c-abl inhibition mitigates diet-induced obesity through improving insulin sensitivity of subcutaneous fat in mice

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1 Dietologi (7) :9 9 DOI.7/s--- ARTICLE -Al inhiition mitigtes iet-inue oesity through improving insulin sensitivity of suutneous ft in mie Rong Wu, & Jin-gung Sun, & Ji-qiu Wng & Binhu Li & Qingsong Liu & Gung Ning & Wnzhu Jin & Zengqing Yun,,7 Reeive: Otoer /Aepte: Deemer /Pulishe online: Jnury 7 # Springer-Verlg Berlin Heielerg 7 Astrt Aims/hypothesis High-energy iets re mong the min uses of the glol epiemi of metoli isorers, inluing oesity n type ietes. The mehnisms of highenergy-iet-inue metoli isorers re omplex n lrgely unknown. The non-reeptor tyrosine kinse -Al plys n importnt role in ipogenesis in vitro ut its role in vivo in the regultion of metolism is still elusive. Hene, Rong Wu n Jin-gung Sun ontriute eqully to this work. Eletroni supplementry mteril The online version of this rtile (oi:.7/s---) ontins peer-reviewe ut uneite supplementry mteril, whih is ville to uthorise users. Wnzhu Jin jinw@ioz..n Zengqing Yun zqyun@ip..n 7 Stte Key Lortory of Brin n Cognitive Sienes, Institute of Biophysis, Chinese Aemy of Sienes, Dtun Ro, Choyng Distrit, Beijing, Chin College of Life Sienes, University of Chinese Aemy of Sienes, Beijing, Chin Sino-Dnish Center Neurosiene Progrm, University of Chinese Aemy of Sienes, Beijing, Chin Ruijin Hospitl Affilite to Shnghi Jiotong University Shool of Meiine, Shnghi, Chin High Mgneti Fiel Lortory, Chinese Aemy of Sienes, Hefei, Anhui, Chin Key Lortory of Animl Eology n Conservtion Biology, Institute of Zoology, Chinese Aemy of Sienes, Beihen West Ro, Choyng Distrit, Beijing, Chin Center of Alzheimerʼs Disese, Beijing Institute for Brin Disorers, Beijing, Chin we sought to ress the role of -Al in iet-inue oesity n oesity-ssoite insulin resistne. Methos The expression of -Al in ifferent ft tissues from oese humns or mie fe high-ft iet (HFD) were first nlyse y western lotting n quntittive PCR. We employe onitionl eletion of the -Al gene (lso known s Al) in ipose tissue using Fp-Cre n -week-ol mie were fe with either how iet (CD) or n HFD. Age-mthe wil-type mie were trete with the -Al inhiitor nilotini or with vehile n expose to either CD or HFD, followe y nlysis of oy mss, ft mss, gluose n insulin tolerne. Histologil stining, ELISA n iohemil nlysis were use to lrify etils of hnges in physiology n moleulr signlling. Results -Al ws highly expresse in suutneous ft from oese humns n HFD-inue oese mie. Conitionl knokout of -Al in ipose tissue improve insulin sensitivity n mitigte HFD-inue oy mss gin, hyperglyemi n hyperinsulinemi. Consistently, tretment with nilotini signifintly reue ft mss n improve insulin sensitivity in HFD-fe mie. Further iohemil nlyses suggeste tht -Al inhiition improve whole-oy insulin sensitivity y reuing HFD-triggere insulin resistne n inresing iponetin in suutneous ft. Conlusions/interprettion Our finings efine new iologil role for -Al in the regultion of iet-inue oesity through improving insulin sensitivity of suutneous ft. This suggests it my eome novel therpeuti trget in the tretment of metoli isorers. Keywors Aiponetin. -Al. Insulin resistne. Nilotini. Oesity

2 Dietologi (7) :9 9 9 Arevitions BAT Brown ipose tissue CD Chow iet KO Conitionl knokout CML Chroni myelogenous leukemi ER Enoplsmi retiulum ewat Epiyml white ipose tissue GLM Generl liner moel HFD High-ft iet ITT Insulin tolerne test JNK -Jun mino-terminl kinse PDGFR Pltelet-erive growth ftor reeptor PIK Phosphtiylinositol -kinse RBP- Retinol-ining protein swat Suutneous white ipose tissue WT Wil-type Introution Oesity n type ietes hve eome glol pnemi n re mong the min uses of moriity n mortlity [, ]. Energy-ense foo n seentry western lifestyle result in surplus of energy store in the ipose tissue, leing to the evelopment of oesity n metoli isorers []. In these metoli isorers, tissues suh s musle, liver n ft eome less responsive or even resistnt to insulin []. Insulin resistne is funmentl pthogeni ftor shre y myri of metoli isorers inluing oesity n type ietes[]. Upon onsumption of high-ft iet (HFD), multiple ftors ontriute to insulin resistne, inluing NEFA, hormones n inflmmtion []. The istriution of oy ft is lso ritil eterminnt of insulin sensitivity. Len iniviuls with minly suutneous istriution of ft re more insulin sensitive thn len iniviuls who hve