Leptolide improves insulin resistance in diet-induced obese mice

ma ine d ugs A icle Lep olide Imp o es Insulin Resis ance in Die -Induced Obese Mice Pablo Villa-Pé ez 1, Me cedes Cue o 2, Ana R. Díaz-Ma e o 3ID , Ca men D. Loba ón1ID , Al edo Mo eno 1, Ge mán Pe domo 4and I ene Cóza -Cas ellano 1,*ID 1Ins i u o de Biología y Gené ica Molecula , Uni e si y o Valladolid-CSIC, Valladolid 47005, Spain; [email p o ec ed] (P.V.); [email p o ec ed] (C.D.L.); [email p o ec ed] (A.M.) 2Ins i u o de P oduc os Na u ales y Ag obiología (CSIC), La Laguna 38206, Spain; [email p o ec ed] 3Ins i u o Uni e si a io de Bioo gánica “A. González”, Uni e si y o La Laguna, La Laguna 38206, Spain; [email p o ec ed] 4School o Nu se y, Uni e si y o Bu gos, Bu gos 09001, Spain; gmpe [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +34-983184005; Fax: +34-983423588 Recei ed: 18 July 2017; Accep ed: 13 Sep embe 2017; Published: 15 Sep embe 2017 Abs ac : Type 2 diabe es (T2DM) is a complex disease linked o panc ea ic be a-cell ailu e and insulin esis ance. Cu en an idiabe ic ea men egimens o T2DM include insulin sensi ize s and insulin sec e agogues. We ha e p e iously demons a ed ha lep olide, a membe o he u anocemb anolides amily, p omo es panc ea ic be a-cell p oli e a ion in mice. Conside ing he bene icial e ec s o lep olide in diabe ic mice, in his s udy, we aimed o add ess he capabili y o lep olide o imp o e insulin esis ance associa ed wi h he pa hology o obesi y. To his end, we es ed he hypo hesis ha lep olide should p o ec agains a y acid-induced insulin esis ance in hepa ocy es. In a ime-dependen manne , lep olide (0.1 µ M) augmen ed insulin-s imula ed phospho yla ion o p o ein kinase B (PKB) by wo- old abo e ehicle- ea ed HepG2 cells. In addi ion, lep olide (0.1 µ M) coun e ac ed palmi a e-induced insulin esis ance by augmen ing by ou - old insulin-s imula ed phospho yla ion o PKB in HepG2 cells. In i o , acu e in ape i oneal adminis a ion o lep olide (0.1 mg/kg and 1 mg/kg) imp o ed glucose ole ance and insulin sensi i i y in lean mice. Likewise, p olonged lep olide ea men (0.1 mg/kg) in die -induced obese mice imp o ed insulin sensi i i y. These e ec s we e pa alleled wi h an ~50% inc eased o insulin-s imula ed phospho yla ion o PKB in li e and skele al muscle and educed ci cula ing p o-in lamma o y cy okines in obese mice. We concluded ha lep olide signi ican ly imp o es insulin sensi i i y in i o and in obese mice, sugges ing ha lep olide may be ano he po en ial ea men o T2DM. Keywo ds: lep olide; insulin esis ance; obesi y; ype 2 diabe es; HepG2 cells 1. In oduc ion Insulin esis ance is one o he hallma ks o ype 2 diabe es (T2DM) and obesi y. Imp o emen o insulin sensi i i y is an indispensable s ep o alle ia e T2DM. The i s phase o T2DM is cha ac e ized by panc ea ic be a-cell compensa ion, displaying hype insulinemia in esponse o insulin esis ance. Be a-cell o e wo king equen ly end in dys unc ion and cell dea h. A his poin , dec eased blood insulin le els exace ba e he onse o T2DM due o bo h insulin de iciency and esis ance [1]. To his day, pha macological managemen o T2DM pa ien s aims o achie e he bes possible glycemic con ol, while a oiding hypoglycemia. Howe e , he na u al his o y o T2DM includes mul iple dys unc ions a ec ing he α -cells, β -cells, li e , skele al muscle, adipose issue, he gas oin es inal ac , kidney and b ain, wha has been e med he ominous oc e [ 2 ]. This complex Ma . D ugs 2017,15, 289; doi:10.3390/md15090289 www.mdpi.com/jou nal/ma ined ugs Ma . D ugs 2017,15, 289 2 o 14 scena io o he pa hophysiology o T2DM equi es a shi in he cu en pa adigm o he ea men o he disease. A keys one in he managemen o diabe es is nu i ional he apy along wi h egula physical ac i i y. Conside ing ha mos people wi h T2DM a e o e weigh o obese, weigh loss is ecommended o imp o e glycemic con ol [ 3 ]. In addi ion, egula physical ac i i y has demons a ed signi ican heal h bene i s [ 4 ]. When li es yle in e en ions ail in he app op ia e con ol o glucose homeos asis, T2DM is ini ially ea ed by mono he apy wi h o al agen s, and e en ually, i may equi e he combina ion o mul iple d ugs. In his way, insulin sensi ize s and insulin sec e agogues a e sa e he apies o T2DM ea men . Among he insulin sensi ize s, me o min is success ully used as a i s line pha maco he apy o he ea men o T2DM pa ien s. The majo e ec o his d ug is he acu e inhibi ion