTRP GFP CTRP Mp mrna (fol) 6 kd kd 7 kd 5 kd 7 kd Positive ells (%) BCL/ (fol).8. BAX/ (fol)..6 AMPKα efiieny ounterte the protetive effets of CTRP in vivo Susequently, we investigte whether the morphologil hnges oserve in rts fter overexpression of CTRP were reverse y AMPKα efiieny. Intrmyoril injetion of Aα resulte in the ownregultion of AMPKα in the herts (Fig. 9). Rts in the αctrp group exhiite n ggrvte phenotype, s inite y erese hert weight in reltion to tii length, restrite LVID, reue FS n ugmente hemoynmi ltertion (Fig. 9 f). The preise mehnism y whih CTRP tivtes AMPKα A previous stuy emonstrte tht the reeptor of CTRP my e G proteinouple reeptor (GPCR) n tht CTRP inrese the level of AMP in TM Leyig ells [6]. We foun tht CTRP inrese AMP in H9 ells (Fig. ). AMP is proue y enylte ylse; therefore, n enylte ylse inhiitor ( 5 Ao) ws use to investigte the possile role. The inhiitor ( μmol/l, h) loke CTRPinue AMPKα tivtion (Fig. ). Next, PKA inhiitor n smll interfering RNA of EPAC Fig. 6 CTRP tivte AMPKα n Akt in the ieti herts (n =6).Whiters, ontrolgfp; light grey rs, ontrolctrp; lk rs, GFP; rk grey rs, CTRP. p <.5for inite omprisons. ACC, etylcoa roxylse; NRF, nuler ftore p5relte ftor ; p, phosphorylte; T, totl., ontrol (no ietes) pampkα TAMPKα pacc TACC pakt TAkt NRF ppkc TPKC PPARα GFP CTRP GFP CTRP 6 kd 6 kd 8 kd 8 kd 6 kd 6 kd 68 kd 8 kd 8 kd 55 kd 7 kd Protein level (fol) Protein level (fol) pampkα pacc pakt NRF ppkc PPARα

8 Dietologi (7) 6:6 7 pampkα TAMPKα pakt TAkt ROS (fol) 5 Time fter CTRP (min) kd CTRP 6 kd 6 kd 6 kd 7 kd ROS (fol) pampkα (fol) pakt (fol) Time (min) CTRP Akt i 5 e tretment PBS Vehile CTRP CTRP p67phox/ (fol) p67phox 67 kd 7 kd CTRP CTRP Vehile PBS f Tnfα mrna (fol) CTRP g CTRP Tnfα mrna (fol) Akt i h Mp mrna (fol)..6.8 CTRP. i Mp mrna (fol).6.8 CTRP Akt i Fig. 7 CTRP suppresse inue proution of ROS n inflmmtion in H9 ells. () CTRP( μg/ml) tivte AMPKα n Akt (n = ).() Representtive fluoresene mirosopy imges of DCFDA fluoresene. Sle r, μm. () AMPKα efiieny offset the protetion ffore y CTRP ginst ROS inue y ( h) (n =6).() (siep) were use. Knokown of EPAC ut not inhiition of PKA prevente CTRPinue AMPKα tivtion (Fig. n ESM Fig. 5). Ativtion of AMPKα ws inhiite y the MEK inhiitor U6 (Fig. ). Then, we exmine how MEK tivtion is relte to the tivtion of AMPKα. However, CMKK II inhiitor (STO69) h no effet on the tivtion of AMPKα (Fig. ). CTRP tivte liver kinse B (LKB) n this tivtion ws prevente y enylte ylse inhiition, EPAC efiieny or MEK inhiition (Fig. ). Moreover, the protetion provie y CTRP ginst inue ell loss ws lso olishey 5 Ao, EPAC efiieny n U6 (Fig. ). Disussion To our knowlege, this is the first report to emonstrte tht knokown of CTRP in riomyoytes results in oxitive stress, inflmmtion n ell eth n tht overexpression of CTRP in the hert protets ginst ietesrelte ri Akt inhiition (Akt i ) i not ffet the proution of ROS (n =6). (e) Protein level of P67phox in H9 ells (n =5).(f i) AMPKα efiieny ut not Akt i offset the protetion ffore y CTRP ginst inflmmtion inue y in H9 ells ( h) (n =6).p <.5 for inite omprisons. p, phosphorylte; T, totl ysfuntion, oxitive mge, inflmmtion n ell eth in vivo. In this stuy, CTRP lso ttenute inue proution of ROS n inflmmtion, n reue inue ell loss in vitro. Moreover, we foun tht the protetive effet of CTRP ws meite y tivtion of AMPKα n tht AMPK efiieny oul offset the protetive effet of CTRP in vivo. For the first time, we foun tht CTRP tivte AMPKα vi the AMP EPAC MEK LKB pthwys. Previous stuies hve note the ssoition etween CTRP n metoli n riovsulr iseses. Cirulting CTRP ws signifintly erese in mie fe highft iet for weeks [6]. Li et l lso foun tht the levels of CTRP were erese in epiiyml ipose tissue of rts in moel of type ietes [7]. Furthermore, rosssetionl stuy revele tht erese CTRP levels were losely ssoite with oesity in humns [8]. sistent with these finings, we lso showe tht CTRP levels in ieti rt herts were signifintly erese. CTRP efiieny t seline n result in oxitive stress, inflmmtion n ell eth, repitulting the phenotype of in vitro.