more viserl ft [7]. Differenes in the hrteristis of ipose tissue from these two epots might explin in prt why the metoli effets re ifferent in viserl n suutneous ft. Viserl ft expresses more genes enoing seretory proteins, suh s resistin n retinol-ining protein (RBP- ), whih re responsile for insulin resistne. Suutneous ft releses more iponetin thn viserl ft []. The non-reeptor tyrosine kinse -Al is uiquitously expresse n meites multiple signlling ses governing the ell yle n ell hesion, prolifertion n poptosis [9 ]. Vrious -Al inhiitors hve een use to tret hroni myelogenous leukemi (CML) []. One suh rug, imtini, ws use to ontrol CML through trgeting the Br Al fusion protein [ ]. Interestingly, imtini tretment inue signifint regression in type ietes in CML ptients [7]. Consistently, two reent stuies showe tht imtini improve insulin sensitivity in the peripherl tissues vi inresing plsm iponetin levels [, 9]. In ition, imtini ws lso foun to inrese the survivl of insulin-prouing et ells vi inution of phosphtiylinositol -kinse signlling or promotion of NKX. (lso known s NKX-) nglut- (lso known s SLCA) gene expression [, ]. A reent stuy inite tht imtini regultes insulin homeostsis through inhiiting the pltelet-erive growth ftor reeptor (PDGFR), whih is lso trget of imtini []. A proteomi nlysis revele tht -Al ws potentil key regultor of ipogenesis vi regultion of the expression n tivity of peroxisome prolifertor tivtor reeptor γ (PPARγ), the mster ipogeni regultor ontrolling ipogenesis [, ]. These finings inite tht -Al prtiiptes in ipoytes metolism regultion in vitro. However, the in vivo role of -Al in ipose tissue n whether -Al inhiition n improve oesity-ssoite insulin sensitivity remins to e further explore. Stuies se on geneti moels n/or speifi -Al kinse inhiitors re urgently neee to onfirm the role of -Al in insulin homeostsis. In this stuy, we employe onitionl ltion of -Al in ipose tissue n speifi inhiitor to test our hypothesis tht -Al plys n importnt role in ietinue oesity n oesity-ssoite insulin resistne. Methos Animls All mie use h C7BL/J kgroun. Mie were house t C uner h light rk yle n h free ess to wter. All mie were mintine in the Animl Cre Fility t the Institute of Biophysis, Chinese Aemy of Sienes, Beijing. Six-week-ol mie were rnomise to feeing with how iet (CD) or HFD (% of totl energy from ft). All the experiments were lin to group ssignment n outome ssessment. All experiments involving nimls were pprove y n onforme to the guielines of the institutionl niml re n ethis ommittee t the Institute of Biophysis of the Chinese Aemy of Sienes (Beijing, Chin). Genertion of mie with onitionl knokout of -Al in ipose tissue -Al flox/flox mie ( gift from Y. Cng, Zhejing University, Hnzhou, Chin; -Al is lso known s Al)were rosse with Fp-Cre mie ( gift from W. Jin, Institute of Zoology, Chinese Aemy of Sienes, Beijing, Chin). The offspring mle -Al flox/flox ::Fp-Cre +/ (-Al onitionl knokout [KO]) mie n -Al flox/flox (wil-type [WT]) mie were use in the experiments. GTTs n insulin tolerne tests After eing fste for h n h, respetively, mie were sujete to GTTs n insulin tolerne tests (ITTs) (see ESM Methos for etils).

3 9 Dietologi (7) :9 9 Energy intke, expeniture n tivity mesurements See ESM Methos for etils of metoli mesurements me in mie. Nilotini tretment Six-week-ol C7BL/J mle mie upon CD or HFD were rnomise to reeive orlly ministere nilotini (7 mg/kg) or vehile t h intervls for weeks. Histologil nlysis Mie were fste for h, nesthetise with ether n then kille efore loo n tissue olletion. Metolism-relte tissues were stine with hemtoxylin n eosin (see ESM Methos for etils). ELISA Serum insulin n ipokines were mesure y ELISA (see ESM Methos for etils). Rel-time quntittive RT-PCR Rel-time quntittive RT-PCR ws use to etermine reltive gene expression levels. After tretments, tissues, inluing ipose tissue, liver n musle, were issete n totl RNA ws extrte. For further etils, see ESM Methos. Tp ws use s housekeeping gene use for input normlistion. Detete genes n their primers re shown in ESM Tle. Immunolotting Tissues were issete, lyse in lysis uffer n sujete to western lotting n protein quntifition. The proteins were etete with ntioies ginst -Al, phospho-- Al, Akt, phospho-akt, -Jun mino-terminl kinse (JNK), phospho-jnk, Grp7, GAPDH n β-tuulin. For etils, see ESM Methos. The intensity of the western lot ns ws etermine using ImgeJ softwre (NIH, Bethes, MD, USA). Humn stuy Viserl n suutneous ft ws otine from ptients t Ruijin Hospitl, Shnghi Jio Tong University Shool of Meiine, for etermintion of -Al protein levels in ipose tissue. The ipose tissues from oese iniviuls (BMI > kg/m ) were otine from hospitlise iniviuls unergoing weight-loss surgery. The ontrol ipose tissues from mthe norml-weight iniviuls (BMI < kg/m ) were otine from hospitlise ptients unergoing surgery for gllstones or herni, without fever n other ritil onitions. All iniviuls were from the estern re of Chin. Detils of ptients re given in ESM Tle. The ipose issues were sujete to immunolotting with -Al ntioy. This stuy ws pprove y the Institutionl Review Bor of the Ruijin Hospitl, Shnghi Jio Tong University Shool of Meiine, n ws in orne with the priniple of the Helsinki Delrtion II. Written informe onsent ws otine from eh prtiipnt. Sttistil nlysis Sttistil nlyses were performe y ANOVA followe y Tukey s post ho test or y two-tile Stuent s t test. All vlues re expresse s mens ± SEM. p <., p <. np <. enote the signifine threshols. There ws no exlusion of ny t, smples or nimls. Results -Al expression is inrese in suutneous white ipose tissue from oese humns n HFD-inue oese mie Despite previous stuies showing tht -Al ws tivte uring ipogenesis, the expression of -Al in oese humn n mouse ipose tissue hs never een investigte. Here, we foun tht the protein levels of -Al were mrkely inrese in suutneous ft from oese humns. However, there ws no signifint ifferene in viserl ft (Fig.,). Interestingly, the protein levels of -Al in suutneous ft were positively relte to BMI (Fig. n ESM Tle). Vis Su BMI (kg/m ² ) e ewat swat Norml Oese -Al β-tuulin -Al β-tuulin R ² = Suutneous -Al protein level (fol) CD HFD -Al β-tuulin -Al β-tuulin -Al/β-tuulin (fol) -Al mrna (fol) f-al/β-tuulin (fol) Vis Su Liver Musle ewat swat.... ewat swat Fig. -Al expression is inrese in swat from oese humns n HFD-inue oese mie. (, ) Representtive immunolots of lystes of viserl (Vis) n suutneous (Su) ft from norml or oese humns with the ntioies inite () n the level of -Al normlise to β-tuulin (). Dt re mens ± SEM (norml n =,oesen =7). White rs, norml; lk rs, oese. p <. vs norml (Stuent s t test). () The reltive protein levels of -Al in humn suutneous ft were positively relte to BMI. () The mrna levels of -Al in ifferent tissues from mie fe with CD or HFD (n = per group). White rs, CD; lk rs, HFD. p <. vs CD (Stuent s t test). (e, f) Representtive immunolots of lystes of ewat n swat from CD-fe mie n HFDinue oese mie with the ntioies inite (e) n the levels of - Al normlise to β-tuulin (f). Dt re expresse s mens ± SEM (n = per group). White rs, CD; lk rs, HFD. p <. vs CD (Stuent s t test)

4 Dietologi (7) :9 9 9 Similr finings were oserve in HFD-inue oese mie. The mrna levels of -Al inrese in suutneous white ipose tissue (swat) (Fig. ). Immunolotting lso showe n inrese -Al protein level in swat ut no signifint ifferene in epiyml white ipose tissue (ewat) (Fig. e,f). Together, these results inite tht suutneous -Al protein levels re o-relte with oesity. KO of -Al in ipose tissue protets ginst HFD-inue oesity Our fining tht -Al ws seletively inrese in oese humn n mouse suutneous ft le us to exmine whether KO of -Al in ipose tissue protets ginst HFDinue oesity. Fp-Cre hs een wiely use to trget ipose tissue [, ]. We rosse -Al flox/flox mie with Fp-Cre mie to generte offspring. -Al KO mie (-Al flox/flox ::Fp-Cre +/ )nwtmie(-al flox/flox )were fe with HFD or CD. Interestingly, Fp-Cre-meite -Al eletion in ipose tissue signifintly erese -Al protein level only in swat n not in other ipose tissues (ESM Fig. ). Therefore, Fp-Cre meite -Al eletion provies n iel moel for stuying the role of -Al in suutneous ft. We foun tht Fp-Cre meite -Al KO remrkly reue HFD-inue oy mss gin, while no hnge ws oserve in mie on CD (Fig. ). MRI nlysis showe tht HFD signifintly inue len n ft mss gin in mie, while -Al KO signifintly reue these gins (Fig.,). Interestingly, -Al KO h no effet on the len or ft mss in CD-fe mie, initing tht Fp-Cre meite -Al intivtion i not use evelopmentl efets (Fig.,). Similrly, speifi -Al eletion in ipose tissue reue the mss of swat, rown ipose tissue (BAT) n liver, ut not ewat, uner HFD feeing onitions (Fig. g); there ws no signifint hnge uner CD (Fig. g). Histologil nlysis revele tht -Al onitionl eletion in ipose tissue mrkely ttenute HFD-inue ipoyte hypertrophy n