o hepa ic gluconeogenesis h ough mechanisms in ol ing he di ec inhibi ion o he mi ochond ial espi a o y-chain complex I, leading o ac i a ion o AMP-ac i a ed p o ein kinase (AMPK) [ 5 ]. The pleio opic e ec s o me o min lead o lowe as ing blood glucose and insulin le els wi h minimal isk o hypoglycemia [6]. When glycemic con ol canno be achie ed using me o min as mono he apy, a second line o d ugs is added o me o min ea men . Among hese ea men s, sul onylu eas imp o e insulin sec e ion by he egula ion o he ATP-sensi i e po assium channels in he plasma memb ane o panc ea ic β -cells [ 7 ]. Thiazolidinediones, agonis s o nuclea pe oxisome p oli e a o -ac i a ed ecep o gamma (PPAR γ ), inc ease insulin sensi i i y in li e and skele al muscle [ 8 ]. Glinides s imula e insulin sec e ion apidly and o a sho pe iod when needed [ 9 ]. Inc e ins, such as glucagon-like pep ide 1 (GLP-1) ecep o agonis s, gas ic inhibi o y polypep ide/glucose-dependen insulino opic pep ide (GIP) and dipep idyl-pep idase-4 (DPP-4) inhibi o s, inc ease insulin sec e ion and egula e glucose homeos asis [ 10 ]. As he disease p og esses, he β -cell unc ion declines, and insulin he apy become necessa y. The side e ec s o hypoglycemia and weigh gain limi he e icacy o his he apy [ 11 ]. Howe e , no el insulin p epa a ions and deli e y sys ems a e a ailable o op imize insulin he apy [ 12 ]. Rega ding new glucose-lowe ing he apies, sodium glucose co anspo e 2 (SGLT2) ecep o inhibi o s lowe blood glucose le els in an insulin-independen manne by inc easing enal glucose exc e ion [ 13 ]. P omising new pha macological a ge s a e cu en ly unde e alua ion, such as si uin agonis s, which enhance insulin sec e ion and/o insulin sensi i i y; o he inhibi o s o p o ein y osine phospha ase 1B, which p olong he ac ion o insulin [10]. Fu anocemb anolides a e polyoxygena ed di e penoids, isola ed om co als, in which a u anic ing and a β -lac one subuni a e inse ed in a cemb ane skele on [ 14 ]. Lep olide is a membe o he u anocemb anolide amily, which was isola ed o he i s ime by Gu ié ez e al. in 2005 [ 14 ] om he oc oco als Lep ogo gia alba and Lep ogo gia igida on he Paci ic coas o Panama. I has been p oposed ha membe s o his amily, such as pukalide, may unc ion in na u e as a de ensi e oxin agains po en ial oc oco al p eda o s [15,16]. The po en ial pha macological use o his amily o compounds is la gely unexplo ed. Al hough, ew examples ha e been epo ed. Among hem, lopho oxin is a neu omuscula oxin ha binds selec i ely and i e e sibly wi hin he ace ylcholine- ecogni ion si e o nico inic ace ylcholine ecep o s, he eby p e en ing ace ylcholine om ac i a ing i s ecep o [ 17 , 18 ]. The an ip oli e a i e and cy o oxic ac i i ies o some o hese compounds ha e been s udied agains he cell lines L-929, K-562, HeLA, MDA-MB-231, A-549, HT-29 and P388 showing weak an ip oli e a i e and cy o oxic p ope ies [ 19 – 21 ]. Fu he mo e, he an iplasmodial ac i i y o six u anocemb anolides and he i egula pseudop e olide isola ed om specimens o Lep ogo gia alba and L. igida was e alua ed, and among hem, lep olide and pukalide showed no biological ac i i y agains he pa asi e [14]. Lep olide, among o he membe s o i s amily, has al eady been shown o inc ease panc ea ic be a-cell p oli e a ion in i o , in INS1cells (Insulin sec e ing be a cell de i ed line) and p ima y cul u es o oden panc ea ic isle s [ 22 ]. In addi ion, epoxypukalide, ano he molecule o his amily, has been shown o imp o e be a-cell p o ec ion in i o and in i o , in oden isle s and in a STZ (s ep ozo ocin)-induced model o diabe es, espec i ely [ 22 , 23 ]. In e es ingly, epoxypukalide also Ma . D ugs 2017,15, 289 3 o 14 alle ia es glucose in ole ance in a p eclinical model o ype 1 diabe es [ 23 ]. Thus, u anocemb anolides appea o be a ac i e molecules o main ain unc ional be a-cell mass and glycemic con ol. In his wo k, we ha e ex ended ou ini ial indings and explo ed he capabili y o lep olide o imp o e insulin sensi i i y. To his end, we ha e assessed he capaci y o lep olide o enhance insulin signaling in insulin- esis an hepa ocy es and in he li e and skele al muscle o die -induced obese mice. 2. Ma e ials and Me hods Lep olide pu i ica ion, cha ac e iza ion and molecula s uc u e we e desc ibed p e iously [ 14 ]. B ie ly, c ude ex ac s om oc oco als we e subjec ed o ac iona ion. Lep olide was ini ially isola ed as a no el compound wi h