9 Dietologi (7) 6:6 7 Cell viility (%) tretment (ys) Cell viility (%) CTRP Cell viility (%) CTRP Akt i Cell viility (%)..8. TNFα CTRP e Cell viility (%)..8. TNFα CTRP Akt i f Cell viility (%)..8. H O CTRP Fig. 8 CTRP loke inue ell eth in H9 ells. () CTRP inrese ell viility of H9 ells (n = 6). Cirles, PBS; squres, CTRP. () AMPKα efiieny offset the protetion ffore y CTRP ginst (7 h)inue eth of H9 ells (n =6). () The Akt/ kinse inhiitor (Akt i )(μmol/l for 7 h) h no effet on eth of H9 ells expose to for 7 h (n =6).()AMPKα efiieny offset This oservtion prompte us to investigte whether optimising CTRP levels in ieti herts oul ttenute ietesrelte ri injury. As expete, overexpression of CTRP in the herts or systemi ministrtion of CTRP oul llevite ietesrelte ri ysfuntion, e P/t mx (mmhg/s) Ampkα mrna (fol) LVID (mm) 6 (weeks) HW/TL (mg/m) FS (%) f P/tmin (mmhg/s) Fig. 9 AMPKα efiieny offset the protetive effets of CTRP in vivo. () mrna level of Ampkα (n = 6). () Rtio of hert weight (HW) to tii length (TL) (n =). (, ) Altertion of FS () n LVID () (n =8). (e, f) Altertion of hemoynmi mesurements (n =8). For( f): white rs, ontrol; lk rs, GFP; grey rs, CTRP. p <.5 for inite omprisons the protetion of CTRP ginst eth of H9 ells use y TNFα (5 ng/ml for h) (n =6). (e) Akt i ( μmol/l for 7 h) h no effet on eth of H9 ells use y TNFα (5 ng/ml for h) (n = 6). (f) AMPKα efiieny offset the protetion ffore y CTRP ginst eth of H9 ells inue y H O ( μmol/l for h) (n = 6). p <.5 for inite omprisons initing tht CTRP is promising trget for the tretment of. Intriguingly, overexpression of CTRP in norml herts i not le to ny normlities, initing tht genese therpy trgeting CTRP is potentil strtegy. Aumulting eviene emonstrtes tht n exponentil inrese in oxitive stress promotes the proess of [9, ]. An inrese in ROS, oserve in myoytes trete with [] n in riomyoytes isolte from ieti mie [9, ], use lipi peroxition n DNA mge [, ], eventully leing to funtionl normlities of the hert. Therefore, it is importnt to ientify suppressors of oxitive stress in ieti herts. In ontrst to previous fining tht CTRP inue ROS in vsulr smooth musle ells [], here, we provie roust eviene suggesting tht CTRP protete ginst ietesinue oxitive mge in vivo n loke inue proution of ROS in vitro. This ws onfirme y our fining tht knokown of CTRP inrese ROS levels in H9 ells. Inflmmtion is nother hrteristi of. Dieti herts isply inrese levels of TNFα n ILβ [5], whih n result in ri ysfuntion [6] n ffet ri ontrtility [7]. CTRP hs een emonstrte to suppress inflmmtion. Kopp et l foun tht CTRP serve s n enogenous ntgonist of lipopolyshrie (LPS) n loke the proution of TNFα, IL6 n MCP[9]. Furthermore, CTRP suppresse systemi inflmmtion in mie trete with LPS [9]. The inhiitory effets of CTRP were further onfirme y stuies using CTRP trnsgeni nimls, showing tht mie overexpressing CTRP h lower irulting levels of TNFα fter highft feeing ompre with wiltype