heptostetosis (Fig. h). Interestingly, there ws no signifint hnge in the size of ipoytes from ewat when ompring WT n -Al KO mie uner HFD (Fig. h). Tken together, these results inite tht speifi eletion of -Al in ipose tissue protets ginst HFDinue oesity. Sine the speifi -Al eletion in ipose tissue signifintly reue lipi umultion in BAT (Fig. h), we wonere whether KO of -Al oul protet mie ginst HFDinue oesity through upregultion of energy expeniture. Ativte BAT inreses the onsumption of ftty is n gluose, whih les to inrese energy expeniture [7]. However, we foun tht there ws no signifint ifferene in BAT levels of thermogenesis-relte gene Up n ftty i oxition-relte genes etween WT n KO mie (Fig. ). Aitionlly, there were no signifint hnges in oxygen onsumption reltive to len mss uner either CD or HFD onition (Fig. ). Aoring to the stnr protool of energy Boy mss (g) ewat mss (g) h Age (weeks)..... WT+CD KO+CD WT+HFD KO+HFD swat mss (g) Len mss (g) expeniture nlysis etween groups with ifferent oy mss [, 9], we performe stringent sttistil test (generl liner moel [GLM] n ANCOVA). We foun tht -Al KO in ipoytes lso h no signifint effet on energy expeniture uner either HFD or CD onition (Fig. e,f). Furthermore, there ws no phenotypi ifferene in loomotive ility etween these mie (Fig. g). We next sought to etermine whether speifi eletion of -Al in ipose tissue reue foo intke. Interestingly, -Al KO h no effet on foo intke uner CD ut slightly inrese the foo intke reltive to oy mss uner HFD onition (Fig. h). Furthermore, uner HFD tretment, the serum leptin level ws signifintly higher in WT mie n this level ws reue y -Al KO (Fig. i). Tken BAT mss (g) Ft mss (g) e f g Liver mss (g) ewat swat BAT Liver Fig. Conitionl eletion of -Al in ipose tissue protets ginst HFD-inue oesity in mie. () BoymssofWTn-Al flox/ flox ::Fp-Cre (KO) mle mie fe with CD or HFD. WT+CD, n =; KO+CD, n = 7; WT+HFD, n = ; KO+HFD, n =. (, ) Len n ft mss mesure y MRI. WT+CD, n = ; KO+CD, n =9; WT+HFD, n = ; KO+HFD, n =9.( g)mssofewat(), swat (e), BAT (f) n liver (g). WT+CD, n = ; KO+CD, n = 9; WT+HFD, n = 7; KO+HFD, n =. In grphs, t re mens ± SEM. Blk rs n imons, WT+CD; lue rs n tringles, KO+CD; green rs n squres, WT+HFD; re rs n irles, KO+HFD. p <., p <. n p <. for inite omprisons (ANOVA). (h) Hemtoxylin n eosin stining of ewat, swat, BAT n liver. Sle r, μm (ewat, swat, liver) or μm (BAT)

5 9 Dietologi (7) :9 9 Averge VO ( ml h - kg - ) g Bem rek (ounts/h) mrna (fol) Up Prm Cpt Cox,, Light Drk Time (h) h Foo intke (g/y) e 7 Fig. Aipose-speifi eletion of -Al hs no effet on energy lne in oese mie. () Reltive gene expression in BAT from inite groups (n = per group). ( ) Oxygen onsumption of WT n -Al KO mle mie fe with CD () orhfd() n verge oxygen onsumption uring light n rk perio (). WT+CD, n = ; KO+CD, n =9; WT+HFD, n = ; KO+HFD, n =9. (e, f) Sttistil nlysis y GLM n ANCOVA of the energy expeniture (EE) of -Al KO n WT mie fe with CD (e) or HFD (f). (g) Loomotive tivity of -Al KO n WT mie fe with CD or HFD. VO ( ml h - kg - ) EE (kj/y) Time (h).... HFD KO 9 7 p=.97 Len mss (g) Foo intke (g [oy mss] - y - ) ( ml h - kg - ) VO EE (kj/y) i Leptin (ng/ml) 7 f..... Time (h) HFD - + KO 9 9 p=.7 Len mss (g) WT+CD, n = ; KO+CD, n =9;WT+HFD,n =;KO+HFD,n =. (h) Dily foo intke in solute vlues n normlise y oy mss. WT+CD, n = ; KO+CD, n =9;WT+HFD, n =;KO+HFD,n =9. (i) Leptin levels in serum from the inite group of mie. WT+CD, n = ; KO+CD, n = ; WT+HFD, n = ; KO+HFD, n =. For grphs, t re mens ± SEM, exept for (e) n(f). Blk rs n imons, WT+CD; lue rs n tringles, KO+CD; green rs n squres, WT+HFD; re rs n irles, KO++HFD. p <. n p <. for inite omprisons (ANOVA) together, speifi eletion of -Al in ipose tissue protets ginst HFD-inue oesity n this oes not our through reution of energy intke. Deletion of -Al in ipoytes protets ginst HFDinue insulin resistne To efine the reltionship etween whole-oy insulin sensitivity n the evelopment of oesity, we performe GTTs n ITTs t ifferent time points fter ietry intervention. As expete, the HFD le to gluose intolerne n insulin resistne in WT mie t weeks ol (Fig.,),nothgluoseintolernen insulin resistne were progressively exerte in WT mie following HFD tretment (Fig.,). At week following HFD tretment, the WT mie evelope hyperglyemi, hyperinsulinemi n ompenstory hypertrophy of pnreti islets (Fig. e g). Compre with WT mie, -Al KO mie showe signifint mitigtion of the HFD-inue phenotypes