an iplasmodial ac i i y, and i s s uc u e was de e mined by NMR and con i med by single-c ys al X- ay c ys allog aphy. 2.1. Cell Cul u e HepG2 cells we e ob ained om he Ame ican Type Cul u e Collec ion (ATCC, Manassas, VA, USA; #HB-8065). The cell line was o iginally isola ed om a li e hepa ocellula ca cinoma o a 15-yea -old Caucasian male. Cells we e g ow h in DMEM (1X) supplemen ed wi h 4.5 g/L D-glucose, 0.6 g/L L-glu amine, 0.1 g/L sodium py u a e and 10% e al bo ine se um. In o de o analyze he e ec s o lep olide on he in acellula insulin signaling pa hway, HepG2 cells we e ea ed wi h 0.1 µ M lep olide o ehicle (DMSO) du ing 24 h in medium wi hou se um. A e wa ds, 100 nM human insulin (Sigma, S . Louis, MO, USA) was added, and HepG2 cells we e collec ed a e 0, 5, 10, 15 and 30 min. To analyze he e ec s o lep olide in he se ing o esis ance, HepG2 cells we e ea ed wi h 0.2 mM palmi a e and 0.1 µ M lep olide in se um- ee medium o 24 h. A e wa ds, 100 nM human insulin (Sigma, S . Louis, MO, USA) was added, and 15 min la e , HepG2 cells we e collec ed. 2.2. Animal P ocedu es C57Bl6J male mice we e pu chase om Cha les Ri e Labo a o y (Écully, F ance). Male mice we e chosen o me abolic pheno yping o a oid he po en ial a iabili y ela ed o es ous cycle. Expe imen al p ocedu es we e app o ed by he Animal Ca e and Use Commi ee o he Uni e si y o Valladolid (UVa), Valladolid, Spain, in acco dance wi h he Eu opean and Spanish Guidelines o he Ca e and Use o Mammals in Resea ch. Mice we e ed wi h s anda d oden chow and wa e ad libi um in en ila ed cages in a 12:12-h ligh /da k cycle. “Acu e” adminis a ion o lep olide was pe o med in 12-week-old males ed a s anda d die (SD) (33% p o ein; 58% ca bohyd a e; 9% a ) (#V1535, Ssni , Soes , Ge many) a he indica ed doses (0.1 mg/kg and 1 mg/kg o body weigh ). “Ch onic” adminis a ion o lep olide was pe o med in 6-week-old male mice ed a 60% kcal high a die (HFD) (20% p o ein; 20% ca bohyd a e; 60% a ) (#D12492, Resea ch Die s, New B unswick, NJ, USA) o 10 weeks. A e 6 weeks o eeding wi h he HFD, mice we e andomly di ided in o wo g oups, which we e ea ed wi h once-daily ip injec ion o lep olide (0.1 mg/kg o body weigh ) o ehicle (DMSO) o ano he 4 weeks. All mice we e main ained on HFD du ing he 4-week ea men . The day be o e sac i ice, mice we e as ed o e nigh , ollowed by an ip insulin o saline injec ion, and 10 min la e , mice we e eu hanized o li e and skele al muscle issues dissec ion as desc ibed p e iously [24]. Fas ing o non- as ing blood was collec ed om he ail ein in o capilla y ubes p ecoa ed wi h po assium-EDTA (Sa s ed , Nümb ech , Ge many) o he p epa a ion o plasma o he de e mina ion o blood glucose le els using he B eeze 2 glucome e (Baye , Le e kusen, Ge many) as p e iously desc ibed [ 24 ]. Insulin le els we e measu ed using he ul asensi i e mouse ELISA assay (Me codia, Uppsala, Sweden). T iglyce ides we e measu ed using a iglyce ides ki (Biosys ems, Ba celona, Spain). Cy okines and lep in we e measu ed using Bio-Plex Luminex Immunoassays (Bio-Rad, He cules, CA, Ma . D ugs 2017,15, 289 4 o 14 USA). The de ec ion limi s o TNF- α , IL-1, IL6, insulin and lep in we e 4.0 pg/mL, 1.6 pg/mL, 0.25 pg/mL, 0.025 pg/mL and 4.9 pg/mL espec i ely. 2.3. Glucose and Insulin Tole ance Tes s, Glucose Decay and HOMA Indexes The in ape i oneal glucose ole ance es (ip-GTT) was pe o med, 30 min a e “acu e” ea men o 4 weeks a e “ch onic” ea men , as p e iously desc ibed [ 24 ]. B ie ly, mice we e as ed o e nigh (15 h) ollowing he ecommended s anda d ope a ing p ocedu e o pheno yping mice by he Eumo phia Conso ium [ 25 ]. A e wa ds, mice we e in ape i oneally injec ed wi h 2 g glucose/kg o body weigh . Blood glucose le els we e de e mined a 0, 15, 30, 60 and 120 min and plo ed as a unc ion o ime. Likewise, he insulin ole ance es (ip-ITT) was pe o med, 30 min a e “acu e” ea men o 4 weeks a e “ch onic” ea men , as p e iously desc ibed [ 24 ]. Fo ip-ITT, non- as ed mice we e injec ed (1 U/kg o body weigh ) wi h insulin (Lilly, Indianapolils, IN, USA). Blood glucose le els we e de e mined a 0, 15, 30, 60 and 90 min and plo ed as a unc ion o ime. Ip-GTT and ip-ITT expe imen s we e pe o med using he same g oup o mice. The imeline o he expe imen s was i s he ip-GTT assays. Then, we le mice eco e o h ee days, and ip-ITT expe imen s we e pe o med. Glucose decay was calcula ed om he glucose measu emen s ob ained du ing he insulin ole ance es . The measu emen s we e con e ed in o na u al loga i hm (Ln); he slope was calcula ed using linea eg ession ( ime × Ln[glucose]) and mul iplied by 100 o ob ain he glucose decay cons an a e pe minu e (%/min). The homeos asis model assessmen (HOMA) es ima es s eady s a e be a cell unc ion (%B), insulin sensi i i y (%S), he in e se o %S and he insulin esis ance (HOMA-IR). The HOMA Calcula o so wa e is eely a ailable a he Uni e si y o Ox o d, Uni ed Kingdom, a he web page www.d u.ox. ac.uk/homacalcula o . 