10 Dietologi (7) 6:6 7 5 Fig. The preise mehnism y whih CTRP tivtes AMPKα.() AMP level in H9 ells trete with CTRP for h (n =6).() plkbnp AMPKα levels in H9 ells trete with 5 Ao ( speifi enylte ylse inhiitor, μmol/l) or U6 ( MEK inhiitor, μmol/l) for h.() Cell viility of H9 ells (n =6).p <.5 for inite omprisons. p, phosphorylte; T, totl AMP levels (pmol/ml) Vehile CTRP plkb TLKB pampkα TAMPKα plkb/tlkb (fol) CTRP Vehile ' 5' Ao U6 siep 5 kd 5 kd 6 kd 6 kd 7 kd Cell viility (%) CTRP 5 Ao siep U6 pampkα/tampkα (fol) CTRP Vehile ' 5' Ao U6 siep littermte ontrols [8]. sistent with these finings, in our stuy, ttenution of inflmmtion fter CTRP tretment ws oserve in the ieti rt herts. We lso foun tht CTRP oul mitigte inue inflmmtion in vitro n tht knokown of CTRP inrese Tnfα n Mp levels in vitro. However, Kopp et l reporte tht CTRP lso ffete the level of IL6 [8], whih, in our urrent stuy, ws unffete y CTRP. Cell eth is key feture of STZinue riomyopthy [9]. Previous stuies foun tht CTRP inhiite poptosis in ishemi mouse herts in vivo [5] n suppresse poptosis of prostte ells in vitro []. sistent with these previous reports, we foun tht CTRP signifintly inhiite ietesinue ell eth in vivo n inue poptosis in vitro. In vitro, CTRP lso loke ell eth inue y TNFα or H O. Tken together, these oservtions emonstrte the protetive role of CTRP ginst ell eth in ieti herts. Reltively little is known out how CTRP exerts its protetive effets. CTRP is reporte to stimulte prolifertion vi PKC signlling pthwys [] ut we file to oserve n ltertion in the PKC pthwy in ieti herts. In ontrst to stuy showing tht CTRP h no effet on AMPKα [5], we foun tht CTRP tivte Akt n AMPKα in vivo n in vitro, whih ws onsistent with previous finings [, ]. Bse on our results, we hypothesise tht the effets of CTRP re meite y Akt or AMPKα. Notly, our stuy inite tht AMPKα efiieny, not inhiition of Akt, olishes the protetion provie y CTRP ginst oxitive stress, inflmmtion n ell eth. This is inonsistent with previous fining emonstrting tht CTRP protete mesenhyml stem ells ginst poptosis y inresing Akt phosphoryltion []. We further showe tht the inhiitory effets of CTRP ginst ietesrelte ri ysfuntion were offset y AMPK efiieny in vivo, initing tht the protetive effets of CTRP were meite y AMPKα. A ruil question rise y our t in this stuy onerns the pthwy through whih CTRP tivtes AMPKα.CTRP promotes the proution of testosterone vi the AMP PKA pthwy [6]. The t in our stuy lso emonstrte tht CTRP oul inrese AMP, s shown y inrese AMP levels fter CTRP ministrtion n the fining tht n enylte ylse inhiitor loke CTRPinue AMPKα tivtion. However, the oservtion tht PKA inhiitor i not ffet AMPKα tivtion use y CTRP inite tht PKA ws not involve in the CTRPmeite AMPKα tivtion. AMP lso tivte EPAC, whih iretly tivtes Rp n inues the tivtion of MEK []. We showe tht knokown of EPAC or inhiition of MEK loke CTRP meite AMPKα tivtion, further initing the involvement of the AMP EPAC MEK pthwy in AMPKα tivtion inue y CTRP. sistent with previous fining tht MEK tivte LKB [, 5], wellknown regultor of AMPKα, the hnge in LKB in our stuy orrelte iretly with AMPKα, suggesting role for LKB in the tivtion of AMPKα. Inhiition of CMKK II h no signifint effet on the phosphoryltion of AMPKα, initing the ispensle role of CMKK II in this proess. Altogether, our t emonstrte tht CTRP tivte AMPKα vi the AMP EPAC MEK LKB pthwys. In summry, this stuy ientifie CTRP s ritil negtive regultor of vi tivtion of AMPKα n ttenution of oxitive stress, inflmmtion n ell eth. Our stuy provies eviene tht CTRP oul e promising therpeuti pproh for the tretment of.