n improve insulin sensitivity uring HFD (Fig. f). In ition, HFD-inue pnreti hypertrophy ws lso mrkely meliorte in -Al KO mie (Fig. g). Insulin sensitivity hs een reporte to e losely relte to phosphtiylinositol -kinse (PIK) Akt signlling. Upon insulin stimultion, the intrinsi tyrosine kinse of the insulin reeptor les to reeptor utophosphoryltion t multiple tyrosine resiues n the phosphoryltion of the insulin reeptor sustrtes (IRS/) les to the tivtion of PIK Akt signlling pthwy n regultion of gluose homeostsis []. Hene, we further explore whether -Al onitionl eletion ltere the levels of phosphorylte Akt in ipose tissue. Immunolotting nlysis revele tht insulin tretment oviously inrese Akt phosphoryltion in ipose tissue n tht HFD tretment signifintly reue the Akt phosphoryltion (Fig. h). Interestingly, we foun tht Fp-Cre meite - Al eletion reue -Al expression n resue Akt phosphoryltion in swat ut not in ewat or BAT (Fig. h n ESM Fig. ). Colletively, KO of -Al in ipoytes protets mie ginst HFD-inue insulin resistne. Nilotini, speifi -Al inhiitor, protets ginst HFDinue oesity We wnte to test whether the ove effet of -Al in oesity evelopment ws epenent on its kinse

6 Dietologi (7) :9 9 9 Fig. KO of -Al improves whole-oy insulin sensitivity. (, ) GTT results for WT n -Al KO mie fe with CD or HFD t weeks () n weeks () ofge(n = per group). The AUC for the GTT is lso shown. (, ) ITT results for WT n -Al KO mie fe with CD or HFD t weeks () n weeks () ofge(n = per group). The AUC for the ITT is lso shown. (e, f) Bloo gluose (e, n = ) n insulin levels (f, n = 9), in the fste stte, of WT n -Al KO mie fe with CD or HFD for weeks. (g) Hemtoxylin n eosin stining of pnreses from WT n -Al KO mie fe with CD or HFD for weeks. Sle r, μm. (h) Immunolot of lystes from inite tissues with the ntioies inite. Quntifition of phosphorylte Akt in the inite tissues is shown. For grphs, t re mens ± SEM. Blk rs n imons, WT+CD; lue rs n tringles, KO+CD; green rs n squres, WT+HFD; re rs n irles, KO+HFD. p <.,p <.n p <. for inite omprisons (ANOVA). In the line grphs in ( ), p vlues re for re irles vs green squres t the inite time points Bloo gluose (mmol/l) Bloo gluose (mmol/l) e Fsting gluose (mmol/l) 9 9 h Akt -Al ewat Insulin HFD Al KO p-akt β-tuulin f Fsting insulin (pmol/l) Bloo gluose (mmol/l) Bloo gluose (mmol/l) swat g CD HFD WT KO BAT p-akt/akt (fol) Insulin p-akt/akt (fol) Insulin p-akt/akt (fol) Insulin tivity n to guge the potentil therpeuti effet of -Al inhiition on metoli isorers. To o this, we ministere nilotini, -Al kinse inhiitor, orlly t h intervls to HFD-inue oese mie. We foun tht nilotini tretment signifintly reue HFD-inue oy mss gin (Fig. ). MRI nlysis showe tht nilotini tretment erese ft mss in oth CD- n HFD-fe mie (Fig. ), while it hs no effet on the len mss (Fig. ). Further exmintion showe tht nilotini tretment le to remrkle erese in the mss of ll ft tissues inluing ewat, swat n BAT (Fig. g). Histologil nlysis revele tht lipi roplets in ewat, swat, BAT n liver from nilotini-trete mie were smller thn those in tissues from ontrol mie uner HFD tretment (Fig. h). As with -Al KO, nilotini tretment file to lter oxygen onsumption n energy expeniture in either HFD- or CD-fe mie (Fig. i l). Interestingly, nilotini tretment reue the oy mss (Fig. ) espite n inrese in foo intke uner the HFD onition (Fig. m). Nilotini tretment lso reue serum leptin levels in CD- n HFD-fe mie (Fig. n). Summrising these finings, nilotini reues nutrient storge n protets ginst HFD-inue oesity. Nilotini improves whole-oy insulin sensitivity We performe GTTs n ITTs to ssess whether nilotini tretment improves insulin sensitivity. When mie were ge weeks, the HFD use gluose intolerne, while nilotini tretment improve the phenotype (Fig. ). In line with the GTT results,