2.4. Wes e n-Blo Analysis HepG2 cells we e p eincuba ed in he p esence o absence o lep olide a he abo e indica ed imes and doses. A he end o he incuba ion pe iod, cul u e media we e disca ded and cells collec ed and washed wi h ice-cold PBS, ollowed by homogeniza ion in lysis bu e (20 mmol/L T is · HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% ( ol/ ol) T i on X-100, 2.5 mmol/L sodium py ophospha e, 1 mmol/L µ -glyce ophospha e, 1 mmol/L Na 3 VO 4 , 1 µ g/mL leupep in and 1 mmol/L phenylme hylsul onyl luo ide) plus p o ease inhibi o s (P o ease Inhibi o Cock ail; Sigma). A e 10 min on ice, ex ac s we e sonica ed and cen i uga ed a 18,000 × g o 10 min a 4 ◦ C. Pelle s we e disca ded, and solubilized p o eins (~20–40 µ g/sample) we e esol ed by 10% SDS-PAGE o an i-p-PKB (Se 473) (1:1000; Cell Signaling, Dan e s, MA, USA) and elec o ans e ed on o poly inylidene di luo ide il e s (PDVF Immobilon-P memb ane (Millipo e, Bille ica, MA, USA)) o immunoblo ing by con en ional means. A e p obing wi h speci ic an ibodies, he memb anes we e s ipped and ep obed wi h an ibody agains ac in (1:3000; Sigma) and PKB (1:1000; Cell Signaling). Signals we e de ec ed by chemiluminescence (Immun-S a Wes e n Chemiluminescence Ki ; Bio-Rad, Mad id, Spain), and band densi ome y was quan i ied wi h Image J so wa e (Na ional Ins i u es o Heal h, Be hesda, MD, USA). Fo animal issues, li e and skele al muscle om mice, s imula ed o no wi h insulin, we e homogenized wi h a poly on (OMNI, Kennesaw, GA, USA) in cell lysis bu e (Cell Signaling, USA) in he p esence o p o ease/phospha ase inhibi o s. As desc ibed o HepG2 cells, ~40–60 µ g/sample we e esol ed in 10%-SDS PAGE o an i-pPKB, PKB and ac in. 2.5. S a is ical Analysis S a is ical analysis o da a was pe o med using he G aphPad P ism So wa e 6.0 (La Jolla, CA, USA). Dis ibu ions we e checked wi h he Kolmogo o –Smi no es . Da a a e p esen ed as he means ± S.E.M. Homogenei y o a iance was pe o med using he Le ene es . Compa isons be ween wo g oups we e done using he unpai ed S uden ’s - es (i homogenei y o a iance) o he Welch es Ma . D ugs 2017,15, 289 5 o 14 (i he e ogenei y o a iance) when a a iable was dis ibu ed no mally; in he case o a non-pa ame ic a iable, he Mann–Whi ney U- es was used. Compa isons be ween mo e han wo g oups we e done using he one-way ANOVA o he K uskal–Wallis es when a a iable was dis ibu ed no mally o non-no mally, espec i ely. Fo pos -hoc analyses, he Bon e oni es o Dunne ’s es was used i he e was homogenei y o he e ogenei y o a iance, espec i ely. Di e ences we e conside ed signi ican a p< 0.05. 3. Resul s 3.1. Lep olide Imp o es Insulin Signaling in HepG2 Cells To s udy he e ec s o lep olide in basal and insulin esis ance condi ions, we ha e used a human hepa oma cell line (HepG2). To dis ega d he possibili y o a cy o oxic e ec o lep olide on HepG2 cells, 750,000 cells we e pla ed and ea ed wi h ehicle o lep olide o 24 h (n= 4 independen expe imen s). Li ing cells we e collec ed a e wa ds, and p o eins we e ex ac ed and quan i ied showing no di e ences in p o ein con en in lep olide- e sus ehicle- ea ed cells (4.42 ± 0.53 µ g/ µ L e sus 4.30 ± 0.77 µ g/ µ L). Vehicle- ea ed cells showed a ime-dependen ac i a ion (5 min) o he insulin signaling pa hway a e insulin s imula ion. Lep olide (0.1 µ M) inc eased insulin signaling ~1.3–2.0- old compa ed o ehicle- ea ed cells (Figu e 1). This imp o emen in insulin sensi i i y is sus ained unde palmi a e-induced insulin esis ance (Figu e 2). HepG2 cells we e ea ed wi h 0.2 mM palmi ic acid o 24 h o cause insulin esis ance, a he same ime cells we e ea ed wi h ehicle o 0.1 µ M lep olide. Con ol cells showed an inc eased p-PKB/PKB a io in basal condi ions, which was impai ed unde insulin esis ance condi ions. In e es ingly, lep olide ea men no only inc eases insulin signaling in basal condi ions, bu also coun e ac ed palmi a e-induced insulin esis ance (Figu e 2). These esul s highligh ha lep olide is a molecule wi h he capaci y o enhancing sensi i i y a basal condi ions and palmi a e-induced insulin esis ance in HepG2 cells. Ma .D ugs2017,15,289 5o 14 Dunne ’s es wasusedi  he ewashomogenei yo he e ogenei yo  a iance, espec i ely. Di e enceswe econside edsigni ican a p<0.05. 