11 6 Dietologi (7) 6:6 7 Dt vilility The t tht support the finings of this stuy re ville from the orresponing uthor upon resonle request. Funing This work ws supporte y grnts from the Ntionl Nturl Siene Fountion of Chin (no. 87, 8756, 87) n the Key Projet of the Ntionl Nturl Siene Fountion (no. 85). Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. triution sttement ZGM, YPY, SCX, WYW, CRX n Q QW performe the stuy, nlyse n interprete the t n wrote the mnusript. ZGM, YPY, SCX, WYW n XZ ontriute to quisition of t n mnusript preprtion n revision. ZGM n QZT oneive the hypothesis, prtiipte in the experimentl esign, t interprettion n mnusript preprtion n revision. HHL n JN ontriute to t olletion n interprettion of t n mnusript rfting. All uthors pprove the finl version of the mnusript. QZT is the gurntor of this work. Referenes. Bouin S, Ael ED (7) Dieti riomyopthy revisite. Cirultion 5:. Bugger H, Ael ED () Moleulr mehnisms of ieti riomyopthy. 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12 Dietologi (7) 6: Feng H, Wng JY, Zheng M et l (6) CTRP promotes energy proution y inuing mitohonril ROS n upexpression of PGClph in vsulr smooth musle ells. Exp Cell Res : Westermnn D, Rutshow S, Jger S et l (7) triutions of inflmmtion n ri mtrix metlloproteinse tivity to ri filure in ieti riomyopthy: the role of ngiotensin type reeptor ntgonism. Dietes 56: Brynt D, Beker L, Rihrson J et l (998) Cri filure in trnsgeni mie with myoril expression of tumor nerosis ftorlph. Cirultion 97: Yokoym T, V L, Rossen RD, Durnte W, Hzrik P, Mnn DL (99) Cellulr sis for the negtive inotropi effets of tumor nerosis ftorlph in the ult mmmlin hert. J Clin Invest 9: 8. Kopp A, Bl M, Buehler C et l () Cq/TNFrelte protein represents novel n enogenous lipopolyshrie ntgonist of the ipose tissue. Enorinology 5: Fiorliso F, Li B, Ltini R et l () Myoyte eth in streptozotoininue ietes in rts in ngiotensin II epenent. L Investig 8:5 57. Hou Q, Lin J, Hung W, Li M, Feng J, Mo X (5) CTRP Stimultes prolifertion n ntipoptosis of prostte ells through PKC signling pthwys. Plos One, e6. Kim JY, Min JY, Bek JM et l (5) CTRP ts s negtive regultor of osteolstogenesis through AMPKFosNFAT signling in vitro n RANKLinue lvril one estrution in vivo. Bone 79: 5. Hou M, Liu J, Liu F, Liu K, Yu B () Cq tumor nerosis ftorrelte protein protets mesenhyml stem ells ginst hypoxin serum eprivtioninue poptosis through the phosphoinositie kinse/akt pthwy. Int J Mol Me :97. e Rooij J, Zwrtkruis FJ, Verheijen MH et l (998) Ep is Rp guninenuleotieexhnge ftor iretly tivte y yli AMP. Nture 96:7 77. Spkot GP, Kieloh A, Lizno JM et l () Phosphoryltion of the protein kinse mutte in PeutzJeghers ner synrome, LKB/STK, t Ser y p9(rsk) n AMPepenent protein kinse, ut not its frnesyltion t Cys(), is essentil for LKB to suppress ell vrowth. J Biol Chem 76: Fu D, Wkyshi Y, LippinottShwrtz J, Aris IM () Bile i stimultes heptoyte polriztion through AMPEp MEKLKBAMPK pthwy. Pro Ntl A Si U S A 8: 8