7 j ml h- 9 Dietologi (7) :9 9 Boy mss (g) h CD+vehile CD+nilotini HFD+vehile HFD+nilotini k EE (kj/y) 7 7 Age (weeks) p=. 7 9 Len mss (g) Len mss (g) ewat swat BAT Liver EE (kj/y) 9 e f g 7 p= Len mss (g) Fig. Nilotini protets ginst HFD-inue oesity. () Boy mss of mie trete with nilotini (7 mg/kg) or vehile plus CD or HFD. CD+vehile, n = ; CD+nilotini, n = ; HFD+vehile, n = ; HFD+ nilotini, n =.(, ) Boylen() n ft () mss mesure y MRI (n = 9 per group). ( g) MssofeWAT(), swat (e), BAT (f) n liver (g). CD++vehile, n = ; CD+nilotini, n = ; HFD+vehile, n =; HFD+nilotini, n =. (h) Hemtoxylin n eosin stining of ewat, swat, BAT n liver. Sle r, μm for ewat, swat n liver or μm for BAT. (i, j) Oxygen onsumption of inite group of mie (i) n verge oxygen onsumption uring light n rk perio (j) (n = 9 per group). (k, l) Sttistil nlysis of energy expeniture (EE) y GLM l Ft mss (g) ewat mss (g)... i.. VO ( ml h kg - ) - m Foo intke (g/y) swat mss (g)... Time (h) HFD Nilotini BAT mss (g) ( kg - ) VO Averge Foo intke (g [oy mss] - y - ) n Leptin (ng/ml) Light Liver mss (g)... HFD - - Nilotini Drk + + n ANCOVA of ifferent groups (n = 9 per group). (m) Dily foo intke in solute vlues n normlise y oy mss (n = 9 per group, p <. for inite omprison; Stuent s t test). (n) Leptin levels in serum (n = per group). For grphs, t re mens ± SEM, exept for (k) n(l). Blk rs n imons, CD+vehile; lue rs n tringles, CD+nilotini; green rs n squres, HFD+vehile; re rs n irles, HFD+nilotini. p <., p <. n p <. for inite omprisons (ANOVA, unless inite otherwise). In the line grphs in (), p vlues re for re irles vs green squres t the inite time points ITT nlysis revele inrese insulin sensitivity fter nilotini tretment uner the HFD onition; no signifint ifferene ws foun uner the CD onition (Fig. ). Notly, fter weeks of HFD, there ws no signifint hnge in pnreti morphology etween ontrol n nilotini tretment (Fig. ). Further iohemil nlysis showe tht nilotini tretment resue HFD-reue Akt phosphoryltion only in swat n not in other tissues (Fig. ). This t inites tht -Al plys n importnt role in the regultion of suutneous ft insulin sensitivity uner HFD hllenge. Together, -Al kinse inhiition in suutneous ft oul improve insulin sensitivity n ontriute to weight reution. -Al inhiition improves insulin sensitivity y inresing iponetin levels Multiple ftors ontriute to high-energy iet-inue insulin resistne, inluing inflmmtion, enoplsmi retiulum (ER) stress n ipokines [,, ]. To eipher the mehnism unerlying the role of -Al in insulin resistne, we first stuie whether -Al inhiition ffete the inflmmtion in ipose tissues. Different types of ipose tissue onfer ifferent inflmmtion pility (e.g. there is reltively higher immunologil response in ewat thn in swat or BAT [,, ]). We foun tht the expression levels of F/ (lso known s Agre), Mp n Pi- (lso known s Serpine) were mrkely inrese in ewat uner HFD hllenge (ESM Fig. f). Compre with -Al KO (ESM Fig. ), nilotini oul signifintly reue the expression of F/, Mp n Pi- in ewat, ut not in swat or BAT, uner the HFD onition (ESM Fig. f). The filure of -Al KO (when ompre with nilotini) to inhiit HFD-inue inflmmtion might e ue to low eletion effiieny of -Al in ewat when using the Fp-Cre

8 Dietologi (7) : Bloo gluose (mmol/l) Akt Insulin p-akt/akt (fol) 9 Bloo gluose (mmol/l) 9 HFD CD Vehile Nilotini ewat swat BAT Liver Musle Insulin HFD Nilotini p-akt Insulin Insulin Insulin Insulin Fig. -Al inhiition improves insulin sensitivity in vivo. () GTT nlysis of nilotini- (7 mg/kg) or vehile-trete -week-ol mie fe with CD or HFD. The AUC for the GTT is lso shown (n = 7 or per group). () ITT results for nilotini- (7 mg/kg) or vehile-trete mie fe with CD or HFD. The AUC for the ITT is lso shown (n = per group). () Hemtoxylin n eosin stining of pnreses from mie trete with nilotini (7 mg/kg) or vehile plus CD or HFD for weeks. Sle r, μm. () Immunolot of lystes from inite tissues with p-akt/akt (fol) p-akt/akt (fol) p-akt/akt (fol) the ntioies inite. Quntifition of phosphorylte Akt in the inite tissues is shown. For grphs, t re mens ± SEM. Blk rs n imons, CD+vehile; lue rs n tringles, CD+nilotini; green rs n squres, HFD+vehile; re rs n irles, HFD+nilotini. p <., p <. n p <. for inite omprisons (ANOVA). In the line grphs in () n(), p vlues re for re irles vs green squres t the inite time points p-akt/akt (fol) promoter (ESM Fig.,g). The nlysis of inflmmtion in ifferent tissues suggests tht the effet of Fp-Cre-meite -Al eletion might e inepenent of inflmmtion. JNK is the puttive trget kinse of inflmmtion ftors n its phosphoryltion/tivtion plys n importnt role in the evelopment of inflmmtion-inue insulin resistne [, ]. We therefore exmine the phosphoryltion of JNK in ipose tissues from ifferent moels uner HFD hllenge. We foun tht HFD inrese JNK phosphoryltion in oth ewat n swat n tht -Al onitionl eletion mrginlly inhiite JNK phosphoryltion in swat ut not in ewat, whih might e ue to the lower effiieny of -Al eletion in ewat ompre with swat (ESM Fig. g). Consistently, nilotini signifintly inhiite JNK/ phosphoryltion in oth swat n ewat uner the HFD onition (ESM Fig. h). However, our previous t show tht oth -Al KO n tretment with nilotini improve insulin sensitivity in suutneous ft ut not in ewat or BAT (Figs h n ). Hene, we rgue tht the improvement in insulin sensitivity rought out y -Al KO or inhiition might not e ue to reution in HFD-inue inflmmtion. Reently, ER stress hs emerge s new plyer in oesity n type ietes n onsierle numer of stuies hve highlighte its role in insulin resistne []. However, there ws no signifint ifferene in expression of Grp7, n ER stress mrker, etween ontrols n either -Al KO mie or nilotini-trete mie uner eithercdorhfdhllenge (ESM Fig. g,h). These t inite tht ER stress oes not ontriute to -Al-inue insulin resistne. Aipose tissues lso serete multiple ipokines, suh s iponetin n resistin, to regulte insulin sensitivity. For exmple, iponetin inreses insulin sensitivity n is minly serete from suutneous ft [7 9]. Two reent stuies foun tht imtini improves insulin sensitivity in the peripherl tissues vi inresing plsm iponetin levels [, 9]. Consistent with this, we foun tht -Al KO or -Al inhiition signifintly resue HFD-inue iponetin reution (Fig. 7,). Resistin, minly serete from viserl ft, is ssoite with the evelopment of insulin resistne n type ietes mellitus, [, ]. We oserve tht HFD inrese plsm resistin levels ut tht -Al KO or nilotini tretment file to reue its levels (Fig. 7,). Tken together, we rgue tht -Al inhiition might improve insulin sensitivity minly through promoting iponetin seretion from suutneous ipose tissue uner HFD tretment (Fig. 7e). However, the etile mehnism of -Al-regulte iponetin nees further stuie. Disussion In this work, we revele ritil role for -Al in oesity through its regultion of insulin response. Geneti eletion of -Al in ipose tissue or phrmologil inhiition of - Al y nilotini mrkely ttenute HFD-inue oesity

9 9 Dietologi (7) :9 9 Aiponetin (μg/ml) HFD - - KO + + Aiponetin (μg/ml) HFD - - Nilotini + + Resistin (ng/ml) HFD - - KO + + Resistin (ng/ml) HFD - - Nilotini + + e Wil type/vehile Oese Insulin resistnt Aiponetin -Al Len Insulin sensitive -Al HFD HFD -Al Nilotini -Al -Al KO/nilotini Aiponetin Viserl ft/ewat Suutneous ft/swat Fig. 7 -Al inhiition inreses iponetin levels in vivo. (, ) Aiponetin () n resistin () levels in serum from WT n -Al KO mle mie fe with CD or HFD. Blk rs, WT+CD, n =; lue rs, KO+CD, n = ; green rs, WT+HFD, n = ; re rs, KO+HFD, n =. (, ) Aiponetin () n resistin () levels in serum from nilotini- (7 mg/kg) or vehile-trete mie fe with CD or HFD. Blk rs, CD+vehile, n = ; lue rs, CD+nilotini, n = ; green rs, Reue weight Insulin sensitive HFD+vehile, n = ; re rs, HFD+nilotini, n =. p <., p <. n p <. for inite omprisons (ANOVA). (e) Moel epiting the role of -Al in oesity n oesity-ssoite insulin resistne. The re rrow represents the evelopment of oesity/insulin resistne uner HFD n the lue rrow represents the ttenution of etrimentl effets of HFD on weight/insulin sensitivity vi -Al KO or -Al inhiition n insulin resistne in vivo. Interestingly, we oserve tht oth -Al speifi eletion n nilotini tretment reue HFD-inue insulin resistne in suutneous ft through restoring the iponetin level. Thus, our finings rgue tht suutneous -Al is importnt for whole-oy insulin sensitivity n suggest -Al s therpeuti trget in the tretment of metoli isorers. White ipose tissue is istriute throughout the oy n is ivie into two mjor types: viserl ft n suutneous ft, eh possessing unique ell-utonomous properties []. In ontrst to viserl white ipose tissue, whih is etrimentl to metoli homeostsis, swat is enefiil for metolism y improving insulin sensitivity []. The expression levels of resistin n RBP- re high n there is high istriution of mrophges, T ells n nturl killer ells in viserl white ipose tissue [,, ]. Interestingly, there is high expression level of iponetin in suutneous ft [7 9]. In ition, it hs een reporte tht the ntilipolyti effet of insulin is greter in suutneous ipose tissue thn in viserl ipose tissue [], implying tht suutneous ipose tissue might ply profoun role in the regultion of insulin sensitivity n oesity. Interestingly, we foun tht -Al inhiition seletively inrese the seretion of iponetin in suutneous ft. This provies new eviene for the ritil role of suutneous ft in mintining whole-oy insulin sensitivity. The non-reeptor tyrosine kinse -Al is tivte y ellulr stress, suh s oxitive stress n DNA mge []. There is eviene showing tht -Al plys entrl role in CML n neuroegenertive iseses [, ]. Reently, extensive stuies hve linke -Al to islet et ell survivl n ipogenesis [,,, ]. By utilising geneti ltion n phrmologil inhiition, we foun new role for -Al in regulting iet-inue oesity n insulin resistne in vivo. To stuy the role of -Al in ipose tissue, Fp-Cre ws use to rete ipose-speifi eletion in vivo. Fp-Cre hs een extensively use to trget ipose tissue [, ]. However, reent stuies showe tht Fp-Cre lines lk tissue speifiity [, ]. We refully exmine the expression levels of -Al in metolism-relte tissues, inluing ipose tissue, liver, skeletl musle n pnres. We oserve tht Fp-Cre-meite -Al KO inee h low eletion effiieny in ewat, swat, BAT n liver t the mrna level, ut t the protein level signifint reution of -Al ws only oserve in swat (ESM Fig.). In ition, -Al KO oul not lok HFD-inue ewat mss gin n ipoyte size

10 Dietologi (7) : (Fig. h). Hene, Fp-Cre meite -Al speifi eletion might provie n iel moel with whih to stuy the role of -Al in suutneous ft. Notly, espite Fp-Cre hving low effiieny for eletion of -Al in BAT, oth geneti eletion of -Al n phrmologil inhiition of -Al mrkely reue lipi umultion in BAT (Figs h, h). We exmine fetures relting to BAT tivtion, inluing energy expeniture n Up expression. Unexpetely, neither geneti eletion nor nilotini tretment h ny effet on these funtions (Figs f n i l). Hene, we rgue tht the reue ipoyte size might e ue to whole-oy insulin sensitivity. Lst, we explore the mehnism y whih -Al eletion or -Al inhiition improves insulin sensitivity. HFD is known to inue low-gre inflmmtion in ipose tissue [7] n - Al inhiition hs een shown to erese the proinflmmtory response in mrophges []. However, we foun tht -Al speifi eletion i not lok HFD-inue inflmmtion in ipose tissue (ESM Fig. ), initing tht inflmmtion might not e the mjor ftor involve in -Almeite insulin resistne. We then sought to etermine whether ipokines ply role in -Al-meite insulin resistne. Interestingly, we foun tht iponetin, minly serete from suutneous ipose tissue [7 9], ws signifintly inrese y -Al speifi eletion or -Al inhiition uner HFD hllenge. However, there ws no signifint hnge in the level of resistin, whih is minly serete from viserl ft [, ]. Colletively, we propose tht -Al promotes insulin resistne n oesity y regulting iponetin seretion in suutneous ft in the presene of HFD hllenge (Fig. 7e). Tken together, our stuy highlights the importnt role of - Al in oesity-ssoite insulin resistne. Inhiiting the tivtion of -Al or speifilly reuing its expression in suutneous ft might e promising therpeuti option for the tretment of oesity n relte metoli isorers. Aknowlegements We thnk the memers of the Yun lortory for ritil reing of the mnusript n helpful isussion. Dt vilility All t supporting the finings of this stuy re ville within the rtile n its ESM. Funing This work ws supporte y the Ntionl Siene Fountion of Chin (Grnt no., n ), the Ntionl Bsi Reserh Progrm of Chin (97-CB97 n DFA99) n Cross-isiplinry Collortive Tems Progrm for Siene, Tehnology n Innovtion (-) from the Chinese Aemy of Sienes. Contriution sttement RW n ZY esigne the stuy n rfte the mnusript. RW, GN, WJ n ZY ontriute to the stuy esign n revise the rtile s intelletul ontent. RW n J-GS performe most of the experiments. J-QW helpe to ollet humn tissue smples n quire t. BL n QL helpe to perform the nilotini tretment of mie n quire t. RW, J-GS, WJ n ZYnlyse the t. J-GS, J- QW, BL n QL revise the rtile n ll uthors pprove the finl version. ZY is responsile for the integrity of this work. Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. Referenes. Ng M, Fleming T, Roinson M et l () Glol, regionl, n ntionl prevlene of overweight n oesity in hilren n ults uring 9-: systemti nlysis for the Glol Buren of Disese Stuy. Lnet :7 7. Lozno R, Nghvi M, Foremn K et l () Glol n regionl mortlity from uses of eth for ge groups in 99 n : systemti nlysis for the Glol Buren of Disese Stuy. Lnet :9. Zimmet P, Thoms CR () Genotype, oesity n riovsulr isese hs tehnil n soil vnement outstrippe evolution? J Intern Me :. Sltiel AR, Khn CR () Insulin signlling n the regultion of gluose n lipi metolism. Nture :799. Ekel RH, Gruny SM, Zimmet PZ () The metoli synrome. Lnet :. Khn SE, Hull RL, Utzshneier KM () Mehnisms linking oesity to insulin resistne n type ietes. Nture : 7. 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