3.Resul s 3.1.Lep olideImp o esInsulinSignalinginHepG2Cells Tos udy hee ec so lep olideinbasalandinsulin esis ancecondi ions,weha eusedahuman hepa omacellline(HepG2).Todis ega d hepossibili yo acy o oxice ec o lep olideonHepG2 cells,750,000cellswe epla edand ea edwi h ehicleo lep olide o 24h(n=4independen  expe imen s).Li ingcellswe ecollec eda e wa ds,andp o einswe eex ac edandquan i ied showingnodi e encesinp o eincon en inlep olide‐ e sus ehicle‐ ea edcells(4.42±0.53μg/μL e sus4.30±0.77μg/μL).Vehicle‐ ea edcellsshoweda ime‐dependen ac i a ion(5min)o  he insulinsignalingpa hwaya e insulins imula ion.Lep olide(0.1μM)inc easedinsulinsignaling ~1.3–2.0‐ oldcompa ed o ehicle‐ ea edcells(Figu e1).Thisimp o emen ininsulinsensi i i yis sus ainedunde palmi a e‐inducedinsulin esis ance(Figu e2).HepG2cellswe e ea edwi h 0.2mMpalmi icacid o 24h ocauseinsulin esis ance,a  hesame imecellswe e ea edwi h ehicleo 0.1μMlep olide.Con olcellsshowedaninc easedp‐PKB/PKB a ioinbasalcondi ions, whichwasimpai edunde insulin esis ancecondi ions.In e es ingly,lep olide ea men no only inc easesinsulinsignalinginbasalcondi ions,bu alsocoun e ac edpalmi a e‐inducedinsulin esis ance(Figu e2).These esul shighligh  ha lep olideisamoleculewi h hecapaci yo  enhancingsensi i i ya basalcondi ionsandpalmi a e‐inducedinsulin esis anceinHepG2cells.  Figu e1.Lep olideaugmen sinsulinsignalinginHepG2cells.Wes e nblo analysiso PKBand p‐PKBinHepG2cells ea edwi hlep olide(0.1μM)o  ehicleands imula edwi h100mMinsulin o 5,10,15and30min.Lep olideac iononHepG2cellsenhancedinsulinsignalinga all imepoin s. Valuesa e hemeans±S.E.M.o n=6pe condi ion.They‐axis ep esen s he a ioo phospho yla ed e sus o alp o einina bi a yuni s.Ac inwasde ec edasaloadingcon ol.*p<0.05 e sus =0 byANOVA; $ p<0.05 e sus ehiclebyANOVA. Figu e 1. Lep olide augmen s insulin signaling in HepG2 cells. Wes e n blo analysis o PKB and p-PKB in HepG2 cells ea ed wi h lep olide (0.1 µ M) o ehicle and s imula ed wi h 100 mM insulin o 5, 10, 15 and 30 min. Lep olide ac ion on HepG2 cells enhanced insulin signaling a all ime poin s. Values a e he means ± S.E.M. o n= 6 pe condi ion. The y-axis ep esen s he a io o phospho yla ed e sus o al p o ein in a bi a y uni s. Ac in was de ec ed as a loading con ol. * p< 0.05 e sus = 0 by ANOVA; $p< 0.05 e sus ehicle by ANOVA. Ma . D ugs 2017,15, 289 6 o 14 Ma .D ugs2017,15,289 6o 14  Figu e2.Lep olidep e en spalmi a e‐inducedinsulin esis anceinHepG2cells.Wes e nblo  analysiso PKBandp‐PKBinHepG2cells ea edwi hlep olide(0.1μM)o  ehicle,in hep esence o absenceo palmi a e.Cellswe es imula edwi h100mMinsulin o 15min.Lep olideac ionon HepG2cellsenhancedinsulinsignaling,p e en ingpalmi a e’snega i ee ec .Valuesa e he means±S.E.M.o n=4pe condi ion.They‐axis ep esen s he a ioo phospho yla ed e sus o al p o einina bi a yuni s.Ac inwasde ec edasaloadingcon ol.*p<0.05 e susnon‐s imula ed insulincellsbyANOVA. 3.2.Acu eT ea men wi hLep olideImp o esGlucoseTole anceandInsulinSensi i i yinLeanMice Toco obo a eou  indingsin i o,weacu elyinjec edlep olidea  wodi e en concen a ions (0.1and1mg/kg)inC57BL6Jmalemice.Eachdoseo lep olideandi s espec i econ olwe e adminis e edaspa o independen expe imen s,and heywe epe o medonsepa a edays. Vehicleda awe epooled ombo hexpe imen s.Lep olidewasinjec ed30minbe o eip‐GTTin as edmice( imepoin −30).A  hesame ime,plasmaglucosele elswe eassessed( imepoin −30). A e wa ds,aboluso glucosewasadminis e edin ape i oneallyasdesc ibedin heMa e ialsand Me hodsSec ion.Plasmaglucosele elswe emoni o eda 15,30,60,90and120mina e glucose challenge.AsshowninFigu e3A,B,bo hlep olideconcen a ionsdisplayedimp o edglucose ole ance.Inadi e en g oupo expe imen s,lep olidewasinjec edp e ious oip‐ITTshowing inc easedinsulinsensi i i ya bo hlep olideconcen a ions(Figu e3C,D).Likewise,acu e adminis a iono lep olide(0.1and1mg/kg)imp o edinsulinsensi i i yinleanmice(Figu e3C,D). Consis en wi h hesee ec so lep olideonwhole‐bodyglucosehomeos asis,non‐ as ingplasma glucosele elswe e educed(p=0.06)inmice ea edwi hahighdoseo lep olide(Figu e3E,F). Collec i ely, hese esul sdemons a e ha bo hdoseso lep olideha eaposi i ee ec on imp o inginsulinsensi i i yandglucosehomeos asis.In iewo  heseda a,weha echosen he lowes doseo lep olide ope o m henex expe imen sindie ‐inducedobesemice. Figu e 2. Lep olide p e en s palmi a e-induced insulin esis ance in HepG2 cells. Wes e n blo analysis o PKB and p-PKB in HepG2 cells ea ed wi h lep olide (0.1 µ M) o ehicle, in he p esence o absence o palmi a e. Cells we e s imula ed wi h 100 mM insulin o 15 min. Lep olide ac ion on HepG2 cells enhanced insulin signaling, p e en ing palmi a e’s nega i e e ec . Values a e he means ± S.E.M. o n = 4 pe condi ion. The y-axis ep esen s he a io o phospho yla ed e sus o al p o ein in a bi a y uni s. Ac in was de ec ed as a loading con ol. * p< 0.05 e sus non-s imula ed insulin cells by ANOVA. 3.2. Acu e T ea men wi h Lep olide Imp o es Glucose Tole ance and Insulin Sensi i i y in Lean Mice To co obo a e ou indings in i o , we acu ely injec ed lep olide a wo di e en concen a ions (0.1 and 1 mg/kg) in C57BL6J male mice. Each dose o lep olide and i s espec i e con ol we e adminis e ed as pa o independen expe imen s, and hey we e pe o med on sepa a e days. Vehicle da a we e pooled om bo h expe imen s. Lep olide was injec ed 30 min be o e ip-GTT in as ed mice ( ime poin − 30). A he same ime, plasma glucose le els we e assessed ( ime poin − 30). A e wa ds, a bolus o glucose was adminis e ed in ape i oneally as desc ibed in he Ma e ials and Me hods Sec ion. Plasma glucose le els we e moni o ed a 15, 30, 60, 90 and 120 min a e glucose challenge. As shown in Figu e 3A,B, bo h lep olide concen a ions displayed imp o ed glucose ole ance. In a di e en g oup o expe imen s, lep olide was injec ed p e ious o ip-ITT showing inc eased insulin sensi i i y a bo h lep olide concen a ions (Figu e 3C,D). Likewise, acu e adminis a ion o lep olide (0.1 and 1 mg/kg) imp o ed insulin sensi i i y in lean mice (Figu e 3C,D). Consis en wi h hese e ec s o lep olide on whole-body glucose homeos asis, non- as ing plasma glucose le els we e educed (p= 0.06) in mice ea ed wi h a high dose o lep olide (Figu e 3E,F). Collec i ely, hese esul s demons a e ha bo h doses o lep olide ha e a posi i e e ec on imp o ing insulin sensi i i y and glucose homeos asis. In iew o hese da a, we ha e chosen he lowes dose o lep olide o pe o m he nex expe imen s in die -induced obese mice. Ma . D ugs 2017,15, 289 7 o 14 Ma .D ugs2017,15,289 7o 14   Figu e3.Acu elep olideadminis a ionimp o esglucose ole anceandinsulinsensi i i yinlean mice.C57Bl6J12‐week‐oldmales edas anda ddie we einjec edwi h ehicleo lep olide30min be o e hein ape i onealglucose ole ance es (ip‐GTT)o  heinsulin ole ance es (ip‐ITT). (A)Glucose ole ance es o miceinjec edwi hlep olide(0.1mg/kglep olideo 1mg/kg)o  ehicle. (B)A eaunde  hecu eo  heip‐GTT.Glucose ole ancewasimp o edusingbo hlep olide concen a ionscompa ed o he ehicle.(C)Insulin ole ance es o miceinjec edwi hlep olide (0.1mg/kglep olideo 1mg/kg)o  ehicle.(D)A eaunde  hecu eo  heip‐ITT.Insulinsensi i i y wasimp o edusingbo hlep olideconcen a ionscompa ed o ehicle.(E)Fas ingand (F)non‐ as ingbloodglucosele elsa e  hi yminu eso lep olideadminis a ion.Fas ingglucose le elswe eunchanged;meanwhile,only1mg/kglep olideimp o ednon‐ as ingglucose.Valuesa e hemeans±S.E.M.o n=12( ehicle);n=6(lep olide0.1mg/kg);n=6(lep olide1mg/kg).*p<0.05 e sus ehiclebyANOVA.  AB CD AUC (A bi a y uni s) Vehicle Lep olide 0.1 mg/kg Lep olide 1 mg/kg Glucose (mg/dL) EF Vehicle Lep olide 0.1 mg/kg Lep olide 1 mg/kg Figu e 3. Acu e lep olide adminis a ion imp o es glucose ole ance and insulin sensi i i y in lean mice. C57Bl6J 12-week-old males ed a s anda d die we e injec ed wi h ehicle o lep olide 30 min be o e he in ape i oneal glucose ole ance es (ip-GTT) o he insulin ole ance es (ip-ITT). ( A ) Glucose ole ance es o mice injec ed wi h lep olide (0.1 mg/kg lep olide o 1 mg/kg) o ehicle. ( B ) A ea unde he cu e o he ip-GTT. Glucose ole ance was imp o ed using bo h lep olide concen a ions compa ed o he ehicle. ( C ) Insulin ole ance es o mice injec ed wi h lep olide (0.1 mg/kg lep olide o 1 mg/kg) o ehicle. ( D ) A ea unde he cu e o he ip-ITT. Insulin sensi i i y was imp o ed using bo h lep olide concen a ions compa ed o ehicle. ( E ) Fas ing and ( F ) non- as ing blood glucose le els a e hi y minu es o lep olide adminis a ion. Fas ing glucose le els we e unchanged; meanwhile, only 1 mg/kg lep olide imp o ed non- as ing glucose. Values a e he means ± S.E.M. o n= 12 ( ehicle); n = 6 (lep olide 0.1 mg/kg); n = 6 (lep olide 1 mg/kg). * p< 0.05 e sus ehicle by ANOVA. 3.3. P olonged Lep olide Adminis a ion Imp o es Li e and Muscle Insulin Resis ance in Die -Induced Obese Mice Nex , we es ed he hypo hesis ha adminis a ion o lep olide imp o es insulin sensi i i y in obese mice. To his end, C57BL6J mice we e ed a high a die (60% kcal o a ) o en weeks. Ma . D ugs 2017,15, 289 8 o 14 The las ou weeks, mice we e in ape i oneally injec ed wi h lep olide (0.1 mg/kg) once a day. A he end o he ea men , insulin sensi i i y was assessed by ip-ITT and he HOMA index in all mice. To in es iga e he impac o lep olide in he in acellula insulin signaling pa hway in li e and skele al muscle issues, one-hal o mice (con ol and lep olide g oups) we e injec ed wi h insulin o 10 min, whe eas he o he hal was injec ed wi h saline. A e wa ds, mice we e eu hanized and sac i iced o he dissec ion o li e o skele al muscle issues. Da a in Figu es 4–6we e pooled om wo disc e e expe imen s using wo di e en g oups o mice. P olonged adminis a ion o lep olide imp o ed glucose ole ance (Figu e 4A,B) and insulin sensi i i y (Figu e 4C,D) in obese mice. Consis en ly, glucose decay du ing he ip-ITT du ing he i s 30 min was imp o ed in he lep olide- ea ed g oup (Figu e 4E). Fu he mo e, he insulin sensi i i y index (%S) was signi ican ly inc eased (Figu e 4F), which was pa alleled wi h a educed HOMA-IR index (p= 0.06; Figu e 4G), al hough body weigh was no signi ican ly dec eased (Figu e 4H). In addi ion, as ing and non- as ing plasma insulin le els we e educed in lep olide- ea ed g oup mice (Figu e 4I,J), which is consis en wi h he no ion o imp o ing insulin sensi i i y. Finally, plasma iglyce ide le els we e signi ican ly educed in obese mice ea ed wi h lep olide (Figu e 4K), which nicely co ela es wi h a 30% educ ion in li e iglyce ide con en in lep olide- e sus ehicle- ea ed mice (Figu e 4L). Taken oge he , hese da a demons a e ha ch onic lep olide adminis a ion coun e ac ed insulin esis ance and imp o ed lipid me abolism in die -induced obese mice. To gain insigh in o he molecula mechanisms by which lep olide imp o ed insulin esis ance, we analyzed le els o ci cula ing p o-in lamma o y cy okines. As shown in Figu e 5, ch onic lep olide ea men was associa ed wi h a non-s a is ically-signi ican dec ease in blood le els o IL-1 β and TNF- α , bu IL-6 le els emained unchanged (Figu e 5A–C). Likewise, lep olide signi ican ly educed he weigh gain du ing he las week o he ea men (Figu e 5D), in pa allel wi h educed plasma lep in le els in obese mice (Figu e 5F). Ma .D ugs2017,15,289 8o 14 3.3.P olongedLep olideAdminis a ionImp o esLi e andMuscleInsulinResis anceinDie ‐Induced ObeseMice Nex ,we es ed hehypo hesis ha adminis a iono lep olideimp o esinsulinsensi i i yin obesemice.To hisend,C57BL6Jmicewe e edahigh a die (60%kcalo  a ) o  enweeks.The las  ou weeks,micewe ein ape i oneallyinjec edwi hlep olide(0.1mg/kg)onceaday.A  he endo  he ea men ,insulinsensi i i ywasassessedbyip‐ITTand heHOMAindexinallmice.To in es iga e heimpac o lep olidein hein acellula insulinsignalingpa hwayinli e andskele al muscle issues,one‐hal o mice(con olandlep olideg oups)we einjec edwi hinsulin o 10min, whe eas heo he hal wasinjec edwi hsaline.A e wa ds,micewe eeu hanizedandsac i iced o  hedissec iono li e o skele almuscle issues.Da ainFigu es4–6we epooled om wodisc e e expe imen susing wodi e en g oupso mice. P olongedadminis a iono lep olideimp o edglucose ole ance(Figu e4A,B)andinsulin sensi i i y(Figu e4C,D)inobesemice.Consis en ly,glucosedecaydu ing heip‐ITTdu ing he i s  30minwasimp o edin helep olide‐ ea edg oup(Figu e4E).Fu he mo e, heinsulinsensi i i y index(%S)wassigni ican lyinc eased(Figu e4F),whichwaspa alleledwi ha educedHOMA‐IR index(p=0.06;Figu e4G),al houghbodyweigh wasno signi ican lydec eased(Figu e4H).In addi ion, as ingandnon‐ as ingplasmainsulinle elswe e educedinlep olide‐ ea edg oupmice (Figu e4I,J),whichisconsis en wi h heno iono imp o inginsulinsensi i i y.Finally,plasma iglyce idele elswe esigni ican ly educedinobesemice ea edwi hlep olide(Figu e4K),which nicelyco ela eswi ha30% educ ioninli e  iglyce idecon en inlep olide‐ e sus ehicle‐ ea ed mice(Figu e4L).Taken oge he , heseda ademons a e ha ch oniclep olideadminis a ion coun e ac edinsulin esis anceandimp o edlipidme abolismindie ‐inducedobesemice. Togaininsigh in o hemolecula mechanismsbywhichlep olideimp o edinsulin esis ance, weanalyzedle elso ci cula ingp o‐in lamma o ycy okines.AsshowninFigu e5,ch oniclep olide ea men wasassocia edwi hanon‐s a is ically‐signi ican dec easeinbloodle elso IL‐1βand TNF‐α,bu IL‐6le els emainedunchanged(Figu e5A–C).Likewise,lep olidesigni ican ly educed heweigh gaindu ing helas weeko  he ea men (Figu e5D),inpa allelwi h educedplasma lep inle elsinobesemice(Figu e5F).  AC B DE Vehicle Lep olide 0 100 200 300 * Vehicle Lep olide 0 2000 4000 6000 * Vehicle Lep olide 0.0 0.5 1.0 1.5 2.0 * F Glucose (mg/dL) AUC (A bi a y Uni s) Vehicle Lep olide 0 10000 20000 30000 40000 50000 * Figu e 4. Con . Ma . D ugs 2017,15, 289 9 o 14 Ma .D ugs2017,15,289 9o 14  Figu e4.P olongedlep olideadminis a ionimp o esinsulinsensi i i yinap eclinicalmodelo  insulin esis ance.C57BL6Jmalemicewe e edanHFD o  enweeks.Thelas  ou weeks,micewe e injec edin ape i oneallywi hlep olideo salineonceaday.A e wa ds,insulinsensi i i yand plasmale elso insulinand iglyce ideswe eassessed.(A)Glucose ole ance es o mice ea ed wi h ehicleo 0.1mg/kglep olide.(B)A eaunde  hecu eo  heip‐GTT.Glucose ole ance imp o edinlep olide‐compa ed o ehicle‐ ea edmice.(C)Insulin ole ance es o mice ea ed wi h ehicleo 0.1mg/kglep olide.(D)A eaunde  hecu eo  heip‐ITT.Insulinsensi i i y imp o edinlep olide‐ compa ed o ehicle‐ ea edmice.(E)Glucosedecaya e  i s 30mino  insulininjec iondu ingip‐ITT.(F)Insulinsensi i i yindex(%S).(G)HOMAindex.Insulinsensi i i y andHOMAindexesshowedimp o edinsulinsensi i i yinHFD+lep olidemice.(H)Fas ingbody weigh .(I)Fas ingand(J)non‐ as ingplasmainsulinle els.Insulinle elswe enon‐signi ican ly dec eased.(K)Fas ingplasma iglyce idesle elswe edec easedinmice ea edwi hlep olide. (L)Li e TG( iglyce ide)con en wasdec easedinpa allelwi hplasma iglyce idele els.Values a e hemeans±S.E.M.o n=12pe g oup.*p<0.05 e sus ehiclebyS uden ’s ‐ es ;$p<0.05 e sus SDbyANOVA. To u he in es iga e heimpac o p olongedadminis a iono lep olideonobesemice, he ac i a iono  hein acellula insulinsignalingpa hwaywasassessedin heli e andskele almuscle o obesemice.AsshowninFigu e6,lep olideimp o eda basalandinsulin‐s imula edcondi ions, wi h hephospho yla iono PKBinli e andskele almuscle issue.These esul sa eingood ag eemen wi h hee ec o lep olideoninsulinsensi i i yinobesemice. H G Fas ing Insulin (mg/dL) Non-Fas ing Insulin (mg/dL) Vehicle Lep olide 0 50 100 150 * I J Li e TG (mg/g li e ) HOMA2 IR Weigh Fas ing (g) SD Vehicle Lep olide 0 10 20 30 40 $$ KL Figu e 4. P olonged lep olide adminis a ion imp o es insulin sensi i i y in a p eclinical model o insulin esis ance. C57BL6J male mice we e ed an HFD o en weeks. The las ou weeks, mice we e injec ed in ape i oneally wi h lep olide o saline once a day. A e wa ds, insulin sensi i i y and plasma le els o insulin and iglyce ides we e assessed. ( A ) Glucose ole ance es o mice ea ed wi h ehicle o 0.1 mg/kg lep olide. ( B ) A ea unde he cu e o he ip-GTT. Glucose ole ance imp o ed in lep olide- compa ed o ehicle- ea ed mice. ( C ) Insulin ole ance es o mice ea ed wi h ehicle o 0.1 mg/kg lep olide. ( D ) A ea unde he cu e o he ip-ITT. Insulin sensi i i y imp o ed in lep olide- compa ed o ehicle- ea ed mice. ( E ) Glucose decay a e i s 30 min o insulin injec ion du ing ip-ITT. ( F ) Insulin sensi i i y index (%S). ( G ) HOMA index. Insulin sensi i i y and HOMA indexes showed imp o ed insulin sensi i i y in HFD + lep olide mice. ( H ) Fas ing body weigh . ( I ) Fas ing and ( J ) non- as ing plasma insulin le els. Insulin le els we e non-signi ican ly dec eased. ( K ) Fas ing plasma iglyce ides le els we e dec eased in mice ea ed wi h lep olide. ( L ) Li e TG ( iglyce ide) con en was dec eased in pa allel wi h plasma iglyce ide le els. Values a e he means ± S.E.M. o n= 12 pe g oup. * p< 0.05 e sus ehicle by S uden ’s - es ; $p< 0.05 e sus SD by ANOVA. To u he in es iga e he impac o p olonged adminis a ion o lep olide on obese mice, he ac i a ion o he in acellula insulin signaling pa hway was assessed in he li e and skele al muscle o obese mice. As shown in Figu e 6, lep olide imp o ed a basal and insulin-s imula ed condi ions, wi h he phospho yla ion o PKB in li e and skele al muscle issue. These esul s a e in good ag eemen wi h he e ec o lep olide on insulin sensi i i y in obese mice.